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Sample records for igm enzyme immunoassay

  1. Enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence.

    PubMed Central

    Crouch, C F

    1995-01-01

    AIMS--To evaluate the clinical performance of enzyme immunoassays for IgG and IgM antibodies to Toxoplasma gondii based on enhanced chemiluminescence. METHODS--Classification of routine clinical samples from the originating laboratories was compared with that obtained using the chemiluminescence based assays. Resolution of discordant results was achieved by testing in alternative enzyme immunoassays (IgM) or by an independent laboratory using the dye test (IgG). RESULTS--Compared with resolved data, the IgM assay was found to be highly specific (100%) with a cut off selected to give optimal performance with respect to both the early detection of specific IgM and the detection of persistent levels of specific IgM (sensitivity 98%). Compared with resolved data, the IgG assay was shown to have a sensitivity and a specificity of 99.4%. CONCLUSIONS--The Amerlite Toxo IgM assay possesses high levels of sensitivity and specificity. Assay interference due to rheumatoid factor like substances is not a problem. The Amerlite Toxo IgG assay possesses good sensitivity and specificity, but is less sensitive for the detection of seroconversion than methods detecting both IgG and IgM. PMID:7560174

  2. Evaluation of enzyme immunoassay for anti-HBc IgM in the diagnosis of acute hepatitis B virus infection.

    PubMed

    Govindarajan, S; Ashcavai, M; Chau, K H; Nevalainen, D E; Peters, R L

    1984-09-01

    Corzyme-MTM (Abbott Laboratories, North Chicago, IL), a newly introduced kit for the measurement of serum IgM antihepatitis B core antigen by enzyme immunoassay, was evaluated for the diagnosis of acute B-viral hepatitis (AVH-B). The study included 175 acute viral hepatitis patients with transient hepatitis B surface antigen (HBsAg). Sera from 160 were tested on multiple occasions until their HBsAg cleared. IgM anti-HBc was found in 171 of 175 patients (98.4%) during the acute phase. The serum samples from 42 patients with liver biopsy-proven chronic active hepatitis, type B (CAH-B), and 18 patients with persistent hepatitis, type B (PH-B), were analyzed for the presence of IgM anti-HBc, using the same technic. None of the sera from 42 patients with CAH-B and only 2 of the 18 patients with PHB had IgM anti-HBc. Thus, the measuring IgM anti-HBc using Corzyme-M kit is helpful in the diagnosis of AVH-B and in the discrimination of acute from chronic HBV infections. PMID:6380271

  3. Diagnostic Accuracy of the InBios Scrub Typhus Detect Enzyme-Linked Immunoassay for the Detection of IgM Antibodies in Northern Thailand

    PubMed Central

    Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Nawtaisong, Pruksa; Kantipong, Pacharee; Laongnualpanich, Achara; Day, Nicholas P. J.

    2015-01-01

    In this study, we examined the diagnostic accuracy of the InBios Scrub Typhus Detect IgM enzyme-linked immunosorbent assay (ELISA) and determined the optimal diagnostic optical density (OD) cutoffs for screening and diagnostic applications based on prospectively collected, characterized samples from undifferentiated febrile illness patients in northern Thailand. Direct comparisons with the serological gold standard, indirect immunofluorescence assay (IFA), revealed strong statistical correlation of ELISA OD values and IFA IgM titers. Determination of the optimal ELISA cutoff for seroepidemiology or screening purposes compared to the corresponding IFA reciprocal titer of 400 as previously described for Thailand was 0.60 OD, which corresponded to a sensitivity (Sn) of 84% and a specificity (Sp) of 98%. The diagnostic performance against the improved and more-stringent scrub typhus infection criteria (STIC), correcting for low false-positive IFA titers, resulted in an Sn of 93% and an Sp of 91% at an ELISA cutoff of 0.5 OD. This diagnostic ELISA cutoff corresponds to IFA reciprocal titers of 1,600 to 3,200, which greatly reduces the false-positive rates associated with low-positive IFA titers. These data are in congruence with the recently improved serodiagnostic positivity criteria using the Bayesian latent class modeling approach. In summary, the InBios Scrub Typhus Detect IgM ELISA is affordable and easy-to-use, with adequate diagnostic accuracy for screening and diagnostic purposes, and should be considered an improved alternative to the gold standard IFA for acute diagnosis. For broader application, regional cutoff validation and antigenic composition for consistent diagnostic accuracy should be considered. PMID:26656118

  4. Homogeneous enzyme immunoassay for netilmicin.

    PubMed Central

    Wenk, M; Hemmann, R; Follath, F

    1982-01-01

    A newly developed homogeneous enzyme immunoassay for the determination of netilmicin in serum was evaluated and compared with a radioenzymatic assay. A total of 102 serum samples from patients treated with netilmicin were measured by both methods. This comparison showed an excellent correlation (r = 0.993). The enzyme immunoassay has proved to be precise, accurate, and specific. Because of its rapidity and the ease of performance, this method is a useful alternative to current assays for monitoring serum netilmicin concentrations. PMID:6760807

  5. Enzyme immunoassays in diagnostic medicine

    PubMed Central

    Voller, A.; Bidwell, D. E.; Bartlett, Ann

    1976-01-01

    Serological methods are playing an increasingly important role in the diagnosis and epidemiological assessment of diseases. Simple, inexpensive methods for large-scale application are urgently needed. The enzyme immunoassay methods developed recently and reviewed here hold great promise for application in a wide variety of conditions. Under laboratory conditions they can be as sensitive as radio-immunoassay, but they can also be adapted as simple field screening procedures. These methods are based on the use of antibodies or antigens that are linked to an insoluble carrier surface. This is then used to “capture” the relevant antigen or antibody in the test solution and the complex is detected by means of an enzyme-labelled antibody or antigen. The degradation of the enzyme substrate, measured photometrically, is proportional to the concentration of the unknown “antibody” or “antigen” in the test solution. The application of these techniques to endocrinology, immunopathology, haematology, microbiology, and parasitology is reviewed. PMID:1085667

  6. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, Matthew R.; Bednarski, Mark D.; Gruber, Patrick R.

    1997-01-01

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups.

  7. Enzyme linked immunoassay with stabilized polymer saccharide enzyme conjugates

    DOEpatents

    Callstrom, M.R.; Bednarski, M.D.; Gruber, P.R.

    1997-11-25

    An improvement in enzyme linked immunoassays is disclosed wherein the enzyme is in the form of a water soluble polymer saccharide conjugate which is stable in hostile environments. The conjugate comprises the enzyme which is linked to the polymer at multiple points through saccharide linker groups. 19 figs.

  8. Neutralization enzyme immunoassay for influenza virus.

    PubMed Central

    Benne, C A; Harmsen, M; De Jong, J C; Kraaijeveld, C A

    1994-01-01

    A neutralization enzyme immunoassay (N-EIA) was developed for the detection of antibody titer rises in sera of patients infected with influenza A (H3N2) virus. In this N-EIA, a selected strain of influenza A (H3N2) virus was added to monolayers of LLC-MK2 cells in microtiter plates. After 24 h, the replicated virus could be demonstrated with a virus-specific enzyme-labeled monoclonal antibody. Preincubation of the influenza virus with convalescent-phase sera of patients infected with influenza A (H3N2) virus resulted 1 day later in decreased absorbance values that could be used for calculation of neutralization titers. From use of paired serum samples from 10 patients with a history of flu-like symptoms, the results obtained with N-EIA correlated well (r = 0.83) with those of the standard hemagglutination inhibition test. PMID:8027355

  9. Application of enzyme immunoassays to coagulation testing.

    PubMed

    Amiral, J; Adalbert, B; Adam, M

    1984-09-01

    Enzyme immunoassays are very useful for the detection of low concentrations of coagulation proteins and pathological markers in plasma. Analytes in the ng/mL range are measurable with good reproducibility with intra- and interassay CVs of less than 5% to 10%. "Sandwich" methods have been developed for von Willebrand factor (plasma concentration about 8 micrograms/mL, Factor IX (5 micrograms/mL), protein C (4 micrograms/mL), and Factor X (10 micrograms/mL). However, this technique is only suitable for macromolecules; for low-molecular-mass peptides such as fibrinopeptide A a competitive method is used. Normal concentrations of fibrinopeptide A are below 3 ng/mL, with greater values suggesting in vivo generation of thrombin; thus this test is quite useful in detecting thrombosis. Reagents for both the sandwich and competitive methods are commercially available and cost effective, and have a longer shelf-life than those for radioimmunoassays. PMID:6380814

  10. Enzyme immunoassay for carminic acid in foods.

    PubMed

    Yoshida, A; Takagaki, Y; Nishimune, T

    1995-01-01

    A competitive enzyme immunoassay (EIA) for carminic acid was investigated. Monoclonal anticarminic acid antibody was obtained from A/J mice immunized with carminic acid-human immunoglobulin G (IgG) conjugate. Carminic acid was extracted with distilled water from beverage, jelly, candy, pasta sauce, yogurt, or ice cream samples. Ham or fish paste samples were digested with pronase, then carminic acid was extracted from samples with sodium hydroxide solution. The extract was diluted more than 10-fold with 1% gelatin in borate buffer solution. Microtiter plates were coated with carminic acid-bovine serum albumin (BSA) conjugate or just BSA. Goat anti-mouse IgG(H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 0.3-10 ng/mL, and the detection limit was 0.2 micrograms/g original sample. Recoveries of carminic acid by this assay were > 95% for milk beverage and jelly, and > 85% for yogurt and fish paste. Carminic acid was detected in 7 of 26 red-colored commercial food products and ranged from 3.5 to 356 micrograms/g. This EIA system also responded to the structural analogue of carminic acid, laccaic acid. PMID:7756895

  11. DETECTION OF ROTAVIRUS WITH A NEW POLYCLONAL ANTIBODY ENZYME IMMUNOASSAY (ROTAZYME 2) AND A COMMERCIAL LATEX AGGLUTINATION TEXT (ROTALEX): COMPARISON WITH A MONOCLONAL ANTIBODY ENZYME IMMUNOASSAY

    EPA Science Inventory

    A total of 176 human fecal specimens were examined for the presence of rotavirus using four different assays: a monoclonal antibody enzyme immunoassay; the original polyclonal antibody enzyme immunoassay marketed by Abbott Laboratories, Chicago, IL (Rotazyme I); a modification of...

  12. Cross-Reactivity of Rapid Salmonella Typhi IgM Immunoassay in Dengue Fever Without Co-Existing Infection

    PubMed Central

    Ali, Farhan; Satti, Siddique Akbar

    2015-01-01

    Introduction: Dengue fever is endemic in developing nations worldwide with as many as 500,000 annual cases of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). A prompt and accurate diagnosis early in the disease course is essential for prompt identification and treatment of severe complications of the dengue virus infection (DVI). We identified cross-reactivity of a rapid IgM test for typhoid fever in patients with febrile illnesses that were determined to be due to dengue virus. Methods: All patients with documented DVI during a recent epidemic in Pakistan also underwent diagnostic testing for Salmonella enterica serovar Typhi. The diagnosis of DVI was made based on clinical findings and the positive results for dengue non-structural protein 1 antigen (NS1Ag) and/or dengue IgM antibody (anti-D IgM) during the acute phase of febrile illness. Patients with positive test results for Salmonella typhi (S. Typhi) IgM also had their blood cultures done. Results: In the group of 322 patients with clinical and serological evidence of DVI, 107 also tested positive for S. Typhi IgM. Blood cultures were negative for S. Typhi bacteria in all patients. Principal disease features included fever, headache, myalgia, retro-orbital pain, and a rash accompanied by thrombocytopenia and leukopenia. Comparisons of clinical and routine laboratory findings between the S. Typhi-positive and negative groups showed no significant differences. Patients testing positive for both NS1Ag and anti-D IgM were significantly more likely to test positive for S. Typhi IgM, even in the absence of typhoid fever. No routine antibiotics were used and all patients survived. Conclusion: One-third of a large group of patients with primary DVI also demonstrated false positive results for typhoid fever. Cross-reactivity of a rapid immunoassay for typhoid fever has not been previously reported in DVI or any other flavivirus infections. Until these findings can be further

  13. Ability of TESTPACK ROTAVIRUS enzyme immunoassay to diagnose rotavirus gastroenteritis.

    PubMed Central

    Chernesky, M; Castriciano, S; Mahony, J; Spiewak, M; Schaefer, L

    1988-01-01

    TESTPACK ROTAVIRUS, a simple 10-min enzyme immunoassay, was compared with electron microscopy and Pathfinder enzyme immunoassay on feces from 172 patients of various ages with gastroenteritis. The percent sensitivities and specificities before blocking with antiserum were as follows: TESTPACK, 100% sensitivity and 99% specificity; Pathfinder, 95% sensitivity and 98% specificity. After blocking, the sensitivity and specificity, respectively, were 100% and 100% for TESTPACK and 95% and 99% for Pathfinder. TESTPACK ROTAVIRUS was more sensitive, but not significantly, than Pathfinder (P greater than 0.1) and the direct electron microscopy technique (P greater than 0.1). PMID:3069866

  14. DETECTION OF NORWALK VIRUS IN STOOLS BY ENZYME IMMUNOASSAY

    EPA Science Inventory

    The development of a solid-phase microtiter enzyme immunoassay (EIA) for detection of Norwalk virus antigen in stool samples is described. The EIA was compared with a previously developed radioimmunoassay (RIA) for detection of Norwalk virus antigen in stools obtained from 30 vol...

  15. Application Of Laser Fluorimetry To Enzyme-Linked Immunoassay

    NASA Astrophysics Data System (ADS)

    Hinsberg, William D.; Milby, Kristin H.; Lidofsky, Steven D.; Zare, Richard N.

    1981-09-01

    An enzyme-linked sandwich immunoassay for insulin is described. Horseradish peroxidase is employed as an enzyme label for antibody, and enzyme activity is measured via the fluorogenic substrate, p-hydroxyphenylacetic acid. The product is detected by excitation of fluorescence with the 325 nm line of a cw helium-cadmium ion laser on-line with reverse phase high performance liquid chromatography. The method requires a total incubation time of 45 minutes, and the limit of insulin detection is 1.1 μU/ml (6.6 pM). This assay is applicable to the analysis of human serum samples.

  16. Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

    NASA Astrophysics Data System (ADS)

    Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

    1999-04-01

    Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

  17. Evaluation of the Captia enzyme immunoassays for detection of immunoglobulins G and M to Treponema pallidum in syphilis.

    PubMed Central

    Lefevre, J C; Bertrand, M A; Bauriaud, R

    1990-01-01

    Two new enzyme-linked immunosorbent assays (ELISA), one for the measurement of immunoglobulin G (IgG) (Captia Syphilis-G) and one for the measurement of IgM (Captia Syphilis-M), were evaluated for detecting antibodies to Treponema pallidum. Serum samples from 169 patients, 96 with various stages of untreated syphilis, 63 with treated syphilis, and 10 who were noninfected, were investigated. All sera were also examined by traditional treponemal and cardiolipin tests and by the fluorescent treponemal antibody absorption (FTA-ABS) test for 19S(IgM). The overall sensitivity of Captia Syphilis-G was 98.3%. The IgG ELISA was very sensitive (100%) in all stages of untreated syphilis, except in primary syphilis (82%). In all diagnostic groups of syphilis, the reactivity of Captia Syphilis-M was similar to that of the 19S(IgM) FTA-ABS test, except in reinfections, in which the IgM capture ELISA was less sensitive. False-positive IgM capture ELISA results were not found in the 10 neonates born to mothers adequately treated for syphilis. However, of six serum samples containing rheumatoid factor, two were reactive in the Captia Syphilis-M test but not in the 19S(IgM) FTA-ABS test. This indicated that the specificity of the IgM capture ELISA was not absolute. All serum samples from treated patients were reactive in the IgG ELISA, but only 15 samples were reactive in the IgM capture ELISA, which appeared to be as effective as the 19S(IgM) FTA-ABS test in monitoring the effect of treatment. Simultaneous measurement of IgG and IgM antibodies for T. pallidum by the Captia immunoassays appears to be an efficient and simple method for confirming the diagnosis of syphilis as well as for indicating whether active disease is present. PMID:2203809

  18. Immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays are analytical methods that employ antibodies or molecules derived from antibodies for the essential binding reactions. The choice of immunoassay system for food safety analysis depends on the analyte, the matrix, and the requirements of the analysis (speed, throughput, sensitivity, spe...

  19. Immunoassays

    NASA Astrophysics Data System (ADS)

    Hsieh, Y.-H. Peggy

    Immunochemistry is a relatively new science that has developed rapidly in the last few decades. One of the most useful analytical developments associated with this new science is immunoassay. Originally immunoassays were developed in medical settings to facilitate the study of immunology, particularly the antibody-antigen interaction. Immunoassays now are finding widespread applications outside the clinical field because they are appropriate for a wide range of analytes ranging from proteins to small organic molecules. In the food analysis area, immunoassays are widely used for chemical residue analysis, identification of bacteria and viruses, and detection of proteins in food and agricultural products. Protein detection is important for determination of allergens and meat species content, seafood species identification, and detection of genetically modified plant tissues. While immunoassays of all formats are too numerous to cover completely in this chapter, there are several procedures that have become standard for food analysis because of their specificity, sensitivity, and simplicity.

  20. Immunoassay using /sup 125/I- or enzyme-labeled protein A and antigen-coated tubes

    SciTech Connect

    Gee, A.P.; Langone, J.J.

    1981-09-15

    Antigen-coated plastic tubes were used with /sup 125/I- or enzyme-labeled stapylococcal protein A in a general immunoassay method for antigens and haptens. Protein A reacts with immunoglobulin G(IgG) regardless of antibody specificity at sites distal to the antigen combining site and does not inhibit the immune reaction. It therefore serves as a general tracer and its use eliminates the need to purify and to label individual components for each assay. Macromolecular antigens were bound to polystyrene or polypropylene tubes by direct passive absorption. Haptens with free carboxyl groups were bound covalently to poly-L-lysine and these conjugates passively absorbed to the tube surface. Optimal assay conditions were established for the quantitative determination of immunoglobulins and the folate derivatives, methotrexate and 5-methyltetrahydrofolate, using /sup 125/I-labeled protein A or protein A labeled with alkaline phosphatase. The method has been used to estimate levels of IgG, IgA, Igm, and IgE in serum in volumes up to 1 ml.

  1. DEVELOPMENT OF A CLASS-SELECTIVE ENZYME IMMUNOASSAY FOR URINARY PHENOLIC GLUCURONIDES. (R825433)

    EPA Science Inventory

    Class-selective immunoassays for the measurement of glucuronides in human urine can aid evaluation of human exposure to complex mixtures of xenobiotics. Therefore, an enzyme immunoassay (EIA) for the group-selective detection of phenolic Rapid dioxin screening of milk and water by enzyme immunoassay

    SciTech Connect

    Harrison, R.O.; Carlson, R.E.; Shirkhan, H.

    1995-12-01

    A simple and easy to use enzyme immunoassay (EIA) system has been developed for rapid screening of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2378D). This EIA has been adapted to analysis of water and milk using an automated system for extraction of liquid samples. Water analysis can be performed directly following extraction and solvent exchange with no extract clean-up. The same automated system is used for milk extraction and the extract is then cleaned chromatographically using the automated FMS Dioxin-Prep{trademark} System. Sensitivity for 2378D in the EIA is approximately 100 pg per analysis. Thus sensitivity to 10 ppt 2378D (whole weight basis) in milk is possible using only 50 ml or less of sample and sensitivity to 0.1 ppt 2378D in water is possible using 1-2 liters of sample. Total time for sample preparation and analysis is about 3 hours for water and 4.5 hours for milk.

  2. Bioluminescent enzyme immunoassay for the detection of norovirus capsid antigen.

    PubMed

    Sakamaki, Nozomi; Ohiro, Yoshiyuki; Ito, Mitsuki; Makinodan, Mitsuru; Ohta, Tsubasa; Suzuki, Wataru; Takayasu, Susumu; Tsuge, Harufumi

    2012-12-01

    An ultrasensitive and fully automated bioluminescent enzyme immunoassay (BLEIA) was developed for the detection of norovirus (NV) capsid antigen. In the evaluation tests with recombinant virus-like particles, the BLEIA demonstrated broad reactivity against several NV genotypes (genotypes 1, 3, 4, 7, 8, and 12 in genogroup I [GI] and genotypes 1, 2, 3, 4, 5, 6, 12, and 13 in GII), a wide dose-response range from 0.25 pg/ml to 10,000 pg/ml, and good reproducibility with low coefficients of variation (CVs) (within-run CVs of <2.8%, between-day CVs of <3.7%). In the evaluation tests with NV-positive fecal samples, a good correlation (y = 0.66x - 3.21, r = 0.84) between the BLEIA and real-time quantitative reverse transcription-PCR was obtained. Furthermore, in the dilution test with NV specimens, the analytical sensitivity of NV was estimated to be 10(5) to 10(6) copies/g of fecal sample, indicating that the analytical sensitivity of the BLEIA is comparable to that of commercially available molecular methods. All assay steps are fully automated, the turnaround time is 46 min, and the throughput of the assay is 120 tests/h. These results indicate that the BLEIA is potentially useful for the rapid diagnosis of NV in epidemic and sporadic gastroenteritis. PMID:23081816

  3. Enzyme immunoassay for measuring 25-hydroxyvitamin D3 in serum.

    PubMed

    Lind, C; Chen, J; Byrjalsen, I

    1997-06-01

    We developed a rapid, competitive enzyme immunoassay (EIA) for measuring 25-hydroxyvitamin D3 [25(OH)D3] in serum. The EIA was based upon 25(OH)D3-3-hemisuccinate covalently coupled to secondary amino groups grafted onto the polystyrene surface of microtiter wells. Optimal coupling conditions were established, and we found that inclusion of 40 mumol/L chloramine T, an agent not previously described for use in coupling to these plates, resulted in both more reproducible coupling as well as more than a twofold increase in the coupling efficiency. Before EIA, 25(OH)D3 was extracted from the serum samples by acetonitrile, and the redissolved extract was incubated with polyclonal rabbit antibody raised against 1,25-dihydroxyvitamin D3-3-hemisuccinate conjugated to bovine serum albumin. Peroxidase-labeled antibody raised in goat against rabbit immunoglobulins was used for detection. The detection limit of the EIA was 4.4 micrograms/L; recovery 102%; on-plate CV 11%; within-run CV including extraction 12%, and between-run CV 15%. There was no clinically important cross-reactivity with other vitamin D metabolites, and results obtained by the EIA were compared with results obtained by a previously described RIA. PMID:9191544

  4. Optimization of condition for conjugation of enrofloxacin to enzymes in chemiluminescence enzyme immunoassay

    NASA Astrophysics Data System (ADS)

    Yu, Songcheng; Yu, Fei; Zhang, Hongquan; Qu, Lingbo; Wu, Yongjun

    2014-06-01

    In this study, in order to find out a proper method for conjugation of enrofloxacin to label enzymes, two methods were compared and carbodiimide condensation was proved to be better. The results showed that the binding ratio of enrofloxacin and alkaline phosphatase (ALP) was 8:1 and that of enrofloxacin and horseradish peroxidase (HRP) was 5:1. This indicated that conjugate synthesized by carbodiimide condensation was fit for chemiluminescence enzyme immunoassay (CLEIA). Furthermore, data revealed that dialysis time was an important parameter for conjugation and 6 days was best. Buffer to dilute conjugate had little effect on CLEIA. The storage condition for conjugates was also studied and it was shown that the conjugate was stable at 4 °C with no additive up to 30 days. These data were valuable for establishing CLEIA to quantify enrofloxacin.

  5. Determination of Babesia microti seroprevalence in blood donor populations using an investigational enzyme immunoassay

    PubMed Central

    Levin, Andrew E; Williamson, Phillip C; Erwin, James L; Cyrus, Sherri; Bloch, Evan M; Shaz, Beth H; Kessler, Debra; Telford, Sam R; Krause, Peter J; Wormser, Gary P; Ni, Xiaoyan; Wang, Haihong; Krueger, Neil X; Caglioti, Sally; Busch, Michael P

    2014-01-01

    Background Transfusion-transmitted babesiosis caused by Babesia microti has emerged as a significant risk to the US blood supply. This study estimated the prevalence of B. microti antibodies in blood donors using an investigational enzyme immunoassay (EIA). Study Design and Methods A peptide-based EIA that detects both immunoglobulin (Ig)G and IgM antibodies to B. microti was developed and validated. Donor samples randomly selected from areas defined as high-risk endemic, lower-risk endemic, and nonendemic for B. microti were deidentified and tested using the investigational EIA. Samples that were EIA repeat reactive were further tested by B. microti immunofluorescent assay (IFA), polymerase chain reaction (PCR) on red blood cell lysates, and peripheral blood smear examination. A random subset of 1272 samples from high-risk endemic areas was tested by IFA, PCR, and peripheral blood smear in parallel with EIA. Results Among 15,000 donations tested with the investigational B. microti EIA, EIA repeat-reactive rates were 1.08% (54/5000) in a high-risk endemic area, 0.74% (37/5000) in a lower-risk area, and 0.40% (20/5000) in a nonendemic area. After application of a revised cutoff, these values were reduced to 0.92%, (46/5000), 0.54% (27/5000), and 0.16% (8/5000). Overall concordance between EIA and IFA among donor samples was 99.34%. One seropositive sample was positive by PCR. Conclusion The seroprevalence of B. microti in blood donors in a high-risk area measured by an investigational EIA was approximately 1%. The EIA shows promise as an efficient high-throughput blood donor screening assay for B. microti. PMID:24995863

  6. Development of a synchronous enzyme-reaction system for a highly sensitive enzyme immunoassay.

    PubMed

    Inouye, Kuniyo; Ueno, Iori; Yokoyama, Shin-ichi; Sakaki, Toshiyuki

    2002-01-01

    A synchronous enzyme-reaction system using water-soluble formazan and a non-enzymatic electron mediator was developed and applied to an enzyme immunoassay (EIA). The reaction system consists of four steps: (I) dephosphorylation of NADP(+) to produce NAD(+) by alkaline phosphatase (ALP), (II) reduction of NAD(+) to produce NADH with oxidation of ethanol to yield acetaldehyde by alcohol dehydrogenase (ADH), (III) reduction of water-soluble tetrazolium salt (WST-1) to produce formazan by NADH via 1-methoxy-5-methyl-phenazinium methyl sulfate (PMS), and (IV) re-reduction of NAD(+) to produce NADH by ADH. During each cycle, one molecule of tetrazolium is converted to one molecule of formazan. The concentration of formazan during the reaction was given by second-order polynomials of the reaction time. Kinetic studies strongly suggested that the synchronous enzyme-reaction system had the potential to detect an analyte at the attomole level in EIA. On the basis of the kinetic studies, optimal conditions for EIA incorporating the synchronous system were examined. NADP(+) was purified thoroughly to remove minor traces of NAD(+) in the preparation, and an ADH preparation contaminated with the lowest level of ALP activity was used. When the synchronous system was applied to a sandwich-type EIA for human C-reactive protein, the protein was detected with a sensitivity of 50 attomole per well of a micro-titer plate (0.1 ml) in a 1-h reaction. In addition, EIA with water-soluble formazan showed a more quantitative and sensitive result than that with insoluble formazan. These findings indicated that the (WST-1)-PMS system introduced in this study has a great potential for highly sensitive enzyme immunoassay. PMID:11754740

  7. Enzyme immunoassay-based survey of prevalence of gentamicin in serum of marketed swine.

    PubMed

    Berkowitz, D B; Webert, D W

    1986-01-01

    Sera from 3182 swine from a national sampling were tested in the gentamicin enzyme immunoassay. Of the sera tested, 6 (0.19%) contained gentamicin. Only 1 serum may have been associated with muscle levels above the tolerance. During the survey, a single analyst processed 300 samples daily. The immunoassay survey was an effective and economical method of obtaining information on the prevalence of a residue. PMID:3522536

  8. Heparin interferes with the radioenzymatic and homogeneous enzyme immunoassays for aminoglycosides

    SciTech Connect

    Krogstad, D.J.; Granich, G.G.; Murray, P.R.; Pfaller, M.A.; Valdes, R.

    1981-07-01

    Heparin interferes with measurement of aminoglycosides in serum by biological, radioenzymatic, and homogeneous enzyme immunoassay techniques, but not with radioimmunoassay. At concentrations greater than or equal to 10/sup 5/ and greater than or equal to 3 X 10/sup 6/ USP units/L, respectively, it interferes with the radioenzymatic assay by inhibiting the gentamicin 3-acetyltransferase and kanamycin 6'-acetyltransferase enzymes used in the assay. It interferes with the homogeneous enzyme immunoassays for gentamicin and tobramycin (at concentrations greater than or equal to 10/sup 5/ and greater than or equal to10/sup 4/ USP units/L, respectively), but not with the commercially available homogeneous enzyme immunoassays for other drugs. Heparin interference with the homogeneous enzyme immunoassay for aminoglycosides requires both the heparin polyanion and glucose-6-phosphate dehydrogenase bound to a cationic aminoglycoside. This interference can be reproduced with dextran sulfate (but not dextran), and does not occur with free enzyme (glucose-6-phosphate dehydrogenase) alone. Heparin interference with these two assays and at concentrations that may be present in intravenous infusions or in seriously underfilled blood-collection tubes is described. (JMT)

  9. Semi-automated continuous-flow enzyme immunoassay for antiepileptic drugs in serum.

    PubMed

    Hayashi, S; Kurooka, S; Arisue, K; Kohda, K; Hayashi, C

    1983-10-01

    We have developed a semi-automated method for measuring five kinds of antiepileptic drugs in serum by successfully adapting commercial competitive-binding enzyme immunoassay kits (MARKIT; Dainippon) for use with a continuous-flow analyzer (Technicon AutoAnalyzer II equipped with a dialyzer). The free enzyme-labeled drug is automatically separated by a microfilter from the competitive immunoreaction mixture between labeled and unlabeled drug for anti-drug immunoglobulin coupled to bacterial cell walls. The concentrations of the antiepileptic drugs in serum samples can be determined by automated measurement of enzyme activity of the enzyme-labeled drugs. Results of the semi-automated method correlated well with those obtained by manual enzyme immunoassay, gas-liquid chromatography, and "high-pressure" liquid chromatography. The correlation coefficients were all greater than 0.95, showing the practicality of this method for therapeutic monitoring of antiepileptic drugs. PMID:6352086

  10. Buprenorphine detection in urine using liquid chromatography-high-resolution mass spectrometry: comparison with cloned enzyme donor immunoassay (ThermoFisher) and homogeneous enzyme immunoassay (immunalysis).

    PubMed

    Belsey, Sarah L; Couchman, Lewis; Flanagan, Robert J

    2014-09-01

    A sensitive liquid chromatographic-high-resolution mass spectrometric (LC-HR-MS) assay for buprenorphine and its urinary metabolites has been developed that requires minimal sample preparation. The results obtained have been compared with those given by (i) cloned enzyme donor immunoassay (CEDIA) and (ii) homogeneous enzyme immunoassay (HEIA) in the analysis of patient urines submitted for buprenorphine analysis. Centrifuged urine (100 µL) was diluted with internal standard solution (25 µL) + LC eluent (875 µL), and 50 µL of the prepared sample were analyzed (Accucore Phenyl-Hexyl column). MS detection was in alternating positive and negative mode using heated electrospray ionization (ThermoFisher Q Exactive). Intra- and inter-assay accuracy and precision were 104-128 and <11%, respectively, at 5 µg/L. Limits of detection were 1.3 µg/L (buprenorphine, norbuprenorphine and buprenorphine glucuronide) and 2.5 µg/L (norbuprenorphine glucuronide). Immunoassay sensitivity and selectivity were 97 and 100% (HEIA) and 99 and 84% (CEDIA), respectively, compared with LC-HR-MS. In 120 patient urines, norbuprenorphine glucuronide was easily the most abundant analyte except when adulteration with buprenorphine had occurred. The median immunoreactive buprenorphine species present (unhydrolysed urine) were 7.5 and 13% for HEIA and CEDIA, respectively. However, codeine, dihydrocodeine, morphine and morphine-3-glucuronide did not interfere in the HEIA assay. PMID:24925983

  11. ENZYME-LINKED IMMUNOASSAYS FOR THE DETECTION OF MICROBIAL ANTIGENS AND THEIR ANTIBODIES

    EPA Science Inventory

    The advantages of enzyme-immunoassay (EIA) over radioactive assay techniques are mainly convenience in use, in that the labelled immunoreagents are stable for long periods, and the precautions and disposal procedures required for radioisotopes are unnecessary. In addition, the us...

  12. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay.

    PubMed

    Blacksell, Stuart D; Lim, Cherry; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L; Paris, Daniel H; Limmathurotsakul, Direk; Day, Nicholas P J

    2016-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  13. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay

    PubMed Central

    Blacksell, Stuart D.; Tanganuchitcharnchai, Ampai; Jintaworn, Suthatip; Kantipong, Pacharee; Richards, Allen L.; Day, Nicholas P. J.

    2016-01-01

    The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus. PMID:27008880

  14. Rapid and Sensitive Chemiluminescent Enzyme Immunoassay for the Determination of Neomycin Residues in Milk.

    PubMed

    Luo, Peng Jie; Zhang, Jian Bo; Wang, Hua Li; Chen, Xia; Wu, Nan; Zhao, Yun Feng; Wang, Xiao Mei; Zhang, Hong; Zhang, Ji Yue; Zhu, Lei; Jiang, Wen Xiao

    2016-05-01

    Immunoassays greatly contribute to veterinary drug residue analysis. However, there are few reports on detecting neomycin residues by immunoassay. Here, a rapid and sensitive chemiluminescent enzyme immunoassay (CLIEA) was successfully developed for neomycin residue analysis. CLIEA demonstrated good cross-reactivity for neomycin, and the IC50 value was 2.4 ng/mL in buffer. The average recovery range was 88.5%-105.4% for spiked samples (10, 50, and 100 μg/kg), and the coefficient of variation was in the range of 7.5%-14.5%. The limit of detection of CLEIA was 9.4 μg/kg, and this method was compared with the liquid chromatography-tandem mass spectrometry method using naturally contaminated samples, producing a correlation coefficient of >0.95. We demonstrate a reliable CLIEA for the rapid screening of neomycin in milk. PMID:27353712

  15. Evaluation of a recombinant rhoptry protein 2 enzyme-linked immunoassay for the diagnosis of toxoplasmosis acquired during pregnancy

    PubMed Central

    Capobiango, Jaqueline Dario; Pagliari, Sthefany; Pasquali, Aline Kuhn Sbruzzi; Nino, Beatriz; Ferreira, Fernanda Pinto; Monica, Thaís Cabral; Tschurtschenthaler, Nely Norder; Navarro, Italmar Teodorico; Garcia, João Luis; Mitsuka-Breganó, Regina; Reiche, Edna Maria Vissoci

    2015-01-01

    The aim of this study was to evaluate an enzyme-linked immunoassay with recombinant rhoptry protein 2 (ELISA-rROP2) for its ability to detectToxoplasma gondii ROP2-specific IgG in samples from pregnant women. The study included 236 samples that were divided into groups according to serological screening profiles for toxoplasmosis: unexposed (n = 65), probable acute infection (n = 48), possible acute infection (n = 58) and exposed to the parasite (n = 65). When an indirect immunofluorescence assay forT. gondii-specific IgG was considered as a reference test, the ELISA-rROP2 had a sensitivity of 61.8%, specificity of 62.8%, predictive positive value of 76.6% and predictive negative value of 45.4% (p = 0.0002). The ELISA-rROP2 reacted with 62.5% of the samples from pregnant women with probable acute infection and 40% of the samples from pregnant women with previous exposure (p = 0.0180). Seropositivity was observed in 50/57 (87.7%) pregnant women with possible infection. The results underscored that T. gondii rROP2 is recognised by specific IgG antibodies in both the acute and chronic phases of toxoplasmosis acquired during pregnancy. However, the sensitivity of the ELISA-rROP2 was higher in the pregnant women with probable and possible acute infections and IgM reactivity. PMID:26517651

  16. Influence of hydrophobic and hydrophilic spacer-containing enzyme conjugates on functional parameters of steroid immunoassay.

    PubMed

    Nara, Seema; Tripathi, Vinay; Chaube, Shail K; Rangari, Kiran; Singh, Harpal; Kariya, Kiran P; Shrivastav, Tulsidas G

    2008-02-01

    Introduction of spacers in coating steroid antigen or enzyme conjugates or immunogen is known to exert an influence on the sensitivity of steroid enzyme immunoassays. We have introduced hydrophobic and hydrophilic spacers between enzyme and steroid moieties and studied their effects on functional parameters of enzyme immunoassays, using cortisol as a model steroid. Cortisol-3-O-carboxymethyloxime-bovine serum albumin (F-3-O-CMO-BSA) was used as immunogen to raise the antiserum in New Zealand white rabbits. Three enzyme conjugates were prepared using cortisol-21-hemisuccinate (F-21-HS) as carboxylic derivative of cortisol and horseradish peroxidase (HRP) as an enzyme label. These were F-21-HS-HRP (without spacer), F-21-HS-adipic acid dihydrazide-HRP (adipic acid dihydrazide as hydrophobic spacer), and F-21-HS-urea-HRP (urea as hydrophilic spacer). The influence of hydrophobic and hydrophilic spacers on the functional parameters of assays such as lower detection limit, ED50, and specificity was studied with reference to enzyme conjugate without spacer. The results of the present investigation revealed that the presence of a hydrophilic spacer in the enzyme conjugate decreases the lower detection limit, decreases the ED50, and marginally improves the specificity of assays. These improvements in functional parameters of assays may be due to the decreased magnitude of the overall hydrophobic interactions existing between the spacer in enzyme conjugate and the antigen binding site of the antibody. PMID:18023401

  17. Improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles

    NASA Astrophysics Data System (ADS)

    Syrvatka, Vasyl J.; Slyvchuk, Yurij I.; Rozgoni, Ivan I.; Gevkan, Ivan I.; Overchuk, Marta O.

    2014-02-01

    Modern routine enzyme immunoassays for detection and quantification of biomolecules have several disadvantages such as high cost, insufficient sensitivity, complexity and long-term execution. The surface plasmon resonance of silver nanoparticles gives reasons of creating new in the basis of simple, highly sensitive and low cost colorimetric assays that can be applied to the detection of small molecules, DNA, proteins and pollutants. The main aim of the study was the improving of enzyme immunoassay for detection and quantification of the target molecules using silver nanoparticles. For this purpose we developed method for synthesis of silver nanoparticles with hyaluronic acid and studied possibility of use these nanoparticles in direct determination of target molecules concentration (in particular proteins) and for improving of enzyme immunoassay. As model we used conventional enzyme immunoassays for determination of progesterone and estradiol concentration. We obtained the possibility to produce silver nanoparticles with hyaluronan homogeneous in size between 10 and 12 nm, soluble and stable in water during long term of storage using modified procedure of silver nanoparticles synthesis. New method allows to obtain silver nanoparticles with strong optical properties at the higher concentrations - 60-90 μg/ml with the peak of absorbance at the wavelength 400 nm. Therefore surface plasmon resonance of silver nanoparticles with hyaluronan and ultraviolet-visible spectroscopy provide an opportunity for rapid determination of target molecules concentration (especial protein). We used silver nanoparticles as enzyme carriers and signal enhancers. Our preliminary data show that silver nanoparticles increased absorbance of samples that allows improving upper limit of determination of estradiol and progesterone concentration.

  18. [The validation of an enzyme immunoassay for the detection of antibodies to bovine leukosis virus].

    PubMed

    Olechnowitz, A F; Miko, A; Koepernik, H; Starick, E; Fröbe, I

    1990-01-01

    Validation of an enzyme immuno-assay for detection of antibodies against bovine leucosis virus is described in this paper. Internal standardisation of the test was done by means of a negative control serum. With absolute extinction of the negative control serum between 100 and 200 mE, a serum sample is rated positive, if its extinction is 1.5 times above the control. The methodological sensitivity of the enzyme immuno-assay described has proved to be four times as high as that of the immunodiffusion test. The results recorded at five diagnostic laboratories suggested a sensitivity of the test of 97.6 percent (92.1 to 100 percent) and a specificity of 98.1 percent (94.4 to 100 percent). The high efficiency of the test can be confirmed by immunoblotting. PMID:2167052

  19. Microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species in blood.

    PubMed Central

    Fujita, S; Lasker, B A; Lott, T J; Reiss, E; Morrison, C J

    1995-01-01

    We developed a microtitration plate enzyme immunoassay to detect PCR-amplified DNA from Candida species. Nucleotide sequences derived from the internal transcribed spacer (ITS) region of fungal rDNA were used to develop species-specific oligonucleotide probes for Candida albicans, C. tropicalis, C. parapsilosis, and C. krusei. No cross-hybridization was detected with any other fungal, bacterial, or human DNAs tested. In contrast, a C. (Torulopsis) glabrata probe cross-reacted with Saccharomyces cerevisiae DNA but with no other DNAs tested. Genomic DNA purified from C. albicans blastoconidia suspended in blood was amplified by PCR with fungus-specific universal primers ITS3 and ITS4. With the C. albicans-specific probe labeled with digoxigenin, a biotinylated capture probe, and streptavidin-coated microtitration plates, amplified DNA from a few as two C. albicans cells per 0.2 ml of blood could be detected by enzyme immunoassay. PMID:7790469

  1. Progress in enzyme immunoassays: production of reagents, experimental design, and interpretation*

    PubMed Central

    Kurstak, Edouard

    1985-01-01

    Enzyme immunoassays represent in many cases the preferred procedure for the detection of antigens or corresponding antibodies. However, many of the current procedures are performed suboptimally. This article reviews the available designs, auxiliary recognition systems, production and purification of antibodies, conjugation procedures, solid-phase materials, recording and interpretation of results, and quality control and standardization of procedures to improve the reproducibility of tests. PMID:3910300

  2. Cytomegalovirus Antibody in Cerebrospinal Fluid of Schizophrenic Patients Detected by Enzyme Immunoassay

    NASA Astrophysics Data System (ADS)

    Fuller Torrey, E.; Yolken, Robert H.; Winfrey, C. Jack

    1982-05-01

    By means of enzyme immunoassay techniques to detect the presence of antibody to cytomegalovirus, the cerebrospinal fluid of 178 patients with schizophrenia, 17 patients with bipolar disorders, and 11 other psychiatric patients was compared with that of 79 neurological patients and 41 normal control subjects. The cerebrospinal fluid of 20 of the schizophrenic patients and 3 of the patients with bipolar disorders showed significant increases in immunoglobulin M antibody to cytomegalovirus; no difference was found in patients on or off psychotropic medications.

  3. Automation of the enzyme immunoassay for the serodiagnosis of infectious diseases in cattle

    SciTech Connect

    Seawright, G.L.; Sanders, W.M.; Bryson, M.

    1980-01-01

    The enzyme immunoassay (EIA) is a versatile and highly sensitive new tool that can be used to detect a wide variety of infectious diseases or toxic agents and other low molecular weight compound. The paper describes the present state of development of the EIA-modified Technicon Autoanalyzer II instrumentation, standardization of quality control criteria for the automated EIA and standardization of the diagnostic decision process. (ACR)

  4. Incorporation of different bridge length linkers in enzyme and its use in the preparation of enzyme conjugates for immunoassay.

    PubMed

    Shrivastav, Tulsidas G

    2004-01-01

    An enzyme horseradish peroxidase (HRP), as a starting material, has been used to introduce different bridge length linkers, and its use in the preparation of enzyme conjugates for immunoassay is described. HRP was conjugated to adipic acid dihydrazide (ADH), gamma amino butyric acid (GABA), followed by ADH and 6-amino caproic acid (6ACA) followed by ADH. The different bridge length linkers-incorporated enzyme was coupled to a carboxylic derivative of cortisol. Four enzyme conjugates with different bridge length were prepared, such as cortisol-21-hemisuccinate-HRP (cortisol-21-HS-HRP), cortisol-21-HS-ADH-HRP, cortisol-21-HS-ADH-GABA-HRP, and cortisol-21-HS-ADH-6ACA-HRP. The influence of linker on sensitivity and specificity of the cortisol assay was studied. The study revealed that incorporation of a linker between hapten and enzyme increases the sensitivity and specificity of the assay. PMID:15461384

  5. Determination of egg proteins in food products by enzyme immunoassay.

    PubMed

    Yeung, J M; Newsome, W H; Abbott, M A

    2000-01-01

    An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of egg proteins in foods. The polyclonal antibodies developed were specific to whole egg proteins and did not cross-react with any of the 38 nuts, legumes, or other common food ingredients tested. The concentrations of egg proteins that will inhibit 50% of antibody-antigen binding, IC50, were 3-7 ng/mL, and the linear range was 0.5-62.5 ng/mL. The detection limit was 0.2 ppm for various foods. Recoveries ranged from 67 to 96%. The intra- and inter-assay coefficients of variation in this procedure were 10-13% for ice cream spiked at 0.8 and 1.6 ppm. The ELISA has been applied to ice creams, noodles, pasta, and breads. Egg proteins were identified in all declared egg products, and no false positives were found. PMID:10693015

  6. Evaluation of the PANBIO Brucella Immunoglobulin G (IgG) and IgM Enzyme-Linked Immunosorbent Assays for Diagnosis of Human Brucellosis

    PubMed Central

    Araj, George F.; Kattar, Mireille M.; Fattouh, Layla G.; Bajakian, Kayane O.; Kobeissi, Sara A.

    2005-01-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  7. Evaluation of the PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays for diagnosis of human brucellosis.

    PubMed

    Araj, George F; Kattar, Mireille M; Fattouh, Layla G; Bajakian, Kayane O; Kobeissi, Sara A

    2005-11-01

    PANBIO Brucella immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) were assessed against Brucella standard agglutination tube and Coombs tests. The sensitivities of ELISA IgG and IgM were 91% and 100%, respectively, while the specificity was 100% for both. These ELISAs are simple, rapid, and reliable for the diagnosis of human brucellosis. PMID:16275951

  8. Validation of a monoclonal enzyme immunoassay for the determination of carbofuran in fruits and vegetables.

    PubMed

    Moreno, M J; Abad, A; Pelegrí, R; Marínez, M J; Sáez, A; Gamón, M; Montoya, A

    2001-04-01

    The N-methylcarbamate pesticide carbofuran is a very important insecticide used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) to determine this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 740 ng/L and with a dynamic range between 200 and 3100 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbofuran at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop, mean recoveries in the 43.9--90.7% range were obtained for purified samples and in the 90.1--121.6% range for crude extracts. The carbofuran immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbofuran at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was very good (y = 0.90x + 2.66, r(2)() = 0.958, n = 25), with HPLC being more precise than ELISA (mean coefficients of variation of 4.1 and 11.5%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.02x + 10.44, r(2)() = 0.933, n = 29). Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbofuran in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases the sample throughput. PMID:11308315

  9. A sensitive surface-enhanced Raman scattering enzyme-catalyzed immunoassay of respiratory syncytial virus.

    PubMed

    Zhan, Lei; Zhen, Shu Jun; Wan, Xiao Yan; Gao, Peng Fei; Huang, Cheng Zhi

    2016-02-01

    Respiratory viruses have become a major global health challenge which would benefit from advances in screening methods for early diagnosis. Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections. Here we present a novel surface-enhanced Raman scattering (SERS) enzyme-catalyzed immunoassay of RSV by employing peroxidase substrate 3, 3'-5, 5'-tetramethylbenzidine (TMB) as Raman molecule. Horseradish peroxidase (HRP) attached to the detection antibody in a novel sandwich immunoassay catalyzes the oxidation of TMB by H2O2 to give a radical cation (TMB(+)), which could be easily adsorbed on the negatively charged surface of silver nanoparticles (AgNPs) through electrostatic interaction, inducing the aggregation of AgNPs and thus giving a strong SERS signal. A linear relationship was obtained between the Raman intensity and the amount of RSV in the range from 0.5 to 20 pg/mL, and the minimum detectable concentration of this SERS-based enzyme immunoassay was 0.05 pg/mL, which was 20 times lower than that found in the colorimetric method. PMID:26653454

  10. A competitive chemiluminescence enzyme immunoassay for rapid and sensitive determination of enrofloxacin

    NASA Astrophysics Data System (ADS)

    Yu, Fei; Wu, Yongjun; Yu, Songcheng; Zhang, Huili; Zhang, Hongquan; Qu, Lingbo; Harrington, Peter de B.

    With alkaline phosphatase (ALP)-adamantane (AMPPD) system as the chemiluminescence (CL) detection system, a highly sensitive, specific and simple competitive chemiluminescence enzyme immunoassay (CLEIA) was developed for the measurement of enrofloxacin (ENR). The physicochemical parameters, such as the chemiluminescent assay mediums, the dilution buffer of ENR-McAb, the volume of dilution buffer, the monoclonal antibody concentration, the incubation time, and other relevant variables of the immunoassay have been optimized. Under the optimal conditions, the detection linear range of 350-1000 pg/mL and the detection limit of 0.24 ng/mL were provided by the proposed method. The relative standard deviations were less than 15% for both intra and inter-assay precision. This method has been successfully applied to determine ENR in spiked samples with the recovery of 103%-96%. It showed that CLEIA was a good potential method in the analysis of residues of veterinary drugs after treatment of related diseases.

  11. Distribution of phosphoneuroprotein 14 (PNP 14) in vertebrates: its levels as determined by enzyme immunoassay.

    PubMed

    Nakajo, S; Tsukada, K; Kameyama, H; Furuyama, Y; Nakaya, K

    1996-11-25

    We have established an enzyme immunoassay for phosphoneuroprotein 14 (PNP 14) which is mainly localized in the cytoplasmic matrix in presynaptic axon terminals and which is phosphorylated in vivo, as well as in vitro. Fab' prepared from rabbit IgG antibodies against bovine PNP 14 was conjugated with maleimide-horseradish peroxidase. The enzyme-conjugated Fab' was used as a second antibody in a sandwich enzyme immunoassay. This assay was able accurately to quantify 0.5-100 ng of rat PNP 14, as well as bovine PNP 14, and it was used for the determination of concentrations of PNP 14 in various rat tissues, neuroblastoma cells, and brains of other vertebrates. The concentrations of PNP 14 in the rat cerebrum, cerebellum, and testis were 1.1, 1.0, and 0.28 micrograms/mg of protein, respectively, and those in other tissues examined were less than 0.1 microgram/mg of protein. PNP 14 was also found in cultured cells, such as rat pheochromocytoma PC12 cells, NG108-15 cells, which are a hybrid between a mouse neuroblastoma and a rat glioma, mouse neuroblastoma Neuro-2a cells, and human neuroblastoma IMR32 cells. Furthermore, PNP 14-specific immunoreactivity was evaluated in the brains of various vertebrates, such as fish, frog, snake and chicken by immunoblot and enzyme immunoassay. The results revealed the immunoreactivity in the brains of all vertebrates examined and the levels were determined to be 0.6-2.1 micrograms bovine PNP 14 equivalents per mg of protein, suggesting that PNP 14 might be an essential component of the central nervous systems of vertebrates. PMID:9001721

  12. Characterization of Treponema pallidum Particle Agglutination Assay-Negative Sera following Screening by Treponemal Total Antibody Enzyme Immunoassays

    PubMed Central

    Maple, P. A. C.; Ratcliffe, D.; Smit, E.

    2010-01-01

    Following a laboratory audit, a significant number of Treponema pallidum particle agglutination assay (TPPA)-negative sera were identified when TPPA was used as a confirmatory assay of syphilis enzyme immunoassay (EIA) screening-reactive sera (SSRS). Sera giving such discrepant results were further characterized to assess their significance. A panel of 226 sera was tested by the Abbott Murex ICE Syphilis EIA and then by the Newmarket Syphilis EIA II. TPPA testing was performed on 223 sera. Further testing by the Venereal Disease Research Laboratory (VDRL) test, the Mercia Syphilis IgM EIA, the fluorescent treponemal antibody (FTA-ABS) assay, and INNO-LIA immunoblotting was undertaken in discrepant cases. One hundred eighty-seven of 223 (83.8%) SSRS were TPPA reactive, while 26 (11.6%) sera which were reactive in both the ICE and Newmarket EIAs were nonreactive by TPPA. The majority (68%) of the TPPA-discrepant sera were from HIV-positive patients and did not represent early acute cases, based on previous or follow-up samples, which were available for 22/26 samples. FTA-ABS testing was performed on 24 of these sera; 14 (58.3%) were FTA-ABS positive, and 10 (41.7%) were FTA-ABS negative. Twenty-one of these 26 sera were tested by INNO-LIA, and an additional 4 FTA-ABS-negative samples were positive. In this study, significant numbers (18/26) of SSRS- and TPPA-negative sera were shown by further FTA-ABS and LIA (line immunoblot assay) testing to be positive. The reason why certain sera are negative by TPPA but reactive by treponemal EIA and other syphilis confirmatory assays is not clear, and these initial findings should be further explored. PMID:20844087

  13. Typing of human rotavirus VP4 by an enzyme immunoassay using monoclonal antibodies.

    PubMed Central

    Coulson, B S

    1993-01-01

    Two different neutralization specificities exist on the outer capsid of group A rotaviruses. At least seven VP7 (G) antigenic types are distinguishable among human rotaviruses. Four distinct antigenic (P) types of human rotavirus VP4 corresponding to separate rotavirus gene 4 groups have been described. The aim of this study was to identify P types in clinical specimens by developing an enzyme immunoassay, using P-type-specific neutralizing monoclonal antibodies (N-MAbs). Three N-MAbs primarily or solely recognizing each of P types 4, 6, and 8 and binding to VP4 or its subunit VP5* were derived. These N-MAbs served as detector antibodies in an enzyme immunoassay P-typing system similar to that in use for G typing. P-type specificity was highest when the G-type specificity of the capture antiserum was matched to the G type of the rotavirus in the test sample. The method correctly identified the P types of 13 well-characterized, cell culture-adapted human rotaviruses and was used to classify a further six strains. P typing of 118 rotavirus-positive stools gave results consistent with the P type inferred from the G type for 98 (83%) samples. Twelve (10%) of the stools showed no reaction with any N-MAb and eight (7%) samples were untypeable because of cross-reactivity between N-MAbs or high background readings. This P-typing enzyme immunoassay system is economical and amenable to large-scale use in epidemiological studies. Its use will facilitate assessment of the distribution of P types worldwide and of the role of VP4 in eliciting protective immune responses. PMID:7678015

  14. [Detection of IgE specific for egg yolk by enzyme immunoassay. Description of a case].

    PubMed

    Carrillo Díaz, T; Cuevas Agustín, M; Moneo Goiri, I; Ibáñez Sandín, M D; Ureña Vilardell, V

    1986-01-01

    Food allergy is a common disease in our country, especially affecting atopic children. Egg-white hypersensitivity is frequently found in these patients. However, egg-yolk hypersensitivity is not usually reported in patients with egg allergy. This article describes a young patient with egg-yolk hypersensitivity, a 12 year old female patient with a medical history of contact urticaria, angioedema and severe acute bronchospasm shortly after the intake of small amounts of egg-yolk. All these episodes required treatment in emergency care units because of the severity of the symptoms. The patient did not describe any other food hypersensitivity and remained symptom-free after the intake of boiled or fried egg-white. She had clinical symptoms of grass pollen hypersensitivity and was therefore on specific immunotherapy at the time of the study. The skin prick-tests were positive to grass pollen and egg-yolk and were negative to mites, moulds, animal dander and to the common food tested (milk, fish, peanut, almond and hazel-nut). Total serum IgE was 1.160 UL/ml. The patient had a positive RAST to egg-white (0.0 PRU/ml) as well as to egg-yolk (8.6 PRU/ml). Furthermore, an indirect enzyme immunoassay as well as a reverse enzyme immunoassay also revealed the presence of specific IgE antibodies. The reverse enzyme immunoassay uses microtiter plates as a solid surface. These plates are coated with a monospecific antihuman IgE antibody. Thereafter, the serum samples are incubated overnight in the wells. After several washings, the presence of specific antibodies is revealed by means of a peroxidase conjugated allergen.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3515886

  15. Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum.

    PubMed

    Chow, Siu-Kei; Atienza, Ederlyn E; Cook, Linda; Prince, Harry; Slev, Patricia; Lapé-Nixon, Mary; Jerome, Keith R

    2016-08-01

    Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection. PMID:27280621

  16. An enzyme immunoassay for rat growth hormone - Applications to the study of growth hormone variants

    NASA Technical Reports Server (NTRS)

    Farrington, Marianne A.; Hymer, W. C.

    1987-01-01

    A sensitive and specific competitive enzyme immunoassay for rat growth hormone (GH) is described and its use in the detection of GH variants is demonstrated. In the present assay, soluble GH and GH adsorbed to a solid-phase support compete for monkey anti-GH antibody binding sites. The immobilized antibody-GH complex is detected and quantified using goat antimonkey immunoglobin G covalently conjugated to horseradish peroxidase. It is noted that the assay can be performed in 27 hours and that sensitivities in the range of 0.19 to 25 ng can be obtained in the region of 10 to 90 percent binding.

  17. Application of magnetic nanoparticles in full-automated chemiluminescent enzyme immunoassay

    NASA Astrophysics Data System (ADS)

    Xie, Xiaomao; Ohnishi, Noriyuki; Takahashi, Yuki; Kondo, Akihiko

    2009-05-01

    The magnetic nanoparticles (MNPs) Therma-Max™ were used as a carrier to develop an automated sandwich chemiluminescent enzyme immunoassay (CLEIA) to detect thyroid-stimulating hormone (TSH) in a sensitive and specific way. The Therma-Max™ particles allow for automation because, unlike magnetic microspheres, they are completely dispersed in aqueous solution and allow for accurate automatic handling. Signal intensities detected with MNPs were 8-fold higher than those found with conventional micron-sized magnetic particles. A reproducibility study suggests that these particles allow for a stable detection method, as the coefficient of variation (CV) is less than 6% ( n=10).

  18. Cross-Reactivity of the PLATELIA CANDIDA Antigen Detection Enzyme Immunoassay with Fungal Antigen Extracts

    PubMed Central

    Rimek, Dagmar; Singh, Jagpal; Kappe, Reinhard

    2003-01-01

    We studied the specificity of the PLATELIA CANDIDA Ag enzyme immunoassay by using 130 isolates of 63 clinically relevant fungal species. Antigen extracts of seven Candida spp. (Candida albicans, C. dubliniensis, C. famata, C. glabrata, C. guilliermondii, C. lusitaniae, and C. tropicalis) repeatedly yielded positive reactions (>0.5 ng/ml). Geotrichum candidum and Fusarium verticillioides were found to yield borderline-positive reactions (0.25 to 0.50 ng/ml). Antigen preparations from the other 54 fungal species, including yeasts, molds, dermatophytes, and dimorphic fungi, did not cross-react in the assay. PMID:12843102

  19. A competitive enzyme immunoassay for the quantitative detection of cocaine from banknotes and latent fingermarks.

    PubMed

    van der Heide, Susan; Garcia Calavia, Paula; Hardwick, Sheila; Hudson, Simon; Wolff, Kim; Russell, David A

    2015-05-01

    A sensitive and versatile competitive enzyme immunoassay (cEIA) has been developed for the quantitative detection of cocaine in complex forensic samples. Polyclonal anti-cocaine antibody was purified from serum and deposited onto microtiter plates. The concentration of the cocaine antibody adsorbed onto the plates, and the dilution of the cocaine-HRP hapten were both studied to achieve an optimised immunoassay. The method was successfully used to quantify cocaine in extracts taken from both paper currency and latent fingermarks. The limit of detection (LOD) of 0.162ngmL(-1) achieved with the assay compares favourably to that of conventional chromatography-mass spectroscopy techniques, with an appropriate sensitivity for the quantification of cocaine at the low concentrations present in some forensic samples. The cEIA was directly compared to LC-MS for the analysis of ten UK banknote samples. The results obtained from both techniques were statistically similar, suggesting that the immunoassay was unaffected by cross-reactivity with potentially interfering compounds. The cEIA was used also for the detection of cocaine in extracts from latent fingermarks. The results obtained were compared to the cocaine concentrations detected in oral fluid sampled from the same individual. Using the cEIA, we have shown, for the first time, that endogeneously excreted cocaine can be detected and quantified from a single latent fingermark. Additionally, it has been shown that the presence of cocaine, at similar concentrations, in more than one latent fingermark from the same individual can be linked with those concentrations found in oral fluid. These results show that detection of drugs in latent fingermarks could directly indicate whether an individual has consumed the drug. The specificity and feasibility of measuring low concentrations of cocaine in complex forensic samples demonstrate the effectiveness and robustness of the assay. The immunoassay presents a simple and cost

  20. The management of isolated positive syphilis enzyme immunoassay results in HIV-negative patients attending a sexual health clinic.

    PubMed

    Thorley, Nicola; Adebayo, Michael; Smit, Erasmus; Radcliffe, Keith

    2016-08-01

    An unconfirmed positive treponemal enzyme immunoassay (enzyme immunoassay positive, Treponema pallidum particle agglutination negative and rapid plasma reagin negative) presents a clinical challenge to distinguish early syphilis infection from false-positive results. These cases are referred for syphilis line assay (INNO-LIA) and recalled for repeat syphilis serology. We performed a retrospective audit to establish the proportion of HIV-negative cases with unconfirmed positive enzyme immunoassay results, the proportion of these cases that received an INNO-LIA test and repeat syphilis serology testing and reviewed the clinical outcomes; 0.35% (80/22687) cases had an unconfirmed positive treponemal enzyme immunoassay result. Repeat syphilis serology was performed in 80% (64/80) cases, but no additional cases of syphilis were identified. Eighty-eight per cent (70/80) received an INNO-LIA test; 14% (5/37) unconfirmed enzyme immunoassay-positive cases with no prior history of syphilis were confirmed on INNO-LIA assay, supporting a diagnosis of latent syphilis. As a confirmatory treponemal test, the INNO-LIA assay may be more useful than repeat syphilis serological testing. PMID:26637236

  1. Monoclonal antibody capture enzyme immunoassay for detection of Paracoccidioides brasiliensis antibodies in paracoccidioidomycosis.

    PubMed Central

    Camargo, Z P; Gesztesi, J L; Saraiva, E C; Taborda, C P; Vicentini, A P; Lopes, J D

    1994-01-01

    Four murine monoclonal antibodies (MAbs 17C, 21A, 21F, and 32B) raised against the 43-kDa glycoprotein of Paracoccidioides brasiliensis were tested in a capture enzyme immunoassay (EIA) for the detection of specific human anti-gp43 immunoglobulin G in patients with paracoccidioidomycosis (PCM). All MAbs reacted similarly in the assay. These MAbs, which detected anti-gp43 at levels of as low as 500 pg/ml, were demonstrated to specifically recognize at least two different epitopes in gp43 binding assays. Specific antibodies in the sera of patients with active PCM were detected at dilutions of as high as 1:819,200, and the reactivities of patient sera, as measured by optical densities, were found to be significantly higher than those of control sera. The comparison between classical ELISA and our capture enzyme immunoassay showed that both sensitivity and specificity were greatly improved by the latter. These MAbs represent the first specific reagents to P. brasiliensis described for use in serological tests for PCM. Images PMID:7814469

  2. A rapid fluorometric enzyme immunoassay for the determination of neonatal TSH from blood spots.

    PubMed

    Tuuminen, T; Rakkolainen, A E; Welin, M G; Weber, T H; Nylander, P L; Käpyaho, K I

    1991-10-31

    We describe a novel method for the detection of thyrotropin from dried blood spots using a horseradish peroxidase-labelled sandwich enzyme immunoassay with fluorometric detection. The detection limit of the present assay is 1.25 mIU/l with within-run and between-run imprecision being in the range 5.2 to 11.4%. The results of the assay correlate well with two commercial methods: an enzyme immunoassay (r = 0.93) and a time-resolved fluorescence assay (r = 0.90). The blood spot values also show a good correlation (r = 0.93) with respective values obtained from plasma using a commercial immunoradiometric method. The assay may also be performed colorimetrically with sensitivity similar to the fluorometric assay. However, the latter provides a wider dynamic range with an upper limit of 400 mIU/l while the colorimetric method reaches a plateau at 25 mIU/l. Due to its simplicity and rapid performance (3 h), the fluorometric assay is suitable for the routine screening of congenital hypothyroidism. PMID:1814645

  3. Enhanced competitive chemiluminescent enzyme immunoassay for the trace detection of insecticide triazophos.

    PubMed

    Jin, Maojun; Shao, Hua; Jin, Fen; Gui, Wenjun; Shi, Xiaomei; Wang, Jing; Zhu, Guonian

    2012-05-01

    A direct competitive chemiluminescent enzyme immunoassay (CLEIA) for triazophos was developed, which was based on the anti-THHe IgG monoclonal antibody and a heterogeneous enzyme tracer (THHu-HRP). Several components of chemiluminescent enhanced solution (CES) were optimized. The results showed that 1 mM of p-iodo-phenol, 0.625 mM of luminol, and 4 mM of H(2)O(2) had the best performance. Based on the study of CES, the influence of several factors (assay buffer, blocking substance, and solvent) on the immunoassay was investigated. The sensitivity for detection, IC(50) value was 0.87 ng/mL at a practical working concentration range between 0.04 ng/mL and 5 ng/mL and the limit of detection for triazophos was 0.063 ng/mL. The average recovery of triazophos added to lettuce, carrot, apple, water, and soil were 78.71%, 67.52%, 118.3%, 117.2%, and 122.0%, respectively. Finally, comparison between the methods of CLEIA and high-performance liquid chromatography-tandem mass spectrum (HPLC-MS/MS) was performed. The results obtained from CLEIA were in agreement with those of HPLC-MS/MS. PMID:22490114

  4. Development of sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in plasma.

    PubMed

    Zehnacker, Laura; Nevers, Marie-Claire; Sinou, Véronique; Parzy, Dominique; Créminon, Christophe; Parzy, Daniel; Azoulay, Stéphane

    2015-10-01

    Despite significant progress in prevention and therapy, malaria is still one of the world's leading major diseases due to its high morbidity and mortality. Recommended treatments by the World Health Organization include the use of artemisinin and artemisinin derivative-based combination therapies. To allow efficient patient monitoring during antimalarial therapy without the use of expensive apparatus, we developed a sensitive direct chemiluminescent enzyme immunoassay for the determination of dihydroartemisinin in biological fluids. To produce specific antibodies against dihydroartemisinin (DHA), a synthetic DHA derivative was coupled to bovine serum albumin as the immunogen. In parallel, a new, rapid, and efficient procedure to covalently link glycoprotein to all amine-containing molecules has been established and the enzyme tracer was prepared by chemically coupling the DHA derivative in combination with SBP rather than the more commonly used HRP. It allowed us to develop, after optimization of the luminescent reagent, a sensitive and stable luminescent EIA, with a LLOQ of 90 pg mL(-1). This assay compares favorably with the most efficient HPLC methods previously reported with a LLOQ close to 1 ng mL(-1) and shows good precision and efficiency since recovery from human plasma spiked with DHA ranged between 91 and 103%, with coefficients of variation of <13%. To date, no immunoassay for DHA has been applied to plasma analysis and this EIA should be very useful in all clinical laboratories for rapid and cost-effective analysis. PMID:26280205

  5. Monoclonal enzyme immunoassay for the analysis of carbaryl in fruits and vegetables without sample cleanup.

    PubMed

    Abad, A; Moreno, M J; Pelegrí, R; Martínez, M I; Sáez, A; Gamón, M; Montoya, A

    2001-04-01

    The N-methylcarbamate pesticide carbaryl is one of the most important insecticides used worldwide. In the present work, the validation of a monoclonal antibody-based enzyme immunoassay (ELISA) for the determination of this compound in fruits and vegetables is described. The immunoassay is a competitive heterologous ELISA in the antibody-coated format, with an I(50) value for standards in buffer of 101.0 +/- 26.9 ng/L and with a dynamic range between 31.6 and 364.0 ng/L. For recovery studies, peppers, cucumbers, strawberries, tomatoes, potatoes, oranges, and apples were spiked with carbaryl at 10, 50, and 200 ppb. After liquid extraction, analyses were performed by ELISA on both extracts purified on solid-phase extraction (SPE) columns and crude, nonpurified extracts. Depending on the crop and the fortification level, recoveries in the 59.0--120.0% range were obtained for purified samples and in the 70.0--137.7% range for crude extracts. The carbaryl immunoassay performance was further validated with respect to high-performance liquid chromatography (HPLC) with postcolumn derivatization and fluorescence detection (EPA Method 531.1). Samples were spiked with carbaryl at several concentrations and analyzed as blind samples by ELISA and HPLC after SPE cleanup. The correlation between methods was excellent (y = 1.04x + 0.71, r(2) = 0.992, n = 33), with HPLC being more precise than ELISA (mean coefficients of variation of 5.2 and 12.0%, respectively). The immunoassay was then applied to the analysis of nonpurified extracts of the same samples. Results also compared very well with those obtained by HPLC on purified samples (y = 1.28x - 0.59, r(2) = 0.987, n = 33) while maintaining similar precision. Therefore, the developed immunoassay is a suitable method for the quantitative and reliable determination of carbaryl in fruits and vegetables even without sample cleanup, which saves time and money and considerably increases sample throughput. PMID:11308314

  6. Development and application of a sensitive enzyme immunoassay for 7-N-(p-hydroxyphenyl)mitomycin C.

    PubMed

    Fujiwara, K; Nakamura, H; Kitagawa, T; Nakamura, N; Saito, A; Hara, K

    1984-09-01

    An antibody specific for 7-N-(p-hydroxyphenyl)mitomycin C (M-83) was developed and used in a simple and sensitive enzyme immunoassay for detection of this anticancer drug in serum. The imino group of M-83 was covalently coupled to mercaptosuccinyl groups introduced into bovine serum albumin with a cross-linker, N-maleoylaminobutyric acid. The resulting conjugate was then used for the production of a specific antibody to M-83 in rabbits. Enzyme labeling of M-83 was performed using beta-D-galactosidase (EC 3.2.1.23) via m-maleoylbenzoic acid. Antibody production was of sufficiently high titer in rabbits to allow the development of a highly sensitive enzyme immunoassay for the free drug which accurately measures 15 pg of M-83 per assay tube. This assay was monospecific to M-83 and showed almost no cross-reactivity with a variety of other mitomycin analogues (mitomycin A, B, and C; porfiromycin; and acetylmitomycin C). Using this assay, preliminary pharmacokinetic study was undertaken using serial serum samples obtained from a patient who received the drug i.v., revealing a biphasic fashion of the kinetics, with an alpha-serum half-life of 9.7 min and a beta-serum half life of 80 min. An i.v. injection of M-83 into rats and assay of serum concentration revealed a similar biphasic response with an alpha-serum half-life of 8.3 min and a beta-serum half-life of 62 min. PMID:6430558

  7. Comparison of three enzyme immunoassays for measuring 17beta-estradiol in flushed dairy manure wastewater.

    PubMed

    Hanselman, Travis A; Graetz, Donald A; Wilkie, Ann C

    2004-01-01

    Natural steroidal estrogens are an environmental concern because low nanogram per liter concentrations in water can adversely affect aquatic vertebrate species by disrupting the normal function of their endocrine systems. There is a critical need to accurately measure estrogens in dairy wastes, a potential source of estrogens such as 17beta-estradiol, to assess the risk of estrogen contamination of agricultural drainage waters resulting from land application. Commercially available enzyme immunoassay (EIA) kits have been used for measuring 17beta-estradiol in livestock manure, but it is not known if different EIAs provide similar results. We compared three EIAs by measuring 17beta-estradiol in two samples of flushed dairy manure wastewater (FDMW). The measured concentrations of 17beta-estradiol in FDMW differed according to the immunoassay used. The differences were attributed to a matrix interference associated with coextracted humic substances. Future research should develop methods that enable routine measurement of 17beta-estradiol in livestock wastes by more conclusive analytical techniques such as gas chromatography-mass spectrometry. PMID:15356254

  8. Determination of thiabendazole in fruits and vegetables by competitive-inhibition enzyme immunoassay.

    PubMed

    Bushway, R J; Young, B E; Paradis, L R; Perkins, L B

    1994-01-01

    An enzyme immunoassay (EIA) was developed for analysis of thiabendazole (TBZ) in fruits and vegetables. A commercial kit using a polyclonal antibody for benomyl-carbendazim was used. Total analysis time, including sample preparation, was 35 min. As many as 8 samples can be analyzed simultaneously, with a limit of quantitation of 9 ppb. The assay's dynamic range ran from 9 to 304 ppb TBZ. Intra-assay coefficients of variation (CVs) ranged from 5.0 to 9.6% for standards and 11 to 30% for samples. Interassay CVs varied from 4.4 to 15% for standards and from 10 to 24% for samples. Average recovery from 29 samples spiked at 50-50,000 ppb was 116%. A total of 107 food products comprising 12 different fruits and vegetables and their processed products were analyzed for their TBZ content by EIA and liquid chromatography (LC). Of these samples, 84 were positive for TBZ by both methods, with a correlation coefficient (r) of 0.989. Eight samples had none detected by either technique, and 15 were positive for carbendazim. Concentrations of TBZ ranged from 11 to 94,000 ppb. The immunoassay with methanol sonication shows promise as a rapid screening method for TBZ in foods. PMID:7950423

  9. Diagnostic accuracy of an IgM enzyme-linked immunosorbent assay and comparison with 2 polymerase chain reactions for early diagnosis of human leptospirosis.

    PubMed

    Vanasco, N B; Jacob, P; Landolt, N; Chiani, Y; Schmeling, M F; Cudos, C; Tarabla, H; Lottersberger, J

    2016-04-01

    Enzyme-linked immunosorbent assay (ELISA) tests and polymerase chain reaction (PCR) may play a key role for early detection and treatment of human leptospirosis in developing countries. The aims of this study were to develop and validate an IgM ELISA under field conditions and to compare the diagnostic accuracy among IgG, IgM ELISAs, conventional PCR (cPCR), and real-time PCR (rtPCR) for early detection of human leptospirosis. Overall accuracy of IgM ELISA was sensitivity of 87.9%, specificity of 97.0%, and area under the curve of 0.940. When the 4 methods were compared, IgM ELISA showed the greatest diagnostic accuracy (J=0.6) followed by rtPCR (J=0.4), cPCR (J=0.2) and IgG ELISA (J=0.1). Our results support the use of IgM ELISA and rtPCR for early diagnosis of the disease. Moreover, due to their high specificity, they could be also useful to replace or supplement microscopic agglutination test as a confirmatory test, allowing more confirmations. PMID:26867967

  10. Detection of Pesticides and Pesticide Metabolites Using the Cross Reactivity of Enzyme Immunoassays

    USGS Publications Warehouse

    Thurman, E.M.; Aga, D.S.

    2001-01-01

    Enzyme immunoassay is an important environmental analysis method that may be used to identify many pesticide analytes in water samples. Because of similarities in chemical structure between various members of a pesticide class, there often may be an unwanted response that is characterized by a percentage of cross reactivity. Also, there may be cross reactivity caused by degradation products of the target analyte that may be present in the sample. In this paper, the concept of cross reactivity caused by degradation products or by nontarget analytes is explored as a tool for identification of metabolites or structurally similar compounds not previously known to be present in water samples. Two examples are examined in this paper from various water quality studies. They are alachlor and its metabolite, alachlor ethane sulfonic acid, and atrazine and its class members, prometryn and propazine. A method for using cross reactivity for the detection of these compounds is explained in this paper.

  11. Evaluation of enzyme immunoassays in the diagnosis of camel (Camelus dromedarius) trypanosomiasis: a preliminary investigation.

    PubMed Central

    Rae, P. F.; Thrusfield, M. V.; Higgins, A.; Aitken, C. G.; Jones, T. W.; Luckins, A. G.

    1989-01-01

    Three enzyme immunoassays were used for the serodiagnosis of Trypanosoma evansi in camels in the Sudan in order to evaluate their ability to discriminate between infected and non-infected animals. Two assays were used for the detection of trypanosomal antibodies, one using specific anti-camel IgG conjugate and another using a non-specific Protein A conjugate. The third assay detected the presence of trypanosomal antigens using anti-T. evansi antibodies in a double antibody sandwich assay. Inspection of the frequency distribution of assay results suggested that the ELISA for circulating trypanosomal antibodies using specific antisera and the ELISA for circulating antigens can distinguish between non-infected camels and infected camels exhibiting patent infections or not. The ELISA using Protein A conjugate to bind non-specifically to camel immunoglobulin did not appear to discriminate between infected and non-infected animals. PMID:2703023

  12. Improvement of an enzyme immunoassay for the determination of mercury (II)

    SciTech Connect

    Marx, A.; Kroetz, E.; Hock, B.

    1998-07-01

    Three systems were tested for the optimization of a heterogeneous noncompetitive enzyme immunoassay (EIA) for the determination of Hg (II). The sensitivity of the nonoptimized Hg-EIA with a detection limit of 2.1 {micro}g/L Hg (II) was improved by an avidin-biotin-complex (ABC) amplification system to a 2-fold lower detection limit (1.1 {micro}g/L Hg (II)). A conventional competitive EIA with the competition reaction between bound and free Hg (II) for antibody (ab) binding sites yielded a detection limit of 1.0 {micro}g/L Hg (II). Further improvement of sensitivity could be achieved by a competitive displacement EIA. In this case ab molecules bound to immobilized haptens are displaced in the next step by free Hg (II). The detection limit of the displacement approach is 0.4 {micro}g/L Hg (II).

  13. Clinical and microbiological effects of subgingival antimicrobial irrigation with citric acid as evaluated by an enzyme immunoassay and culture analysis.

    PubMed

    Renvert, S; Dahlén, G; Snyder, B

    1997-04-01

    The purpose of the present study was to compare an enzyme immunoassay with culture samples from untreated and non-surgically treated periodontal pockets and to assess the clinical and microbiological effects of citric acid irrigation as a supplement to scaling and root planing. The enzyme immunoassay used in this study is a chairside diagnostic tool aimed at identifying the presence of P. gingivalis, P. intermedia, and A. actinomycetemcomitans. Six sites with pocket depths > or = 6 mm in each of 16 patients were monitored for 24 weeks using clinical and microbiological parameters. In two out of the six sites, scaling and root planing was supplemented with subgingival citric acid irrigation of the pocket after completion of the mechanical treatment. The sensitivity of the immunoassay in relation to culture was calculated to 85.5% and the specificity to 90.2%. The immunoassay corresponded to a detection level of 10(4) as estimated by culture. Sites treated with a combination of scaling and irrigation with citric acid demonstrated a similar healing pattern as sites treated with scaling and root planing alone. The profile of the marker bacteria was almost parallel for the two groups. The results of this investigation thus indicated that the immunoassay can be used as a screening tool for selected periodontal pathogens and that adjunctive irrigation with citric acid has no measurable clinical or microbiological effects. PMID:9150039

  14. Enzyme immunoassays as a method for quantifying hair reproductive hormones in two felid species

    PubMed Central

    Terwissen, C. V.; Mastromonaco, G. F.; Murray, D. L.

    2014-01-01

    Non-invasive monitoring of wild felid reproductive states is important, given that many species reproduce poorly in captivity. Despite extensive work in faecal hormone analysis in felids, continued development of techniques is necessary, particularly with wild populations. In this study, our aims were as follows: (i) biochemical validation of enzyme immunoassays for estrogen, testosterone and progesterone in Canada lynx and domestic cat hair extracts; (ii) assessment of the use of hair reproductive hormones to differentiate between reproductive states (intact, estrus, pregnant and spayed/neutered), using domestic cats as a model; and (iii) assessment of the use of hair reproductive hormones to differentiate between age and sex, accounting for potential regional variability in wild lynx populations. Analysis of hair hormone levels showed prospective value in detecting pregnancy states, with pregnant domestic cats having higher levels of progesterone than spayed females. However, intact and pregnant cats did not differ in progesterone levels. Yet, two female domestic cats had higher levels of hair progesterone following a 38-day oral progestin treatment, perhaps providing a preliminary pharmacological validation of the method. Estrogen and testosterone did not differ statistically according to reproductive states of domestic cats, although intact males had higher levels of hair testosterone than neutered males. When we applied these techniques to lynx fur, we determined that hormone levels were not sufficiently precise to differentiate age classes. Hair reproductive hormone ratios differed between sexes, with the estrogen-to-progesterone ratio demonstrating the highest accuracy in differentiating males from females. Hair hormone levels differed regionally for wild lynx, indicating that spatial variability should be a consideration in wildlife hormone studies spanning large spatial scales. We conclude that use of hair hormone analysis by enzyme immunoassay may

  15. Rapid chemiluminescent sandwich enzyme immunoassay capable of consecutively quantifying multiple tumor markers in a sample.

    PubMed

    Kim, Julie; Kim, Jennie; Rho, Tae Ho D; Lee, Ji Hoon

    2014-11-01

    Using the role of p-iodophenol in enzyme assay, enhanced 1,1'-oxalyldiimidazole chemiluminescent enzyme immunoassays (ODI-CLEIAs) were developed to consecutively quantify trace levels of triple tumor markers, such as alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), and prostate specific antigen (PSA) in a sample. Due to the high sensitivity of enhanced ODI-CLEIAs, it was possible to fix the incubation times (1) to capture a tumor marker with two antibodies, which are primary antibody immobilized on the surface of polystyrene strip-well and detection antibody-conjugated horseradish peroxidase (HRP), and (2) to form resorufin with the addition of substrates (e.g., Amplex Red, H2O2) in order to quantify triple markers in human serum. Enhanced ODI-CLEIAs capable of consecutively and rapidly quantifying triple markers with the same incubation time were more sensitive than conventional enzyme-linked immunosorbent assay (ELISA) capable of separately and slowly quantifying them with different incubation times. In addition, accuracy, precision, and recovery of enhanced ODI CLEIAs in the presence of p-iodophenol were acceptable within statistical error range. PMID:25127571

  16. Second generation competitive enzyme immunoassay for detection of bovine antibody to Brucella abortus.

    PubMed

    Nielsen, K; Smith, P; Yu, W L; Elmgren, C; Nicoletti, P; Perez, B; Bermudez, R; Renteria, T

    2007-09-20

    A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed. This assay was different from previously developed CELISAs in that the detection reagent used was a recombinant combination of the receptor portions of protein A and protein G, labelled with horseradish peroxidase. This eliminates the need for polyclonal anti-mouse-enzyme conjugate reagents for detection thus allowing for true standardization. The assay utilized a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3 but which did not react with protein A/G. This monoclonal antibody was used to compete with antibody in the bovine test serum. Binding of bovine antibody to the smooth lipopolysaccharide antigen was then measured directly with the protein A/G enzyme conjugate. In this case, development of colour in the reaction was indicative of the presence of bovine antibody. The performance characteristics, sensitivity, specificity and exclusion of B. abortus S19 vaccinated animals, of the assay were very similar to those of the classical CELISA. PMID:17467200

  17. The TUBEX typhoid test based on particle-inhibition immunoassay detects IgM but not IgG anti-O9 antibodies.

    PubMed

    Tam, Frankie Chi Hang; Lim, Pak Leong

    2003-11-01

    A serological test kit (TUBEX, IDL Biotech, Sweden) developed recently for the diagnosis of typhoid fever detects antibodies to the Salmonella enterica serovar Typhi lipopolysaccharide (LPS) O9 antigen. The antibodies are detected by their ability to inhibit the interaction between two types of reagent particles: (a). indicator latex microspheres sensitized with an anti-O9 monoclonal antibody, and (b). magnetic microspheres sensitized with S. typhi LPS. Following rapid mixing of the serum with these reagents and sedimentation of the magnetic particles by magnetic force, the concentration of indicator particles left in suspension provides a measure of the inhibition. Whereas it was previously assumed that both IgM and IgG antibodies could inhibit in the system, the present study reveals, surprisingly, that only the IgM antibodies do. It is not clear why IgG anti-O9 antibodies, both of mouse and human origin, do not inhibit, although these can bind to the LPS-sensitized magnetic particles as efficiently as the IgM antibodies. In addition, they can also inhibit very well in another detection system (ELISA) which uses a similar assay format and the same antibody and antigen reagents. Increasing the size of the LPS-sensitized microspheres made no difference; microscopic analysis of the TUBEX reaction mixture revealed that while the indicator particles bound abundantly to the IgG-aggregated LPS-sensitized particles, forming large clumps, these only formed a very light decoration on the IgM-aggregated particles. Thus, the TUBEX system is ideally suited for use in the diagnosis of infections as it allows IgM antibodies to be detected easily and rapidly from whole sera. PMID:14604543

  18. Enzyme immunoassay for rat growth hormone: applications to the study of growth hormone variants

    SciTech Connect

    Farrington, M.A.; Hymer, W.C.

    1987-06-29

    A sensitive and specific competitive enzyme immunoassay (EIA) for rat growth hormone was developed. In this assay soluble growth hormone and growth hormone adsorbed to a solid-phase support compete for monkey anti-growth hormone antibody binding sites. The immobilized antibody-growth hormone complex is detected and quantified using goat anti-monkey immunoglobin G covalently conjugated to horse radish peroxidase. Therefore, a high concentration of soluble growth hormone in the sample will result in low absorbance detection from the colored products of the enzyme reaction. Assay parameters were optimized by investigating the concentration of reagents and the reaction kinetics in each of the assay steps. The assay can be performed in 27 hours. A sensitivity range of 0.19 ng to 25 ng in the region of 10 to 90% binding was obtained. Near 50% binding (3 ng) the intraassay coefficient of variation (CV) was 5.54% and the interassay CV was 5.33%. The correlation coefficient (r/sup 2/) between radioimmunoassay and EIA was 0.956 and followed the curve Y = 0.78X + 1.0. 9 references, 6 figures.

  19. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin

    SciTech Connect

    Delaney, C.J.; Opheim, K.E.; Smith, A.L.; Plorde, J.J.

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay.

  20. Performance characteristics of bioassay, radioenzymatic assay, homogeneous enzyme immunoassay, and high-performance liquid chromatographic determination of serum gentamicin.

    PubMed Central

    Delaney, C J; Opheim, K E; Smith, A L; Plorde, J J

    1982-01-01

    We compared the accuracy, precision, and between-method error of the microbiological assay, the radioenzymatic assay, the homogeneous enzyme immunoassay, and the high-performance liquid chromatographic assay for the quantitation of gentamicin in serum. Precision and accuracy were evaluated by reference samples prepared to contain 0.0 to 32.7 micrograms of gentamicin per ml. Correlations between the methods utilized patient sera with gentamicin concentrations ranging from 0.6 to 13.3 micrograms/ml. All methods were reliable within acceptable limits for routine clinical use; intermethod correlation coefficients exceeded 0.96. Relative to the microbiological assay, the alternative methods offer the advantage of rapid analysis. The elapsed times for acquiring data on a set of 10 specimens under routine operating conditions were 0.5 h by the enzyme immunoassay, 4 h by the radioenzymatic assay, 5 h by the high-performance liquid chromatographic assay, and 10 h by the microbiological assay. PMID:7044297

  1. High sensitivity chemiluminescence enzyme immunoassay for detecting staphylococcal enterotoxin A in multi-matrices.

    PubMed

    Zhang, Chunmei; Liu, Zhijia; Li, Yongming; Li, Qi; Song, Chaojun; Xu, Zhuwei; Zhang, Yun; Zhang, Yusi; Ma, Ying; Sun, Yuanjie; Chen, Lihua; Fang, Liang; Yang, Angang; Yang, Kun; Jin, Boquan

    2013-09-24

    In this study, detection of staphylococcal enterotoxin A (SEA) in multi-matrices using a highly sensitive and specific microplate chemiluminescence enzyme immunoassay (CLEIA) has been established. A pair of monoclonal antibodies (mAbs) was selected from 37 anti-SEA mAbs by pairwise analysis, and the experimental conditions of the CLEIA were optimized. This CLEIA exhibited high performance with a wide dynamic range from 6.4 pg mL(-1) to 1600 pg mL(-1), and the measured low limit of detection (LOD) was 3.2 pg mL(-1). No cross-reactivity was observed when this method was applied to test SEB, SEC1, and SED. It has also been successfully applied for analyzing SEA in a variety of environmental, biological, and clinical matrices, such as sewage, tap water, river water, roast beef, peanut butter, cured ham, 10% nonfat dry milk, milk, orange juice, human urine, and serum. Thus, the highly sensitive and SEA-specific CLEIA should make it attractive for quantifying SEA in public health and diagnosis in near future. PMID:24016577

  2. Determination of mite allergens in house dust using the enzyme immunoassay.

    PubMed

    Prester, Ljerka; Brcić Karaconji, Irena; Macan, Jelena

    2007-12-01

    The aim of this study was to determine the level of two major mite allergens Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1) in 30 urban homes in Zagreb, Croatia, using the enzyme immunoassay with two monoclonal antibodies which has been established as the reference method for indoor allergen analysis. Dust samples were taken by vacuuming a carpeted area and collected on cellulose filters. The ranges of Der p 1 and Der f 1 were (0.1-12.5) microg g-1 (median 0.32 microg g-1) and (0.1-31.2) microg g-1 (median 0.35 microg g-1), respectively. Der p 1 and Der f 1 (>2 microg g-1) associated with increased risk of sensitization to mite allergens were found in approximately 16% homes for each allergen. The sum of allergen (Der p 1 + Der f 1) exceeded the lower threshold in 27% of homes. Analytical evaluation of the ELISA assay showed satisfactory results for precision (intra-assay CV <6.9%, inter-assay CV<13.3%), accuracy (91% to 93%), and sensitivity (2 ng mL-1). The ELISA assay for the measurement of dust mite allergens demonstrated very good analytical characteristics for routine laboratory use, and will provide the essential basis for our future studies of various indoor allergens. PMID:18063526

  3. Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

    PubMed

    Skraba, Cissiara Manetti; Pedroso, Raíssa Bocchi; Fiorini, Adriana; Rosado, Fábio Rogério; Aristides, Sandra Mara Alessi; Lonardoni, Maria Valdrinez Campana; Teixeira, Jorge Juarez Vieira; Silveira, Thaís Gomes Verzignassi

    2014-04-01

    This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients. PMID:24485589

  4. Improved reliability of a serum alpha-fetoprotein enzyme immunoassay compared with a radioimmunoassay.

    PubMed

    Peleg, L; Slor, H; Cordova, T; Ayalon, D; Legum, C; Rudick, A

    1986-01-01

    During the 6-month period between September 1982 and February 1983, serum samples were obtained from 850 pregnant women attending the municipal family clinics in Tel Aviv. All serum samples were tested by alpha-fetoprotein (AFP) enzyme immunoassay (EIA), and 650 samples were also tested by AFP radioimmunoassay (RIA). Comparisons were made between the absolute values, the medians, and the multiples of the median (MOM) obtained by each method. The ratios of the 90th percentile to the median and of the median to the 10th percentile were also compared. Inter- and intraassay variation were determined for both methods. Correlating the test results with the birth results showed that both methods detected the four fetuses with a neural tube defect (NTD) and three of nine twin pairs, when using a cutoff point of greater than or equal to 2.5 MOM. Elevated maternal serum AFP (MSAFP) levels were also noted in 32 normal singletons (3.8%). There were no false negative results during this phase of the study. Although the median values were similar for both methods, the mean of the ratios of the median to the 10th percentile and the 90th percentile to the median, showed a reduced spread for the EIA method. PMID:2427476

  5. Screening and diagnostic performance of enzyme immunoassay for antibody to lymphadenopathy-associated virus.

    PubMed Central

    Handsfield, H H; Wandell, M; Goldstein, L; Shriver, K

    1987-01-01

    In a multicenter cooperative study, an enzyme immunoassay (EIA) using purified antigen of lymphadenopathy-associated virus was compared with radioimmune precipitation (RIP) for detection of antibody to human immunodeficiency virus (HIV) in 634 patients with acquired immunodeficiency syndrome or related conditions, 687 apparently healthy persons at risk for HIV infection, 93 controls with cancer or autoimmune diseases, and 10,038 blood or plasma donors. Excluding the donors, the EIA was reactive in 875 (61.9%) of 1,414 subjects; compared with RIP, the sensitivity and specificity of EIA both were 99.8%. There was one false-positive EIA among 148 intravenous drug abusers and two false-negative EIAs among 472 apparently healthy homosexual men; no other discordant results between EIA and RIP occurred in these subjects. The EIA was repeatably reactive in 20 donors (0.2%), among whom 13 (65%) were positive by RIP; none of 529 randomly selected EIA-negative donors was RIP positive. In addition to its utility as a screening test in low-risk populations, the EIA for antibody to lymphadenopathy-associated virus is useful as a diagnostic test in persons with clinical evidence of or at risk for HIV infection. Images PMID:3294890

  6. Detection of Chlamydia trachomatis antigen by enzyme immunoassay: importance of confirmatory testing.

    PubMed Central

    Fonseca, K; Megran, D W; Anand, C M

    1995-01-01

    AIM--To determine when a fluorescence assay for Chlamydia trachomatis elementary bodies in the specimen buffer is of most value as a verification test for genital specimens reactive on screening enzyme immunoassay (EIA). METHOD--Genital swabs from high and medium prevalence populations were tested using EIA. Samples with absorbance values greater than the positive threshold and those within the range of 30% below this value were verified by the MicroTrak direct fluorescence assay (DFA) test. Quotients derived from the threshold value and specimen absorbances were used to establish confidence limits for the EIA. RESULTS--Of 13,283 swabs tested, 474 from the high risk group and 236 from the medium risk group were reactive on EIA and confirmed by DFA. Thirty six (5.9%) patients with confirmed reactive samples would have been missed if the kit criteria alone were followed. When confidence limits were applied to the calculated quotients, only those samples with an EIA quotient of > or = 4.0 were universally confirmed by the DFA. CONCLUSION--A scheme of testing which uses the DFA to verify EIA reactive specimens over a specified range was found to improve the sensitivity and specificity of the EIA screening test. PMID:7730479

  7. Enzyme immunoassay for anti-treponemal IgG: screening or confirmatory test?

    PubMed Central

    Young, H; Moyes, A; McMillan, A; Patterson, J

    1992-01-01

    AIMS: To review the performance of the Venereal Diseases Research Laboratory (VDRL) test and the Treponema pallidum haemagglutination assay (TPHA) as a combined screen for syphilis to provide a baseline for assessing screening by anti-treponemal IGG EIA. METHODS: Between 1980 and 1987 all serum samples were screened by both VDRL and TPHA tests. The FTA-ABS test was also used in suspected early primary syphilis, or when one of the other tests was positive. A positive result in a screening test was confirmed by quantitative testing. From 1988 all specimens were screened with an enzyme immunoassay (Captia Syph G) as a single screening test. RESULTS: Of the 44 primary, 47 secondary, and 38 early latent cases of syphilis, the VDRL and TPHA detected 32 (73%) and 31 (71%) of the primary cases; the combination detected 37 (84%). All 85 cases of cases of secondary and early latent infection were reactive in the TPHA test, whereas the VDRL was reactive in only 68 (80%). EIA had a reported sensitivity of 82% for primary infection. CONCLUSIONS: EIA can be used as a single screening test for detecting early syphilis because its results are comparable with those of the combined VDRL and TPHA tests. The conventional VDRL test should not be used as a single screening test. PMID:1740512

  8. Recombinant antigen-based enzyme immunoassay for screening of Treponema pallidum antibodies in blood bank routine.

    PubMed Central

    Zrein, M; Maure, I; Boursier, F; Soufflet, L

    1995-01-01

    This work reports a comparison of an enzyme immunoassay (EIA) using two major Treponema pallidum recombinant antigens with a T. pallidum hemagglutination (TPHA) assay and a nontreponemal Venereal Disease Reference Laboratory (VDRL) test. A total of 1,822 normal donor serum samples was tested for cardiolipin and T. pallidum antibodies, respectively, by the VDRL assay and EIA. Among these samples, 440 were further tested by TPHA technology. Four samples were found positive by EIA, while all were reported to be negative by both TPHA and VDRL routine assays. Subsequent testing of EIA-positive samples confirmed 100% (four of four samples) and 25% (one of four samples) positive results, respectively, by immunofluorescence assay and a Western blot (immunoblot) syphilis kit. The sensitivity of the recombinant EIA was estimated at virtually 100% with a reference panel of 50 syphilitic samples. According to this study, the newly developed EIA kit shows 100% sensitivity combined to a specificity greater than 99.8% for detecting treponemal immunoglobulin G antibodies in blood bank syphilis screening. PMID:7751351

  9. Detection of galactomannan antigenemia by enzyme immunoassay in experimental invasive aspergillosis.

    PubMed Central

    de Repentigny, L; Boushira, M; Ste-Marie, L; Bosisio, G

    1987-01-01

    A sensitive enzyme immunoassay (EIA) for galactomannan antigenemia that avoids the use of radioisotopes was devised. Three carbohydrate-rich antigenic fractions were purified from Aspergillus fumigatus 2085: a cold alkali extract (CA) from mycelium, an acetone-precipitated pyridine extract (APSK-66) from mycelium, and a methanol precipitate from culture filtrate. CA and APSK-66 were further purified by gel filtration and ion-exchange chromatography, respectively. An acid hydrolysate of CA contained only mannose and galactose, as determined by gas-liquid chromatography. Rabbit antisera were raised against conidia, mycelia, and cell walls of A. fumigatus. By indirect EIA, the best immunoglobulin G response (1/8,000) was obtained against CA in rabbits immunized intravenously with cell walls. Antigenemia was detected by indirect EIA inhibition in heat-dissociated sera of four immunosuppressed rabbits that were infected intravenously but was absent in two uninfected controls. The circulating antigen was resistant to pronase, was adsorbed onto concanavalin A, and had a molecular size of 50 to 100 kilodaltons. PMID:3294887

  10. Competitive enzyme-linked immunoassay for sialoglycoprotein of edible bird's nest in food and cosmetics.

    PubMed

    Zhang, Shiwei; Lai, Xintian; Liu, Xiaoqing; Li, Yun; Li, Bifang; Huang, Xiuli; Zhang, Qinlei; Chen, Wei; Lin, Lin; Yang, Guowu

    2012-04-11

    The proliferation of fake and inferior edible bird's nest (EBN) products has recently become an increasingly serious concern. To identify and classify EBN products, a competitive enzyme-linked immunoassay (ELISA) was developed to quantitate sialoglycoprotein in EBN used in food and cosmetic applications. The characteristic sialoglycoprotein in EBN was found, extracted, purified, and analyzed. Sialoglycoprotein, considered the main carrier of sialic acid in EBN, consisted of 106 and 128 kDa proteins. A monoclonal antibody that could recognize both proteins was prepared. The heat-treated process did not change the affinity of sialoglycoprotein with the antibody. An optimized ELISA method was established with a cross-reactivity of less than 0.1% and an IC(50) of 3.3 μg/mL. On the basis of different food and cosmetic samples, the limits of detection (LOD) were 10-18 μg/g. Recoveries of fortified samples at levels of 20 and 80 μg/g ranged from 81.5 to 96.5%, respectively. The coefficients of variation were less than 8.0%. PMID:22439641

  11. Single-antibody in situ enzyme immunoassay for infectivity titration of hepatitis A virus.

    PubMed

    Borovec, S; Uren, E

    1997-10-01

    Hepatitis A virus (HAV) establishes a persistent infection in cultured cells, with minimal effect on host cell metabolism. As a result, the virus produces very little, if any, cytopathic effect (CPE), even with cell culture-adapted strains. This feature precludes the use of a plaque or standard endpoint assay (using CPE as an indicator of infection) for the titration of infectious virus. The radioimmunofocus assay (RIFA) is the standard method for HAV titration, though this method is labour intensive and requires the use of radioisotopes. To this end, a single-antibody in situ enzyme immunoassay (EIA) has been developed, using binding of a perioxidase-labelled monoclonal antibody to fixed cell monolayers as an indicator of infection. This novel assay is highly reproducible, can be read by eye, and is suitable for high throughput situations. Furthermore, the assay has been validated against the RIFA making it suitable for use in studies validating the safety of therapeutic biologicals for human use. PMID:9395142

  12. Detection of Snake Venom in Post-Antivenom Samples by Dissociation Treatment Followed by Enzyme Immunoassay

    PubMed Central

    Maduwage, Kalana P.; O’Leary, Margaret A.; Silva, Anjana; Isbister, Geoffrey K.

    2016-01-01

    Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell’s viper venom or Australian elapid venom measured by EIA. In confirmed Russell’s viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell’s viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell’s viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom. PMID:27136587

  13. Establishment of new cloned enzyme donor immunoassays (CEDIA) for haloperidol and bromperidol.

    PubMed

    Yasui-Furukori, Norio; Saito, Manabu; Furukori, Hanako; Inoue, Yoshimasa; Someya, Toshiyuki; Kaneko, Sunao; Tateishi, Tomonori

    2004-06-01

    The authors have developed and verified the precision and accuracy of new automated cloned enzyme donor immunoassays (CEDIA) for haloperidol and bromperidol, and cross-validations have been performed with conventional semiautomated EIA kits (MARKIT-M) and high-performance liquid chromatographic (HPLC) methods. The CEDIA method provides a quick (about 10 minutes) assay for haloperidol or bromperidol, requiring no serum/plasma pretreatment or predilution. The CEDIA haloperidol/bromperidol assay showed little or no cross reactivity with either their metabolites or many drugs commonly coprescribed. MARKIT-M revealed considerable cross reactivity values proportional to the spiked amounts of reduced metabolites. Precision, accuracy, recovery, and linearity testing for the CEDIA assay were all sufficient for clinical use. Significant linear correlations were found between CEDIA and HPLC in measuring haloperidol (CEDIA = 1.06 x HPLC + 0.869; n = 44, rs = 0.913, P < 0.001) and bromperidol (CEDIA = 1.06 x HPLC + 0.606; n = 56, rs = 0.914, P < 0.001) concentrations. This study has, therefore, demonstrated that the CEDIA assay has a quick run time with high precision and accuracy, and this method is a useful tool for the TDM of haloperidol or bromperidol. PMID:15167638

  14. [Value of prostate-specific antigen measurements with newly developed enzyme immunoassay (MARKIT-M PA)].

    PubMed

    Arai, Y; Onishi, H; Oishi, K; Takeuchi, H; Yoshida, O

    1992-10-01

    Serum prostate-specific antigen (PSA) levels in patients with prostate cancer and benign prostate hypertrophy (BPH) were investigated with a newly developed enzyme immunoassay (MARKIT-M PA, Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). Sensitivity of the assay system is 0.5 ng/ml and the detection range is 0.5-100 ng/ml. There was a high linear correlation (r = 0.987) between the assay and MARKIT-F PA, and values obtained with the assay were almost equal to those yielded by MARKIT-F PA assay. Using the BPH group as a negative control, the upper cut-off value in BPH patients was determined to be 3.6 ng/ml. Of the 48 patients with untreated prostate cancer, 77% was detectable by means of MARKIT-M PA assay. Using the BPH group as a negative control, specificity and efficiency were 93% and 86%, respectively. In another group of 27 BPH patients whose blood samples were taken immediately after digital prostatic examination, PSA was elevated in 15%. During follow-up of prostate cancer patients, PSA was elevated in 82% at the time of clinically detectable progression. In 15 patients whose disease was clinically well controlled, all levels of PSA were observed to be negative. These findings suggests that detection of serum PSA with this assay is of great use both in the diagnosis and monitoring of prostate cancer patients. PMID:1282772

  15. The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay

    PubMed Central

    Chen, Ge; Yang, Lihua; Jin, Maojun; Du, Pengfei; Zhang, Chan; Wang, Jian; Shao, Hua; Jin, Fen; Zheng, Lufei; Wang, Shanshan; She, Yongxin; Wang, Jing

    2015-01-01

    A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples. PMID:26218576

  16. Fieldable, real-time enzyme immunoassay kits for drugs on surfaces

    NASA Astrophysics Data System (ADS)

    Chiappini, Michele W.; Wendel, Gregory J.; Duquette, Peter H.; Hamilton, Martha J.; Chudzik, Stephen J.; Chappa, Ralph A.

    1994-03-01

    Immunoassays (e.g., RIA, EIA) have been demonstrated to be useful for rapid, convenient detection and semiquantitative analysis of drugs. Thermedics Detection, Inc. manufactures a rapid, sensitive, self-contained, disposable, EIA device, developed by Bio-Metric Systems, Inc., designed to allow untrained personnel to perform in field situations. This format has been developed for drugs in biological fluids and on surfaces. The analyte in the test sample competes with an enzyme-analyte conjugate for a limited number of immobilized antibody sites. The AccuPRESS Test format can detect analytes at 10 ppb in biological fluids, water, and soil, and on surfaces, such as suitcases, vehicles, tables and hands, with positive results indicated by clearly visible color development within 5 minutes. This format is designed to have all dry components and to have an ambient shelf life of greater than one year. The format is available for cocaine and opiate derivatives, including heroin, and is readily adaptable for use with numerous other drugs, explosives, and environmental pollutants.

  17. The Rapid Screening of Triazophos Residues in Agricultural Products by Chemiluminescent Enzyme Immunoassay.

    PubMed

    Chen, Ge; Yang, Lihua; Jin, Maojun; Du, Pengfei; Zhang, Chan; Wang, Jian; Shao, Hua; Jin, Fen; Zheng, Lufei; Wang, Shanshan; She, Yongxin; Wang, Jing

    2015-01-01

    A highly sensitive chemiluminescent enzyme immunoassay (CLEIA) method was developed in this study for efficient screening of triazophos residues in a large number of samples. Based on the maximum residue limits (MRLs) set by China and CAC for triazophos in different agro-products, the representative apple, orange, cabbage, zucchini, and rice samples were selected as spiked samples, and the triazophos at the concentrations of the MRL values were spiked to blank samples. Subsequently, the five samples with the spiked triazophos standard were measured by CLEIA 100 times, and the detection results indicated that the correction factors of the apple, orange, cabbage, zucchini, and rice were determined as 0.79, 0.66, 0.85, 0.76, and 0.91, respectively. In this experiment, 1500 real samples were detected by both the CLEIA and the GC-MS methods. With the GC-MS method, 1462 samples were identified as negative samples and 38 samples as positive samples. Based on the correction factors, the false positive rate of the CLEIA method was 0.13%, and false negative rate was 0. The results showed that the established CLEIA method could be used to screen a large number of real samples. PMID:26218576

  18. Detection of Snake Venom in Post-Antivenom Samples by Dissociation Treatment Followed by Enzyme Immunoassay.

    PubMed

    Maduwage, Kalana P; O'Leary, Margaret A; Silva, Anjana; Isbister, Geoffrey K

    2016-01-01

    Venom detection is crucial for confirmation of envenomation and snake type in snake-bite patients. Enzyme immunoassay (EIA) is used to detect venom, but antivenom in samples prevents venom detection. We aimed to detect snake venom in post-antivenom samples after dissociating venom-antivenom complexes with glycine-HCl (pH 2.2) and heating for 30 min at 950 °C. Serum samples underwent dissociation treatment and then Russell's viper venom or Australian elapid venom measured by EIA. In confirmed Russell's viper bites with venom detected pre-antivenom (positive controls), no venom was detected in untreated post-antivenom samples, but was after dissociation treatment. In 104 non-envenomed patients (negative controls), no venom was detected after dissociation treatment. In suspected Russell's viper bites, ten patients with no pre-antivenom samples had venom detected in post-antivenom samples after dissociation treatment. In 20 patients with no venom detected pre-antivenom, 13 had venom detected post-antivenom after dissociation treatment. In another 85 suspected Russell's viper bites with no venom detected pre-antivenom, 50 had venom detected after dissociation treatment. Dissociation treatment was also successful for Australian snake envenomation including taipan, mulga, tiger snake and brown snake. Snake venom can be detected by EIA in post-antivenom samples after dissociation treatment allowing confirmation of diagnosis of envenomation post-antivenom. PMID:27136587

  19. Rapid serodiagnosis of typhoid fever by dot enzyme immunoassay in an endemic area.

    PubMed

    Choo, K E; Oppenheimer, S J; Ismail, A B; Ong, K H

    1994-07-01

    A dot enzyme immunoassay (EIA) using 50-kD outer-membrane proteins (OMPs) of Salmonella typhi was compared with the Widal test for the serodiagnosis of typhoid fever in 109 febrile children admitted to a hospital in an endemic area. In the culture-positive typhoid group, the initial dot EIA was positive in 40 of 42 cases and the initial Widal test was positive in 41. In the culture-negative clinical typhoid group, both the dot EIA and the Widal test were positive in 17 of 18 cases. In the nontyphoidal fever group, the dot EIA was negative in all of 49 cases and the Widal test was negative in 44. With culture used as the gold standard, the dot EIA is as sensitive as the Widal test (95% vs. 98%), has a similar high negative predictive value (96% vs. 98%), and is more specific (75% vs. 67%). In addition, the dot EIA offers the advantages of simplicity, speed, early diagnosis, economy, and flexibility (i.e., other diagnostic tests can be conducted simultaneously). PMID:7948526

  20. Which one of the two common reporter systems is more suitable for chemiluminescent enzyme immunoassay: alkaline phosphatase or horseradish peroxidase?

    PubMed

    Yu, Songcheng; Yu, Fei; Liu, Lie; Zhang, Hongquan; Zhang, Zhenzhong; Qu, Lingbo; Wu, Yongjun

    2016-05-01

    Alkaline phosphatase and horseradish peroxidase are the most commonly used reporter systems in chemiluminescent enzyme immunoassay (CLEIA). Which one, therefore, would be better when establishing a CLEIA method for a new target substance? There was no standard answer. In this study, both reporters were compared systematically including luminescence kinetics, conjugation methods, optimal condition and detection performance, using two common drugs, SD-methoxy-pyrimidine and enrofloxacin, as determination objects. The results revealed that there was much difference between the luminescence kinetics of the two systems. However, there was little difference between these systems when detecting the same substance, including in optimal conditions and determination of performance. Both reporters were suitable for establishing chemiluminescent enzyme immunoassays. Therefore, the choice of alkaline phosphatase or horseradish peroxidase as the reporter system in chemiluminescent enzyme immunoassays depends on availability. Conversely, these two report systems could be applied in simultaneous analysis of multicomponents due to their different optical behaviors and similar performances. But attention should be paid to conjugation method and coating buffer, which affected the luminescent intensity of different determination targets. PMID:26552992

  1. Development of a Highly Sensitive and Specific Blastomycosis Antibody Enzyme Immunoassay Using Blastomyces dermatitidis Surface Protein BAD-1

    PubMed Central

    Smedema, Melinda L.; Durkin, Michelle M.; Brandhorst, T. Tristan; Hage, Chadi A.; Connolly, Patricia A.; Leland, Diane S.; Davis, Thomas E.; Klein, Bruce S.; Wheat, L. Joseph

    2014-01-01

    Serologic tests for antibodies to Blastomyces dermatitidis are not thought to be useful for the diagnosis of blastomycosis, in part due to the low sensitivity of immunodiffusion and complement fixation. Earlier studies have shown that the enzyme immunoassay improves the sensitivity of antibody detection for the diagnosis of blastomycosis. Microplates coated with the B. dermatitidis surface protein BAD-1 were used for testing sera from patients with proven blastomycosis or histoplasmosis and controls. Semiquantification was accomplished by using standards containing human anti-B. dermatitidis antibodies. The antibodies were detected in 87.8% of the patients with blastomycosis by the enzyme immunoassay compared to 15.0% by immunodiffusion. The specificities were 99.2% for patients with nonfungal infections and healthy subjects and 94.0% for patients with histoplasmosis. The results were highly reproducible on repeat testing. When combined with antigen testing, antibody testing improved the sensitivity from 87.8% to 97.6%. Enzyme immunoassay detection of antibodies against BAD-1 is highly specific, has greatly improved sensitivity over immunodiffusion, and may identify cases with negative results by antigen testing. This assay has the potential to aid in the diagnosis of blastomycosis. PMID:24285817

  2. Anti-TNP monoclonal antibodies as reagents for enzyme immunoassay (ELISA).

    PubMed

    Léo, P; Ucelli, P; Augusto, E F; Oliveira, M S; Tamashiro, W M

    2000-12-01

    The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG1, and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h(-1) and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays. PMID:11152399

  3. An ultrasensitive and universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification.

    PubMed

    Wang, Guang-Li; Shu, Jun-Xian; Dong, Yu-Ming; Wu, Xiu-Ming; Li, Zai-Jun

    2015-04-15

    An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01pg/mL to 1.0ng/mL with a low detection limit of 6.0fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications. PMID:25437365

  4. [Measurement of serum PA values by a newly developed enzyme immunoassay].

    PubMed

    Kuriyama, M; Esaki, N; Shinoda, I; Ito, S; Yamada, S; Tokuyama, K; Deguchi, T; Takahashi, Y; Kawada, Y; Akimoto, S

    1993-02-01

    Serum prostate-specific antigen (PA) values detected by a newly developed enzyme immunoassay (EIA, MARKIT-M PA) as a successor of MARKIT-F PA, which has been a leading kit in Japan, were evaluated for its role in the diagnosis of cancer of the prostate and follow-up of the patients afflicted with the disease. The system is one-step sandwich type EIA using horseradish peroxidase as a tracer and has 0.50-100 ng/ml of detectable range with small amount of sample volume (25 microliters) and reliable quality control data. Furthermore, serum PA values detected by the assay were almost equivocal to those detected by MARKIT-F PA. Serum PA values in prostate cancer patients (n = 122) were statistically higher than those in normal males (n = 90), urological malignancies other than prostate cancer (n = 48) or benign prostatic hypertrophy (BPH, n = 73). Even in the patients with stage A and B prostate cancer, serum PA values were observed to be statistically higher than those in BPH cases. If 3.6 ng/ml was used, which is normal value in MARKIT-F PA, as a cut-off value and BPH cases as a control, the sensitivity, specificity and efficacy for diagnosis of prostate cancer were 77.9, 91.8 and 83.1%, respectively, which showed the best results during the range examined. Serially determined serum PA values in following up the patients with prostate cancer were confirmed to be highly effective to evaluate treatment responses. These results suggest that MARKIT-M PA is thought to be one of the best tool for determination of serum PA values. PMID:7681886

  5. Enzyme immunoassay for swine trichinellosis using antigens purified by immunoaffinity chromatography

    SciTech Connect

    Seawright, G.L.; Despommier, D.; Zimmermann, W.; Isenstein, R.S.

    1983-11-01

    Various preparations of crude and a purified preparation of Trichinella spiralis antigens were compared in a rapid, micro-enzyme immunoassay (EIA) for detecting trichinellosis in swine. The crude antigen preparations (XM-300 or S/sub 3/ fraction) were lipid-free, cell-free fractions of muscle larvae, and the purified antigen was prepared by immunoaffinity chromatography of the soluble fraction of stichocyte secretory granules from rat muscle larvae. The antigens were tested against normal and immune swine sera for sensitivity and specificity, and for their ability to detect seroconversions early in the immune response. Tests of sequential sera from experimentally-infected pigs showed that the column antigen produced lower absorbances with pre-infection sera and, from 18 days post-infection, higher absorbances with positive sera. From 21-28 days post-infection, absorbances and S/N ratios with column antigen were nearly twice those with XM-300. Column antigen detected antibodies more often than XM-300 antigen in sera collected prior to the appearance of larvae. Crude antigen did not distinguish all true negatives from weakly positives in a study involving 100 sera from muscle digestion-negative pigs and 75 sera from experimentally infected pigs, whereas the column antigen distinguished all negatives from positives. In a larger scale test of the column antigen, 1130 pigs from Puerto Rico were tested in the micro-EIA test. Puerto Rico has no endogenous trichinellosis, and all 1130 pigs were shown to be muscle digestion negative. These results show that the column antigen out-performs the crude antigens in sensitivity, specificity, and early detection. The column antigen is therefore a major improvement in the EIA for swine trichinellosis.

  6. Development and evaluation of an enzyme immunoassay for rapid diagnosis of rabies in humans and animals.

    PubMed

    Vasanth, Joel P; Madhusudana, S N; Abhilash, K V; Suja, M S; Muhamuda, K

    2004-10-01

    The presently advocated tests for rapid diagnosis of rabies such as fluorescent antibody test (FAT) is expensive and requires expertise to carry out and interpret the results. In this study we have developed and evaluated a simple enzyme immuno-assay (EIA) to detect rabies antigen in the brain specimens of animals and humans. We have also evaluated the utility of this test in ante mortem diagnosis of human rabies. The brain homogenates of suspected rabid animals (n=250), humans (n=16) and clinical samples like saliva (n=16) and cerebrospinal fluid (CSF, n=16) applied on to ELISA plates coated with rabies antinucleoprotein antibody and the absorbed rabies nucleoprotein antigen was detected using biotinylated anti-nucleoprotein antibody followed by treatment with streptavidin peroxidase conjugate and colour development with OPD. Rabies infected and normal mouse brain homogenates were used as positive and negative controls respectively. The results of this test was evaluated with fluorescent antibody technique (for brain samples) and mice inoculation test (for saliva and CSF samples). A distinct dark brown color was seen in positive control and all positive samples and there was no color development in negative control and samples. The concordance between FAT and EIA was 98.4%. With brain samples, 83.3% with saliva and 91.6% with CSF samples. The specificity of the test was found to be 100%. It can be concluded that the EIA described here is a sensitive, specific and rapid test for post mortem diagnosis of rabies in animals and humans. The utility of this test for ante mortem diagnosis of rabies needs to be further evaluated. PMID:16295401

  7. Accuracy of immunoglobulin M immunoassay for diagnosis of chlamydial infections in infants and adults.

    PubMed Central

    Mahony, J B; Chernesky, M A; Bromberg, K; Schachter, J

    1986-01-01

    An improved solid-phase enzyme immunoassay (EIA) with Chlamydia trachomatis L2 434/Bu elementary bodies was developed for the measurement of immunoglobulin M (IgM) antibody to C. trachomatis in serum. Comparison of EIA and microimmunofluorescence IgM antibody titers of 156 serum samples revealed an EIA sensitivity and specificity of 100% for infants, but reduced sensitivity (85%) and specificity (76%) for sera from adults. Sera containing IgM class rheumatoid factor produced false-positive IgM results which could easily be eliminated by pretreatment of the sera with anti-human IgG. Analysis of sera from infants with chlamydial infections revealed that 17 of 17 infants with C. trachomatis pneumonia had high IgM antibody titers (geometric mean titer, 1:64,812), whereas two infants with conjunctivitis only lacked detectable IgM antibody. EIA detected IgM antibody to several serovar groups in serum, including serovars B, BDE, FG, and J. IgM antibody to C. trachomatis in serum was detected as early as 5 days after the infection that was acquired at delivery and persisted for 3 months. The availability of an EIA possessing good sensitivity and specificity for the detection of IgM antibody to C. trachomatis may permit more laboratories to diagnose perinatal chlamydial infections. PMID:3533983

  8. Enzyme immunoassay using a reusable extended-gate field-effect-transistor sensor with a ferrocenylalkanethiol-modified gold electrode.

    PubMed

    Kamahori, Masao; Ishige, Yu; Shimoda, Maki

    2008-09-01

    A reusable extended-gate field-effect transistor (FET) sensor with an 11-ferrocenyl-1-undecanethiol (11-FUT) modified gold electrode was developed for applying to enzyme immunoassay. It was found that the 11-FUT modified FET sensor detected a thiol compound 50 times or more repeatedly after a treatment with a 5% hydrogen peroxide solution. The gate-voltage shift of the FET sensor showed a fairly good linearity (R(2) = 0.998) within a range from 10(-2) to 10(-6) M on the concentration of 6-hydroxyl-1-hexanethiol, which is a thiol compound, at a Nernstian response of 58.5 mV/decade. The FET-based immunoassay was constructed by combining the 11-FUT modified-FET sensor with the enzyme-linked immunosorbent assay (ELISA), in which the enzyme chemistry of acetylcholinesterase (AChE) was used to generate a thiol compound. The 11-FUT modified FET sensor with an AC voltage at 1 MHz superimposed onto the reference electrode detected the AChE-catalyzed product corresponding to a serum concentration of interleukin 1beta from 10 to 5000 pg/mL. In addition, all measurements were successfully performed by using the same FET-sensor chip after a treatment with a 5% hydrogen peroxide solution. PMID:18781015

  9. Evaluation of a homogenous enzyme immunoassay for the detection of synthetic cannabinoids in urine

    PubMed Central

    Barnes, Allan J.; Young, Sheena; Spinelli, Eliani; Martin, Thomas M.; Klette, Kevin L.; Huestis, Marilyn A.

    2014-01-01

    Introduction The recent emergence and widespread availability of many new synthetic cannabinoids support the need for an accurate and high-throughput urine screen for these new designer drugs. We evaluated performance of the immunalysis homogeneous enzyme immunoassay (HEIA) to sensitively, selectively, and rapidly identify urinary synthetic cannabinoids. Methods 2443 authentic urine samples were analyzed with the HEIA that targets JWH-018 N-pentanoic acid, and a validated LC-MS/MS method for 29 synthetic cannabinoids and metabolites. Semiquantitative HEIA results were obtained, permitting performance evaluation at and around three cutoffs (5, 10 and 20 μg/L), and diagnostic sensitivity, specificity and efficiency determination. Performance challenges at ±25 and ±50% of each cutoff level, cross-reactivity and interferences also were evaluated. Results Sensitivity, specificity, and efficiency of the immunalysis HEIA K2 Spice kit with the manufacturer's recommended 10 μg/L cutoff were 75.6%, 99.6% and 96.8%, respectively, as compared to the reference LC-MS/MS method with limits of detection of 0.1 -10 μg/L. Performance at 5 μg/L was 92.2%, 98.1% and 97.4%, and for the 20 μg/L cutoff were 62.9%, 99.7% and 95.4%. Semi-quantitative results for in-house prepared standards were obtained from 2.5-30 μg/L, and documented acceptable linearity from 5-25 μg/L, with inter-day imprecision <30% (n = 17). Thirteen of 74 synthetic cannabinoids evaluated were classified as highly cross-reactive (≥50% at 10 μg/L); 4 showed moderate cross-reactivity (10–50% at 10 μg/L), 30 low cross-reactivity (<10% at 500 μg/L), and 27 <1% cross-reactivity at 500 μg/L. There was no interference from 102 investigated compounds. Only a mixture containing 1000 μg/L each of buprenorphine/norbuprenorphine produced a positive result above our proposed cutoff (5 μg/L) but below the manufacturer's recommended cutoff concentration (10 μg/L). Conclusion The Immunalysis HEIA K2 Spice kit

  10. Evaluation of a novel chemiluminescent microplate enzyme immunoassay for hepatitis B surface antigen detection.

    PubMed

    Yang, Lin; Song, Liu-Wei; Fang, Lin-Lin; Wu, Yong; Ge, Sheng-Xiang; Li, Hui; Yuan, Quan; Zhang, Jun; Xia, Ning-Shao

    2016-02-01

    Hepatitis B virus surface antigen (HBsAg) is an important biomarker used in the diagnosis of hepatitis B virus (HBV) infection, but false-negative results are still reported in the detection of HBsAg using commercial assays. In this study, we evaluated the qualitative properties of a novel HBsAg chemiluminescence enzyme immunoassay (CLEIA) assay--WTultra. WHO standard sample dilution series and samples from low-level HBsAg carriers (<1 ng/mL) were used to evaluate the sensitivity of the WTultra assay. Boston Biomedica, Inc. (BBI) hepatitis B seroconversion panels were used to assess the ability of the WTultra assay to detect the window period. In addition, dilution series of 22 serum samples with different genotypes, serotypes and HBsAg mutations were used to assess the WTultra assay, and these were compared with other commercial assays. The lower detection limit of the WTultra assay was 0.012 IU/mL, and it showed a high sensitivity (97.52%, 95% CI, 94.95-99.00) in the detection of 282 low-level HBsAg carriers (<1 ng/mL). In samples with various HBV genotypes, serotypes and HBsAg mutations, the WTultra assay yielded 117 positive results in 132 samples, which was significantly higher than the results with the other four commercial assays (89, 83, 65 and 45, respectively, p<0.01). In the assays of mutant strains, the WTultra assay detected 82 positive results in 90 samples, which was significantly better than the results for the Hepanostika HBsAg Ultra (58 positive) and Architect (55 positive) (p<0.01) assays, which in turn were significantly better than the Murex V.3 (41 positive, p=0.026) and AxSYM V2 (29 positive, p<0.01) assays. However, in the detection of 42 samples of wild-type strains with various genotypes and serotypes, no significant differences were observed among the WTultra (35 positive), Architect (28 positive) and Hepanostika HBsAg Ultra (31 positive) assays. However, the WTultra assay detected significantly more samples than the Murex V.3 (24

  11. Enzyme immunoassay for the detection of antibody to hepatitis E virus based on synthetic peptides.

    PubMed

    Favorov, M O; Khudyakov, Y E; Fields, H A; Khudyakova, N S; Padhye, N; Alter, M J; Mast, E; Polish, L; Yashina, T L; Yarasheva, D M

    1994-02-01

    Five synthetic peptides were prepared based on the nucleotide sequence of open reading frames 2 and 3 encoded in the hepatitis E virus (HEV) genome and were used to develop an enzyme immunoassay (EIA) for the detection of anti-HEV activity in sera. Three different approaches were employed to ascertain the optimal preparation of these peptides as an immunodiagnostic reagent, including (1) a mixture of unconjugated peptides, (2) conjugating individual peptides to bovine serum albumin (BSA) followed by mixing each conjugate at various concentrations, and (3) mixing the peptides before conjugation with BSA to create an artificial antigen complex. The third method was superior in discriminating anti-HEV activity in sera previously tested by Western blot (WB). A frequency distribution of optical density values demonstrated that the peptide-based EIA was able to readily discriminate anti-HEV positive sera from sera devoid of anti-HEV activity. To confirm anti-HEV activity a neutralization test was developed using a mixture of 5 unconjugated peptides. With the exception of sera containing high levels of anti-HEV activity, all sera were neutralized greater than 50%. Strong sera required a higher dilution before a 50% neutralization was achieved. The sensitivity of the WB compared to EIA was 89.5% with and overall concordance of 94.8%. The peptide-EIA was used to determine anti-HEV activity in sera collected from various populations worldwide. In six outbreaks of ET-NANB hepatitis in various geographic regions, anti-HEV activity was demonstrated in 78-100% of cases. The peptide-EIA also detected anti-HEV activity in 14 out of 14 follow-up sera obtained 4-6 months after onset of disease and in 2 of 2 of these patients 5 yr after the acute episode. Anti-HEV activity was found in 8.5% of sera obtain from a healthy population residing in an HEV endemic region and 0.5% in two non-endemic regions (P < 0.001). These data demonstrate that a synthetic peptide-based EIA is sensitive

  12. The measurement of triclosan in water using a magnetic particle enzyme immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle-based immunoassay to determine triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) in drinking water and wastewater was developed. Rabbit antiserum was produced by immunizing the rabbit with 6-(5-chloro-2-(2,4-dichlorophenoxy)phenoxy)hexanoic acid-keyhole limpet hemocya...

  13. Dried venous blood samples for the detection and quantification of measles IgG using a commercial enzyme immunoassay.

    PubMed Central

    Riddell, Michaela A.; Byrnes, Graham B.; Leydon, Jennie A.; Kelly, Heath A.

    2003-01-01

    OBJECTIVES: To determine whether samples of dried venous blood (DVB) were an acceptable alternative to serum for detecting measles-specific IgG in a commercial enzyme immunoassay. METHODS: Paired samples of serum and DVB were collected from 98 suspected cases of measles and 1153 schoolchildren in Victoria, Australia. All samples were tested using the Dade Behring Enzygnost Anti-Measles-Virus/IgG immunoassay. DVB samples were eluted using either the sample buffer provided with the kit or 5% dry milk powder in phosphate-buffered saline-Tween 20. FINDINGS: DVB samples eluted by sample buffer showed significantly better linear correlation to the serum samples than did DVB samples eluted in 5% dry milk in phosphate-buffered saline-Tween 20. To improve the comparability of serum and DVB samples an adjustment factor of 1.28 was applied to the optical density (OD) values of DVB. This adjustment also enabled quantification of the titre of measles IgG in mIU/ml directly from the OD value using the alpha calculation as specified by the kit protocol. For DVB samples stored for less than six months at 4 degrees C, the assay showed an overall sensitivity of 98.4% and a specificity of 97.2% compared with the results of serum testing. CONCLUSION: These results illustrate the potential for DVB samples to be widely used with the Dade Behring enzyme immunoassay system for determining the immunity of the individual and the population to the measles virus. PMID:14758429

  14. Naked-eye detection as a universal approach to lower the limit of detection of enzyme-linked immunoassays.

    PubMed

    O'Connor, Erin F; Paterson, Sureyya; de la Rica, Roberto

    2016-05-01

    Colorimetric biosensors for the detection of analytes with the naked eye are required in environmental monitoring, point-of-care diagnostics, and analyses in resources constrained settings, where detection instruments may not be available. However, instrument-based detection methods are usually more adequate for detecting small variations in the signal compared to naked-eye detection schemes, and consequently the limit of detection of the latter is usually higher than the former. Here, we demonstrate that the limit of detection of colorimetric enzyme-linked immunoassays can be decreased several orders of magnitude when using naked-eye detection instead of a spectrophotometer for detecting the signal. The key step to lower the limit of detection is adding a small volume of chromogenic substrate during the signal generation step. This generates highly colored solutions that can be easily visualized with the naked eye and recorded with the camera of a mobile phone. The proposed method does not require expensive equipment or complex protocols to enhance the signal, and therefore it is a universal approach to lower the limit of detection of colorimetric enzyme-linked immunoassays. PMID:26970749

  15. Enhanced Colorimetric Immunoassay Accompanying with Enzyme Cascade Amplification Strategy for Ultrasensitive Detection of Low-Abundance Protein

    PubMed Central

    Gao, Zhuangqiang; Hou, Li; Xu, Mingdi; Tang, Dianping

    2014-01-01

    Methods based on enzyme labels have been developed for colorimetric immunoassays, but most involve poor sensitivity and are unsuitable for routine use. Herein, we design an enhanced colorimetric immunoassay for prostate-specific antigen (PSA) coupling with an enzyme-cascade-amplification strategy (ECAS-CIA). In the presence of target PSA, the labeled alkaline phosphatase on secondary antibody catalyzes the formation of palladium nanostructures, which catalyze 3,3′,5,5′-tetramethylbenzidine-H2O2 system to produce the colored products, thus resulting in the signal cascade amplification. Results indicated that the ECAS-CIA presents good responses toward PSA, and allows detection of PSA at a concentration as low as 0.05 ng mL−1. Intra- and inter-assay coefficients of variation are below 9.5% and 10.7%, respectively. Additionally, the methodology is validated for analysis of clinical serum specimens with consistent results obtained by PSA ELISA kit. Importantly, the ECAS-CIA opens a new horizon for protein diagnostics and biosecurity. PMID:24509941

  16. Use of sequential enzyme immunoassay and direct fluorescent antibody tests for detection of Chlamydia trachomatis infections in women.

    PubMed Central

    Schwebke, J R; Stamm, W E; Handsfield, H H

    1990-01-01

    Endocervical infections due to Chlamydia trachomatis remain difficult to diagnose due to the lack of an inexpensive, rapid, and accurate test. We evaluated an alternative strategy for diagnosis in which initial screening was performed with an enzyme immunoassay (Chlamydiazyme) followed by a direct fluorescent antibody (DFA) test on specimens in which the Chlamydiazyme optical density (OD) reading fell in an intermediate zone. Lowering the Chlamydiazme OD ratio (specimen to control) used to define a positive test from 1.0 (the ratio suggested by the manufacturer) to 0.3 raised the sensitivity of Chlamydiazyme from 73 to 83%. Confirmation of those specimens having OD ratios of 0.3 to 0.99 by DFA testing increased the specificity of Chlamydiazyme from 95 to 100%. This strategy necessitated performance of the DFA test on 5% of the specimens. Lowering the cutoff OD ratio below 0.3 increased the sensitivity even further but required DFA testing on greater than 25% of the specimens. Use of an adjusted positive cutoff value for defining positive enzyme immunoassays followed by DFA confirmation for intermediate-zone readings may be a feasible approach for some laboratories that lack cell culture facilities. PMID:2254422

  17. Multiplexed Electrochemical Immunoassay of Phosphorylated Proteins Based on Enzyme-Functionalized Gold Nanorod Labels and Electric Field-Driven Acceleration

    SciTech Connect

    Du, Dan; Wang, Jun; Lu, Donglai; Dohnalkova, Alice; Lin, Yuehe

    2011-09-09

    A multiplexed electrochemical immunoassay integrating enzyme amplification and electric field-driven strategy was developed for fast and sensitive quantification of phosphorylated p53 at Ser392 (phospho-p53 392), Ser15 (phospho-p53 15), Ser46 (phospho-p53 46) and total p53 simultaneously. The disposable sensor array has four spatially separated working electrodes and each of them is modified with different capture antibody, which enables simultaneous immunoassay to be conducted without cross-talk between adjacent electrodes. The enhanced sensitivity was achieved by multi-enzymes amplification strategy using gold nanorods (AuNRs) as nanocarrier for co-immobilization of horseradish peroxidase (HRP) and detection antibody (Ab2) at high ratio of HRP/Ab2, which produced an amplified electrocatalytic response by the reduction of HRP oxidized thionine in the presence of hydrogen peroxide. The immunoreaction processes were accelerated by applying +0.4 V for 3 min and then -0.2 V for 1.5 min, thus the whole sandwich immunoreactions could be completed in less than 5 min. The disposable immunosensor array shows excellent promise for clinical screening of phosphorylated proteins and convenient point-of-care diagnostics.

  18. Validation of a second generation competitive enzyme immunoassay (CELISA) for the diagnosis of brucellosis in various species of domestic animals.

    PubMed

    Nielsen, K; Smith, P; Yu, W L; Elmgren, C; Halbert, G; Nicoletti, P; Perez, B; Conde, S; Samartino, L; Nicola, A; Bermudez, R; Renteria, T

    2008-10-15

    A second generation competitive enzyme immunoassay (CELISA) for detection of bovine antibody to Brucella abortus was developed to eliminate reagent variables in the assay. This assay was different from earlier CELISA formats in that it used recombinant protein A and protein G immunoglobulin receptors (PAG), labelled with horseradish peroxidase, thus eliminating the requirement for polyclonal anti-mouse-enzyme conjugate for detection. This allowed standardization of the assay. The CELISA uses a monoclonal antibody specific for a common epitope of the O-polysaccharide (OPS) of smooth lipopolysaccharide (SLPS) derived from B. abortus S1119.3. This antibody did not react with PAG. This monoclonal antibody was used to compete with antibody in the bovine test serum to the smooth lipopolysaccharide (SLPS) antigen. Reaction of bovine antibody was then measured directly with the PAG enzyme conjugate. In this case, development of colour in the reaction indicated a positive reaction. The performance characteristics of the new CELISA, sensitivity, specificity and exclusion of antibody of B. abortus S19 vaccinated animals, were very similar to those of the classical CELISA and to the indirect enzyme immunoassay (IELISA) when using sera deemed positive by isolation of the bacterium, either from individual animals or from some animals on the premises. All sera were tested by the buffered antigen plate agglutination test (BPAT) and the complement fixation test (CFT). Only samples positive on both BPAT and CFT were considered as positive and only samples negative on both tests were used considered negative. Sufficient samples from cattle, swine, sheep and goats to validate the test were included based on OIE guidelines suggesting inclusion of a minimum of 300 positive and 1000 negative samples. PMID:18771805

  19. A single-step enzyme immunoassay capillary sensor composed of functional multilayer coatings for the diagnosis of marker proteins.

    PubMed

    Funano, Shun-ichi; Sugahara, Masato; Henares, Terence G; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

    2015-03-01

    A single-step, easy-to-use enzyme immunoassay capillary sensor, composed of functional multilayer coatings, was developed in this study. The coatings were composed of substrate-immobilized hydrophobic coating, hydrogel coating, and soluble coating containing an enzyme-labeled antibody. The response mechanism involved a spontaneous immunoreaction triggered by capillary action-mediated introduction of a sample antigen solution and subsequent separation of unreacted enzyme-labeled antibodies and antigen-enzyme-labeled antibody complexes by the molecular sieving effect of the hydrogel. An enzyme reaction at the substrate-immobilized hydrophobic coating/hydrogel coating interface resulted in a protein-selective fluorescence response. An antigen concentration-dependent response was obtained for diagnostic marker protein samples (hemoglobin A1c (HbA1c), 7.14-16.7 mg mL(-1); alpha-fetoprotein (AFP), 1.4-140 ng mL(-1); C-reactive protein (CRP), 0.5-10 μg mL(-1)) that cover a clinically important concentration range. The successful measurement of CRP in diluted serum samples demonstrated the application of this capillary sensor. PMID:25599100

  20. Development of a highly sensitive bioluminescent enzyme immunoassay for hepatitis B virus surface antigen capable of detecting divergent mutants.

    PubMed

    Minekawa, Takayuki; Takehara, Shizuka; Takahashi, Masaharu; Okamoto, Hiroaki

    2013-08-01

    Hepatitis B virus (HBV) infections are sometimes overlooked when using commercial kits to measure hepatitis B virus surface antigen (HBsAg) due to their low sensitivities and reactivities to mutant strains of various genotypes. We developed an ultrasensitive bioluminescent enzyme immunoassay (BLEIA) for HBsAg using firefly luciferase, which is adaptable to a variety of HBsAg mutants, by combining four monoclonal antibodies with a polyclonal antibody against HBsAg. The measurement of seroconversion panels showed trace amounts of HBsAg during the early infection phase by the BLEIA because of its high sensitivity of 5 mIU/ml. The BLEIA detected HBsAg as early as did PCR in five of seven series and from 2.1 to 9.4 days earlier than commercial immunoassay methods. During the late infection phase, the BLEIA successfully detected HBsAg even 40 days after the disappearance of HBV DNA and the emergence of antibodies against HBsAg. The HBsAg BLEIA successfully detected all 13 recombinant HBsAg and 45 types of HBsAg mutants with various mutations within amino acids 90 to 164 in the S gene product. Some specimens had higher values determined by the BLEIA than those by a commercial chemiluminescent immunoassay; this suggests that such discrepancies were caused by the dissociation of preS1/preS2 peptides from the particle surface. With its highly sensitive detection of low-titer HBsAg, including various mutants, the HBsAg BLEIA is considered to be useful for the early diagnosis and prevention of HBV infection because of the shorter window of infection prior to detection, which facilitates early prediction of recurrence in HBV-infected individuals. PMID:23761660

  1. Evaluation of performances of three DNA enzyme immunoassays for detection of Helicobacter pylori PCR products from biopsy specimens.

    PubMed Central

    Monteiro, L; Cabrita, J; Mégraud, F

    1997-01-01

    PCR is recognized as a promising method for the detection of Helicobacter pylori in gastric biopsy specimens. However, detection of PCR products by gel electrophoresis is difficult to implement in routine clinical laboratories. The aim of this study was to compare three new DNA enzyme immunoassays with the standard method in their ability to detect PCR products. The three assays were based on the amplification of a fragment of the ureC gene of H. pylori and a colorimetric hybridization assay. The first assay (GEN-ETI-K DNA enzyme immunoassay; Sorin, Sallugia, Italy) was based on the hybridization of amplified DNA with a probe bound in microtiter wells and detected with labelled anti-DNA antibody. The second assay (Pylori-prob; Biocode, Sclessin, Belgium) comprised a solid-phase sandwich hybridization system with a specific biotinylated probe being used for detection. Finally, the third assay (PCR enzyme-linked immunosorbent assay; Boehringer, Mannheim, Germany) was based on the hybridization of amplified DNA labelled with digoxigenin as a probe (used as a coating in microtiter wells) and detected with antidigoxigenin-peroxidase as conjugate. The sensitivity of the colorimetric assay was evaluated by using amplification products from PCR assays performed on several 10-fold dilutions of DNA from H. pylori CIP 101260, and the specificity was assessed with different urease-positive bacteria. Biopsy specimens from 199 patients were tested; 106 were classified as H. pylori positive, and 93 were classified as H. pylori negative by culture and/or histological examination as the "gold standard." The receiving operating characteristic curve was used to determine the best cutoff point for each assay. The detection of PCR products by colorimetric hybridization increases the sensitivity up to 100-fold compared to that with gel electrophoresis. The results are rapid (4 h) and easy to interpret and can be automated. PMID:9350762

  2. A genetically engineered fusion protein with horseradish peroxidase as a marker enzyme for use in competitive immunoassays.

    PubMed

    Grigorenko, V; Andreeva, I; Börchers, T; Spener, F; Egorov, A

    2001-03-15

    Horseradish peroxidase is one of the most widely used marker enzymes in immunoassays. Several disadvantages are encountered upon chemical conjugation of peroxidase with antibodies or antigens, as are low reproducibility and undefined stoichiometry. We here describe for the first time the production of a recombinant fusion of a protein analyte with horseradish peroxidase in Escherichia coli, employing refolding of inclusion bodies and reconstitution with heme. The genetic fusion approach enables preparation of conjugates with 1:1 stoichiometry and defined structure. As a protein analyte, the human heart fatty acid binding protein (H-FABP) was chosen, which is a new and sensitive marker for acute myocardial infarction. The recombinant conjugate was fully active [650 U/mg with 2,2-azino-bis(3-ethyl-thiazoline-6-sulfonate) as substrate] and obtained in a yield of 12 mg/L of E. coli culture, which is better than that for recombinant peroxidase alone. The competitive immunoassay that was developed with the recombinant conjugate requires fewer incubation steps than the traditional sandwich ELISA format. It permitted the detection of H-FABP directly in plasma in the range of 10-1500 ng/mL which is the relevant range for clinical decision-making. PMID:11305642

  3. Amplified inhibition of the electrochemical signal of ferrocene by enzyme-functionalized graphene oxide nanoprobe for ultrasensitive immunoassay.

    PubMed

    Lai, Guosong; Cheng, Hui; Xin, Dinghong; Zhang, Haili; Yu, Aimin

    2016-01-01

    A nanoprobe-induced signal inhibition mechanism was designed for ultrasensitive electrochemical immunoassay at a chitosan-ferrocene (CS-Fc) based immunosensor. The nanoprobe was prepared by covalently loading signal antibody and high-content horseradish peroxidase (HRP) on the graphene oxide (GO) nanocarrier. The immunosensor was prepared through the stepwise assembly of gold nanoparticles (Au NPs) and capture antibody at a CS-Fc modified electrode. After sandwich immunoreaction, the GO-HRP nanoprobes were quantitatively captured onto the immunosensor surface and thus induced the production of a layer of insoluble film through the enzymatically catalytic reaction of the HRP labels. Both the dielectric immunocomplex formed on the immunosensor surface and the enzymatic precipitate with low electroconductivity led to the electrochemical signal decease of the Fc indicator, which was greatly amplified by the multi-enzyme signal amplification of the nanoprobe. Based on this amplified signal inhibition mechanism, a new ultrasensitive electrochemical immunoassay method was developed. Using carcinoembryonic antigen as a model analyte, this method showed a wide linear range over 5 orders of magnitude with a detection limit down to 0.54 pg/mL. Besides, the immunosensor showed good specificity, acceptable reproducibility and stability as well as satisfactory reliability for the serum sample analysis. PMID:26703270

  4. A prospective field evaluation of an enzyme immunoassay: Detection of eastern equine encephalomyelitis virus antigen in pools of Culiseta melanura

    USGS Publications Warehouse

    Scott, T.W.; Olson, J.G.; Lewis, T.E.; Carpenter, J.W.; Lorenz, L.H.; Lembeck, L.A.; Joseph, S.R.; Pagac, B.B.

    1987-01-01

    A prospective field study was conducted to determine the sensitivity and specificity of an enzyme immunoassay (EIA) compared to virus isolation in cell culture for the detection of eastern equine encephalomyelitis (EEE) virus in naturally infected mosquitoes. A total of 10,811 adult female Culiseta melanura were collected in light traps during 1985 from four locations in Maryland. Eastern equine encephalomyelitis virus was isolated from 5 of 495 mosquito pools in African green monkey kidney and baby hamster kidney cell cultures. All five virus-infected pools were detected by the EIA, and all 490 uninfected pools were correctly scored as not containing virus. The EIA did not produce false positive or false negative results. Results support the assertion of previous researchers that the antigen detection EIA is a rapid, sensitive, specific, and simple alternative to traditional bioassays for the detection of EEE virus in mosquitoes.

  5. [Enzyme immunoassay for detection of porcine circovirus type 2, by using the recombinant capsid protein ORF-2].

    PubMed

    Shkaeva, M A; Bogdanova, V S; Tsibezov, V V; Gibadulin, R A; Musienko, M I; Alekseev, K P; Grebennikova, T V; Verkhovskiĭ, O A; Zaberezhnyĭ, A D; Aliper, T I

    2006-01-01

    Recombinant antigen ORF2 from porcine circovirus type 2 (PCV-2) was produced, by using the baculovirus expression system, with histidine tags to allow purification by metal-chelate affinity chromatography. The purity of the protein was verified by polyacrylamide gel electrophoresis; and its immunospecificity was confirmed by the immunoblotting test using reference PCV-2-positive and PCV-2-negative porcine sera and monoclonal antibodies. The protein was used as an antigen to develop an indirect enzyme immunoassay (EIA) of PCV-2 antibodies. EIA was shown to have a high sensitivity and specificity as compared with indirect immunofluorescence test. Porcine serum samples from 15 pig-breeding farms of the Russian Federation were studied. Seropositive samples were found in all age pig groups in all the farms, The number of seropositive animals was shown to be directly related to its age. PMID:17087066

  6. A novel protease activity assay method based on an engineered autoinhibited protein using an enzyme-linked immunoassay.

    PubMed

    Yoon, Hyun Kyung; Yoo, Tae Hyeon

    2013-12-01

    Proteases are involved in various biological phenomena, and their aberrant activity can be an important indicator of disease. Thus, various methods have been developed to analyze the activities of proteases, but their wide application has been hampered because each method has drawbacks. In this report, we propose a new protease assay method based on an engineered autoinhibited protein and enzyme-linked immunoassay (ELISA) in which a protease of interest activates the autoinhibited protein and the signal is amplified via ELISA. Using this concept a sensitive assay method for MMP2 and caspase-3 was developed. The limit of detection for the two proteases was as low as 7 pM for MMP2 and 0.1 pM for caspase-3. The autoinhibited protein is designed modularly, and the new platform is general enough for the development of assay methods for other proteases with minimal modification. PMID:24106734

  7. TRICLOSAN AND METHYL-TRICLOSAN MONITORING STUDY IN THE NORTHEAST OF SPAIN USING A MAGNETIC PARTICLE ENZYME IMMUNOASSAY AND CONFIRMATORY ANALYSIS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The occurrence of triclosan in the water environment around a Mediterranean region was investigated. Triclosan and methyl-triclosan content of ninety five environmental samples were screened using a magnetic particle enzyme immunoassay. Positive samples were confirmed by solid phase extraction (SPE...

  8. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay.

    PubMed

    Adungo, Ferdinard; Yu, Fuxun; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu; Morita, Kouichi

    2016-08-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  9. Development and Characterization of Monoclonal Antibodies to Yellow Fever Virus and Application in Antigen Detection and IgM Capture Enzyme-Linked Immunosorbent Assay

    PubMed Central

    Adungo, Ferdinard; Kamau, David; Inoue, Shingo; Hayasaka, Daisuke; Posadas-Herrera, Guillermo; Sang, Rosemary; Mwau, Matilu

    2016-01-01

    Yellow fever (YF) is an acute hemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in an Escherichia coli expression system. Both the recombinant protein and sucrose gradient-purified YF vaccine virus 17D (YF-17D) were used to immunize BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterized by indirect enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue virus or Japanese encephalitis virus. Epitope mapping showed strong binding of three MAbs to amino acid positions 1 to 51, while two MAbs mapped to amino acid positions 52 to 135 of the envelope protein. The remaining three MAbs did not show reactivity to envelope fragments. The established MAbs exert no neutralization against wild-type YF and 17D viruses (titer of <10 for both strains). The applicability of MAbs 8H3 and 3F4 was further evaluated using IgM capture ELISA. A total of 49 serum samples were analyzed, among which 12 positive patient and vaccinee samples were correctly identified. Using serum samples that were 2-fold serially diluted, the IgM capture ELISA was able to detect all YF-positive samples. Furthermore, MAb-based antigen detection ELISA enabled the detection of virus in culture supernatants containing titers of about 1,000 focus-forming units. PMID:27307452

  10. Enzyme-triggered tyramine-enzyme repeats on prussian blue-gold hybrid nanostructures for highly sensitive electrochemical immunoassay of tissue polypeptide antigen.

    PubMed

    Xu, Tisen; Zhang, Haiying; Li, Xuegui; Xie, Zhaohui; Li, Xiangyong

    2015-11-15

    A novel sandwich-type electrochemical immunoassay with sensitivity enhancement was developed for quantitative detection of tissue polypeptide antigen (TPA) by coupling with target-induced tyramine signal amplification on prussian blue-gold hybrid nanostructures. The immunosensor was prepared through immobilizing anti-TPA capture antibody on a cleaned screen-printed carbon electrode (SPCE). Prussian blue-gold hybrid nanostructures (PBGNS) labeled with horseradish peroxidase (HRP) and detection antibody were utilized as the signal-transduction tags. Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the electrode. The carried HRP could trigger the formation of tyramine-HRP repeats on the PBGNS in the presence of H2O2. Using the doped prussian blue as the electron mediator, the conjugated HRP could catalyze the reduction of H2O2. Under the optimal conditions, the catalytic currents increased with the increasing target TPA in the dynamic range from 1.0 pg mL(-1) to 100 ng mL(-1) with a detection limit of 0.3 pg mL(-1). The reproducibility and specificity of the electrochemical immunoassay were acceptable. In addition, the contents of target TPA in nine human serum specimens were evaluated by using the developed electrochemical immunosensor, and the obtained results correlated well with those from commercially enzyme-linked immunosorbent assay (ELISA) method with a correlation coefficient of 0.9975. PMID:26067328

  11. Concordance of four commercial enzyme immunoassay and three immunoblot formats for the detection of Lyme borreliosis antibodies in human serum: the two-tier approach remains.

    PubMed

    Dickeson, David J; Chen, Sharon C-A; Sintchenko, Vitali G

    2016-04-01

    Serological tests show considerable variation in their ability to correctly diagnose Lyme borreliosis (LB). This study compared four commercially available screening enzyme immunoassays (EIA) for the detection of LB IgG using either whole cell lysate (WCL) antigens, purified proteins or recombinant antigens with the second-tier whole cell sonicate (WCS) western immunoblots or recombinant antigen line blots. A consensus between three EIA results from 222 patient sera was designated as a point of comparison for each method which gave 66 positive and 156 negative results. The positive predictive values (PPV) of WCL EIA were 40% for the MarDx Diagnostics Borrelia burgdorferi EIA 'combined' IgG and IgM (Trinity Biotech) and 55% for the EUROIMMUN plus VlsE IgG. These were significantly lower PPVs than that produced by the recombinant antigen-based EIA NovaLisa Borrelia burgdorferi IgG-ELISA (NovaTec Immunodiagnostica) and the EUROIMMUN Anti-Borrelia Select ELISA IgG (90% and 100%, respectively; p = 0.02). The WCS western immunoblot using B. burgdorferi and B. afzelii separately showed a high PPV of 91% but its positive agreement with consensus EIA result was only 65%. Another WCL western immunoblot with purified extracts of Osp C and VlsE, the Trinity Biotech EU Lyme + VlsE IgG Western Blot had a PPV of 92% while the recombinant line blot from EUROIMMUN, the Anti-Borrelia (IgG) EUROLINE-RN-AT, demonstrated a significantly reduced PPV of 70% with some non-specific reactions in sera containing antibodies to Leptospira species, Helicobacter pylori and Treponema pallidum. The use of recombinant antigens in EIA for LB IgG screening significantly improves the predictive values of serological results above those of WCL antigen EIA. Second tier WCS western immunoblots offer high PPVs, especially with added specific purified proteins, more so than in one recombinant line blot. PMID:27020501

  12. Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

    PubMed Central

    Stibbs, H H; Samadpour, M; Manning, J F

    1988-01-01

    An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy. PMID:3183015

  13. Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species

    PubMed Central

    Sircar, Subhankar; Saurabh, Sharad; Gulati, Baldev R.; Singh, Neeraj; Singh, Arvind Kumar; Joshi, Vinay G.; Banyai, Krisztian; Dhama, Kuldeep

    2016-01-01

    We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831–0.956 at 95% CI = 0.711–1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings. PMID:27391106

  14. Rapid determination of recent cocaine use with magnetic particles-based enzyme immunoassays in serum, saliva, and urine fluids.

    PubMed

    Vidal, Juan C; Bertolín, Juan R; Bonel, Laura; Asturias, Laura; Arcos-Martínez, M Julia; Castillo, Juan R

    2016-06-01

    Cocaine is one of the most worldwide used illicit drugs. We report a magnetic particles-based enzyme-linked immunoassay (mpEIA) method for the rapid and sensitive determination of cocaine (COC) in saliva, urine and serum samples. Under optimized conditions, the limits of detections were 0.09ngmL(-1) (urine), 0.15ngmL(-1) (saliva), and 0.06ngmL(-1) COC (human serum). Sensitivities were in the range EC50=0.6-2.5ngmL(-1) COC. The cross-reactivity with the principal metabolite benzoylecgonine (BZE) was only 1.6%. Recovering percentages of doped samples (0, 10, 50, and 100ngmL(-1) of COC) ranged from about 86-111%. Some advantages of the developed mpEIA over conventional ELISA kits are faster incubations, improved reproducibility, and consumption of lower amounts of antibody and enzyme conjugates due to the use of magnetic beads. The reported method was validated following the guidelines on bioanalytical methods of the European Medicines Agency (2011). Unmetabolized COC detection has a great interest in pharmacological, pharmacokinetics, and toxicokinetics studies, and can be used to detect a very recent COC use (1-6h). PMID:27003120

  15. Serological evaluation of thin-layer immunoassay-enzyme-linked immunosorbent assay for antibody detection in human trichinellosis.

    PubMed

    Gómez-Priego, A; Crecencio-Rosales, L; de-La-Rosa, J L

    2000-09-01

    A new immunoenzymatic test, named the thin-layer immunoassay-enzyme-linked immunosorbent assay (TIA-ELISA), was evaluated for antibody detection in human trichinellosis using excretion and secretion products prepared from Trichinella spiralis muscle larvae. Serum samples from people with positive muscle biopsies or symptoms compatible with the disease (n = 8 or 26, respectively), all reactive in enzyme-linked immunoelectrotransfer blot assay (EITB), as well as 67 serum samples from healthy, EITB-negative people, were tested in an ELISA and TIA-ELISA. TIA-ELISA was performed in polystyrene plastic petri dishes by adding dots of 10 microl each of antigen (7 microg/ml) followed by adding diluted serum and the conjugate. Finally, the substrate mixed with agar was added to develop the reaction. Enzymatic by-products were easily detected by the naked eye as defined dots. Sensitivity and specificity were 76 and 94% for ELISA, and both parameters were 91% for TIA-ELISA. The kappa correlation indices for both tests in relation to EITB were 0.73 and 0.80, respectively. The TIA-ELISA can be carried out with common laboratory equipment in 3 h and uses lower quantities of antigen than EITB and ELISA. Since TIA-ELISA is easy to perform, cheap, sensitive, and specific, the test could be an acceptable alternative to use in clinical laboratories lacking specialized equipment needed for ELISA and EITB and in field studies for antibody detection in human trichinellosis. PMID:10973459

  16. Comparison of sensor structures for the signal amplification of surface plasmon resonance immunoassay using enzyme precipitation

    NASA Astrophysics Data System (ADS)

    Yang, Chih-Tsung; Thierry, Benjamin

    2015-12-01

    Surface plasmon resonance (SPR) biosensing has been successfully applied for the label-free detection of a broad range of bioanalytes ranging from bacteria, cell, exosome, protein and nucleic acids. When it comes to the detection of small molecules or analytes found at low concentration, amplification schemes are desirable to enhance binding signals and in turn increase sensitivity. A number of SPR signal amplification schemes have been developed and validated; however, little effort has been devoted to understanding the effect of the SPR sensor structures on the amplification of binding signals and therefore on the overall sensing performance. The physical phenomenon of long-range SPR (LRSPR) relies on the propagation of coupled surface plasmonic waves on the opposite sides of a nanoscale-thick noble metal film suspended between two dielectrics with similar refractive indices. Importantly, as compared with commonly used conventional SPR (cSPR), LRSPR is not only characterized by a longer penetration depth of the plasmonic waves in the analyzed medium but also by a greater sensitivity to bulk refractive index changes. In this work, an immunoassay signal amplification platform based on horseradish peroxidase (HRP) catalyzed precipitation was utilized to investigate the sensing performance with regards to cSPR and LRSPR. The enzymatic precipitation of 3, 3'-diaminobenzidine tetrahydrochloride (DAB)/H2O2 was used to amplify SPR signals. The structure-function relationship of cSPR and LRSPR sensors is presented for both standard refractometric measurements and the enzymatic precipitation scheme. Experimental data shows that despite its inherent higher sensitivity to bulk refractive index changes and higher figure of merit, LRSPR was characterized by a lower angular sensitivity in the model enzymatic amplification scheme used here.

  17. Anti-idiotypic nanobody-alkaline phosphatase fusion proteins: Development of a one-step competitive enzyme immunoassay for fumonisin B1 detection in cereal.

    PubMed

    Shu, Mei; Xu, Yang; Liu, Xing; Li, Yanping; He, Qinghua; Tu, Zhui; Fu, Jinheng; Gee, Shirley J; Hammock, Bruce D

    2016-06-14

    A rapid and sensitive one-step competitive enzyme immunoassay for the detection of FB1 was developed. The anti-idiotypic nanobody-alkaline phosphatase (Ab2β-Nb-AP) was validated by the AP enzyme activity and the properties of bounding to anti-FB1-mAb (3F11) through colorimetric and chemiluminescence analyses. The 50% inhibitory concentration and the detection limit (LOD) of colorimetric enzyme-linked immunosorbent assay (ELISA) for FB1 were 2.69 and 0.35 ng mL(-1), respectively, with a linear range of 0.93-7.73 ng mL(-1). The LOD of the chemiluminescence ELISA (CLIA) was 0.12 ng mL(-1), and the IC50 was 0.89 ± 0.09 ng mL(-1) with a linear range of 0.29-2.68 ng mL(-1). Compared with LC-MS/MS, the results of this assay indicated the reliability of the Ab2β-Nb-AP fusion protein based one-step competitive immunoassay for monitoring FB1 contamination in cereals. The Ab2β-Nb-AP fusion proteins have the potential to replace chemically-coupled probes in competitive enzyme immunoassay systems. PMID:27181644

  18. Competitive chemiluminescent enzyme immunoassay for vitamin B12 analysis in human milk

    Technology Transfer Automated Retrieval System (TEKTRAN)

    BACKGROUND Few accurate data exist on the concentration of vitamin B12 in human milk. Binding of the vitamin to haptocorrin (HC) can interfere with the assay if not removed by pretreatment, and very low values can occur in women with poor B12 status. This study evaluated two competitive enzyme bind...

  19. Evaluation of three commercial enzyme immunoassays compared with the 13C urea breath test for detection of Helicobacter pylori infection.

    PubMed Central

    Marchildon, P A; Ciota, L M; Zamaniyan, F Z; Peacock, J S; Graham, D Y

    1996-01-01

    The diagnostic significance of the serological detection of antibodies to Helicobacter pylori has been established by numerous investigators. Reports of the clinical reliabilities of commercial enzyme immunoassay (EIA) kits available for this purpose vary as a result of the different H. pylori antigen sources and reference methods used. The 13C urea breath test (UBT) has been shown to be an extremely accurate and reliable method of detecting H. pylori infection. We used the 13C urea breath test as the confirmatory method for H. pylori status to evaluate three commercially available EIA kits designed to detect immunoglobulin G antibodies to H. pylori. These kits were the HM-CAP EIA kit (Enteric Products, Inc.), the PYLORI STAT EIA kit (BioWhittaker, Inc.), and the G.A.P. kit (Bio-Rad Laboratories/Biomerica, Inc.). The evaluations were performed in a double-blind manner with samples from 473 clinically characterized patients. This group included patients with symptomatic gastrointestinal disorders as well as nonsymptomatic volunteers. The sensitivities of the kits were as follows: HM-CAP, 98.4%; PYLORI STAT, 99.2%; and G.A.P., 100%. The specificities were as follows: HM-CAP, 96.4%; PYLORI STAT, 90.1%; and G.A.P., 26.0%. Although the HM-CAP and PYLORI STAT kits performed comparably, the G.A.P. test yielded significantly more false-positive results and an unacceptably high number of indeterminate results. PMID:8727892

  20. Comparison of an enzyme immunoassay for the detection of Helicobacter pylori antigens in the faeces with the urea breath test

    PubMed Central

    Shepherd, A.; Williams, C.; Doherty, C.; Hossack, M.; Preston, T.; McColl, K.; Weaver, L.

    2000-01-01

    BACKGROUND—Current diagnostic tests for Helicobacter pylori are invasive (endoscopy) or indirect (urea breath test, serology).
AIMS—To evaluate a new enzyme immunoassay (EIA) which detects H pylori antigens in faeces, by comparing its sensitivity and specificity in children with the 13C urea breath test (UBT).
METHODS—A total of 119 children underwent a UBT and provided a faecal sample for antigen testing within seven days. After an overnight fast each child provided a pretest breath sample, and samples at 30 and 40 minutes after ingestion of 100 mg 13C labelled urea. 13C enrichment of breath was measured by isotope ratio mass spectrometry. Faeces were stored at −70°C until antigen testing, using the EIA. Samples were read spectrophotometrically at 450 nm and results were interpreted using recommended cut offs of optical density <0.14 as negative, ⩾0.16 as positive, with ⩾0.14 and <0.16 representing equivocal results. Sensitivity and specificity were calculated using the manufacturer's cut off compared with UBT.
RESULTS—Sensitivity and specificity were 88% and 82%, respectively. Negative and positive predictive values were 97% and 58%.
CONCLUSIONS—The EIA is an alternative, non-invasive, and easy to use method for the detection of H pylori in children. Its high negative predictive value suggests a role in screening out uninfected children.

 PMID:10952653

  1. Effectiveness of saliva collection and enzyme-immunoassay for the quantification of cortisol in socially housed baboons.

    PubMed

    Pearson, Brandon L; Judge, Peter G; Reeder, Deeann M

    2008-12-01

    Circulating cortisol levels are often used to assess the biological stress response in captive primates. Some methods commonly used to collect blood samples may alter the stress response. As such, noninvasive means to analyze cortisol levels are increasingly being developed. We adapted an existing collection method to simultaneously obtain saliva from multiple socially living hamadryas baboons (Papio hamadryas hamadryas) and validated an enzyme-immunoassay kit to quantify cortisol within the saliva samples. Over a period of 12 months, saliva samples were regularly collected from approximately half of the 18-member colony, representing younger monkeys who were more willing to participate. The assay met the four criteria typically used to assess the effectiveness of a new analytical technique: parallelism, precision, accuracy, and sensitivity. Cortisol levels were also proportional to those expected given published plasma levels of cortisol in baboons. Further, salivary cortisol levels increased in individuals following significant stress-related events, such as removal from the group, indicating biological validation. The technique provided a reliable and effective means to assess a physiological indicator of stress in a social group without initiating a stress response owing to handling or sedation, and provided a real-time assessment of cortisol levels and reactivity. PMID:18785637

  2. Rapid Differentiation of Aspergillus Species from Other Medically Important Opportunistic Molds and Yeasts by PCR-Enzyme Immunoassay

    PubMed Central

    de Aguirre, Liliana; Hurst, Steven F.; Choi, Jong Soo; Shin, Jong Hee; Hinrikson, Hans Peter; Morrison, Christine J.

    2004-01-01

    We developed a PCR-based assay to differentiate medically important species of Aspergillus from one another and from other opportunistic molds and yeasts by employing universal, fungus-specific primers and DNA probes in an enzyme immunoassay format (PCR-EIA). Oligonucleotide probes, directed to the internal transcribed spacer 2 region of ribosomal DNA from Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus ustus, and Aspergillus versicolor, differentiated 41 isolates (3 to 9 each of the respective species; P < 0.001) in a PCR-EIA detection matrix and gave no false-positive reactions with 33 species of Acremonium, Exophiala, Candida, Fusarium, Mucor, Paecilomyces, Penicillium, Rhizopus, Scedosporium, Sporothrix, or other aspergilli tested. A single DNA probe to detect all seven of the most medically important Aspergillus species (A. flavus, A. fumigatus, A. nidulans, A. niger, A. terreus, A. ustus, and A. versicolor) was also designed. Identification of Aspergillus species was accomplished within a single day by the PCR-EIA, and as little as 0.5 pg of fungal DNA could be detected by this system. In addition, fungal DNA extracted from tissues of experimentally infected rabbits was successfully amplified and identified using the PCR-EIA system. This method is simple, rapid, and sensitive for the identification of medically important Aspergillus species and for their differentiation from other opportunistic fungi. PMID:15297489

  3. Detection of Antibodies to Pasteurella multocida by capture enzyme immunoassay using a monoclonal antibody against P37 antigen.

    PubMed Central

    Peterson, R R; Deeb, B J; DiGiacomo, R F

    1997-01-01

    As infection with Pasteurella multocida is common in rabbits, an enzyme immunoassay (EIA) was developed for its detection. A murine immunoglobulin G monoclonal antibody was used to capture a 37-kDa polypeptide of P. multocida serotype A:12 in an EIA to detect antibodies to P. multocida. The 37-kDa antigen was selected since it was previously shown to be a major immunogen during P. multocida infection in rabbits. The sensitivity of the P37 EIA, determined with sera from 56 rabbits infected with P. multocida, was 98%. Specificity, evaluated with sera from 62 rabbits from colonies free of P. multocida, was 92%. Titration curves of sera from rabbits immunized with P. multocida serotype A:3 or A:12 coincided, indicating that the P37 EIA was equally efficient in detecting antibodies to the two major serotypes of the organism. Comparison of the P37 EIA with the current serodiagnostic test, a bacterial lysate EIA, revealed relatively good correlation (r = 0.68). However, specificity was greatly improved, as 34% of uninfected rabbits were falsely positive by the lysate EIA whereas only 3% of uninfected rabbits were falsely positive by the P37 EIA. The coefficient of variation for same-day tests was 10%, and that for interday tests was 15%, indicating good reproducibility. The greater sensitivity and specificity of the P37 EIA should significantly enhance diagnostic capability to identify rabbits infected with P. multocida. PMID:8968909

  4. Broad-Specificity Chemiluminescence Enzyme Immunoassay for (Fluoro)quinolones: Hapten Design and Molecular Modeling Study of Antibody Recognition.

    PubMed

    Zeng, Haopeng; Chen, Jiahong; Zhang, Chijian; Huang, Xin-An; Sun, Yuanming; Xu, Zhenlin; Lei, Hongtao

    2016-04-01

    On the basis of the structural features of (fluoro)quinolones (FQs), pazufloxacin was first used as a generic immunizing hapten to raise a broad-specificity antibody. The obtained polyclonal antibody exhibited broad cross-reactivity ranging from 5.19% to 478.77% with 21 FQs. Furthermore, the antibody was able to recognize these FQs below their maximum residue limits (MRLs) in an indirect competitive chemiluminescence enzyme immunoassay (ic-CLEIA), with the limit of detection (LOD) ranging from 0.10 to 33.83 ng/mL. For simply pretreated milk samples with spiked FQs, the ic-CLEIA exhibited an excellent recovery with a range of 84.6-106.9% and an acceptable coefficient of variation below 15%, suggesting its suitability and reliability for the use of a promising tool to detect FQs. Meanwhile, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models, with statistically significant correlation coefficients (q(2)CoMFA = 0.559, r(2)CoMFA = 0.999; q(2)CoMSIA = 0.559, r(2)CoMSIA = 0.994), were established to investigate the antibody recognition mechanism. These two models revealed that in the antibody, the active cavity binding FQs' 7-position substituents worked together with another cavity (binding FQs' 1-position groups) to crucially endow the high cross-reactivity. This investigation will be significant for better exploring the recognition mechanism and for designing new haptens. PMID:26976361

  5. Ultrasensitive enhanced chemiluminescence enzyme immunoassay for the determination of alpha-fetoprotein amplified by double-codified gold nanoparticles labels.

    PubMed

    Yang, Xiao-Yan; Guo, Ying-Shu; Bi, Sai; Zhang, Shu-Sheng

    2009-04-15

    A novel enhanced chemiluminescent (CL) immunoassay for ultrasensitive determination of alpha-fetoprotein (AFP) was reported. The method made full use of 4-(4'-iodo)phenylphenol (IPP) as a new potential signal enhancer and double-codified gold nanoparticles (DC-AuNPs) labels modified with horseradish peroxidase (HRP)-conjugated anti-AFP used for further signal amplification. This protocol involved a sandwich format, in which the antigen in the sample was first captured by the immobilized primary antibody on the surface of magnetic beads, and then recognized by the second antibody labeled with DC-AuNPs. The combination of the remarkable sensitivity of the enhanced CL method and the use of AuNPs as an anti-AFP-HRP carrier for the enzymatic signal amplification, provided a linear response range of AFP from 0.008 to 0.3 ng mL(-1) with an extremely low detection limit of 5 pg mL(-1), much lower than those achieved by the classical enzyme-linked immunosorbent assay (ELISA). This new system can be easily extended to a variety of immunodetection as well as DNA analysis. PMID:19152783

  6. Validation of a cortisol enzyme immunoassay and characterization of salivary cortisol circadian rhythm in chimpanzees (Pan troglodytes).

    PubMed

    Heintz, Matthew R; Santymire, Rachel M; Parr, Lisa A; Lonsdorf, Elizabeth V

    2011-09-01

    Monitoring concentrations of stress hormones is an important tool for behavioral research and conservation for animals both in the wild and captivity. Glucocorticoids can be measured in mammals as an indicator of stress by analyzing blood, feces, urine, hair, feathers, or saliva. The advantages of using saliva for measuring cortisol concentrations are three-fold: it is minimally invasive, multiple samples can be collected from the same individual in a short timeframe, and cortisol has a relatively short response time in saliva as compared with other materials. The purpose of this study was to: (1) conduct an adrenocorticotropic hormone (ACTH) challenge as a physiological validation for an enzyme immunoassay to measure salivary cortisol in chimpanzees and (2) characterize the circadian rhythm of salivary cortisol in chimpanzees. We determined that salivary cortisol concentrations peaked 45 min following the ACTH challenge, which is similar to humans. Also, salivary cortisol concentrations peaked early in the morning and decreased throughout the day. We recommend that saliva collection may be the most effective method of measuring stress reactivity and has the potential to complement behavioral, cognitive, physiological, and welfare studies. PMID:21538448

  7. Enzyme immunoassay for anatoxin-A. Phase 1. Final report, 15 August 1988-15 June 1989

    SciTech Connect

    Amos, R.A.; Behrens, C.; Swan, G.; Chappa, A.

    1989-02-14

    The ability to detect the presence of naturally occurring toxins in the environment is important for the protection of military and civilian personnel. One such toxin of considerable interest is anatoxin a, a secondary metabolite of the cyanobacteria (blue-green algae) Anabena flos-aquae, which has been shown to be extraordinarily toxic. Numerous examples of animal deaths have been reported after drinking from lakes and ponds following blooms of this freshwater algae. This phase I report describes the results of an immunoassay development based on an ELISA format. Two different derivatives of anatoxin a were prepared and they were coupled to carrier proteins for use as immunogens, and to active enzymes for use as conjugates in an ELISA. Polyclonal antibodies to the immunogens have been raised in rabbits, and they have been found to be suitable for use in an ELISA. No current test procedure exists for anatoxin a in freshwater supplies despite the very real hazard presented by this naturally occurring toxin. The Army needs to have the capability of testing for the use of this material in biological warfare as well as for evaluating the suitability of drinking water sources in the field. The wide-spread occurrence of blue-green algae blooms in lakes and ponds indicates that a significant commercial market is available, particularly in the agricultural and water-treatment areas.

  8. IgM anti-histone H-3 antibody associated with undifferentiated rheumatic disease syndromes.

    PubMed

    Molden, D P; Klipple, G L; Peebles, C L; Rubin, R L; Nakamura, R M; Tan, E M

    1986-01-01

    A distinctive type of speckled antinuclear antibody staining pattern was identified by indirect immunofluorescence on mouse kidney substrate in 4.8% of 5,976 specimens analyzed for antinuclear antibodies. This pattern, termed variable large speckles (VLS), consisted of 3-10 nuclear speckles ranging in size from approximately 0.2-2.0 mu. The pattern could be differentiated from other indirect immunofluorescence patterns related to specific antibodies. The predominant immunoglobulin isotype demonstrating the VLS pattern was IgM in 27 of 28 sera examined and IgG in 1 serum. VLS sera had substantial IgM antibodies to histone demonstrated by enzyme immunoassay, and further analysis of representative sera showed predominant antibody activity to histone class 3 (H-3). Adsorption with histone H-3 resulted in decrease or removal of antibody producing the VLS pattern. Available information showed that most patients with IgM antibodies of the VLS pattern had undifferentiated connective tissue disease symptoms. They were characterized by a heterogeneity of chronic symptoms including arthralgias, myalgias, inflammatory polyarthritis, myositis, sicca symptoms, and pleurisy associated with elevation of the erythrocyte sedimentation rate. It remains to be determined whether the IgM anti-histone H-3 profile of these patients is a transient or long-standing serologic characteristic. PMID:2418845

  9. Enzyme immunoassay for the detection of porcine gelatine in edible bird's nests.

    PubMed

    Tukiran, Nur Azira; Ismail, Amin; Mustafa, Shuhaimi; Hamid, Muhajir

    2015-01-01

    Porcine gelatine is a common adulterant found in edible bird's nests (EBNs) used to increase the net weight prior to sale. This study aimed to develop indirect enzyme-linked immunosorbent assays (ELISAs) for porcine gelatine adulteration using anti-peptide polyclonal antibodies. Three indirect ELISAs were developed (PAB1, 2 and 3), which had limits of detection (LODs) of 0.12, 0.10 and 0.11 µg g(-1), respectively. When applied to standard solutions of porcine gelatine, the inter- and intra-assays showed coefficients of variation (CVs) less than 20% and were able to detect at least 0.5 ng µg(-1) (0.05%) porcine gelatine in spiked samples. The proposed ELISA offers attractions for quality control in the EBN industry. PMID:25861981

  10. Development and Application of an Immunoaffinity Column Enzyme Immunoassay for Mycotoxin Zearalenone in Complicated Samples

    PubMed Central

    Li, Peiwu; Zhang, Qi; Li, Ran; Zhang, Wen; Ding, Xiaoxia; Lei, Jiawen; Zhang, Zhaowei

    2014-01-01

    The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC50 against ZEA of 0.02 µg L−1. The mAb 2D3 exhibited a high recognition of ZEA (100%) and β-zearalenol (β-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and β-zearalanol (β-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83–93%) and HPLC (94–108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize. PMID:24465616

  11. Determination of Aspergillus fumigatus allergen 1 in poultry farms using the enzyme immunoassay.

    PubMed

    Prester, Ljerka; Macan, Jelena; Matković, Kristina; Vucemilo, Marija

    2010-06-01

    Poultry farms contain high levels of allergenic fungi, and Aspergillus spp. is the most common genus of moulds. Aspergillus fumigatus antigens are responsible for the development of several respiratory diseases including asthma. The aim of this study was to measure the mass fraction of Asp f 1, a major allergen of Asperillus fumigatus in 37 indoor dust samples collected from four poultry farms in a rural area of the Zagreb County (Croatia) using the enzyme-linked immunosorbent assay. More than 62 % of dust samples had detectable Asp f 1 levels (limit of detection 3.6 ng g(-1)). The overall mean Asp f 1 level was 17.9 ng g(-1) [range (3.8 to 72.4) ng g(-1)]. Satisfactory results were obtained for analytical within-run imprecision (6.7 %), between-run imprecision (10.5 %), and accuracy (91 % to 115 %). Microclimate parameters (air temperature, relative humidity, and velocity) were within the recommended ranges in all poultry farms. This study has shown that Asp f 1 settles on dust at poultry farms and that occupational exposure to this allergen deserves monitoring in livestock buildings. PMID:20587390

  12. Use of enzyme immunoassay for large water-quality surveys of major herbicides

    SciTech Connect

    Thurman, E.M.; Aga, D.S.; Zimmerman, L.R.; Goolsby, D.A.

    1996-10-01

    Commercially available enzyme-linked immunosorbent assay (ELISA) was used for the determination of major herbicides in several large water-quality surveys of surface water, rainwater, and ground water throughout the United States. The ELISA results were compared with gas chromatography/mass spectrometry (GC/MS) for accuracy and cross reactivity. In total, five compounds were analyzed: alachlor, atrazine, cyanazine, metolachlor, and (2,4-dichlorophenoxy) acetic acid (2,4-D). Results indicated that the ELISA and GC/MS results were comparable for cyanazine and metolachlor. The atrazine ELISA correlated well with GC/MS for surface- and ground-water samples from the central United States but did not correlate with samples from Texas where the cotton triazine, prometryn, is used. Results using the alachlor ELISA were poor because of cross reactivity with the metabolite, alachlor ethane-sulfonic acid. The ELISA for (2,4-dichlorophenoxy) acetic acid was insensitive at concentrations that occur in most surface water.

  13. Development and application of an immunoaffinity column enzyme immunoassay for mycotoxin zearalenone in complicated samples.

    PubMed

    Tang, Xiaoqian; Li, Xin; Li, Peiwu; Zhang, Qi; Li, Ran; Zhang, Wen; Ding, Xiaoxia; Lei, Jiawen; Zhang, Zhaowei

    2014-01-01

    The zearalenone (ZEA) monoclonal antibody (mAb) 2D3, one of the highest sensitivity antibodies, was developed. Based on this mAb, it was established of an immunoaffinity column (IAC) coupled with an indirect competitive enzyme-linked immunosorbent assay (icELISA). After optimization, the icELISA allowed an IC50 against ZEA of 0.02 µg L(-1). The mAb 2D3 exhibited a high recognition of ZEA (100%) and β-zearalenol (β-ZOL, 88.2%). Its cross-reactivity with α-zearalenol (α-ZOL) and β-zearalanol (β-ZAL) were found to be 4.4% and 4.6%, respectively. The IAC-icELISA method was employed to analyze ZEA contamination in food samples, compared with high-performance liquid chromatography (HPLC). The spiked assay for ZEA demonstrated the considerable recoveries for IAC-icELISA (83-93%) and HPLC (94-108%) methods. Results showed that the mAb 2D3 and IAC-icELISA method posed potential applications in sensitively determination of ZEA in maize. PMID:24465616

  14. Chronic Chagas Disease Diagnosis: A Comparative Performance of Commercial Enzyme Immunoassay Tests.

    PubMed

    Santos, Fred Luciano Neves; de Souza, Wayner Vieira; Barros, Michelle da Silva; Nakazawa, Mineo; Krieger, Marco Aurélio; Gomes, Yara de Miranda

    2016-05-01

    There is a significant heterogeneity in reported performance of serological assays for Chagas disease diagnosis. The conventional serology testing in laboratory diagnosis and in blood banks is unsatisfactory because of a high number of inconclusive and misclassified results. We aimed to assess the quality of four commercially available enzyme-linked immunosorbent assay tests for their ability to detect Trypanosoma cruzi antibodies in 685 sera samples. Cross-reactivity was assessed by using 748 sera from patients with unrelated diseases. Initially, we found that the reactivity index against T. cruzi antigen was statistically higher in sera from Chagas disease patients compared with those from non-chagasic patients, supporting the notion that all evaluated tests have a good discriminatory ability toward the diagnosis of T. cruzi infection in patients in the chronic phase of the disease. Although all tests were similarly sensitive for diagnosing T. cruzi infection, there were significant variations in terms of specificity and cross-reactivity among them. Indeed, we obtained divergent results when testing sera from patient with unrelated diseases, particularly leishmaniasis, with the levels of cross-reactivity being higher in tests using whole T. cruzi extracts compared with those using recombinant proteins. Our data suggest that all four tests may be used for the laboratory diagnosis and routine blood screening diagnose for Chagas disease. We also emphasize that, despite their general good performance, caution is needed when analyzing the results when these tests are performed in areas where other diseases, particularly leishmaniasis, are endemic. PMID:26976886

  15. Rapid screening of flonicamid residues in environmental and agricultural samples by a sensitive enzyme immunoassay.

    PubMed

    Liu, Zhenjiang; Zhang, Zhen; Zhu, Gangbing; Sun, Jianfan; Zou, Bin; Li, Ming; Wang, Jiagao

    2016-05-01

    A fast and sensitive polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) was developed for the analysis of flonicamid in environmental and agricultural samples. Two haptens of flonicamid differing in spacer arm length were synthesized and conjugated to proteins to be used as immunogens for the production of polyclonal antibodies. To obtain most sensitive combination of antibody/coating antigen, two antibodies were separately screened by homologous and heterologous assays. After optimization, the flonicamid ELISA showed that the 50% inhibitory concentration (IC50 value) was 3.86mgL(-1), and the limit of detection (IC20 value) was 0.032mgL(-1). There was no cross-reactivity to similar tested compounds. The recoveries obtained after the addition of standard flonicamid to the samples, including water, soil, carrot, apple and tomato, ranged from 79.3% to 116.4%. Moreover, the results of the ELISA for the spiked samples were largely consistent with the gas chromatography (R(2)=0.9891). The data showed that the proposed ELISA is an alternative tool for rapid, sensitive and accurate monitoring of flonicamid in environmental and agricultural samples. PMID:26897400

  16. A sensitive and specific two-site enzyme-immunoassay for human calcitonin using monoclonal antibodies.

    PubMed

    Seth, R; Motté, P; Kehely, A; Wimalawansa, S J; Self, C H; Bellet, D; Bohuon, C; MacIntyre, I

    1988-11-01

    A highly sensitive, specific and rapid two-site enzyme-immunometric assay (EIA) for the measurement of immunoreactive (ir) human calcitonin (hCT) in human plasma was developed using high-affinity monoclonal antibodies. The assay was validated in terms of sensitivity, specificity and reproducibility and its performance compared with that of a radioimmunoassay (RIA) employing a polyclonal antiserum. The sensitivity of the overnight EIA (2 pmol/l) was comparable with the long-incubation (7 days) RIA. The overnight RIA had a sensitivity of 10 pmol/l. The inter- and intra-assay variations of the EIA were less than 12%. Some related and non-related peptides were compared with synthetic hCT for cross-reactivity in the assay and were found to be negative. The mean recovery of added synthetic hCT from plasma of normal volunteers was 96%. Both RIA and EIA have been applied to the measurement of ir-hCT in normal volunteers and in patients with medullary carcinoma of the thyroid. In both groups, the level of ir-hCT measured by EIA was found to be lower than that measured by RIA, presumably due to the ability of the more specific EIA to detect only the 'mature' form of the hormone. EIA offers an attractive alternative to the more cumbersome and lengthy RIA in current usage, with the added advantage of employing a non-isotopic label. PMID:3058855

  17. Serum anti-GAGA4 IgM antibodies differentiate relapsing remitting and secondary progressive multiple sclerosis from primary progressive multiple sclerosis and other neurological diseases.

    PubMed

    Brettschneider, Johannes; Jaskowski, Troy D; Tumani, Hayrettin; Abdul, Sana; Husebye, Dee; Seraj, Haniah; Hill, Harry R; Fire, Ella; Spector, Larissa; Yarden, Jennifer; Dotan, Nir; Rose, John W

    2009-12-10

    The serum level of IgM antibodies against Glc(alpha1,4)Glc(alpha) (GAGA4) is higher in relapsing remitting multiple sclerosis (RRMS) compared to other neurological disease (OND) patients and healthy controls (HC). Detecting the level of anti-GAGA4 antibody by enzyme immunoassay and total IgM, we confirmed that anti-GAGA4 IgM can differentiate RRMS from OND patients and HC. Moreover, secondary progressive MS (SPMS) and RRMS patients have similar levels of anti-GAGA4 demonstrating the biomarker's presence throughout the disease. Interestingly, the anti-GAGA4 assay may also differentiate between primary progressive MS (PPMS) and RRMS/SPMS patients, since nearly all PPMS patients were negative for the assay. PMID:19879655

  18. Enzyme-linked immunosorbent assay and colloidal gold immunoassay for ochratoxin A: investigation of analytical conditions and sample matrix on assay performance.

    PubMed

    Wang, Xiang-Hong; Liu, Tao; Xu, Na; Zhang, Yan; Wang, Shuo

    2007-10-01

    A polyclonal antibody against ochratoxin A (OTA) was produced from rabbits immunized with the OTA-BSA conjugate. A competitive direct enzyme-linked immunosorbent assay (cdELISA) and a membrane-base colloidal gold immunoassay in flow-through format were developed for the rapid detection of OTA in various food matrices. In the cdELISA, the concentration causing 50% inhibition was 0.07 ng mL(-1), and the effects of different chemical conditions (ionic strength, pH value, and organic solvent) were studied. The sensitivity of the assay was higher than those previously reported. A simple, rapid, and efficient extraction method was developed and 74-110% recoveries of spiked samples were obtained. Fifty percent methanol extracts of some food samples such as barley, wheat, oat, corn, rice, and raisins could be analyzed directly by immunoassay after dilution in PBS; grape juice and beer samples could be analyzed directly after dilution with PBS; for coffee samples, a more complex method was used to remove the matrix effect effectively. Membrane-based colloidal gold immunoassays had a visual detection limit of 1.0 ng mL(-1) for OTA with a detection time of less than 10 min. For the validation of the cdELISA and membrane-based colloidal gold immunoassay, samples were analyzed by high-performance liquid chromatography. The correlation between data obtained using the microwell assay and HPLC was good (R2 = 0.984). The developed immunoassay methods are suitable for the rapid quantitative or qualitative determination of OTA in food samples. PMID:17668189

  19. Direct detection of influenza virus antigen in nasopharyngeal specimens by direct enzyme immunoassay in comparison with quantitating virus shedding.

    PubMed Central

    Döller, G; Schuy, W; Tjhen, K Y; Stekeler, B; Gerth, H J

    1992-01-01

    We developed a direct enzyme immunoassay [EIA; Enzygnost Influenza A(Ag) and Enzygnost Influenza B(Ag)] for the direct detection of influenza A and B virus antigens in nasopharyngeal secretion specimens (NPS). The test is performed without sonification of specimens, and results are obtained within 4 h. A direct comparison between direct EIA and quantitation of virus shedding for influenza A and B virus antigen detection was carried out. A total of 210 NPS and 98 nasopharyngeal wash specimens (NPW) were investigated. We isolated influenza A viruses from 79 (37.6%) of 210 NPS; of these 79 cell-culture-positive NPS, 70 (88.6%) were also positive by direct EIA. Of 29 (13.8%) NPS from which influenza B virus was isolated, 24 (82.8%) NPS were positive by direct EIA. Virus shedding was determined quantitatively in 48 NPS from patients with influenza A and in 24 NPS from patients with influenza B. Only a crude correlation between optical density values and virus concentrations was observed. Detection of influenza virus antigens in NPS by direct EIA showed sensitivities of 89.7% for influenza A virus and 87.9% for influenza B virus and specificities of 99.3% for influenza A virus and 100% for influenza B virus. With direct EIA, all NPW were negative for influenza A virus, although virus was isolated from 21 (21.4%) NPW. Of 15 NPW from which influenza B virus was isolated, 7 showed positive results in direct EIA. In addition, direct EIA is suitable for detecting influenza A and B viruses in cell cultures before the appearance of any cytopathic effects and can be used as a cell culture confirmation test. PMID:1572972

  20. Comparison of a lateral flow milk progesterone test with enzyme immunoassay as an aid for reproductive status determination in cows.

    PubMed

    Waldmann, A; Raud, A

    2016-03-12

    The lateral flow test (LFT) is an immunochromatographic method that utilises an immunostrip for non-laboratory diagnostic purposes. The present study evaluated a milk progesterone LFT against the enzyme immunoassay (EIA) to confirm oestrus and a non-pregnancy diagnosis. In total, 277 milk samples from 70 cows were analysed, collected on the day of artificial insemination and at 19 days, 21 days and 24 days post insemination. The level of accuracy of the LFT compared with the EIA was 95.0 per cent for milk samples containing <2 ng/ml progesterone and 97.0 per cent for milk samples containing >10 ng/ml progesterone. The validation of oestrus by the LFT was 98.6 per cent accurate using 2 ng/ml progesterone as the EIA estimate for oestrus. The test performance for a non-pregnancy diagnosis was subject to the day of milk sampling, showing the highest accuracy on day 24 post insemination for both tests. When optimised for maximum specificity, and compared with rectal palpation, the LFT had a sensitivity and specificity for non-pregnancy diagnosis on day 24 post insemination of 75.0 per cent and 100.0 per cent, respectively, with an overall accuracy of 84.4 per cent. The corresponding characteristics for the quantitative EIA were 85.0 per cent, 100.0 per cent and 90.6 per cent, respectively. The LFT results compared favourably with the quantitative milk progesterone EIA. PMID:26873072

  1. Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis.

    PubMed

    Dumollard, C; Bailly, S; Perriot, S; Brenier-Pinchart, M P; Saint-Raymond, C; Camara, B; Gangneux, J P; Persat, F; Valot, S; Grenouillet, F; Pelloux, H; Pinel, C; Cornet, M

    2016-05-01

    Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the Virion\\Serion kit (P = 0.04). The Bordier and Bio-Rad kits had similar specificity (P = 0.8), both higher than that of the Virion\\Serion kit (P = 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\\Serion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P = 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis. PMID:26888904

  2. Loop-Mediated Isothermal Amplification Compared to Real-Time PCR and Enzyme Immunoassay for Toxigenic Clostridium difficile Detection

    PubMed Central

    Sural, Preethi; Loomis, Caroline R.; Pesta, Christine; Gonzalez-Krellwitz, Laura; Robinson-Dunn, Barbara; Riska, Paul

    2012-01-01

    Clostridium difficile infection is the primary cause of health care-associated diarrhea. While most laboratories have been using rapid antigen tests for detecting C. difficile toxins, they have poor sensitivity; newer molecular methods offer rapid results with high test sensitivity and specificity. This study was designed to compare the performances of two molecular assays (Meridian illumigene and BD GeneOhm) and two antigen assays (Wampole Quik Chek Complete and TechLab Tox A/B II) to detect toxigenic C. difficile. Fecal specimens from hospitalized patients (n = 139) suspected of having C. difficile infection were tested by the four assays. Nine specimens were positive and 109 were negative by all four methods. After discrepant analysis by toxigenic culture (n = 21), the total numbers of stool specimens classified as positive and negative for toxigenic C. difficile were 21 (15%) and 118 (85%), respectively. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were as follows: GeneOhm (95.2%, 100%, 100%, and 99.2%), illumigene (95.2%, 96.6%, 83.3%, and 99.2%), Tox A/B II (52.4%, 97.5%, 78.6%, and 92.4%), and Quik Chek Complete (47.6%, 100%, 100%, and 91.9%). The illumigene assay performed comparably to the GeneOhm assay with a slight decrease in test specificity; the sensitivities of both far exceeded those of the antigen assays. The clinical characteristics of the concordant and discrepant study patients were similar, including stool consistency and frequency. In the era of rapid molecular-based tests for toxigenic C. difficile, toxin enzyme immunoassays (EIAs) should no longer be considered the standard of care. PMID:22189114

  3. Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in spotted hyenas (Crocuta crocuta).

    PubMed

    Benhaiem, Sarah; Dehnhard, Martin; Bonanni, Roberto; Hofer, Heribert; Goymann, Wolfgang; Eulenberger, Klaus; East, Marion L

    2012-09-01

    The use of enzyme immunoassays (EIAs) to measure faecal glucocorticoid metabolites (fGCM) is a useful non-invasive technique to monitor adrenocortical activity in vertebrates. The first objective of this study was to validate an 'in-house' EIA (cortisol-3-CMO) for the measurement of fGCM concentrations in spotted hyenas. High-performance liquid chromatography (HPLC) was used to characterise fGCM in samples from a captive hyena that received an i.v. injection of [(3)H] cortisol. All HPLC fractions were analysed with the EIA for the presence and quantities of radiolabelled fGCM. Radiolabelled fGCM consisted of substances with a higher polarity than cortisol and substances of lower polarity that eluted between cortisol and corticosterone. Authentic radiolabelled cortisol was not detected. The EIA measured substantial amounts of immunoreactivity corresponding to the radioactive peaks. It also detected a significant increase in fGCMs after an adrenocorticotropic hormone (ACTH) challenge in two other captive animals and a significant increase in fGCMs in a fourth captive animal after anaesthesia. The second objective was to investigate an age effect on fGCM: we conducted pairwise comparisons of fGCM concentrations in individual free-ranging juvenile spotted hyenas when less than 6 months of age and when between 6 and 24 months of age. We expected juveniles to experience a more unpredictable and therefore more stressful environment when younger than when older. When younger, juveniles had significantly higher fGCM concentrations than when they were older. Our results demonstrate that our assay can be used to assess adrenocortical activity in spotted hyenas. PMID:22634955

  4. Development of a versatile enzyme immunoassay for non-invasive assessment of glucocorticoid metabolites in a diversity of taxonomic species.

    PubMed

    Watson, Rebecca; Munro, Coralie; Edwards, Katie L; Norton, Vicki; Brown, Janine L; Walker, Susan L

    2013-06-01

    Endocrinology is a useful tool for conservation biologists and animal managers, and measuring glucocorticoids can help understand biological mechanisms associated with species decline and animal welfare. The current study describes the development and optimization of a glucocorticoid enzyme immunoassay (EIA) to non-invasively assess adrenal activity in a variety of taxa. The antiserum (CJM006) was raised in rabbits to a corticosterone-3-CMO-BSA immunogen and used in a standard competitive EIA system. However, the EIA initially produced results with unacceptably high inter-assay variation, attributed to consistent patterns observed within the optical density of developing plates. To determine the cause of this variability, a number of factors were examined using synthetic corticosterone standard and endogenous faecal extract, including: plate type (Nunc MaxiSorp® II versus Immulon IB plates); the use of non-specific secondary antibody; type (artificial versus natural) and presence (light versus dark) of light during incubation; plate loading temperature (4°C versus room temperature); and substrate reagent temperature (4°C versus room temperature). Results indicated that variability was associated with plate location effects, which were not initially detected because control samples were always run in the same positions across plates. Light and temperature were the two major factors that affected EIA reliability. For this assay, the standard protocol required slight modification, with the optimal protocol using Nunc MaxiSorp® plates, room temperature substrate reagents and dark incubation conditions. Following optimization, this EIA was then validated biochemically for 38 species, through parallel displacement curves and interference assessment tests of faecal and urine samples. Additionally, biological validation was performed opportunistically in a subset of species, with use of this EIA demonstrating significant elevations in faecal glucocorticoid metabolites

  5. Filter Paper Blood Spot Enzyme Linked Immunoassay for Adiponectin and Application in the Evaluation of Determinants of Child Insulin Sensitivity

    PubMed Central

    Martin, Richard M.; Patel, Rita; Oken, Emily; Thompson, Jennifer; Zinovik, Alexander; Kramer, Michael S.; Vilchuck, Konstantin; Bogdanovich, Natalia; Sergeichick, Natalia; Foo, Ying; Gusina, Nina

    2013-01-01

    Background Adiponectin is an adipocyte-derived hormone that acts as a marker of insulin sensitivity. Bloodspot sampling by fingerstick onto filter paper may increase the feasibility of large-scale studies of the determinants of insulin sensitivity. We first describe the validation of an enzyme-linked immunoassay (ELISA) for quantifying adiponectin from dried blood spots and then demonstrate its application in a large trial (PROBIT). Methods We quantified adiponectin from 3-mm diameter discs (≈3 µL of blood) punched from dried blood spots obtained from: i) whole blood standards (validation); and ii) PROBIT trial samples (application) in which paediatricians collected blood spots from 13,879 children aged 11.5 years from 31 sites across Belarus. We examined the distribution of bloodspot adiponectin by demographic and anthropometric factors, fasting insulin and glucose. Results In the validation study, mean intra-assay coefficients of variation (n = 162) were 15%, 13% and 10% for ‘low’ (6.78 µg/ml), ‘medium’ (18.18 µg/ml) and 'high’ (33.13 µg/ml) internal quality control (IQC) samples, respectively; the respective inter-assay values (n = 40) were 23%, 21% and 14%. The correlation coefficient between 50 paired whole bloodspot versus plasma samples, collected simultaneously, was 0.87 (95% CI: 0.78 to 0.93). Recovery of known quantities of adiponectin (between 4.5 to 36 µg/ml) was 100.3–133%. Bloodspot adiponectin was stable for at least 30 months at −80°C. In PROBIT, we successfully quantified fasting adiponectin from dried blood spots in 13,329 of 13,879 (96%) children. Mean adiponectin (standard deviation) concentrations were 17.34 µg/ml (7.54) in boys and 18.41 µg/ml (7.92) in girls and were inversely associated with body mass index, fat mass, triceps and subscapular skin-fold thickness, waist circumference, height and fasting glucose. Conclusions Bloodspot ELISA is suitable for measuring adiponectin in very small volumes of blood

  6. Filter Paper Blood Spot Enzyme Linked Immunoassay for Insulin and Application in the Evaluation of Determinants of Child Insulin Resistance

    PubMed Central

    Martin, Richard M.; Patel, Rita; Zinovik, Alexander; Kramer, Michael S.; Oken, Emily; Vilchuck, Konstantin; Bogdanovich, Natalia; Sergeichick, Natalia; Gunnarsson, Robert; Grufman, Lisa; Foo, Ying; Gusina, Nina

    2012-01-01

    Background In large-scale epidemiology, bloodspot sampling by fingerstick onto filter paper has many advantages, including ease and low costs of collection, processing and transport. We describe the development of an enzyme-linked immunoassay (ELISA) for quantifying insulin from dried blood spots and demonstrate its application in a large trial. Methods We adapted an existing commercial kit (Mercodia Human Insulin ELISA, 10-1113-01) to quantify insulin from two 3-mm diameter discs (≈6 µL of blood) punched from whole blood standards and from trial samples. Paediatricians collected dried blood spots in a follow-up of 13,879 fasted children aged 11.5 years (interquartile range 11.3–11.8 years) from 31 trial sites across Belarus. We quantified bloodspot insulin levels and examined their distribution by demography and anthropometry. Results Mean intra-assay (n = 157) coefficients of variation were 15% and 6% for ‘low’ (6.7 mU/L) and ‘high’ (23.1 mU/L) values, respectively; the respective inter-assay values (n = 33) were 23% and 11%. The intraclass correlation coefficient between 50 paired whole bloodspot versus serum samples, collected simultaneously, was 0.90 (95% confidence interval 0.85 to 0.95). Bloodspot insulin was stable for at least 31 months at −80°C, for one week at +30°C and following four freeze-thaw cycles. Paediatricians collected a median of 8 blood spots from 13,487 (97%) children. The geometric mean insulin (log standard deviation) concentrations amongst 12,812 children were 3.0 mU/L (1.1) in boys and 4.0 mU/L (1.0) in girls and were positively associated with pubertal stage, measures of central and peripheral adiposity, height and fasting glucose. Conclusions Our simple and convenient bloodspot assay is suitable for the measurement of insulin in very small volumes of blood collected on filter paper cards and can be applied to large-scale epidemiology studies of the early-life determinants of circulating insulin. PMID:23056434

  7. An enzyme immunoassay and immunoblot analysis for curculin, a new type of taste-modifying protein: cross-reactivity of curculin and miraculin to both antibodies.

    PubMed

    Nakajo, S; Akabane, T; Nakaya, K; Nakamura, Y; Kurihara, Y

    1992-02-01

    We have developed an enzyme immunoassay method for curculin, a new type of taste-modifying protein. This method can accurately quantify 0.05-20 ng of curculin, a sensitivity about 3000-times that of the psychometric method. The content of curculin in the fruit of Curculigo latifolia increased gradually until 3 weeks after artificial pollination and dramatically at 4 weeks, to finally reach 1.3 mg per fruit. Immunoblot analysis indicated that antiserum to curculin was faintly reactive with miraculin, but not with thaumatin or monellin. PMID:1737052

  8. Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay.

    PubMed Central

    Armstrong, Michael; Liu, Andrew H.; Harbeck, Ronald; Reisdorph, Rick; Rabinovitch, Nathan; Reisdorph, Nichole

    2009-01-01

    A new analytical method suitable for high throughput measurements of LTE4 in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/ tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500 pg/mL in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE4 from healthy adults and children are reported. PMID:19726242

  9. Development and evaluation of an ELISA method for the determination of lipoprotein lipase mass concentration--comparison with a commercial, one-step enzyme immunoassay.

    PubMed

    Antikainen, M; Suurinkeroinen, L; Jauhiainen, M; Ehnholm, C; Taskinen, M R

    1996-07-01

    We developed a non-competitive, enzyme-linked, immunosorbent assay (ELISA) for the quantitation of lipoprotein lipase (LPL) in human postheparin plasma using affinity-purified antihuman milk lipoprotein lipase antibodies produced in chicken eggs and a monoclonal antibody directed against human lipoprotein lipase. We compared our ELISA method with a commercially available sandwich-enzyme immunoassay (Markit-F LPL EIA Kit, Dainippon Pharmaceutical Co, Ltd. Osaka, Japan). The reference values for lipoprotein lipase catalytic activity concentration and mass concentration in healthy Finns were determined. Lipoprotein lipase activity concentration (mean +/- SD) was 297 +/- 112 U/l in women, and mass concentration as measured by the ELISA method was 1058 +/- 367 micrograms/l. The corresponding values for men were 247 +/- 97 U/l and 815 +/- 207 micrograms/l, respectively. Across the whole concentration range of the ELISA method, the control samples' intra- and inter-assay coefficients of variation (CV) were 5.1% and 6.5%, respectively. The correlation between the ELISA and EIA methods was good, r = +0.81. The importance of the correct standardisation of immunoassays is discussed. PMID:8864403

  10. IMMUNOASSAY HUMAN EXPOSURE STUDIES

    EPA Science Inventory

    The Human Exposure Research Branch has developed several enzyme-linked immunosorbent assay (ELISA) methods to support human exposure assessment studies. Immunoassays to detect low levels (<10 ng/mL) of chlorpyrifos in food, track-in dirt and house dust have been applied to sam...

  11. Magnetic bead-based enzyme-chromogenic substrate system for ultrasensitive colorimetric immunoassay accompanying cascade reaction for enzymatic formation of squaric acid-iron(III) chelate.

    PubMed

    Lai, Wenqiang; Tang, Dianping; Zhuang, Junyang; Chen, Guonan; Yang, Huanghao

    2014-05-20

    This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (<1 min) coordinate with the SQA. Formation of the iron-squarate complex causes the color of the solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. On the basis of the SQA-iron(III) system, a new immunoassay protocol with GOx-labeled anti-PSA detection antibody can be designed for the detection of target PSA on capture antibody-functionalized magnetic immunosensing probe, monitored by recording the color or absorbance (λ = 468 nm) of the generated SQA-iron(III) chelate. The absorbance intensity shows to be dependent on the concentration of target PSA. A linear dependence between the absorbance and target PSA concentration is obtained under optimal conditions in the range from 1.0 pg mL(-1) to 30 ng mL(-1) with a detection limit (LOD) of 0.5 pg mL(-1) (0.5 ppt) estimated at the 3Sblank level. The sensitivity displays to be 3-5 orders of magnitude better than those of most commercialized human PSA ELISA kits. In addition, the developed colorimetric immunoassay was validated by assaying 12 human serum samples, receiving in good accordance with those obtained by the commercialized PSA ELISA kit. Importantly, the SQA-based immunosensing system can be further extended for the detection of other low-abundance proteins or biomarkers by controlling the target

  12. Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

    PubMed

    Hunt, Ann R; Hall, Roy A; Kerst, Amy J; Nasci, Roger S; Savage, Harry M; Panella, Nicholas A; Gottfried, Kristy L; Burkhalter, Kristen L; Roehrig, John T

    2002-06-01

    An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay. PMID:12037058

  13. Rapid and sensitive detection of immunoglobulin M (IgM) and IgG antibodies against canine distemper virus by a new recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay.

    PubMed

    von Messling, V; Harder, T C; Moennig, V; Rautenberg, P; Nolte, I; Haas, L

    1999-04-01

    Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (kappa = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of >/=50% showed very good inter-rater agreement (kappa = 0.968) with V-NA titers of >/=1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of >/=1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies

  14. Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

    PubMed Central

    von Messling, Veronika; Harder, Timm C.; Moennig, Volker; Rautenberg, Peter; Nolte, Ingo; Haas, Ludwig

    1999-01-01

    Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the

  15. [Rheumatoid factor activity as a disturbing factor in the serological diagnosis of specific IgM antibodies].

    PubMed

    Lindenschmidt, E G

    1984-04-01

    Rheumatoid factors (RF) are autoantibodies mainly directed against autologous IgG. They belong at most to the IgM class antibodies. It is demonstrated at groups with unsolved hepatitis B, rubella, syphilis and toxoplasmose infection that RF do occur not rarely at these patients even without rheumatoid arthritis. This is probably due to stimulation by antigen-IgG-complexes. During serologic detection of specific IgM antibodies they present an antigen independent mu-specificity. So the test for specific IgM might even loose its diagnostic and possibly therapy indicating value. It is shown how the disturbance by RF can be calculated after adsorption with aggregated IgG. Also RF can be titrated by an enzyme immunoassay (ELISA). With IgG coated latex particles RF can be eliminated prior to the IgM-test. Solid phase techniques which are applied with enzyme-coupled antigen instead of marked anti-IgM cannot be disturbed by RF significantly. PMID:6398795

  16. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    PubMed

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting. PMID:23107315

  17. Comparison of the Directigen Flu A+B Membrane Enzyme Immunoassay with Viral Culture for Rapid Detection of Influenza A and B Viruses in Respiratory Specimens

    PubMed Central

    Cazacu, Andreea C.; Chung, Sooyoung E.; Greer, Jewel; Demmler, Gail J.

    2004-01-01

    The performance of a commercially available, rapid membrane enzyme immunoassay for influenza A and B virus detection was compared to that of viral culture in 4,092 respiratory specimens collected from patients presenting with respiratory symptoms during the 2002-2003 influenza season. The test's overall sensitivity was 43.83%, lower than previously reported but similar for detection of both influenza A and B viruses (42.98 versus 44.76%). However, specificity, 99.74%, was excellent for both influenza A and B viruses (99.82 versus 99.92%). These values make this test a very good confirmatory test when clinical suspicion is high, but a less accurate screening test for large populations. PMID:15297520

  18. Development of a flow-through enzyme immunoassay and application in screening green coffee samples for ochratoxin A with confirmation by high-performance liquid chromatography.

    PubMed

    Sibanda, L; De Saeger, S; Bauters, T G; Nelis, H J; Van Peteghem, C

    2001-10-01

    A flow-through enzyme immunoassay has been developed for the screening of green coffee bean samples for ochratoxin A (OA) and was later used in a survey on OA in green coffee from different countries. The test has a sensitivity of 8 ng/g, and calculated recoveries ranged from 70 to 89% and from 86 to 95% for spiked and naturally contaminated samples, respectively. There were no significant differences in within-day and between-day assay performance (P > 0.05). Green coffee samples (15 Arabica and 7 Robusta) received from an international coffee trader were analyzed for intrinsic fungal contamination, screened for OA, and subsequently confirmed by high-performance liquid chromatography (HPLC). All 22 samples were contaminated by fungal species of the genus Aspergillus, while Penicillium species were isolated from a mere 13.6% of the total number of samples. Isolates were tested for their ability to produce OA, and only 3.9% were positive. There was no correlation between occurrence of OA-producing isolates and levels of OA in contaminated samples. Results of the screening procedure showed that 4 of the 22 samples were contaminated with 8 ng/g or higher. The HPLC method confirmed that the OA levels ranged from 27 to 168 ng/g. A fifth sample, which was shown to be negative during screening, had an OA concentration of 4 ng/g. There were no false negatives or positives recorded, and the flow-through enzyme immunoassay results correlated with those obtained by HPLC. PMID:11601711

  19. Comparison of PremierTM Rotaclone®, ProSpecTTM, and RIDASCREEN® Rotavirus Enzyme Immunoassay Kits for Detection of Rotavirus Antigen in Stool Specimens

    PubMed Central

    Gautam, Rashi; Lyde, Freda; Esona, Mathew D.; Quaye, Osbourne; Bowen, Michael D.

    2015-01-01

    Background Rotaviruses are the major cause of severe dehydrating diarrhea in children throughout the world. Enzyme immunoassays (EIA) have been the standard method for detection of rotavirus in stool specimens since the 1980s. The World Health Organization (WHO) Rotavirus Surveillance Network has proposed including three EIA kits in the WHO-GSM (Global Management System/Système Mondial de Gestion ) catalogue for easy procurement of EIA kits by participating rotavirus surveillance network laboratories. Objectives In this study, we conducted a comparative analysis of 3 commercially available enzyme immunoassay kits: PremierTM Rotaclone® (Meridian Bioscience, Inc.), ProSpecTTM (Oxoid, Ltd.) and RIDASCREEN® (R-biopharm AG) for rotavirus diagnostics. Study design Using reverse-transcriptase-PCR (RT-PCR) as the gold standard, the 3 EIA kits were evaluated by testing a stool panel consisting of 56 rotavirus-positive and 54 rotavirus negative samples. Results The sensitivities of the PremierTM Rotaclone®, ProSpecTTM and RIDASCREEN® kits were 76.8%, 75% and 82.1% respectively, but did not differ significantly. The specificity of all the 3 kits was 100%. The use of RT-PCR as a gold standard lowered the observed sensitivity of all 3 EIA kits but helps to reduce equivocal results that can be seen when another EIA or other non-molecular methods are used as the reference assay in comparison studies. Conclusion Our study found that all three kits are suitable for use by rotavirus surveillance programs. PMID:23850415

  20. Highly sensitive micro-plate enzyme immunoassay screening and NCI-GC-MS confirmation of flunitrazepam and its major metabolite 7-aminoflunitrazepam in hair.

    PubMed

    Negrusz, A; Moore, C; Deitermann, D; Lewis, D; Kaleciak, K; Kronstrand, R; Feeley, B; Niedbala, R S

    1999-10-01

    Flunitrazepam (Rohypnol) is a benzodiazepine used in the treatment of insomnia as a sedative hypnotic and as preanesthetic medication in European countries and Mexico. Although it has no medicinal purpose in the United States, the occurrence of its abuse is increasing. Sexual abuse of both men and women while under the influence of so-called "date-rape" drugs has been the focus of many investigations. Reported date-rape drugs include flunitrazepam (FN), clonazepam, diazepam, oxazepam, gamma-hydroxybutyrate, and many others. FN has been banned in the United States because of its alleged use in such situations. Unfortunately, the detection of FN or its metabolites 7-aminoflunitrazepam (7-AFN) and desmethylflunitrazepam in a single specimen such as urine or blood is difficult in criminal situations because of the likelihood of single-dose ingestion and the length of time since the alleged incident. Hair provides a solution to the second of these problems in that drugs tend to incorporate into hair and remain there for longer periods of time than either urine or blood. There are various techniques for the detection of FN in plasma, blood, and urine, but little work has been done with hair. Hair collection is a virtually noninvasive procedure that can supply information on drug use for several months preceding collection. The objective of this paper was to determine if a commercially available micro-plate enzyme immunoassay system was sufficiently sensitive for the routine screening of 7-AFN in hair by the development of extraction procedures and optimization of the immunoassay kit. Further, this study used the same solid-phase extraction to isolate FN and its major metabolite, 7-AFN, and gas chromatography-mass spectrometry with negative ion chemical ionization for confirmation. Two seven-point standard curves were established ranging from 0.5 pg/mg to 100 pg/mg for 7-AFN and 2.5 pg/mg to 200 pg/mg for FN with respective deuterated internal standards. A replicate

  1. EQCM Immunoassay for Phosphorylated Acetylcholinesterase as a Biomarker for Organophosphate Exposures Based on Selective Zirconia Adsorption and Enzyme-Catalytic Precipitation

    SciTech Connect

    Wang, Hua; Wang, Jun; Choi, Daiwon; Tang, Zhiwen; Wu, Hong; Lin, Yuehe

    2009-03-01

    A zirconia (ZrO2) adsorption-based immunoassay by electrochemical quartz crystal microbalance (EQCM) has been initially developed, aiming at the detection of phosphorylated acetylcholinesterase (AChE) as a potential biomarker for bio-monitoring exposures to organophosphate (OP) pesticides and chemical warfare agents. Hydroxyl-derivatized monolayer was preferably chosen to modify the crystal serving as the template for directing the electro-deposition of ZrO2 film with uniform nanostructures. The resulting ZrO2 film was utilized to selectively capture phosphorylated AChE from the sample media. Horseradish peroxidase (HRP)-labeled anti-AChE antibodies were further employed to recognize the captured phosphorylated protein. Enzyme-catalytic oxidation of the benzidine substrate resulted in the accumulation of insoluble product on the functionalized crystal. Ultrasensitive EQCM quantification by mass-amplified frequency responses as well as rapid qualification by visual color changes of product could be thus achieved. Moreover, 4-chloro-1-naphthol (CN) was comparably studied as an ideal chromogenic substrate for the enzyme-catalytic precipitation. Experimental results show that the developed EQCM technique can allow for the detection of phosphorylated AChE in human plasma. Such an EQCM immunosensing format opens a new door towards the development of simple, sensitive, and field-applicable biosensor for biologically monitoring low-level OP exposures.

  2. Development of a Highly Specific IgM Enzyme-Linked Immunosorbent Assay for Bartonella henselae Using Refined N-Lauroyl-Sarcosine-Insoluble Proteins for Serodiagnosis of Cat Scratch Disease.

    PubMed

    Otsuyama, Ken-Ichiro; Tsuneoka, Hidehiro; Kondou, Kaori; Yanagihara, Masashi; Tokuda, Nobuko; Shirasawa, Bungo; Ichihara, Kiyoshi

    2016-04-01

    The conventional anti-Bartonella henselaeIgM enzyme-linked immunosorbent assay (IgM-ELISA) methods for diagnosing cat scratch disease (CSD) remain poor in both sensitivity and specificity. We sought to develop an IgM-ELISA with improved accuracy in the serodiagnosis of CSD by exploring the antigens that are most suitable for an ELISA. We prepared 5 different protein antigens: antigen I (sonicatedB. henselaewhole-cell antigen), antigen II (N-lauroyl-sarcosine-insoluble antigen), antigen III (processed sarcosine-soluble antigen), and antigen IV and antigen V (sarcosine-insoluble and sarcosine-soluble antigens refined by DEAE-Sepharose Fast Flow ion-exchange chromatography). The IgM antibodies in the sera of 47 patients with clinically suspected CSD (24 definite, 23 suspected) and of 85 healthy individuals were examined by ELISAs using the 5 antigens, and the results were compared with those of an IgM indirect fluorescent antibody assay (IgM-IFA). In a reference panel, which consisted of 5 positive and 5 negative sera, antigen I and antigen III failed to distinguish between the two statuses, whereas the other three antigens succeeded in distinguishing between them. When the cutoff value was set at the 98th percentile of the ELISA index for healthy individuals, the sensitivity of IgM-IFA for the 24 cases of definite CSD was 54%, whereas the sensitivities of the IgM-ELISAs with antigen II, IV, and V were 75%, 83%, and 75%, respectively. The sensitivities of these three IgM-ELISAs for all 47 of the clinically suspected cases were 49%, 64%, and 51%, respectively. In contrast, the sensitivity of IgM-IFA was 28%. These results indicate that the refined sarcosine-insoluble proteins (antigen IV), which possessed the highest specificity among the 5 antigens, are the most appropriate for developing an IgM-ELISA for the highly specific serodiagnosis of CSD. PMID:26865692

  3. Anti-α-glucose-based glycan IgM antibodies predict relapse activity in multiple sclerosis after the first neurological event

    PubMed Central

    Freedman, MS; Laks, J; Dotan, N; Altstock, RT; Dukler, A; Sindic, CJM

    2009-01-01

    Background There is no specific serum-based biomarker for the diagnosis or prognosis of relapsing-remitting multiple sclerosis (RRMS). Objective We investigated whether levels of IgM antibodies to Glc(α1,4)Glc(α) (GAGA4) or to a panel of four glucose-based glycans could differentiate MS from other neurological diseases (OND) or predict risk of early relapse following first presentation (FP) of RRMS. Methods Retrospective analysis of 440 sera samples of three cohorts: A) FP-RRMS (n = 44), OND (n = 44); B) FP-RRMS (n = 167), OND (n = 85); and C) FP (n = 100). Anti-GAGA4 IgM levels were measured by enzyme immunoassay in cohort-A and cohort-B. Cohort-C IgM antibodies to glucosebased glycan panel were measured by immunofluorescence. Results FP-RRMS had higher levels of anti-GAGA4 IgM than OND patients (cohort-A, P = 0.01; cohort-B, P = 0.0001). Sensitivity and specificity were 27% and 97% for cohort-A; and 26% and 90% for cohort-B, respectively. In cohort-C, 58 patients experienced early relapse (<24 months), 31 had late relapse (≥24 months), and 11 did not experience second attack during follow-up. Kaplan– Meier curves demonstrated decrease in time to next relapse for patients positive for the antibody panel (P = 0.02, log rank). Conclusions Serum anti-GAGA4 IgM discerns FP-RRMS patients from OND patients. Higher levels of serum anti-α-glucose IgM in FP patients predict imminent early relapse. PMID:19324980

  4. Development of a nanobody-alkaline phosphatase fusion protein and its application in a highly sensitive direct competitive fluorescence enzyme immunoassay for detection of ochratoxin A in cereal

    PubMed Central

    Liu, Xing; Xu, Yang; Wan, De-bin; Xiong, Yong-hua; He, Zhen-yun; Wang, Xian-xian; Gee, Shirley J.; Ryu, Dojin; Hammock, Bruce D.

    2015-01-01

    A rapid and sensitive direct competitive fluorescence enzyme immunoassay (dc-FEIA) for ochratoxin A (OTA) based on a nanobody (Nb)-alkaline phosphatase (AP) fusion protein was developed. The VHH gene of Nb28 was subcloned into the expression vector pecan45 containing the AP double mutant gene. The Nb28-AP construct was transformed into E. coli BL21(DE3)plysS and soluble expression in bacteria was confirmed by SDS-PAGE and Western blot. Both the Nb properties and AP enzymatic activity were validated by colorimetric and fluorometric analysis. The 50% inhibitory concentration and the detection limit of the dc-FEIA were 0.13 ng/mL and 0.04 ng/mL, respectively, with a linear range of 0.06–0.43 ng/mL. This assay was compared with LC-MS/MS, and the results indicated the reliability of Nb-AP fusion protein-based dc-FEIA for monitoring OTA contamination in cereal. PMID:25531426

  5. Vidas UP-enzyme-linked fluorescent immunoassay based on recombinant phage protein and fluorescence in situ hybridization as alternative methods for detection of Salmonella enterica serovars in meat.

    PubMed

    Zadernowska, Anna; Chajęcka-Wierzchowska, Wioleta; Kłębukowska, Lucyna

    2014-09-01

    Several methods for the rapid and specific detection of Salmonella spp. in meat have been described. This study was conducted to evaluate the capability of the VIDAS(®) UP (SPT [Salmonella Phage Technology]), an enzyme-linked fluorescent immunoassay method, and fluorescence in situ hybridization (FISH) to complement the International Organization for Standardization Method 6579 (ISO) in detecting Salmonella spp. from beef, pork, and poultry meat samples. The meat was inoculated with a mixture of Salmonella spp. on three levels of contamination. It was also checked that the tests did not produce cross-reactions with other Enterobacteriaceae rods. On the basis of the results, the relative specificity, relative accordance, and relative sensitivity of the method were determined. In meat samples, Vidas UP and FISH detection results were in substantial agreement with ISO, with relative specificity, accordance, and sensitivity rates of 90%, 96.3%, and 100%, respectively, for Vidas UP and 100%, 100%, and 99.4%, respectively, for FISH. This is the first report on the evaluation of both Vidas UP and FISH compared to ISO for the rapid detection of Salmonella enterica serovars in meat. PMID:24971928

  6. Mumps virus-specific antibody titers from pre-vaccine era sera: comparison of the plaque reduction neutralization assay and enzyme immunoassays.

    PubMed

    Mauldin, Jeremy; Carbone, Kathryn; Hsu, Henry; Yolken, Robert; Rubin, Steven

    2005-09-01

    Mumps virus-neutralizing antibodies are believed to be the most predictable surrogate marker of protective immunity. However, assays used to detect neutralizing antibodies, such as the plaque reduction neutralization (PRN) assay, are labor- and time-intensive and consequently are often supplanted by the more rapid and inexpensive enzyme immunoassay (EIA) technique. For virus infections for which international antibody standards exist and are bridged to clinical studies of protection (e.g., measles and rubella), the EIA has been successfully used to determine immune surrogate endpoints, yet no such international reference exists for mumps serology. Since both virus-neutralizing and nonneutralizing antibodies are measured in the EIA, in the absence of a mumps serological standard, the EIA may be prone to yielding false-positive results when utilized for assessing surrogate markers of protective immunity. Moreover, since mumps virus-specific antibody titers are generally low in comparison to antibody levels induced by other viruses and EIA procedures often employ relatively high serum dilution factors, the EIA may be prone to yielding false-negative results. To examine these issues, a PRN assay and two commercially available EIA kits were used to evaluate wild-type mumps virus serological responses in human serum samples from the pre-mumps vaccine era. Our results indicate that the PRN assay is a more sensitive and specific method of measuring serological responses to wild-type mumps virus. PMID:16145156

  7. Equivalence of assurance Gold Enzyme Immunoassay for visual or instrumental detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study.

    PubMed

    Feldsine, P T; Mui, L A; Forgey, R L; Kerr, D E

    2000-01-01

    Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Assurance Gold Salmonella Enzyme Immunoassay (EIA) and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the Assurance method and one by the AOAC culture method. The results for Assurance method were read visually and instrumentally with a microplate reader. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between Assurance Gold Salmonella EIA with either visual or instrumental interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The Assurance visual and instrumental options of reading sample reactions produced the same results for 1277 of the 1296 sample and controls analyzed. PMID:10995113

  8. Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum Antigens in Human Fecal Specimens Using the Triage Parasite Panel Enzyme Immunoassay

    PubMed Central

    Garcia, Lynne S.; Shimizu, Robyn Y.; Bernard, Caroline N.

    2000-01-01

    The Triage parasite panel (BIOSITE Diagnostics, San Diego, Calif.) is a new qualitative enzyme immunoassay (EIA) panel for the detection of Giardia lamblia, Entamoeba histolytica/E. dispar, and Cryptosporidium parvum in fresh or fresh, frozen, unfixed human fecal specimens. By using specific antibodies, antigens specific for these organisms are captured and immobilized on a membrane. Panel performance was evaluated with known positive and negative stool specimens (a total of 444 specimens) that were tested by the standard ova and parasite (O&P) examination as the “gold standard,” including staining with both trichrome and modified acid-fast stains. Specimens with discrepant results between the reference and Triage methods were retested by a different method, either EIA or immunofluorescence. A number of samples with discrepant results with the Triage device were confirmed to be true positives. After resolution of discrepant results, the number of positive specimens and the sensitivity and specificity results were as follows: for G. lamblia, 170, 95.9%, and 97.4%, respectively; for E. histolytica/E. dispar, 99, 96.0%, and 99.1%, respectively; and for C. parvum, 60, 98.3%, and 99.7%, respectively. There was no cross-reactivity with other parasites found in stool specimens, including eight different protozoa (128 challenges) and three different helminths (83 challenges). The ability to perform the complete O&P examination should remain an option for those patients with negative parasite panel results but who are still symptomatic. PMID:10970380

  9. Validation of an enzyme-immunoassay for the non-invasive monitoring of faecal testosterone metabolites in male cheetahs (Acinonyx jubatus).

    PubMed

    Pribbenow, Susanne; Wachter, Bettina; Ludwig, Carsten; Weigold, Annika; Dehnhard, Martin

    2016-03-01

    In mammals, the sex hormone testosterone is the major endocrine variable to objectify testicular activity and thus reproductive function in males. Testosterone is involved in the development and function of male reproductive physiology and sex-related behaviour. The development of a reliable androgen enzyme-immunoassay (EIA) to monitor faecal testosterone metabolites (fTM) is a powerful tool to non-invasively assess the gonadal status of males. We validated an epiandrosterone EIA for male cheetahs by performing a testosterone radiometabolism study followed by high-performance liquid chromatography (HPLC) analyses and excluding possible cross-reactivities with androgenic metabolites not derived from testosterone metabolism. The physiological and biological relevance of the epiandrosterone EIA was validated by demonstrating (1) a significant increase in fTM concentrations within one day in response to a testosterone injection, (2) a significant increase in fTM concentrations within one day in response to a gonadotropin-releasing hormone (GnRH) injection, which failed following a placebo injection, and (3) significant differences in fTM concentrations between adult male and adult female cheetahs and between adult and juvenile male cheetahs of a free-ranging population. Finally, we demonstrated stability of fTM concentrations measured in faecal samples exposed to ambient temperatures up to 72h. Our results clearly demonstrate that the epiandrosterone EIA is a reliable non-invasive method to monitor testicular activity in male cheetahs. PMID:26828820

  10. Development of an enzyme immunoassay specific for a core protein epitope of a novel small basement membrane associated heparan sulphate proteoglycan from human kidney.

    PubMed

    Stöcker, G; Stickeler, E; Switalla, S; Fischer, D C; Greiling, H; Haubeck, H D

    1997-02-01

    Heparan sulphate proteoglycans are major components of the glomerular basement membrane and play a key role in their molecular organization and function. Moreover, their presence is essential for the maintenance of the selective permeability of the glomerular basement membrane. Recently, we have isolated and characterized a novel, small basement membrane associated heparan sulphate proteoglycan from human aorta and kidney. Using specific monoclonal antibodies we have shown that the novel heparan sulphate proteoglycan is predominantly located in the glomerular basement membrane, to a lesser extent in the basement membrane of tubuli, and also in the mesangium. Turnover or, in the course of kidney diseases, degradation of heparan sulphate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulphate proteoglycan. Therefore, changes in the structure and function of glomerular basement membranes may be directly detected by measuring the excretion of a component of this basement menbrane, e. g. heparan sulphate proteoglycan into urine. Here we describe the establishment of an enzyme immunoassay for the sensitive detection of the novel, small heparan sulphate proteoglycan in urine. In this assay the specific monoclonal antibody 1F10/B8, which recognizes a core protein epitope, was used to detect the polyanionic heparan sulphate proteoglycan bound to the surface of a cationic charge modified microtitre plate. This assay allows the sensitive and specific detection of the small heparan sulphate proteoglycan, which is released from the glomerular basement membrane into urine during normal turnover and also in the course of kidney diseases. PMID:9056750

  11. Detection and serogroup differentiation of Salmonella spp. in food within 30 hours by enrichment-immunoassay with a T6 monoclonal antibody capture enzyme-linked immunosorbent assay.

    PubMed Central

    Ng, S P; Tsui, C O; Roberts, D; Chau, P Y; Ng, M H

    1996-01-01

    We previously described an antigen capture enzyme-linked immunosorbent assay which makes use of monoclonal antibody T6, which recognizes an epitope on the outer core polysaccharide of Salmonella lipopolysaccharide molecules that is common to almost all Salmonella serovars. In this paper, we show that this assay can detect between 10(5) and 10(7) Salmonella cells per ml even in the presence of excess Escherichia coli. A total of 153 of 154 (99%) serogroup A to E strains and 51 of 78 (71%) serogroup F to 67 strains were reactive as determined by this assay. This corresponds to a detection rate of approximately 98% of all salmonellae known to affect humans. None of the 65 strains of non-Salmonella bacteria tested positive. Taking advantage of the O-factor polysaccharides also present on the antigen captured by the immobilized T6 antibody, we showed that 136 of 154 Salmonella serogroup A to E strains (88%) were correctly differentiated according to their serogroups by use of enzyme conjugates of a panel of O-factor-specific monoclonal antibodies. We evaluated this assay for the detection and serogroup differentiation of salmonellae directly from enrichment cultures of simulated food, eggs, pork, and infant formula milk. All 26 samples which had been contaminated with Salmonella spp. were detected by T6 (100% sensitivity), with only one false-positive result from 101 samples not contaminated by Salmonella spp. (99% specificity). The detection time was substantially reduced to between 17 and 29 h, depending on the enrichment methods used. Since there were no false-negative results, we concluded that this enrichment-immunoassay method can afford rapid screening for Salmonella spp. in food samples. PMID:8779567

  12. Identification of specific antinuclear antibodies in dogs using a line immunoassay and enzyme-linked immunosorbent assay.

    PubMed

    Bremer, Hanna D; Lattwein, Erik; Renneker, Stefanie; Lilliehöök, Inger; Rönnelid, Johan; Hansson-Hamlin, Helene

    2015-12-15

    Circulating antinuclear antibodies (ANA) are commonly present in the systemic autoimmune disease Systemic Lupus Erythematosus (SLE) and in other systemic rheumatic diseases, in humans as well as in dogs. The indirect immunofluorescence (IIF)-ANA test is the standard method for detecting ANA. Further testing for specific ANA with immunoblot techniques or ELISAs is routinely performed in humans to aid in the diagnosis and monitoring of disease. Several specific ANA identified in humans have been identified also in suspected canine SLE but, in contrast to humans, investigation of autoantibodies in canine SLE is mainly restricted to the IIF-ANA test. Our aim was to identify both known and novel specific ANA in dogs and to investigate if different IIF-ANA patterns are associated with different specific ANA in dogs. Sera from 240 dogs with suspicion of autoimmune disease (210 IIF-ANA positive (ANA(pos)) and 30 IIF-ANA negative (ANA(neg))) as well as sera from 27 healthy controls were included. The samples were analysed with a line immunoassay, LIA (Euroline ANA Profile 5, Euroimmun, Lübeck, Germany) and four different ELISAs (Euroimmun). The ANA(pos) dogs were divided in two groups depending on the type of IIF-ANA pattern. Of the 210 ANA(pos) samples 68 were classified as ANA homogenous (ANA(H)) and 141 as ANA speckled (ANA(S)), one sample was not possible to classify. Dogs in the ANA(H) group had, compared to the other groups, most frequently high levels of anti-double stranded deoxyribonucleic acid (dsDNA) and anti-nucleosome ANA. Anti-dsDNA antibodies were confirmed in some dogs with the Crithidia luciliae indirect immunofluorescence test (CLIFT). The frequency of ANA(H) dogs with values above those observed in the healthy group was significantly higher compared to ANA(S) dogs for anti-dsDNA, anti-nucleosome, and anti-histone reactivity. Dogs in the ANA(S) group had, compared to the other groups, most frequently high levels of anti-ribonucleoproteins (RNP) and

  13. Comparison of capture immunoglobulin M (IgM) and IgG enzyme-linked immunosorbent assay (ELISA) and nonstructural protein NS1 serotype-specific IgG ELISA for differentiation of primary and secondary dengue virus infections.

    PubMed

    Shu, Pei-Yun; Chen, Li-Kuang; Chang, Shu-Fen; Yueh, Yi-Yun; Chow, Ling; Chien, Li-Jung; Chin, Chuan; Lin, Ting-Hsiang; Huang, Jyh-Hsiung

    2003-07-01

    We have found that NS1 serotype-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) can be used to differentiate primary and secondary dengue virus infections. This is due to the fact that the NS1-specific IgG antibody cannot be detected before day 9 of illness for primary infection, so the NS1-specific IgG antibodies measured in acute-phase sera must come from previous infection. Comparison of NS1 serotype-specific IgG ELISA with envelope- and membrane-specific capture IgM and IgG ELISA in the differentiation of primary and secondary dengue virus infections showed good correlation (95.90% agreement). Most important, we have found that the serotype of the dengue virus from the majority of patients with primary infection could be correctly identified when convalescent-phase or postinfection sera were analyzed by NS1 serotype-specific IgG ELISA. These findings suggested that NS1 serotype-specific IgG ELISA could be reliably applied for serodiagnosis and seroepidemiological study of dengue virus infection. PMID:12853395

  14. Enhanced immunoassay for porcine circovirus type 2 antibody using enzyme-loaded and quantum dots-embedded shell-core silica nanospheres based on enzyme-linked immunosorbent assay.

    PubMed

    Wu, Long; Li, Xuepu; Shao, Kang; Ye, Shiyi; Liu, Chen; Zhang, Chenjun; Han, Heyou

    2015-08-01

    Boosting the detection sensitivity of enzyme-linked immunosorbent assay (ELISA) is significant to the early clinical diagnosis of various diseases. Here, we developed a versatile immunosensor using silica nanospheres as carriers for sensitive detection of porcine circovirus type 2 (PCV2) antibody. With HRP enzyme covalently immobilized on the silica nanospheres and CdSe nanocrystals embedded inside, these signal probes were successfully utilized in the sensitive detection of PCV2 antibody by ELISA, fluorometry and square-wave voltammetry (SWV). To further demonstrate the performance of the immunosensor, Human IgG (HIgG) was used as a model analyte. Since more HRP and CdSe QDs were loaded, 5-, 200- and 400-fold enhancements in amplified ELISA, fluorometry and voltammetry responses for HIgG could be achieved compared to conventional ELISA. The respective detection limits of theses methods for HIgG were 3.9, 0.1 and 0.05 ng mL(-1) with a RSD below 5% for amplified ELISA, fluorescence and SWV measurements. Additionally, a 100-fold improvement was obtained in the detection sensitivity for PCV2 antibody immunoassay. The versatile immunosensor exhibits good sensitivity, stability and reproducibility, suggesting its potential applications in clinical diagnostics. PMID:26320802

  15. Use of a monoclonal antibody in an enzyme immunoassay for the detection of Entamoeba histolytica in fecal specimens.

    PubMed

    Ungar, B L; Yolken, R H; Quinn, T C

    1985-05-01

    An indirect enzyme-linked immunosorbent assay (ELISA) was developed for the detection of Entamoeba histolytica in human feces, using both a monoclonal antibody and rabbit antisera. It detected from less than 1 to 57 trophozoites of 6 E. histolytica strains. Stool specimens were positive by ELISA in 18 of 22 (82%) patients with E. histolytica and in 3 of 186 (2%) of patients without demonstrable E. histolytica in their stools. The latter included one from a child living near an asymptomatic cyst carrier and another from a traveler with giardiasis who had recently taken antibiotics. One hundred eight of 183 microscopy-and ELISA-negative specimens contained other parasites including Giardia (49 specimens), Endolimax nana (24), Entamoeba coli (21), Iodamoeba butschlii (2), and Entamoeba hartmanni (1). This ELISA for E. histolytica is a simple, sensitive and specific diagnostic tool. PMID:2860814

  16. An enzyme flow immunoassay that uses beta-galactosidase as the label and a cellobiose dehydrogenase biosensor as the label detector.

    PubMed

    Burestedt, E; Nistor, C; Schagerlöf, U; Emnéus, J

    2000-09-01

    The aim was to develop a fast generic enzyme flow immunoassay (EFIA) using a beta-galactosidase (beta-GAL) label in combination with colorimetric detection as well as with a new amperometric biosensor as the label detector. The amperometric biosensor was previously developed within the group for the determination of diphenols in surface water samples. Antigen (Ag, analyte), tracer (Ag*, antigen labeled with beta-GAL), and antibody (Ab) were incubated off-line. After the equilibrium was reached, the sample was introduced into the flow system. The antibody complexes, AgAb and Ag*Ab, were trapped in a protein G column while the free unbound tracer was eluted and detected by an amperometric biosensor downstream after substrate reaction. The enzyme label beta-GAL converted the substrate 4-aminophenyl-beta-D-galactopyranoside (4-APG) into 4-aminophenol (4-AP), which subsequently was detected by a cellobiose dehydrogenase (CDH) modified solid graphite electrode. 4-AP was first oxidized at the electrode surface at +300 mV vs Ag/AgCl, and the formed 4-imino quinone (4-IQ) was reduced back to 4-AP by the CDH in the presence of cellobiose. By combining the EFIA with the CDH biosensor, the overall signal of one tracer molecule is amplified at two occasions, i.e., one enzyme label converts the substrate into many 4-AP molecules, and second these are further amplified by the CDH biosensor. The optimum conditions for the EFIA in terms of the molar ratio between tracer and beta-GAL, temperature, flow rate, etc., was investigated with colorimetric detection, using 2-nitrophenyl-beta-D-galactopyranoside (2-NPG) as the beta-GAL substrate. The performance of both the colorimetric and CDH biosensor detection was investigated and both methods were applied for determination of the model compound atrazine in spiked surface water samples. Detection limits of 0.056 +/- 0.008 and 0.038 +/- 0.007 microg L(-1) and IC50 values of 2.04 +/- 0.294 and 0.42 +/- 0.08 microg L(-1) were obtained for

  17. An ultra-sensitive monoclonal antibody-based competitive enzyme immunoassay for sterigmatocystin in cereal and oil products.

    PubMed

    Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

    2014-01-01

    Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg · mL(-1)) ELISA format, in which the antibody was diluted to 1:80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng · g(-1) for wheat, 0.06 ng · g(-1) for maize, and 0.1 ng · g(-1) for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90-104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

  18. An Ultra-Sensitive Monoclonal Antibody-Based Competitive Enzyme Immunoassay for Sterigmatocystin in Cereal and Oil Products

    PubMed Central

    Li, Min; Li, Peiwu; Wu, Hui; Zhang, Qi; Ma, Fei; Zhang, Zhaowei; Ding, Xiaoxia; Wang, Hengling

    2014-01-01

    Sterigmatocystin (STG), a biosynthesis precursor of aflatoxin B1, is well known for its toxic and carcinogenic effects in humans and animals. STG derivatives and protein conjugates are needed for generation of monoclonal antibodies (mAbs). This work describes a reliable and fast synthesis of novel STG derivatives, based on which novel STG bovine serum albumin conjugates were prepared. With the novel STG bovine serum albumin conjugates, three sensitive and specific mAbs against STG, named VerA 3, VerA 4, and VerA 6, were prepared by semi-solid hypoxanthine/aminopterin/thymidine (HAT) medium using a modified two-step screening procedure. They exhibited high affinity for STG and no cross-reactivity (CR) with aflatoxins B1, B2, G1, G2, and M1. Based on the most sensitive antibody VerA 3, an ultra-sensitive competitive enzyme-linked immunosorbent assay (ELISA) was developed for STG in wheat, maize, and peanuts. Assays were performed in the STG-GA-BSA-coated (0.5 µg·mL−1) ELISA format, in which the antibody was diluted to 1∶80,000. Several physicochemical factors influencing assay performance, such as pH, ionic strength, blocking solution, and diluting solution, were optimized. The final results showed that the assays had the detection limits of 0.08 ng·g−1 for wheat, 0.06 ng·g−1 for maize, and 0.1 ng·g−1 for peanuts, inter-assay and intra-assay variations of less than 10%, and recoveries ranging from 83% to 110%. These recoveries were in good agreement with those obtained by using HPLC-MS/MS method (90–104%), indicating the importance of the mAb VerA 3 in the study of STG in crude agricultural products. PMID:25184275

  19. Profiling of urinary testosterone and luteinizing hormone in exercise-stressed male athletes, using gas chromatography-mass spectrometry and enzyme immunoassay techniques.

    PubMed

    Yap, B K; Kazlauskas, R; Elghazi, K; Johnston, G A; Weatherby, R P

    1996-12-01

    Knowledge of the effects of episodic or short-term exercise-stress on endogenous testosterone and luteinizing hormone levels still remains fragmentary and inconclusive. In this study, an approach based on the absolute concentrations of urinary total testosterone (T), luteinizing hormone (LH) and the T/LH concentration ratios, was used to profile short-term exercise-stress responses in healthy drug-free male athletes. Testosterone and luteinizing hormone concentrations were measured using gas chromatography-mass spectrometry (GC-MS) and microparticle enzyme immunoassay (MEIA) techniques, respectively. Stress profiles derived from exercise-stress at VO2max, 68.1% VO2max and 51.6% VO2max were plotted using the concentrations of T, LH and the ratios of T/LH found under non-stressed and stressed conditions. Significant changes in LH concentrations (p < 0.005) and T/LH ratios (p < 0.005) levels were observed between the pre-stress and post-exercise conditions during acute exercise-stress at VO2max but the T concentration did not show any marked change relative to the non-stressed condition. Whilst exercise-stress appeared to reduce the change in T concentrations between the pre- and post-exercise states compared to that in the non-stressed control condition, the change in LH concentrations showed a moderate increase at submaximal oxygen uptake values. The stress profiles derived from this study facilitated an assessment of the relationship between the endogenous T, LH and T/LH ratio stress-responses over a short period of applied exercise-stress. PMID:9001959

  20. Assessment of Pregnancy Status of Asian Elephants (Elephas maximus) by Measurement of Progestagen and Glucocorticoid and Their Metabolite Concentrations in Serum and Feces, Using Enzyme Immunoassay (EIA)

    PubMed Central

    KAJAYSRI, Jatuporn; NOKKAEW, Weerapun

    2013-01-01

    ABSTRACT The study was to find patterns of progestagen (progesterone and its metabolite) and glucocorticoid and their metabolite concentrations in serum and feces of pregnant Asian elephants (Elephas maximus). The 5 female Asian domestic elephants were naturally mated until pregnancy. After that, blood and feces samples were collected monthly during pregnancy for progestagen, glucocorticoid and their metabolites analysis by enzyme immunoassay (EIA). The results showed the serum progestagen concentration during gestation was 2.11 ± 0.60 to 18.44 ± 2.28 ng/ml. Overall, serum progestagen concentration rose from the 1st month to reach peak in the 11th month, after which it declined to its lowest level in the 22nd month of pregnancy. Fecal progestagen concentration varied from 1.18 ± 0.54 to 3.35 ± 0.45 µg/g during pregnancy. In general, fecal progestagen concentration increased from the 1st month to its highest level in the 12th month. After this, it declined reaching its lowest point in the 22nd month of pregnancy. Glucocorticoid hormones and their metabolite concentrations both in serum and feces fluctuated from low to medium throughout almost the entire pregnancy period and then rapidly increased around the last week before calving. Our study suggests that this profile of progestagen and glucocorticoid hormones and their metabolite concentration levels in serum and feces can be used to assess the pregnancy status of Asian elephants. If serum and fecal progestagen concentrations were found in very low levels and glucocorticoid and their metabolite concentrations were found in very high levels, it was indicated that the cow elephant would calve within 7 days. PMID:24257195

  1. Chemiluminescence enzyme immunoassay for monitoring hepatitis C virus core protein during interferon-alpha2b and ribavirin therapy in patients with genotype 1 and high viral loads.

    PubMed

    Enomoto, Masaru; Nishiguchi, Shuhei; Tamori, Akihiro; Kohmoto, Modoka; Habu, Daiki; Sakaguchi, Hiroki; Takeda, Tadashi; Kawada, Norifumi; Seki, Shuichi; Shiomi, Susumu

    2005-09-01

    This study evaluated an updated chemiluminescence enzyme immunoassay (CLEIA) for hepatitis C virus (HCV) core protein for monitoring viral kinetics during treatment with interferon (IFN)-alpha and ribavirin. Using the CLEIA, serum levels of HCV core protein were measured in 17 patients with genotype 1 and high baseline viral loads during the first 4 weeks of combination therapy. HCV RNA was measured by the Amplicor Monitor test for comparison. At the start of therapy, the median HCV level (interquartile range) was 700 (540-940) kIU/ml of viral RNA and 11,310 (5,528-14,238) fmol/L of core protein. HCV RNA was above the upper limit of the linear range of the Amplicor Monitor test in 13 of the 17 patients, while the core protein level was within the linear range of the CLEIA in all patients. During therapy, the proportion of patients with HCV levels below the cutoff values at each time point was less with the Amplicor Monitor test than with CLEIA. Serum HCV core protein level decreased rapidly during the first 24 hr of therapy and more slowly thereafter, with median exponential decays of 1.08 and 0.046 log10/day, respectively. In the second phase, between day 1 and 28, the median decrease in HCV core protein level was higher in four patients with sustained virologic response (0.13 log10/day) than in 13 patients with no response (0.028 log10/day, P = 0.042). The wide linear range of the HCV core protein assay is appropriate for measuring viral loads during therapy with IFN-alpha and ribavirin. PMID:16032731

  2. Diagnosing Clostridium difficile-associated diarrhea using enzyme immunoassay: the clinical significance of toxin negativity in glutamate dehydrogenase-positive patients

    PubMed Central

    Yuhashi, Kazuhito; Yagihara, Yuka; Misawa, Yoshiki; Sato, Tomoaki; Saito, Ryoichi; Okugawa, Shu; Moriya, Kyoji

    2016-01-01

    Purpose The enzyme immunoassay (EIA) has lower sensitivity for Clostridium difficile toxins A and B than the polymerase chain reaction in the diagnosis of C. difficile-associated diarrhea (CDAD). Furthermore, toxin positivity with EIA performed on C. difficile isolates from stool cultures may be observed even in patients with EIA glutamate dehydrogenase (GDH)-positive and toxin-negative stool specimens. It is unclear whether such patients should be treated as having CDAD. Methods The present study retrospectively compared patient characteristics, treatment, and diarrhea duration among three groups of patients who underwent stool EIA testing for CDAD diagnosis: a toxin-positive stool group (positive stool group; n=39); a toxin-negative stool/toxin-positive isolate group (discrepant negative/positive group, n=14); and a dual toxin-negative stool and isolate group (dual negative group, n=15). All cases included were confirmed to be GDH positive on EIA test. Results Patients’ backgrounds and comorbidities were not significantly different among three groups. No difference was observed among the three groups with regard to antimicrobial drug use before diarrhea onset. Treatment was received by 82.1% of the positive stool group compared to 7.1% of the discrepant positive/negative group and 0% of the dual negative group, while mean diarrhea duration was 10.6 days compared to 7.9 days (P=0.6006) and 3.4 days (P=0.0312), respectively. Conclusion Even without treatment, patients with toxin-negative stool specimens had shorter diarrhea duration than those with toxin-positive stool specimens even with toxin-positive isolates. These findings may suggest a limited need for CDAD treatment for GDH-positive patients and toxin-negative stool specimens. PMID:27313472

  3. Measurement of β-(1,3)-glucan in household dust samples using Limulus amebocyte assay and enzyme immunoassays: an inter-laboratory comparison.

    PubMed

    Brooks, Collin R; Siebers, Rob; Crane, Julian; Noss, Ilka; Wouters, Inge M; Sander, Ingrid; Raulf-Heimsoth, Monika; Thorne, Peter S; Metwali, Nervana; Douwes, Jeroen

    2013-02-01

    Environmental levels of β-(1,3)-glucan, an inflammatory fungal cell wall component, have been suggested to be related to respiratory symptoms. However there is currently little data comparing β-(1,3)-glucan detection methods and/or results obtained in different laboratories. The aim of this study was to compare levels of β-(1,3)-glucans detected in household dust samples (n = 40) using different extraction/detection methods (Limulus amebocyte assay (LAL), inhibition enzyme immunoassay (EIA) and sandwich EIA) in five different laboratories. Dust sample aliquots were sent to participating centres, extracted and analysed for β-(1,3)-glucan according to standard in-house procedures. Significant differences in the levels of β-(1,3)-glucan were observed between all laboratories (geometric mean levels ranging from 15.4 μg g (-1) to 4754 μg g(-1) dust; p < 0.0001) with the exception of those using a similar LAL method. The inhibition EIA used in laboratory D produced mean β-(1,3)-glucan measurements 80-100 times higher than the LAL assays, 4 times higher than the sandwich EIA in the same lab, 17.6 times those obtained with the EIA in lab E and 363 times those obtained in the EIA in laboratory C. Pearson's correlations generally showed significant associations between methods and laboratories, particularly those using similar methodology (R ranging from 0.5 to 0.8; p < 0.001), although some poor and even inverse correlations were observed. Bland-Altman analyses showed moderate to good agreement between most assays, although clear absolute differences were observed. In conclusion, although results obtained with different methods were often significantly correlated and therefore comparable in relative terms, direct comparison of results between laboratories and assays may be inappropriate. PMID:25208705

  4. Sensitive Enzyme Immunoassay with β-d-Galactosidase—Fab Conjugate for Detection of Type A Influenza Virus Antigen in Clinical Specimens

    PubMed Central

    Harmon, Maurice W.; Russo, Lorrie L.; Wilson, Samuel Z.

    1983-01-01

    The most sensitive method for diagnosis of type A influenza virus infection is isolation of the agent in cell culture. However, detection and identification may require several days to complete. This delay in diagnosis prevents effective use of the antiviral agents available for treatment of type A influenza infection. As a rapid diagnostic method, enzyme immunoassay (EIA) is attaining increased usage for direct detection of viral antigen in clinical specimens. Standard EIA techniques, however, are usually not sensitive enough for reliable detection of viral antigen in respiratory secretions. We developed a conjugate consisting of the antigen-binding fragment of goat antirabbit immunoglobulin G coupled to β-d-galactosidase, using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. Other immunoreagents in our EIA consisted of guinea pig and rabbit antisera to influenza A/Brazil/11/78 (H1N1) for microtiter plate coating and primary antiserum, respectively. The sensitivity of this EIA was tested with 60 clinical specimens containing influenza A/England/333/80 (H1N1) which closely resembles A/Brazil. Of 31 initial specimens, collected within 24 h of the onset of symptoms, 27 (87%) were positive, using a fluorgenic substrate, and 18 of 29 (62%) specimens obtained 12 to 60 h after the initial specimens were positive, for a total of 75% (45 of 60). All positive reactions were specific, as shown in a confirmatory test with preimmune and hyperimmune guinea pig globulins. Clinical specimens negative for virus (n = 33) or containing heterologous respiratory viruses (n = 26) were negative in this system. These results indicate that EIA systems can be developed with a sensitivity approaching that required for clinical usefulness. PMID:6403573

  5. Assessment of the stress response in Columbian ground squirrels: laboratory and field validation of an enzyme immunoassay for fecal cortisol metabolites.

    PubMed

    Bosson, Curtis O; Palme, Rupert; Boonstra, Rudy

    2009-01-01

    Stress responses play a critical role in the ecology and demography of wild animals, and the analysis of fecal hormone metabolites is a powerful noninvasive method to assess the role of stress. We characterized the metabolites of injected radiolabeled cortisol in the urine and feces of Columbian ground squirrels and validated an enzyme immunoassay for measuring fecal cortisol metabolites (FCM) with a 5 alpha-3beta,11 beta-diol structure by stimulation and suppression of adrenocortical activity and by evaluation of the circadian pattern of FCM excretion. In addition, we also evaluated the impact of capture, handling, and acclimation to the laboratory on FCM. Cortisol is highly metabolized, with virtually none being excreted, and of the radiolabeled cortisol injected, 31% was recovered in urine and 6.5% in feces. The lag time between cortisol injection and its appearance in urine and feces was 4.5 +/- 0.82 (SE) h and 7.0 +/- 0.53 (SE) h, respectively. FCM levels varied over the day, reflecting circadian variation in endogenous cortisol. Dexamethasone decreased FCM levels by 33%, and ACTH increased them by 255%. Trapping and housing initially increased FCM levels and decreased body mass, but these reversed within 3-7 d, indicating acclimation. Finally, FCM levels were modestly repeatable over time (r=0.57) in wild, live trapped, nonbreeding animals, indicating that FCMs provide a measure of the squirrel's stress-axis state. This assay provides a robust noninvasive assessment of the stress response of the Columbian ground squirrel and will facilitate an integration of its life history and physiology. PMID:19335228

  6. Total pregnancy-associated plasma protein A--a first trimester maternal serum marker for Down's syndrome: clinical and technical assessment of a poly-monoclonal enzyme immunoassay.

    PubMed

    Christiansen, M; Jaliashvili, I

    2003-01-01

    Pregnancy-associated plasma protein A (PAPP-A) is a maternal serum marker of fetal chromosomal disease and a risk marker for adverse outcome. PAPP-A in the circulation exists both as a 2:2 complex (PAPP-A/proMBP) with the proform of eosinophil major basic protein (proMBP) and as dimeric PAPP-A. Non-PAPP-A containing proMBP complexes constitute the bulk of proMBP in maternal serum. We developed and characterized a sandwich enzyme immunoassay for PAPP-A using a polyclonal rabbit anti-PAPP-A/proMBP antibody (SSI 6823) and a monoclonal murine anti-PAPP-A/proMBP antibody (HYB 234-3), reactive with the PAPP-A part of PAPP-A/proMBP. The assay range was 2 mIU/L-500 mIU/L, intra- and inter-assay coefficients of variation <10%. The immunoreactivity eluted ahead of thyroglobulin, Mr 669 kDa, in gel filtration and bound to a heparin column. Serum concentrations of PAPP-A were determined in gestational weeks 5-13 in 167 pregnant women with normal fetuses and 39 women with Down's syndrome (DS) fetuses. The median PAPP-A MoM (multiples of the median in normal controls) in DS pregnancies was 0.30 (quartile range: 0.17-0.54). The PAPP-A logMoMs in DS pregnancies were normally distributed with a mean of -0.5927 and SD of 0.3639. When simulating the performance of PAPP-A and age as markers for DS in population screening a detection rate (DR) of 62% was found for a screen positive rate (SPR) of 5%. Together with beta-HCG and nuchal translucency, two other first trimester markers for fetal DS, a DR 90% could be obtained for an SPR of 5%. PMID:14594321

  7. Variation in the limit-of-detection of the ProSpecT Campylobacter microplate enzyme immunoassay in stools spiked with emerging Campylobacter species.

    PubMed

    Bojanić, Krunoslav; Midwinter, Anne Camilla; Marshall, Jonathan Craig; Rogers, Lynn Elizabeth; Biggs, Patrick Jon; Acke, Els

    2016-08-01

    Campylobacter enteritis in humans is primarily associated with C. jejuni/coli infection. The impact of other Campylobacter spp. is likely to be underestimated due to the bias of culture methods towards Campylobacter jejuni/coli diagnosis. Stool antigen tests are becoming increasingly popular and appear generally less species-specific. A review of independent studies of the ProSpecT® Campylobacter Microplate enzyme immunoassay (EIA) developed for C. jejuni/coli showed comparable diagnostic results to culture methods but the examination of non-jejuni/coli Campylobacter spp. was limited and the limit-of-detection (LOD), where reported, varied between studies. This study investigated LOD of EIA for Campylobacter upsaliensis, Campylobacter hyointestinalis and Campylobacter helveticus spiked in human stools. Multiple stools and Campylobacter isolates were used in three different concentrations (10(4)-10(9)CFU/ml) to reflect sample heterogeneity. All Campylobacter species evaluated were detectable by EIA. Multivariate analysis showed LOD varied between Campylobacter spp. and faecal consistency as fixed effects and individual faecal samples as random effects. EIA showed excellent performance in replicate testing for both within and between batches of reagents, in agreement between visual and spectrophotometric reading of results, and returned no discordance between the bacterial concentrations within independent dilution test runs (positive results with lower but not higher concentrations). This study shows how limitations in experimental procedures lead to an overestimation of consistency and uniformity of LOD for EIA that may not hold under routine use in diagnostic laboratories. Benefits and limitations for clinical practice and the influence on estimates of performance characteristics from detection of multiple Campylobacter spp. by EIA are discussed. PMID:27317896

  8. Validation of an enzyme immunoassay for the measurement of faecal glucocorticoid metabolites in Eurasian (Lynx lynx) and Iberian lynx (Lynx pardinus).

    PubMed

    Pribbenow, Susanne; Jewgenow, Katarina; Vargas, Astrid; Serra, Rodrigo; Naidenko, Sergey; Dehnhard, Martin

    2014-09-15

    Stress hormone levels are important indicator of an animal's well-being, as stress has harmful effects on reproduction, growth and immune function. The development of enzyme immunoassays (EIA) to monitor faecal glucocorticoid metabolites (fGM) contributes a powerful tool to assess an animal's adrenal status non-invasively. We aimed to identify a suitable EIA for monitoring fGM by assessing the suitability of six different EIAs for detecting quantitative changes in fGM concentrations in response to an ACTH challenge test in Eurasian lynx. FGM were characterised in a male Eurasian lynx that received an injection of (3)H-cortisol. Using HPLC analyses radiolabeled metabolites were compared with immunoreactive metabolites. The second aim was to biologically validate the established EIA for monitoring adrenocortical activity of captive Iberian lynxes after a translocation to new enclosures in relation to behaviour. Additionally faecal samples of ten pregnant Iberian lynxes from the peripartal period were analysed. The ACTH challenge revealed an 11β-hydroxyetiocholanolone EIA as the most sensitive assay to reflect acute fGM elevations in the Eurasian lynx. HPLC immunograms demonstrated that the 11β-hydroxyetiocholanolone EIA measured significant amounts of immunoreactivities corresponding to radiolabeled metabolites with strong similarities across both lynx species. Additionally, HPLC and GC-MS analyses confirmed the presence of 11β-hydroxyetiocholanolone in faeces of both, the Eurasian and the Iberian lynx. Longitudinal fGM profiles of Iberian lynx revealed increases in concentrations associated with management events. During the peripartal period, however, fGM concentrations were not significantly elevated. Our results show that the 11β-hydroxyetiocholanolone EIA is a reliable tool to assess fGM in both lynx species. PMID:25066418

  9. Development and validation of a simple, sensitive, second antibody format enzyme immunoassay (EIA) for LH determination in mithun (Bos frontalis) plasma.

    PubMed

    Mondal, Mohan; Dhali, Arindam; Prakash, Bhukya; Rajkhowa, Chandan; Prakash, B S

    2005-01-01

    The objective of this study was to develop and validate a simple and highly sensitive enzyme immunoassay (EIA) for LH determination in mithun plasma on microtitreplates using the biotin-streptavidin amplification system and the second antibody coating technique. Biotin was coupled to LH and used to bridge between streptavidin-peroxidase and immobilized antiserum in competitive assay. The EIA was carried out directly in 20 microL mithun plasma. The LH standards ranging from 6.25 pg/well/20 microL to 400 pg/well/20 microL were prepared in hormone free plasma collected from a mithun on day 3 post calving. The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/mL plasma; the 50 percent relative binding sensitivity was seen at 100 pg/well/20 microL. Plasma volumes for the EIA viz. 10 and 20 microL did not influence the shape of standard curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous mithun plasma LH with bovine LH standards. It showed good parallelism with the bovine standard curve. For the biological validation of the assay, 3 mithuns were used. These were administered 10 microg i.v., with a synthetic analogue of GnRH (Buserelin-Acetate, Intervet, India) and blood samples were collected at 15 min intervals using indwelling jugular catheter beginning 1 h prior to GnRH injection till 8 h post injection. In all animals, sharp increases in LH concentrations were recorded post GnRH administration, which confirms the biological validation of the EIA. In conclusion, the EIA developed for LH determination in mithun blood plasma is sufficiently reliable, economical, and sensitive enough to estimate LH in all physiological variations in mithun. PMID:15794124

  10. Evaluation of a New Dot Blot Enzyme Immunoassay (Directigen Flu A+B) for Simultaneous and Differential Detection of Influenza A and B Virus Antigens from Respiratory Samples

    PubMed Central

    Reina, Jordi; Padilla, Emma; Alonso, Fermin; Ruiz de Gopegui, Enrique; Munar, Maria; Mari, Margarita

    2002-01-01

    We report a prospective evaluation of a new dot blot enzyme immunoassay (EIA) method for the direct, rapid, qualitative, simultaneous, and differential detection of the influenza A (IA) and B (IB) virus antigen in different respiratory samples. The EIA method was compared with the shell vial culture system (MDCK cell line) used with the same samples. We studied 160 samples from 93 (58.1%) pediatric patients (hospital emergency room) and from 67 (41.9%) adult patients (sentinel network). Seventy-four(46.2%) samples were considered positive; of them, 46 (62.2%) were from pediatric patients and 28 (37.8%) were from an adult group (P < 0.05), with overall positive values of 49.9% and 41.7%, respectively. All 74 (100%) of the positive samples were isolated in cell culture versus the 68.9% that were detected as positive by the new EIA method (P < 0.05). Of the 41 samples positive for the IA virus, the EIA detected 34 (82.9%) positive samples; of the 33 samples positive for the IB virus, the EIA detected 17 (51.5%) positive samples (P < 0.05). No false-positive reaction was detected with the EIA method (specificity and positive predictive value of 100%). The overall results obtained in the comparison between the new EIA and the shell vial culture had a sensibility of 82.9% and predictive negative values of 92.4% for the IA virus and 51.5% and 84.3%, respectively, for the IB virus. This evaluation shows sensitivity and specificity percentages for the new EIA method that is acceptable for routine use in IA virus detection. The results obtained were worse for IB virus detection, but this new EIA method is actually the only one with the capacity to differentiate between the two influenza viruses. PMID:12202608

  11. Characterisation and validation of an enzyme-immunoassay for the non-invasive assessment of faecal glucocorticoid metabolites in cheetahs (Acinonyx jubatus).

    PubMed

    Ludwig, C; Wachter, B; Silinski-Mehr, S; Ganswindt, A; Bertschinger, H; Hofer, H; Dehnhard, M

    2013-01-01

    The non-invasive measurement of adrenocortical function in cheetahs is an important tool to assess stress in captive and free-ranging individuals, because stress has been suggested to be one of the causes of poor reproductive performance of captive cheetahs. We tested four enzyme immunoassays (EIA) in two captive cheetahs in Germany using adrenocorticotropic hormone (ACTH) challenges and identified the corticosterone-3-CMO EIA to be most sensitive to the increase in faecal glucocorticoid metabolite (fGCM) concentrations after the ACTH challenge. This EIA performed also well in five captive cheetahs in South Africa. The fGCM concentrations across all seven cheetahs increased within 24h by 681% compared to the baseline levels prior to ACTH. Storage of faecal samples at 0-4°C did not strongly affect fGCM concentrations within 24h, simplifying sample collection when immediate storage at -20°C is not feasible. The two cheetahs in Germany also received an injection of [(3)H]cortisol to characterise fGCMs in faecal extracts using high-performance liquid chromatography (HPLC) immunograms. HPLC fractions were measured for their radioactivity and immunoreactive fGCM concentrations with the corticosterone-3-CMO EIA, respectively. The results revealed a polar peak of radiolabelled cortisol metabolites co-eluting with the major peak of immunoreactive fGCMs. Thus, our EIA measured substantial amounts of fGCMs corresponding to the radioactive peaks. The peaks were of higher polarity than native cortisol and corticosterone, suggesting that the metabolites were conjugated, which was confirmed by solvolysis of the HPLC fractions. Our results show that the corticosterone-3-CMO EIA is a reliable tool to assess fGCMs in cheetahs. PMID:23108105

  12. Comparison of clinical performance of antigen based-enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-PCR for detection of genital Chlamydia trachomatis infection

    PubMed Central

    Nateghi Rostami, Mahmoud; Hossein Rashidi, Batool; Aghsaghloo, Fatemeh; Nazari, Razieh

    2016-01-01

    Background: Chlamydia trachomatis is the most common sexually transmitted bacterial pathogen worldwide. Early detection and treatment of C.trachomatis genital infection prevent serious reproductive complications. Objective: Performances of enzyme immunoassay (EIA) and major outer membrane protein (MOMP)-polymerase chain reaction (PCR) for diagnosis of genital C.trachomatis infection in women were compared. Materials and Methods: In this cross sectional study a total of 518 women volunteers were included (33.67±8.3 yrs) who had been referred to Gynecology clinics of Qom province, Iran, were included. Endocervical swab specimens were collected to detect lipopolysaccharide (LPS) antigen in EIA and to amplify MOMP gene of C.trachomatis in PCR. Results were confirmed using ompI nested-PCR. Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated for performance of the tests. Odds ratios were determined using binary logistic regression analysis. Results: In total, 37 (7.14%) cases were positive by EIA and/or MOMP-PCR. All discrepant results were confirmed by nested-PCR. Sensitivity, specificity, PPV and NPV values of EIA were 59.46%, 100%, 100% and 96.98%, and those of MOMP-PCR were 97.30%, 100%, 100%, 99.79%, respectively. Reproductive complications including 2.7% ectopic pregnancy, 5.4% stillbirth, 5.4% infertility, and 10.8% PROM were recorded. The risk of developing chlamydiosis was increased 4.8-fold in volunteers with cervicitis (p<0.05; OR 4.80; 95% CI 1.25-18.48). Conclusion: C.trachomatis infection should be regarded in women of reproductive ages especially those with cervicitis. Primary screening of women by using the low cost antigen-EIA is recommended; however, due to the low sensitivity of Ag-EIA, verification of the negative results by a DNA amplification method is needed. PMID:27525325

  13. Performance of the fourth-generation Bio-Rad GS HIV Combo Ag/Ab enzyme immunoassay for diagnosis of HIV infection in Southern Africa

    PubMed Central

    Piwowar-Manning, Estelle; Fogel, Jessica M.; Richardson, Paul; Wolf, Shauna; Clarke, William; Marzinke, Mark A.; Fiamma, Agnès; Donnell, Deborah; Kulich, Michal; Mbwambo, Jessie K.K.; Richter, Linda; Gray, Glenda; Sweat, Michael; Coates, Thomas J.; Eshleman, Susan H.

    2015-01-01

    Background Fourth-generation HIV assays detect both antigen and antibody, facilitating detection of acute/early HIV infection. The Bio-Rad GS HIV Combo Ag/Ab assay (Bio-Rad Combo) is an enzyme immunoassay that simultaneously detects HIV p24 antigen and antibodies to HIV-1 and HIV-2 in serum or plasma. Objective To evaluate the performance of the Bio-Rad Combo assay for detection of HIV infection in adults from Southern Africa. Study design Samples were obtained from adults in Soweto and Vulindlela, South Africa and Dar es Salaam, Tanzania (300 HIV-positive samples; 300 HIV-negative samples; 12 samples from individuals previously classified as having acute/early HIV infection). The samples were tested with the Bio-Rad Combo assay. Additional testing was performed to characterize the 12 acute/early samples. Results All 300 HIV-positive samples were reactive using the Bio-Rad Combo assay; false positive test results were obtained for 10 (3.3%) of the HIV-negative samples (sensitivity: 100%, 95% confidence interval [CI]: 98.8–100%); specificity: 96.7%, 95% CI: 94.0–98.4%). The assay detected 10 of the 12 infections classified as acute/early. The two infections that were not detected had viral loads < 400 copies/mL; one of those samples contained antiretroviral drugs consistent with antiretroviral therapy. Conclusions The Bio-Rad Combo assay correctly classified the majority of study specimens. The specificity reported here may be higher than that seen in other settings, since HIV-negative samples were pre-screened using a different fourth-generation test. The assay also had high sensitivity for detection of acute/early infection. False-negative test results may be obtained in individuals who are virally suppressed. PMID:25542477

  14. Validation of an enzyme immunoassay for assessing adrenocortical activity and evaluation of factors that affect levels of fecal glucocorticoid metabolites in two New World primates.

    PubMed

    Rimbach, Rebecca; Heymann, Eckhard W; Link, Andrés; Heistermann, Michael

    2013-09-15

    Non-invasive methods to assess stress hormone output via fecal glucocorticoid metabolites (FGCMs) have become a powerful tool in behavioral studies and conservation biology because they allow exploring the link between behavior, an animal's socio-ecological environment and its adrenocortical activity. However, FGCM levels are influenced by numerous other factors which often confound their interpretation. Thus, before applying these methods, knowledge on the impact of these factors is important. In this study we investigated the effect of (1) time of day, (2) age, (3) sex and (4) female reproductive state on FGCM levels in brown spider monkeys (Ateles hybridus) and red howler monkeys (Alouatta seniculus). Initially, we validated a 11β-hydroxyetiocholanolone enzyme immunoassay for monitoring the physiological stress response via fecal analysis in both species. We determined FGCM levels in fecal samples collected from two and six groups of wild spider monkeys (n=461 samples) and howler monkeys (n=166 samples), respectively. Our analyses revealed a strong effect of time of day on FGCM levels in spider monkeys, but no effect in howler monkeys. Adults of both species had significantly higher FGCM levels than subadults. In neither of the two species we found a sex-effect on FGCM output. Reproductive condition strongly affected FGCM levels in female spider monkeys which showed increasing concentrations with progressing gestation. This was not investigated in female howler monkeys due to an insufficient sample size. Our data indicate that the influence of the tested factors on fecal glucocorticoid metabolite output is species-specific, and that these variables need to be considered when interpreting FGCM levels in the species. PMID:23707497

  15. Optimization of blood collection card method/enzyme-linked immunoassay for monitoring exposure of bottlenose dolphin to brevetoxin-producing red tides.

    PubMed

    Maucher, Jennifer M; Briggs, Lyn; Podmore, Colleen; Ramsdell, John S

    2007-01-15

    Blood collection cards have been successfully used as a tool to monitor brevetoxin (PbTx) exposure in several species, including fish, mice, and rats. Previous methanolic methods used for extracting brevetoxin from blood collection cards have shown dolphin blood to have matrix difficulties in several biological assays. To better biomonitor protected marine mammal species in the Florida area, which is historically prone to unusual mortality events caused by brevetoxin exposure, we have modified the previous extraction method to consistently recover brevetoxin with a known efficiency from dolphin blood collection card samples with minimal matrix interference. A combination of phosphate-buffered saline (PBS) with 6% MeOH and 100% acetonitrile was used to elute blood from the cellulose card and precipitate proteins, respectively. Analysis was performed using a newly developed direct enzyme-linked immunoassay (ELISA), which yields a sample limit of quantification of 1 ng PbTx-3 equiv/mL. This extraction method allowed for linear recovery of PbTx-3 spiked into dolphin blood (1-30 ng/mL) with a consistent recovery rate of 58% and has subsequently been used to monitor brevetoxins in dolphins, as well as sea turtles and manatees, in regions endemic to red tides. In addition, two known metabolites of PbTx-2 were isolated and also found to be detectable using the ELISA. The cysteine conjugate (m/z 1018) and cysteine sulfoxide conjugate (m/z 1034) were found to have linear recoveries of 87% and 66%, respectively. In summary, this method of extracting brevetoxins and their metabolites from blood collection cards, in conjunction with the ELISA detection method, is a simple and reliable way to biomonitor physiologically relevant toxin levels in protected marine animals. PMID:17310722

  16. Survey of immunoassay techniques for biological analysis

    SciTech Connect

    Burtis, C.A.

    1986-10-01

    Immunoassay is a very specific, sensitive, and widely applicable analytical technique. Recent advances in genetic engineering have led to the development of monoclonal antibodies which further improves the specificity of immunoassays. Originally, radioisotopes were used to label the antigens and antibodies used in immunoassays. However, in the last decade, numerous types of immunoassays have been developed which utilize enzymes and fluorescent dyes as labels. Given the technical, safety, health, and disposal problems associated with using radioisotopes, immunoassays that utilize the enzyme and fluorescent labels are rapidly replacing those using radioisotope labels. These newer techniques are as sensitive, are easily automated, have stable reagents, and do not have a disposal problem. 6 refs., 1 fig., 2 tabs.

  17. Measurement of Chlamydia pneumoniae-Specific Immunoglobulin A (IgA) Antibodies by the Microimmunofluorescence (MIF) Method: Comparison of Seven Fluorescein-Labeled Anti-Human IgA Conjugates in an In-House MIF Test Using One Commercial MIF and One Enzyme Immunoassay Kit

    PubMed Central

    Paldanius, Mika; Bloigu, Aini; Leinonen, Maija; Saikku, Pekka

    2003-01-01

    For the serological diagnosis of acute Chlamydia pneumoniae infection, the microimmunofluorescence (MIF) test is the most commonly used method and also the “gold standard” for the measurement of immunoglobulin G (IgG) and IgM antibodies. The role of IgA antibodies in diagnosis has not been established. Commercially available fluorescein-labeled anti-human IgA conjugates have not been systematically compared to each other, and this situation may cause considerable variations in IgA results. Therefore, we tested 261 serum samples from 122 patients with pneumonia for IgA antibodies by using six α-chain-specific anti-IgA conjugates in our in-house MIF test, one commercial MIF test, and one enzyme immunoassay (EIA). Interfering IgG antibodies were removed with Gullsorb reagent before the measurement of IgA antibodies. Altogether, 14 significant IgA antibody increases in serum samples between the acute phase and the convalescent phase were detected by at least one of the conjugates in the MIF test, while no increases were found in the IgA EIA. Only one patient showed a significant IgA antibody increase with all of the fluorescein-labeled conjugates. Five significant titer changes were detected by at least two conjugates, and in nine instances, the titer increase was detected by one conjugate only. The titer agreement indicated by kappa coefficients was very good or good for all of the fluorescein-labeled conjugates and the EIA with low antibody titers but decreased with increasing titers. PMID:12522032

  18. Determination of plasma kisspeptin concentrations during reproductive cycle and different phases of pregnancy in crossbred cows using bovine specific enzyme immunoassay.

    PubMed

    Mondal, Mohan; Baruah, Kishore Kumar; Prakash, Bukkaraya Samudram

    2015-12-01

    Kisspeptin, a decapeptide and potent secretagogue of GnRH has been emerged recently as a master player in the regulation of reproduction in animals. Determination of kisspeptin in peripheral circulation is, therefore, very important for studying the control of its secretion and its role on reproduction in bovine species, the information on which is not available during any physiological state in this species, may probably be due to non-availability of simple assay procedure to measure the hormone. Therefore, the objective of this study was to develop and validate a simple and sufficiently sensitive enzyme immunoassay (EIA) for kisspeptin determination in bovine plasma using the biotin-streptavidin amplification system and second antibody coating technique. Biotin was coupled to kisspeptin and used to bridge between streptavidin-peroxidase and the immobilized kisspeptin antiserum in the competitive assay. The EIA was conducted directly in 100 μl of unknown bovine plasma. Kisspeptin standards ranging from 0.01 to 25.6 ng/100 μl/well were prepared in hormone-free plasma. The lowest detection limit was 0.1 ng/ml plasma. Plasma volumes for the EIA, viz., 50, 100 and 200 μl did not influence the shape of standard curve even though a drop in OD450 was seen with higher plasma volumes. A parallelism test was carried out to compare the endogenous bovine kisspeptin with kisspeptin standard used. It showed good parallelism with the kisspeptin standard curve. For the biological validation of the assay, plasma kisspeptin was measured in blood samples collected from six non-lactating cyclic cows during entire estrous cycle and from 18 pregnant cows during different stages of pregnancy. The mean plasma kisspeptin concentration during different days of the estrous cycle was different (P<0.001). Three peaks of kisspeptin were recorded, one on a day before appearance of preovulatory LH surge, second at day 6 and third one at day 18 of the estrous cycle. Plasma kisspeptin

  19. Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera.

    PubMed

    Thraenhart, O; Ramakrishnan, K

    1989-10-01

    A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high

  20. Quantification of 5-methylcytosine, 5-hydroxymethylcytosine and 5-carboxylcytosine from the blood of cancer patients by an Enzyme-based Immunoassay

    PubMed Central

    Chowdhury, Basudev; Cho, Il-Hoon; Hahn, Noah; Irudayaraj, Joseph

    2014-01-01

    Background Genome-wide aberrations of the classic epigenetic modification 5-methylcytosine (5mC), considered the hallmark of gene silencing, has been implicated to play a pivotal role in mediating carcinogenic transformation of healthy cells. Recently, three epigenetic marks derived from enzymatic oxidization of 5mC namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), have been discovered in the mammalian genome. Growing evidence suggests that these novel bases possess unique regulatory functions and may play critical roles in carcinogenesis. Methods To provide a quantitative basis for these rare epigenetic marks, we have designed a biotin-avidin mediated Enzyme-based Immunoassay (EIA) and evaluated its performance in genomic DNA isolated from blood of patients diagnosed with metastatic forms of lung, pancreatic and bladder cancer, as well as healthy controls. The proposed EIA incorporates spatially optimized biotinylated antibody and a high degree of horseradish-peroxidase (HRP) labeled streptavidin, facilitating signal amplification and sensitive detection. Results We report that the percentages of 5mC, 5hmC and 5caC present in the genomic DNA of blood in healthy controls as 1.025 + 0.081, 0.023 + 0.006 and 0.001 + 0.0002 respectively. We observed a significant (p<0.05) decrease in the mean global percentage of 5hmC in blood of patients with malignant lung cancer (0.013 + 0.003 %) in comparison to healthy controls. Conclusion The precise biological roles of these epigenetic modifications in cancers are still unknown but in the past two years it has become evident that the global 5hmC content is drastically reduced in a variety of cancers. To the best of our knowledge, this is the first report of decreased 5hmC content in the blood of metastatic lung cancer patients and the clinical utility of this observation needs to be further validated in larger sample datasets. PMID:25441900

  1. Suboptimal Detection of Influenza Virus in Adults by the Directigen Flu A+B Enzyme Immunoassay and Correlation of Results with the Number of Antigen-Positive Cells Detected by Cytospin Immunofluorescence

    PubMed Central

    Landry, Marie L.; Ferguson, David

    2003-01-01

    To provide 24-h influenza diagnosis for adults presenting to the emergency department, the Directigen Flu A+B enzyme immunoassay (EIA) was performed in the chemistry laboratory during the night shift. Nasopharyngeal swabs were retested by cytospin-enhanced direct immunofluorescence (DFA; SimulFluor respiratory screen) when the virology laboratory opened. The influenza EIA detected 16 influenza A virus infections, whereas cytospin-enhanced DFA detected 31 influenza A virus infections as well as 3 respiratory syncytial virus, 2 adenovirus, and 1 parainfluenza virus infections. A positive EIA result usually correlated with 50 or more influenza virus cells positive by DFA. PMID:12843105

  2. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans.

    PubMed

    Galula, Jedhan U; Chang, Gwong-Jen J; Chuang, Shih-Te; Chao, Day-Yu

    2016-02-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great

  3. Establishment of an Algorithm Using prM/E- and NS1-Specific IgM Antibody-Capture Enzyme-Linked Immunosorbent Assays in Diagnosis of Japanese Encephalitis Virus and West Nile Virus Infections in Humans

    PubMed Central

    Galula, Jedhan U.; Chang, Gwong-Jen J.

    2015-01-01

    The front-line assay for the presumptive serodiagnosis of acute Japanese encephalitis virus (JEV) and West Nile virus (WNV) infections is the premembrane/envelope (prM/E)-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA). Due to antibody cross-reactivity, MAC-ELISA-positive samples may be confirmed with a time-consuming plaque reduction neutralization test (PRNT). In the present study, we applied a previously developed anti-nonstructural protein 1 (NS1)-specific MAC-ELISA (NS1-MAC-ELISA) on archived acute-phase serum specimens from patients with confirmed JEV and WNV infections and compared the results with prM/E containing virus-like particle-specific MAC-ELISA (VLP-MAC-ELISA). Paired-receiver operating characteristic (ROC) curve analyses revealed no statistical differences in the overall assay performances of the VLP- and NS1-MAC-ELISAs. The two methods had high sensitivities of 100% but slightly lower specificities that ranged between 80% and 100%. When the NS1-MAC-ELISA was used to confirm positive results in the VLP-MAC-ELISA, the specificity of serodiagnosis, especially for JEV infection, was increased to 90% when applied in areas where JEV cocirculates with WNV, or to 100% when applied in areas that were endemic for JEV. The results also showed that using multiple antigens could resolve the cross-reactivity in the assays. Significantly higher positive-to-negative (P/N) values were consistently obtained with the homologous antigens than those with the heterologous antigens. JEV or WNV was reliably identified as the currently infecting flavivirus by a higher ratio of JEV-to-WNV P/N values or vice versa. In summary of the above-described results, the diagnostic algorithm combining the use of multiantigen VLP- and NS1-MAC-ELISAs was developed and can be practically applied to obtain a more specific and reliable result for the serodiagnosis of JEV and WNV infections without the need for PRNT. The developed algorithm should provide great

  4. Target-induced nano-enzyme reactor mediated hole-trapping for high-throughput immunoassay based on a split-type photoelectrochemical detection strategy.

    PubMed

    Zhuang, Junyang; Tang, Dianyong; Lai, Wenqiang; Xu, Mingdi; Tang, Dianping

    2015-09-15

    Photoelectrochemical (PEC) detection is an emerging and promising analytical tool. However, its actual application still faces some challenges like potential damage of biomolecules (caused by itself system) and intrinsic low-throughput detection. To solve the problems, herein we design a novel split-type photoelectrochemical immunoassay (STPIA) for ultrasensitive detection of prostate specific antigen (PSA). Initially, the immunoreaction was performed on a microplate using a secondary antibody/primer-circular DNA-labeled gold nanoparticle as the detection tag. Then, numerously repeated oligonucleotide sequences with many biotin moieties were in situ synthesized on the nanogold tag via RCA reaction. The formed biotin concatamers acted as a powerful scaffold to bind with avidin-alkaline phosphatase (ALP) conjugates and construct a nanoenzyme reactor. By this means, enzymatic hydrolysate (ascorbic acid) was generated to capture the photogenerated holes in the CdS quantum dot-sensitized TiO2 nanotube arrays, resulting in amplification of the photocurrent signal. To elaborate, the microplate-based immunoassay and the high-throughput detection system, a semiautomatic detection cell (installed with a three-electrode system), was employed. Under optimal conditions, the photocurrent increased with the increasing PSA concentration in a dynamic working range from 0.001 to 3 ng mL(-1), with a low detection limit (LOD) of 0.32 pg mL(-1). Meanwhile, the developed split-type photoelectrochemical immunoassay exhibited high specificity and acceptable accuracy for analysis of human serum specimens in comparison with referenced electrochemiluminescence immunoassay method. Importantly, the system was not only suitable for the sandwich-type immunoassay mode, but also utilized for the detection of small molecules (e.g., aflatoxin B1) with a competitive-type assay format. PMID:26291091

  5. Bioelectrochemical Immunoassay of Polychlorinated Biphenyl

    SciTech Connect

    Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

    2008-04-01

    A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP-PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP-PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP-PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 5 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring.

  6. Evaluation of Enzyme Immunoassays and Real-Time PCR for Detecting Shiga Toxin-Producing Escherichia coli in Southern Alberta, Canada

    PubMed Central

    Patterson-Fortin, Laura; Kuo, Julie; Li, Vincent; Boras, Valerie

    2015-01-01

    Two immunoassays (Shiga Toxin Chek and Shiga Toxin Quik Chek) and real-time PCR were used to detect Shiga toxin-producing Escherichia coli. For enriched culture, the sensitivity and specificity of the three methods ranged from 80.0% to 98.2% and 98.0% to 100.0%, respectively. STEC isolates were identified in 2.6% of the 784 samples. PMID:25588656

  7. Triclosan and methyl-triclosan monitoring study in the northeast of Spain using a magnetic particle enzyme immunoassay and confirmatory analysis by gas chromatography mass spectrometry

    NASA Astrophysics Data System (ADS)

    Kantiani, Lina; Farré, Marinella; Asperger, Danijela; Rubio, Fernando; González, Susana; López de Alda, Maria J.; Petrović, Mira; Shelver, Weilin L.; Barceló, Damià

    2008-10-01

    SummaryFor the first time, the occurrence of triclosan and its metabolite methyl-triclosan was investigated in a typical Mediterranean area using a two-step methodology based on screening using a magnetic particle immunoassay (IA) and confirmatory analysis by solid phase extraction (SPE) followed by gas chromatography-mass spectrometry (GC-MS). In this study, 95 environmental samples were analyzed. A commercial immunoassay was assessed for use in the different types of water selected for this study. A large monitoring study was performed on the influent and the effluent of eight wastewater treatment plants (WWTPs), water samples from Ebro and Llobregat rivers, and drinking water. All wastewater samples tested in this study (influents and effluents) showed the presence of triclosan, with concentrations for raw influents being high (10 μg/L as average value). The percentages of triclosan removal for the WWTPs were evaluated (30-70%) along the different treatment processes showing that the best removal rates were obtained by the processes equipped with membrane bioreactors (MBRs). However, important concentrations of triclosan were detected even after treatment by MBRs. The presence of this biocide was confirmed in 50% of the river samples analyzed. Twenty two drinking water samples from the Barcelona city area were investigated, and in this case no triclosan was detected. Due to its properties and the widespread usage of triclosan, there is a need for monitoring and controlling the amounts present in wastewater effluents, river water, drinking water catchments areas, and drinking water. To this end, we present a feasible methodology using a magnetic particle-based immunoassay as a screening, followed by confirmatory analysis using solid phase extraction-gas chromatography-mass spectrometry (SPE-GC-MS).

  8. Colorimetric Immunoassay for Detection of Tumor Markers

    PubMed Central

    Yin, Yongmei; Cao, Ya; Xu, Yuanyuan; Li, Genxi

    2010-01-01

    Tumor markers are substances, usually proteins, produced by the body in response to cancer growth, or by the cancer tissue itself. They can be detected in blood, urine, or tissue samples, and the discovery and detection of tumor markers may provide earlier diagnosis of cancer and improved therapeutic intervention. Colorimetric immunoassays for tumor marker detection have attracted considerable attention, due to their simplicity and high efficiency. The traditionally used colorimetric immunoassays for the detection of tumor markers are based on enzyme-linked immunosorbent assays, and the great achievement of nanotechnology has further opened opportunities for the development of such kind of immunoassays. This paper will summarize recent advances in the field of colorimetric immunoassays for detecting tumor markers, which is aimed to provide an overview in this field, as well as experimental guidance for the learner. PMID:21614193

  9. Identification of a Novel Host-Specific IgM Protease in Streptococcus suis

    PubMed Central

    Seele, Jana; Singpiel, Alena; Spoerry, Christian; von Pawel-Rammingen, Ulrich; Valentin-Weigand, Peter

    2013-01-01

    Streptococcus suis serotype 2 is a highly invasive, extracellular pathogen in pigs with the capacity to cause severe infections in humans. This study was initiated by the finding that IgM degradation products are released after opsonization of S. suis. The objective of this work was to identify the bacterial factor responsible for IgM degradation. The results of this study showed that a member of the IdeS family, designated IdeSsuis (Immunoglobulin M-degrading enzyme of S. suis), is responsible and sufficient for IgM cleavage. Recombinant IdeSsuis was found to degrade only IgM but neither IgG nor IgA. Interestingly, Western blot analysis revealed that IdeSsuis is host specific, as it exclusively cleaves porcine IgM but not IgM from six other species, including a closely related member of the Suidae family. As demonstrated by flow cytometry and immunofluorescence microscopy, IdeSsuis modulates binding of IgM to the bacterial surface. IdeSsuis is the first prokaryotic IgM-specific protease described, indicating that this enzyme is involved in a so-far-unknown mechanism of host-pathogen interaction at an early stage of the host immune response. Furthermore, cleavage of porcine IgM by IdeSsuis is the first identified phenotype reflecting functional adaptation of S. suis to pigs as the main host. PMID:23243300

  10. Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    PubMed Central

    Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Hörkkö, Sohvi

    2012-01-01

    Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875

  11. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  12. The differences in short- and long-term varicella-zoster virus (VZV) immunoglobulin G levels following varicella vaccination of healthcare workers measured by VZV fluorescent-antibody-to-membrane-antigen assay (FAMA), VZV time-resolved fluorescence immunoassay and a VZV purified glycoprotein enzyme immunoassay.

    PubMed

    Maple, P A C; Haedicke, J; Quinlivan, M; Steinberg, S P; Gershon, A A; Brown, K E; Breuer, J

    2016-08-01

    Healthcare workers (HCWs) reporting no history of varicella frequently receive varicella vaccination (vOka) if they test varicella-zoster virus (VZV) immunoglobulin G (IgG) negative. In this study, the utilities of VZV-IgG time-resolved fluorescence immunoassay (VZV-TRFIA) and a commercial VZV-IgG purified glycoprotein enzyme immunoassay (gpEIA) currently used in England for confirming VZV immunity have been compared to the fluorescent-antibody-to-membrane-antigen assay (FAMA). A total of 110 HCWs received two doses of vOka vaccine spaced 6 weeks apart and sera collected pre-vaccination (n = 100), at 6 weeks post-completion of vaccination (n = 86) and at 12-18 months follow-up (n = 73) were analysed. Pre-vaccination, by FAMA, 61·0% sera were VZV IgG negative, and compared to FAMA the sensitivities of VZV-TRFIA and gpEIA were 74·4% [95% confidence interval (CI) 57·9-87·0] and 46·2% (95% CI 30·1-62·8), respectively. Post-completion of vaccination the seroconversion rate by FAMA was 93·7% compared to rates of 95·8% and 70·8% determined by VZV-TRFIA and gpEIA, respectively. At 12-18 months follow-up seropositivity rates by FAMA, VZV-TRFIA and gpEIA were 78·1%, 74·0% and 47·9%, respectively. Compared to FAMA the sensitivities of VZV-TRFIA and gpEIA for measuring VZV IgG following vaccination were 96·4% (95% CI 91·7-98·8) and 74·6% (95% CI 66·5-81·6), respectively. Using both FAMA and VZV-TRFIA to identify healthy adult VZV susceptibles and measure seroconversion showed that vOka vaccination of HCWs is highly immunogenic. PMID:27018820

  13. Evaluation of a commercial enzyme immunoassay kit (RIDASCREEN) for detection of staphylococcal enterotoxins A, B, C, D, and E in foods.

    PubMed Central

    Park, C E; Akhtar, M; Rayman, M K

    1994-01-01

    The RIDASCREEN SET kit (R-Biopharm GmbH, Darmstadt, Germany), a commercial staphylococcal enterotoxin (SE) visual immunoassay kit, was evaluated for its efficacy. The kit utilizes monovalent capture antibodies against SE types A to E (SEA to SEE); therefore, it simultaneously detects and identifies the enterotoxin types. The major advantages of the kit are (i) a high degree of specificity (except for naturally occurring peroxidases, food compositions or ingredients and microbiological products due to growth of nonstaphylococcal microorganisms did not cause false-positive results; additionally, no cross-reactions among reagents of the kits were observed), (ii) excellent sensitivity (minimum detectable limits were 0.20 to 0.30 ng of SEs per ml of extracts of ham, salami, and mushroom and 0.30 to 0.35 ng of SEs per ml of cheese extracts, or 0.50 to 0.75 ng of SEs per g of foods such as noodles, ham, salami, cheese, and turkey), (iii) simplicity (the kit enabled direct assay of SEs in food extracts without the need for lengthy extraction or concentration procedures), (iv) rapidity (it took less than 3 h to complete the analysis of individual enterotoxin types SEA to SEE), and (v) its semiquantitative results (optical density values could be read against a standard curve to estimate the amount of SE in the extract). The RIDASCREEN kit is a convenient, rapid, and reliable tool for the detection and identification of SEs in foods. PMID:8135522

  14. IgM, IgG and IgA rheumatoid factors and circulating immune complexes in patients with AIDS and AIDS-related complex with serological abnormalities.

    PubMed Central

    Procaccia, S; Lazzarin, A; Colucci, A; Gasparini, A; Forcellini, P; Lanzanova, D; Foppa, C U; Novati, R; Zanussi, C

    1987-01-01

    To investigate some humoral aspects which may reflect the involvement of B lymphocytes in the acquired immunodeficiency syndrome (AIDS), we used an enzyme-linked immunoassay (ELISA) to determine the levels of IgM, IgG and IgA rheumatoid factors (RF) in 16 patients suffering from full-blown AIDS and 32 patients with AIDS-related complex (ARC), in the clinical form of lymphoadenopathy syndrome (LAS), compared with 40 healthy, young heterosexual subjects. Both AIDS and ARC patients showed a greater incidence of high IgM RF levels, with mean values significantly higher than controls, but with no differences between the two pathological groups. IgG RF behaviour was similar in the two patient populations and the healthy subjects. IgA RF were significantly raised in AIDS and ARC. Further information on RF was obtained by determination of the immunoglobulin levels of the respective isotypes in the same patients. Mean IgG levels were above normal in AIDS and ARC patients, but the latter group showed a higher incidence of increased values and higher mean levels. The IgA isotype was significantly increased mainly in AIDS patients. The behaviour of IgM was virtually the same in the three groups studied. A difference between AIDS and ARC patients was established by the detection of circulating immune-complexes (IC) by the C1q-binding and CIC-conglutinin assays. IC were significantly high, by both methods, only in the ARC group, but normal or very low in AIDS. These overall findings suggest once again the impairment of B cell function in AIDS, with prevalent hyperactivation in ARC and exhaustion in full-blown AIDS, and apparent preservation, in the latter group, of the antibody responses which are more closely related to the activity of subsets of T helper cells. PMID:3608224

  15. Immunoassays in Biotechnology

    EPA Science Inventory

    Immunoassays have broad applications for a wide variety of important biological compounds and environmental contaminants. Immunoassays can detect the presence of an antigen in the human body, a pollutant in the environment, or a critical antibody in a patient’s serum to develop a...

  16. Increased concentration of two different advanced glycation end-products detected by enzyme immunoassays with new monoclonal antibodies in sera of patients with rheumatoid arthritis

    PubMed Central

    2010-01-01

    Background Levels of pentosidine (representative of advanced glycation end-products) in sera of patients with rheumatoid arthritis are increased when compared with sera of other diagnoses or healthy controls. These levels have been reported to correlate with clinical indices of rheumatoid arthritis activity and with laboratory markers of inflammation. The purpose of this study was to find out if these findings pertain to other advanced glycation end-products. Methods We have developed two immunoassays based on new monoclonal antibodies to advanced glycation end-products. Antibody 103-E3 reacts with an unidentified antigen, formed in the reaction of proteins with ribose, while antibody 8-C1 responds to Nε-(carboxyethyl)lysine. We have used these monoclonal antibodies to measure levels of advanced glycation end-products in sera of patients with rheumatoid arthritis, systemic lupus erythematosus, osteoarthritis, and healthy controls. We calculated the correlations between advanced glycation end-product levels in rheumatoid arthritis sera and the Disease Activity Score 28 (DAS28), age, disease duration, CRP, anti-CCP, rheumatoid factor and treatment with corticosteroids, respectively. Results Levels of both glycation products were significantly higher in sera of patients with rheumatoid arthritis when compared with sera of patients with systemic lupus erythematosus, osteoarthritis, or the healthy controls. Neither the level of Nε-(carboxyethyl)lysine nor the level of the 103-E3 antigen in rheumatoid arthritis sera correlated with the DAS28-scored rheumatoid arthritis activity. The levels of both antigens in rheumatoid arthritis sera did not correlate with age, gender, corticosteroid treatment, or levels of CRP, anti-CCP antibodies, and rheumatoid factor in sera. Conclusions We report highly specific increases in the levels of two advanced glycation end-products in sera of patients with rheumatoid arthritis. This increase could be explained neither by rheumatoid

  17. Kinase Activity Studied in Living Cells Using an Immunoassay

    ERIC Educational Resources Information Center

    Bavec, Aljos?a

    2014-01-01

    This laboratory exercise demonstrates the use of an immunoassay for studying kinase enzyme activity in living cells. The advantage over the classical method, in which students have to isolate the enzyme from cell material and measure its activity in vitro, is that enzyme activity is modulated and measured in living cells, providing a more…

  18. Microfluidic paper-based analytical device for photoelectrochemical immunoassay with multiplex signal amplification using multibranched hybridization chain reaction and PdAu enzyme mimetics.

    PubMed

    Lan, Feifei; Sun, Guoqiang; Liang, Linlin; Ge, Shenguang; Yan, Mei; Yu, Jinghua

    2016-05-15

    promising platform of clinical immunoassay for other biomolecules. PMID:26735876

  19. Detection of Semliki Forest virus in cell culture by use of an enzyme immunoassay with peroxidase-labeled monoclonal antibodies specific for glycoproteins E1 and E2.

    PubMed Central

    van Tiel, F H; Boere, W A; Vinjé, J; Harmsen, T; Benaissa-Trouw, B J; Kraaijeveld, C A; Snippe, H

    1984-01-01

    Four noncompeting monoclonal antibodies (MA) directed against either the E1 (UM 8.64 and 8.139) or E2 (UM 8.55 and 8.73) glycoprotein of Semliki Forest virus were purified and labeled with horseradish peroxidase. Each enzyme-labeled MA was tested alone and in combination with others for its sensitivity to detect virus-infected cells. Semliki Forest virus-infected L cells seeded as monolayers in 96-well plates were screened for the virus after incubation with enzyme-labeled MA and a substrate. In this system single enzyme-labeled MA even at high dilution (10(3.0) to 10(4.5] were able to detect virus-infected cells. The sensitivity of the test could be enhanced by combining two noncompeting MA (10(4.5) to 10(5.0]. Combinations of three and four MA were less effective, due to high absorbance values for noninfected cells. The threshold of virus defection was between 10(5) and 10(6) PFU/ml. This test is sensitive and specific and therefore may be useful for diagnostic purposes. PMID:6386855

  20. Clostridium difficile testing algorithms using glutamate dehydrogenase antigen and C. difficile toxin enzyme immunoassays with C. difficile nucleic acid amplification testing increase diagnostic yield in a tertiary pediatric population.

    PubMed

    Ota, Kaede V; McGowan, Karin L

    2012-04-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens. PMID:22259201

  1. Comparison of Dissociation-Enhanced Lanthanide Fluorescent Immunoassays to Enzyme-Linked Immunosorbent Assays for Detection of Staphylococcal Enterotoxin B, Yersinia pestis-Specific F1 Antigen, and Venezuelan Equine Encephalitis Virus

    PubMed Central

    Smith, Darci R.; Rossi, Cynthia A.; Kijek, Todd M.; Henchal, Erik A.; Ludwig, George V.

    2001-01-01

    The dissociation-enhanced lanthanide fluorescent immunoassays (DELFIA) were developed for the detection of staphylococcal enterotoxin B, Yersinia pestis-specific F1 antigen, and Venezuelan equine encephalitis virus. These assays were compared to previously developed enzyme-linked immunosorbent assays (ELISAs) by determining the sensitivity or limit of detection (LOD), the dynamic range, and the reproducibility of each assay in a number of different sample matrices. The sensitivity and specificity of each assay were then determined by using a small panel of blinded spiked and nonspiked samples. All three DELFIAs demonstrated at least 1 log greater sensitivity than corresponding ELISAs utilizing the same reagents and showed an increase in dynamic range of at least 2 log10 concentrations. This increased LOD resulted in higher sensitivity rates for the DELFIA. The specificity of all of the assays evaluated was 100%, and no sample matrix effects were observed in either format. However, the reproducibility of the DELFIA was poor due to randomly distributed wells exhibiting excessive background signal (hot wells), which occurred throughout the evaluation. As this technology matures, the reproducibility of these assays should improve, as will the ability to identify hot wells. Despite its sensitivity, the logistical burden associated with the DELFIA and the technical expertise required to complete assays and interpret the data limit the application of this technology to reference or large clinical laboratories. PMID:11687442

  2. Clostridium difficile Testing Algorithms Using Glutamate Dehydrogenase Antigen and C. difficile Toxin Enzyme Immunoassays with C. difficile Nucleic Acid Amplification Testing Increase Diagnostic Yield in a Tertiary Pediatric Population

    PubMed Central

    McGowan, Karin L.

    2012-01-01

    We evaluated the performance of the rapid C. diff Quik Chek Complete's glutamate dehydrogenase antigen (GDH) and toxin A/B (CDT) tests in two algorithmic approaches for a tertiary pediatric population: algorithm 1 entailed initial testing with GDH/CDT followed by loop-mediated isothermal amplification (LAMP), and algorithm 2 entailed GDH/CDT followed by cytotoxicity neutralization assay (CCNA) for adjudication of discrepant GDH-positive/CDT-negative results. A true positive (TP) was defined as positivity by CCNA or positivity by LAMP plus another test (GDH, CDT, or the Premier C. difficile toxin A and B enzyme immunoassay [P-EIA]). A total of 141 specimens from 141 patients yielded 27 TPs and 19% prevalence. Sensitivity, specificity, positive predictive value, and negative predictive value were 56%, 100%, 100%, and 90% for P-EIA and 81%, 100%, 100%, and 96% for both algorithm 1 and algorithm 2. In summary, GDH-based algorithms detected C. difficile infections with superior sensitivity compared to P-EIA. The algorithms allowed immediate reporting of half of all TPs, but LAMP or CCNA was required to confirm the presence or absence of toxigenic C. difficile in GDH-positive/CDT-negative specimens. PMID:22259201

  3. Electrochemical immunoassay of benzo[a]pyrene based on dual amplification strategy of electron-accelerated Fe3O4/polyaniline platform and multi-enzyme-functionalized carbon sphere label.

    PubMed

    Lin, Mouhong; Liu, Yingju; Sun, Zihong; Zhang, Shenglai; Yang, Zhuohong; Ni, Chunlin

    2012-04-13

    An electrochemical immunosensor, basing on a dual amplification strategy by employing a biocompatible Fe(3)O(4)/polyaniline/Nafion (Fe(3)O(4)/PANI/Nafion) layer as sensor platform and multi-enzyme-antibody functionalized highly-carbonized spheres (multi-HRP-HCS-Ab(2)) as label, was constructed for sensitive detection of benzo[a]pyrene (BaP). The stable film, Fe(3)O(4)/PANI/Nafion, can not only immobilize biomolecules, but also catalyze the reduction of hydrogen peroxide, indicating an accelerated electron transfer pathway of the platform. The experimental conditions, including the concentration of Nafion, concentration of Fe(3)O(4)/polyaniline (Fe(3)O(4)/PANI), pH of the detection solution and concentrations of biomolecules, were studied in detail. Basing on a competitive immunoassay, the current change was proportional to the logarithm of BaP concentration in the range of 8 pM and 2 nM with the detection limit of 4 pM. The proposed immunosensor exhibited acceptable reproducibility and stability. This new type of dual amplification strategy may provide potential applications for the detection of environmental pollutants. PMID:22444540

  4. Evaluation of third-generation RIDASCREEN enzyme immunoassay for the detection of norovirus antigens in stool samples of hospitalized children in Belém, Pará, Brazil.

    PubMed

    Siqueira, Jones Anderson Monteiro; Linhares, Alexandre da Costa; Oliveira, Darleise de Souza; Soares, Luana da Silva; Lucena, Maria Silvia Sousa; Wanzeller, Ana Lúcia Monteiro; Mascarenhas, Joana D'Arc Pereira; Gabbay, Yvone Benchimol

    2011-12-01

    Noroviruses (NoVs) are major agents of gastroenteritis outbreaks and hospitalization worldwide. This study evaluated the sensitivity and specificity of the commercially available third-generation RIDASCREEN® Norovirus Enzyme Immunoassay (EIA) kit in comparison to the reverse transcription-polymerase chain reaction (RT-PCR) to detect NoVs in hospitalized children with gastroenteritis. An agreement of 88% (81/92) was observed when comparing EIA with RT-PCR. A sensitivity of 92% and a specificity of 83.3% were demonstrated. Eleven samples were positive by 1 method only (4 RT-PCR/7 EIA). Fourteen samples were sequenced and all classified as NoV genogroup GII-4. The 7 positive only by EIA were also evaluated by electron microscopy, and in 3 (42.9%) samples viral particles with a suggestive morphology of NoVs were visualized. These same samples were tested by seminested-RT-PCR with a positivity of 85.7%. The results obtained in this study demonstrated a significant improvement in the sensitivity and specificity of this updated assay. PMID:22001621

  5. Updates in immunoassays: parasitology.

    PubMed

    Josko, Deborah

    2012-01-01

    Although most clinical laboratories use microscopy and routine O&P procedures when identifying parasitic infections, there are several parasites that are better detected through serological means. Toxoplasma, Giardia, and Cryptosporidium were discussed along with immunoassays used for their detection. Immunoassays provide quick results and are less labor intensive than specimen concentration and slide preparation for microscopic examination. These assays are easy to use and provide sensitive and specific results. Some clinical laboratories no longer perform O&Ps in house and refer specimens to reference laboratories for evaluation. By using immunoassays, some of the more common parasites can be identified in a timely manner reducing turn-around times. Some controversy exists over the use of IIF and EIA tests used for ANA testing along with measuring CRPs and PCT as predictors of bacterial sepsis and septic shock. Regardless of the methodology discussed in this series of articles, there are pros and cons to the various immunoassays available. Determining the most appropriate assay based on patient population and volume is governed by the institution and its patients' needs. In conclusion, immunoassays, whether manual or automated, are easy to use, cost effective and allow the medical laboratory professional to provide quick and accurate results to the clinician so the most appropriate treatment can be administered to the patient. The ultimate goal of healthcare professionals is to provide the highest quality of medical care in a timely manner. The use of immunoassays in the clinical laboratory allows the healthcare team to successfully achieve this goal. PMID:22953520

  6. High-voltage isoelectric focusing in ultrathin gels and enzyme-amplified immunoassay: a new method for analysis of cerebrospinal fluid proteins.

    PubMed

    Kjellin, K G; Hallander, L B

    1982-01-01

    A procedure using high-voltage isoelectric focusing (IF) in ultrathin (02. mm) gels and enzyme-amplified immuno-sandwich assay was elaborated to get optimal IF separation conditions, to avoid CSF concentration, e.g. by ultrafiltration preceding IF with the risk of unequal protein losses, to minimize the amounts of CSF and expensive reagents needed, especially antibodies and to shorten the analysis time, including the selective detection of proteins. The high voltage (2000-3000 V/10 cm) and efficient cooling during IF were obtained using ECPS 3000/150 and FBE 3000 (Pharmacia, Sweden). Ampholytes (Pharmalytes) of different pI intervals were used. The CSF and (diluted) serum samples were microdialysed in polyacrylamide gel before IF to minimize band curvature and to obtain optimal resolution. The IF separation was performed in about 1 h. Owing to the rapid fixation of ultrathin gels after IF, full use could be made of the high-voltage resolving capacity. The thin gels also made histochemical techniques applicable. Different immunological identification assays have been tested. An enzyme-amplified (alkaline phosphatase) immuno-sandwich method was found to be very sensitive and selective, and has so far given the best results. Many proteins in the same sample, applied as a line on the gel before IF, could be detected by overlaying antibody-soaked membrane strips. Furthermore, one specific protein could be examined in many samples simultaneously by overlaying or immersion of diluted antibody solutions. A few microlitres of unconcentrated CSF and diluted serum were used for the analysis performed within 1 day. The findings for albumin, transferrin and IgG in CSF and sera from patients with different neurological diseases, especially including cases with "normal" CSF, barrier damage, degenerative and demyelinating disorders, have been compared with the corresponding protein-stained (Coomassie R-250) patterns where the CSF had been concentrated by a special vacuum

  7. Ultrasensitive enzyme-free electrochemical immunoassay for free thyroxine based on three dimensionally ordered macroporous chitosan-Au nanoparticles hybrid film.

    PubMed

    Zhang, Qi; Chen, Xiaojun; Tu, Fulai; Yao, Cheng

    2014-09-15

    The measurement of free thyroxine concentration in serum is considered to be an essential indicator of thyroid function. Here, a novel enzyme-free sandwich electrochemical immunosensor for the detection of FT4 antigen based on the immobilization of primary antibody (Ab1) on three dimensional ordered macroporous chitosan-Au nanoparticles hybrid (3DOM CS-AuNPs) film electrode, and magnetic multiwall carbon nanotubes (MMWCNTs) were used as label of secondary antibody (Ab2). The 3DOM CS-AuNPs film electrode was constructed by one-step electrodeposition of CS-AuNPs composite onto Au electrode with silica opal template. MMWCNTs were prepared by chemical co-precipitation of Fe(2+) and Fe(3+) salts on carboxylated MWCNTs. Ru(bpy)3(2+) labeled anti-FT4 (Ru(bpy)3(2+)-Ab2) was covalently attached to MMWCNTs through the formation of amide bond between the carboxylic groups of MWCNTs and the amine groups of antibody. Under the optimal conditions, FT4 was detected in a concentration range from 0.71 fg mL(-1) to 1.15 pg mL(-1) with a correlation coefficient of 0.998 and a detection limit of 0.20 fg mL(-1). Moreover, the immunosensor showed excellent selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from the commercialized electrochemiluminescent method. PMID:24752149

  8. Enzyme immunoassay and proteomic characterization of troponin I as a marker of mammalian muscle compounds in raw meat and some meat products.

    PubMed

    Zvereva, Elena A; Kovalev, Leonid I; Ivanov, Alexei V; Kovaleva, Marina A; Zherdev, Anatoly V; Shishkin, Sergey S; Lisitsyn, Andrey B; Chernukha, Irina M; Dzantiev, Boris B

    2015-07-01

    The skeletal muscle protein troponin I (TnI) has been characterized as a potential thermally stable and species-specific biomarker of mammalian muscle tissues in raw meat and meat products. This study proposed a technique for the quantification of TnI comprising protein extraction and sandwich enzyme-linked immunosorbent assay (ELISA). The technique is characterized by a TnI detection limit of 4.8 ng/ml with quantifiable concentrations ranging from 8.7 to 52 ng/ml. The method was shown to be suitable for detection of TnI in mammalian (beef, pork, lamb, and horse) meat but not in poultry (chicken, turkey, and duck) meat. In particular, the TnI content in beef was 0.40 3 ± 0.058 mg/g of wet tissue. The TnI estimations obtained for the pork and beef samples using ELISA were comparable to the proteomic analysis results. Thus, the quantitative study of TnI can be a convenient way to assess the mammalian muscle tissue content of various meat products. PMID:25777979

  9. Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.

    PubMed Central

    Merkelbach, S.; Gehlen, J.; Handt, S.; Füzesi, L.

    1997-01-01

    Four different DNA extraction methods were compared to determine their ability to provide DNA for amplification of viral sequences from paraffin-embedded human tissue samples by polymerase chain reaction (PCR). The suitability of extraction methods was assessed using parameters like DNA yield, length of recovered DNA fragments, and duration. Furthermore, the efficiency of amplifying a human single-copy gene, the beta-globin gene, from DNA samples was tested. The best preservation of DNA molecules could be achieved by binding the DNA onto a silica column before further purification. Viral DNA sequences could be amplified by PCR in DNA extracted from routinely processed paraffin blocks from cases with clinically or morphologically suspected cytomegalovirus or Epstein-Barr virus infections. The PCR products were specified by a novel liquid hybridization assay called PCR-enzyme-linked immunosorbent assay. Using this assay, the time-consuming Southern hybridization could be replaced and the time requirement for the detection of PCR products could be reduced from 1 day to 4 hours. The assay system described here represents a reliable, sensitive, and specific method for the detection of viral DNA from paraffin-embedded tissue samples. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:9137080

  10. Validity and Reliability of Enzyme Immunoassays Using Leishmania major or L. infantum Antigens for the Diagnosis of Canine Visceral Leishmaniasis in Brazil

    PubMed Central

    de Arruda, Mauro Maciel; Figueiredo, Fabiano Borges; Cardoso, Fernanda Alvarenga; Hiamamoto, Roberto Mitsuyoshi; Brazuna, Júlia Cristina Macksoud; de Oliveira, Maria Regina Fernandes; Noronha, Elza Ferreira; Romero, Gustavo Adolfo Sierra

    2013-01-01

    Background American visceral leishmaniasis is caused by the protozoan Leishmania infantum. Dogs are the main reservoirs in the domestic transmission cycle. The limited accuracy of diagnostic tests for canine leishmaniasis may contribute to the lack of impact of control measures recommended by the Brazilian Ministry of Health. The objective of this study was to estimate the accuracy of two enzyme-linked immunosorbent assays employing L. major or L. infantum antigens and their reliability between three laboratories of different levels of complexity. Methods A validation study of ELISA techniques using L. major or L. infantum antigens was conducted. Direct visualization of the parasite in hematoxylin/eosin-stained histopathological sections, immunohistochemistry, and isolation of the parasite in culture.were used as gold standard. An animal that was positive in at least one of the tests was defined as infected with L. infantum. Serum samples collected from 1,425 dogs were analyzed. Samples were separated in three aliquots and tested in three different laboratories. Sensitivity, specificity and the area under de ROC curve were calculated and the reliability was evaluated between the participant laboratories. Results The sensitivity was 91.8% and 89.8% for the L. major and L. infantum assays, respectively. The specificity was 83.75% and 82.7% for the L. major and L. infantum assays, respectively. The area under de ROC curve was 0.920 and 0.898 for L. major and L. infantum, respectively. The mean intraclass correlation coefficients between laboratories ranged from 0.890 to 0.948 when L. major was used as antigen, and from 0.818 to 0.879 when L. infantum was used. Interpretation ELISA tests using L. major or L. infantum antigens have similar accuracy and reliability. Our results do not support the substitution of the L. major antigen of the ELISA test currently used for the diagnosis of canine visceral leishmaniasis in Brazil. PMID:23922884

  11. Clinical Performance of a New Soluble CD14-Subtype Immunochromatographic Test for Whole Blood Compared with Chemiluminescent Enzyme Immunoassay: Use of Quantitative Soluble CD14-Subtype Immunochromatographic Tests for the Diagnosis of Sepsis

    PubMed Central

    Sato, Masayuki; Takahashi, Gaku; Shibata, Shigehiro; Onodera, Makoto; Suzuki, Yasushi; Inoue, Yoshihiro; Endo, Shigeatsu

    2015-01-01

    We previously reported that a soluble CD14-subtype (sCD14-ST) immunochromatographic test (ICT) for plasma is more convenient than chemiluminescent enzyme immunoassay (CLEIA), but plasma separation makes bedside measurements difficult. We developed a new sCD14-ST ICT for whole blood and investigated whether quantitative determinations of sCD14-ST by ICT were useful for diagnosing sepsis and severe sepsis/septic shock. We studied 20 patients who fulfilled two or more systemic inflammatory response syndrome (SIRS) criteria and 32 patients who had been diagnosed with sepsis or severe sepsis/septic shock. Whole blood was collected on day 0 (on admission) and day 7, and the sCD14-ST concentration was quantitatively measured by CLEIA and ICT for whole blood. The patients’ Acute Physiology and Chronic Health Evaluation (APACHE) II, Sequential Organ Failure Assessment (SOFA), and Mortality in Emergency Department Sepsis (MEDS) scores were also calculated. The cut-off values obtained by the quantitative measurements made by ICT were 464.5 pg/mL for sepsis and 762.7 pg/mL for severe sepsis/septic shock (P < 0.0001). A Bland–Altman plot showed that no fixed bias or proportional bias was detected between CLEIA and quantitative ICT for whole blood. sCD14-ST concentrations were significantly correlated with APACHE II, SOFA, and MEDS scores (P < 0.0001). These results suggest that the new sCD14-ST ICT for whole blood may be a useful tool for the convenient, rapid bedside diagnosis and treatment of sepsis. PMID:26623644

  12. Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment.

    PubMed

    Tao, Xiaoqi; Chen, Min; Jiang, Haiyang; Shen, Jianzhong; Wang, Zhanhui; Wang, Xia; Wu, Xiaoping; Wen, Kai

    2013-09-01

    A chemiluminescent competitive indirect enzyme-linked immunosorbent assay, based on a mutant single-chain variable fragment (scFv), was developed to detect a broad range of fluoroquinolones (FQs) in fish and shrimp matrices. In this study, the best scFvC4A9H1_mut2 was adopted, which showed 10-fold improved affinity to sarafloxacin (SAR), difloxacin (DIF), and trovafloxacin (TRO), while the affinity to other FQs was fully inherited from wild-type scFvC4A9H1. In the optimized generic test, scFvC4A9H1_mut2 in combination with norfloxacin-ovalbumin conjugate and horseradish peroxidase-labeled anti-c-myc 9E10 antibody showed 50 % binding inhibition (IC50) at 0.12 μg kg(-1) for norfloxacin in buffer. Screening for the class of FQ antibiotics is accomplished using a simple, rapid extraction carried out with ethanol/acetic acid (99:1, v/v). This common extraction was able to detect 20 FQ residues such as s ciprofloxacin (CIP), danofloxacin, DIF, enoxacin, enrofloxacin (ENR), fleroxacin, amifloxacin, flumequine, levofloxacin, lomefloxacin hydrochloride, marbofloxacin, norfloxacin (NOR), ofloxacin, orbifloxacin, pazufloxacin, pefloxacin-d5 (PEF), prulifloxacin, SAR, sparfloxacin, and TRO in fish and shrimp. The limit of detection (LOD) for NOR was 0.2 μg kg(-1) and the LODs for CIP and ENR were all <0.2 μg kg(-1). Values of LODs inferred from the cross-reactivity data will range from approximately 0.23 μg kg(-1) for PEF to 2.1 μg kg(-1) for TRO. Field fish and shrimp samples were analyzed and compared to the results obtained from liquid chromatography tandem mass spectrometric method. All five instances (from 0.25 to 15.6 μg kg(-1)) in which FQs were present at concentrations near or above the assay LOD were identified as positive by the newly developed assay, demonstrating the usefulness of this assay as a screening tool. PMID:23842902

  13. Updates in immunoassays: bacteriology.

    PubMed

    Josko, Deborah

    2012-01-01

    There are many immunoassays available that provide rapid, accurate and sensitive results. The intent of this article was to provide a brief overview of some of the products and methodologies available for clinical use and to discuss some of the principles behind the methodology and instrumentation. In the area of infectious disease, the use of immunoassays ensures rapid turnaround times that will result in the administration of prompt, accurate treatment for the patient. Ultimately, this will improve overall patient outcomes while possibly decreasing the costs associated with increased hospital stay. In conclusion, immunoassays are essentially easy to perform, cost-effective, produce highly sensitive and specific results, and allow the medical laboratory professional the ability to report accurate results in a timely manner. PMID:22953518

  14. Hydrogel nanoparticle based immunoassay

    DOEpatents

    Liotta, Lance A; Luchini, Alessandra; Petricoin, Emanuel F; Espina, Virginia

    2015-04-21

    An immunoassay device incorporating porous polymeric capture nanoparticles within either the sample collection vessel or pre-impregnated into a porous substratum within fluid flow path of the analytical device is presented. This incorporation of capture particles within the immunoassay device improves sensitivity while removing the requirement for pre-processing of samples prior to loading the immunoassay device. A preferred embodiment is coreshell bait containing capture nanoparticles which perform three functions in one step, in solution: a) molecular size sieving, b) target analyte sequestration and concentration, and c) protection from degradation. The polymeric matrix of the capture particles may be made of co-polymeric materials having a structural monomer and an affinity monomer, the affinity monomer having properties that attract the analyte to the capture particle. This device is useful for point of care diagnostic assays for biomedical applications and as field deployable assays for environmental, pathogen and chemical or biological threat identification.

  15. Mutations in activation-induced cytidine deaminase in patients with hyper IgM syndrome.

    PubMed

    Minegishi, Y; Lavoie, A; Cunningham-Rundles, C; Bédard, P M; Hébert, J; Côté, L; Dan, K; Sedlak, D; Buckley, R H; Fischer, A; Durandy, A; Conley, M E

    2000-12-01

    Recent studies have shown that mutations in a newly described RNA editing enzyme, activation-induced cytidine deaminase (AID), can cause an autosomal recessive form of hyper IgM syndrome. To determine the relative frequency of mutations in AID, we evaluated a group of 27 patients with hyper IgM syndrome who did not have defects in CD40 ligand and 23 patients with common variable immunodeficiency. Three different mutations in AID were identified in 18 patients with hyper IgM syndrome, including 14 French Canadians, 2 Lumbee Indians, and a brother and sister from Okinawa. No mutations were found in the remaining 32 patients. In the group of patients with hyper IgM syndrome, the patients with mutations in AID were older at the age of diagnosis, were more likely to have positive isohemagglutinins, and were less likely to have anemia, neutropenia, or thrombocytopenia. Lymphoid hyperplasia was seen in patients with hyper IgM syndrome and normal AID as well as the patients with hyper IgM syndrome and defects in AID. PMID:11112359

  16. A photoacoustic immunoassay for biomarker detection.

    PubMed

    Zhao, Yunfei; Cao, Mingfeng; McClelland, John F; Shao, Zengyi; Lu, Meng

    2016-11-15

    Challenges in protein biomarker analysis include insufficient sensitivity for detecting low-abundance biomarkers, poor measurement reproducibility, and the high costs and large footprints of detection systems. To address these issues, a new detection modality was developed for analyzing protein biomarkers based on the plasmon-enhanced photoacoustic (PA) effect. The detection modality employed a heterogeneous immunoassay scheme and used gold nanoparticles (AuNPs) as the signal reporter. Due to their localized plasmon resonance, AuNPs can strongly interact with intensity-modulated laser excitation and generate strong PA signals, which are subsequently sensed and quantified using a microphone. As an example, the performance of the PA immunoassay was evaluated by detecting the human interleukin 8 chemokine. The PA immunoassay provided approximately 143× lower limit of detection (LOD) than observed with the gold standard enzyme-linked immunosorbent assay - a decrease from 23pg/mL to 0.16pg/mL. In addition to the significant performance improvement in terms of the LOD, the PA immunoassay also offers advantages in terms of compatibility with low-cost instruments and the long-term stability of assay results. PMID:27183276

  17. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W.; Williams, Peter; Krone, Jennifer Reeve

    2005-12-13

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  18. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

    2007-12-04

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  19. Mass spectrometric immunoassay

    DOEpatents

    Nelson, Randall W; Williams, Peter; Krone, Jennifer Reeve

    2013-07-16

    Rapid mass spectrometric immunoassay methods for detecting and/or quantifying antibody and antigen analytes utilizing affinity capture to isolate the analytes and internal reference species (for quantification) followed by mass spectrometric analysis of the isolated analyte/internal reference species. Quantification is obtained by normalizing and calibrating obtained mass spectrum against the mass spectrum obtained for an antibody/antigen of known concentration.

  20. Role of signal-to-cut-off ratios of anti-hepatitis C virus antibody by enzyme immunoassays along with ID-NAT for screening of whole blood donors in India

    PubMed Central

    Arora, Satyam; Doda, Veena

    2016-01-01

    Background: The use of elevated signal-to-cut off ratios (S/CO) as an alternate to further supplemental testing (i.e., RIBA) has been included in the guidelines provided by the Centres for Disease Control and Prevention for HCV diagnostic purposes since 2003. With availability of screening by NAT and non availability of RIBA, further confirmation of HCV infection has been possible at the molecular level (RNA). Aims: To study the role of S/CO ratios of anti hepatitis C virus antibody detection by enzyme immunoassays (EIA) along with ID-NAT for screening of whole blood donors. Methods: In this study we reviewed the donor screening status for anti HCV from January 2013 to May 2014. All the donations were screened for anti HCV with fourth generation ELISA (BioRad Monolisa Ag-Ab Ultra) as well as with ID NAT (Procleix Ultrio). The S/CO ratio of all the anti-HCV reactive samples were analysed for their presence of HCV RNA. Results: On screening 21,115 donors for HCV, 83 donors (0.39%) were found reactive on pilot tube and repeat plasma bag testing (S/Co ratio ≥1) by ELISA. 41 donors were HCV RNA reactive with ID-NAT. 4 samples out of 41 were NAT yields and 37 were concordant reactive with ELISA. The S/Co ratio of anti-HCV reactive samples ranged from 0.9-11.1 [mean = 5.1; SD ± 2.9] whereas S/Co ratio of anti HCV and NAT reactive samples (concordant positives) ranged from 4.1-11.1 [mean 7.3]. In our analysis we found that S/CO ratio of 4 showed positive predictive value (PPV) and sensitivity of 100%. Summary/Conclusions: Our study showed that S/CO of 4 for anti HCV on ELISA would have maximum positive predictive value of having donor with HCV RNA. S/CO ratio of 4 is very close to 3.8 which was the CDC guideline. The presence of anti-HCV does not distinguish between current or past infections but a confirmed anti-HCV-positive result indicates the need for counseling and medical evaluation for HCV infection. PMID:27011676

  1. Comparison of Common Immunoassay Kits for Effective Application in Workplace Drug Urinalysis.

    PubMed

    Liu, R H

    1994-06-01

    Workplace drug urinalysis protocols include an initial immunoassay followed by a confirmation gas chromatography/mass spectrometry (GC/MS) test of immunoassay-positive samples. (Drug categories that are commonly tested include: amphetamines, barbiturates, benzodiazepines, cannabis, cocaine, lysergic acid diethyl amide, methadone, methaqualone, opiates, phencyclidine, and propoxyphene. Not all drug categories are tested by all workplace drug urinalysis programs.) Only those samples that are tested positive by both the initial and the confirmatory procedures can be reported as positive. Thus, when adopting an immunoassay, one must have knowledge of the assay's cross-reacting characteristics and the assay's apparent analyze concentration that corresponds to a specific analyze concentration determined by the GC/MS procedure. The underlying principles of the commonly used radioimmunoassay, enzyme immunoassay, fluorescence polarization immunoassay, and particle immunoassay are outlined. Cross-reacting characteristics of these immunoassays as reported by the manufacturers and independent laboratories are tabulated. This information shown that commercial immunoassay kits for drug categories that are included in workplace drug urinalysis programs are generally more specific than those kits that are for clinical use only. Furthermore, recently manufactured immunoassay kits targeted for use in workplace drug urinalysis programs are more specific than those manufactured earlier. Reported effects of adulterants, such as salt, cleaning agents, etc., on commonly used immunoassays are summarized. Without more comprehensive and systematic studies, it is difficult to make general statements concerning the superiority of one methodology over the others. It is clear, however, that cannabinoid assesses are most susceptible to the influence of adulterants. Reported immunoassay-GC/MS correlation data are reviewed. Significant correlations exist in all cases. The immunoassay apparent

  2. IgG and IgM autoantibody differences in discoid and systemic lupus patients.

    PubMed

    Chong, Benjamin F; Tseng, Lin-chiang; Lee, Thomas; Vasquez, Rebecca; Li, Quan Z; Zhang, Song; Karp, David R; Olsen, Nancy J; Mohan, Chandra

    2012-12-01

    Systemic lupus erythematosus (SLE) patients with discoid lupus erythematosus (DLE) were reported to have milder disease. To test this observation, we used sandwich arrays containing 98 autoantigens to compare autoantibody profiles of SLE subjects without DLE (DLE-SLE+) (N=9), SLE subjects with DLE (DLE+SLE+) (N=10), DLE subjects without SLE (DLE+SLE-) (N=11), and healthy controls (N=11). We validated differentially expressed autoantibodies using immunoassays in DLE-SLE+ (N=18), DLE+SLE+ (N=17), DLE+SLE- (N=23), and healthy subjects (N=22). Arrays showed 15 IgG autoantibodies (10 against nuclear antigens) and 4 IgM autoantibodies that were differentially expressed (q-value<0.05). DLE-SLE+ subjects had higher IgG autoantibodies against double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA), histone H2A and H2B, and SS-A (52 kDa) compared with all other groups including DLE+SLE+ subjects (P<0.05). Immunoassays measuring anti-dsDNA, -ssDNA, and -SS-A (52 kDa) IgG autoantibodies showed similar trends (P<0.05). Healthy and DLE+SLE- subjects expressed higher IgM autoantibodies against alpha beta crystallin, lipopolysaccharide, heat-shock cognate 70, and desmoglein-3 compared with DLE+SLE+ and DLE-SLE+ subjects. IgG:IgM ratios of autoantibodies against nuclear antigens progressively rose from healthy to DLE-SLE+ subjects. In conclusion, lower IgG autoantibodies against nuclear antigens in DLE+SLE+ versus DLE-SLE+ subjects suggest that DLE indicates lower disease severity. Higher IgM autoantibodies against selected antigens in healthy and DLE+SLE- subjects may be nonpathogenic. PMID:22763789

  3. IgG and IgM autoantibody differences in discoid and systemic lupus patients

    PubMed Central

    Chong, Benjamin F.; Tseng, Lin-chiang; Lee, Thomas; Vasquez, Rebecca; Li, Quan Z.; Zhang, Song; Karp, David R.; Olsen, Nancy J.; Mohan, Chandra

    2012-01-01

    Systemic lupus (SLE) patients with discoid lupus (DLE) were reported to have milder disease. To test this observation, we employed sandwich arrays containing 98 autoantigens to compare autoantibody profiles of SLE subjects without DLE (DLE−SLE+) (N=9), SLE subjects with DLE (DLE+SLE+) (N=10), DLE subjects without SLE (DLE+SLE−) (N=11), and healthy controls (N=11). We validated differentially expressed autoantibodies using immunoassays in DLE−SLE+ (N=18), DLE+SLE+ (N=17), DLE+SLE− (N=23), and healthy subjects (N=22). Arrays showed 15 IgG autoantibodies (ten against nuclear antigens) and four IgM autoantibodies that were differentially expressed (q-value<0.05). DLE−SLE+ subjects had higher IgG autoantibodies against dsDNA, ssDNA, dsRNA, histone H2A and H2B, and SS-A (52 kDa) than all other groups including DLE+SLE+ subjects (p<0.05). Immunoassays measuring anti-dsDNA, -ssDNA, and -SS-A (52 kDa) IgG autoantibodies showed similar trends (p<0.05). Healthy and DLE+SLE−subjects expressed higher IgM autoantibodies against alpha beta crystallin, lipopolysaccharide, heat shock cognate 70, and desmoglein-3 than DLE+SLE+ and DLE−SLE+ subjects. IgG:IgM ratios of autoantibodies against nuclear antigens progressively rose from healthy to DLE−SLE+ subjects. In conclusion, lower IgG autoantibodies against nuclear antigens in DLE+SLE+ versus DLE−SLE+ subjects suggest that DLE indicates lower disease severity. Higher IgM autoantibodies against selected antigens in healthy and DLE+SLE−subjects may be non-pathogenic. PMID:22763789

  4. Anti-rabies virus IgM in serum and cerebrospinal fluid from rabid dogs.

    PubMed

    Tingpalapong, M; Hoke, C H; Ward, G S; Burke, D S; Elwell, M R; Lohytyothin, S; Saisombat, S

    1986-12-01

    An anti-rabies IgM antibody capture radio immunoassay was used to test serum and cerebrospinal fluid from 37 dogs held in quarantine for suspicion of rabies. Rabies was confirmed in dogs that died by mouse inoculation and subsequent examination of mouse brains by fluorescent antibody technique to detect rabies antigen. The mean counts per minute (CPM) of iodinated anti-rabies gamma globulin coupled IgM rabies antibody in CSF and serum from rabid dogs were significantly higher than in CSF and serum from non-rabid dogs. Mean CPM from rabid dogs was greater in CSF than in sera, in contrast with non-rabid dogs, from which mean cpm was higher in sera than CSF, suggesting that antibody may have been synthesized in the CSF. To evaluate this test further, a dog was infected by rabies virus, and serial serum and CSF specimens were collected until the time of death. IgM anti-rabies antibody developed in the CSF and serum 29 days following infection, and rose just before the dog died of rabies on day 34. The rabies MAC RIA is potentially useful as a diagnostic method in quarantined dogs with rabies-like illness. Perhaps more importantly, it may be applied to better understand the immunopathogenicity of rabies. PMID:3576284

  5. Updates in immunoassays: virology.

    PubMed

    Josko, Deborah

    2012-01-01

    Virus identification is a challenge to the clinical microbiologist since growing viruses in traditional cell culture is labor intensive, time consuming, and subject to contamination. The advent of rapid and automated immunoassays has eliminated this problem by generating positive results in minutes to hours. For example, testing for infectious mononucleosis can yield a positive result in 3-8 minutes as seen with the Beckman Coulter, Inc. ICON Mono test or in 5-15 minutes with the MONO Mononucleosis Rapid Test Device marketed by ACON Laboratories, Inc. Fully automated immunoassay analyzers provide fast, accurate, sensitive results that aid in a prompt and accurate diagnosis for the patient. Turnaround times are shortened, allowing for timely medical intervention and treatment. The priority in any hospital or medical facility is to treat the patient as quickly and appropriately as possible. By using immunoassays, clinical laboratory professionals are able to report out correct results in a timely manner, ensuring overall positive patient outcomes and improved quality of healthcare. PMID:22953519

  6. Detection of bound residues in soils by sandwich-immunoassay

    SciTech Connect

    Dosch, M.; Weller, M.G.; Niessner, R.

    1995-12-31

    Immunoassays are useful analytical instruments for the detection of many environmental compounds. This method was not introduced for the detection of non-extractable compounds in soil. So-called ``bound residues`` consist of a soil component, e.g. humic acids and an irreversibly bound pollutant. Because of the complexity of those macromolecules conventional analytical methods in general do not work. Enzyme immunoassays, in contrast, seem to have a large potential for applications and further developments in this field. The use of antibodies with high affinity to the analytes makes a selective detection of environmental pollutants possible. With the development of an enzyme-labeled sandwich-immunoassay polycyclic aromatic hydrocarbons (PAHs) irreversibly bound to humic acids were determined for the first time.

  7. Detection of bound residues in soils by sandwich-immunoassay

    NASA Astrophysics Data System (ADS)

    Dosch, M.; Weller, Michael G.; Niessner, Reinhard

    1995-10-01

    Immunoassays are useful analytical instruments for the detection of many environmental compounds. This method is now introduced for the detection of non-extractable compounds in soil. So-called 'bound residues' consist of a soil component, e.g. humic acids, and an irreversibly bound pollutant. Because of the complexity of those macromolecules conventional analytical methods in general do not work. Enzyme immunoassays, in contrast, seem to have a large potential for applications and further developments in this field. The use of antibodies with high affinity to the analytes makes a selective detection of environmental pollutants possible. With the development of an enzyme-labeled sandwich-immunoassay polycyclic aromatic hydrocarbons (PAHs), irreversibly bound to humic acids, were determined for the first time.

  8. Homogeneous Immunoassays: Historical Perspective and Future Promise

    NASA Astrophysics Data System (ADS)

    Ullman, Edwin F.

    1999-06-01

    The founding and growth of Syva Company is examined in the context of its leadership role in the development of homogeneous immunoassays. The simple mix and read protocols of these methods offer advantages in routine analytical and clinical applications. Early homogeneous methods were based on insensitive detection of immunoprecipitation during antigen/antibody binding. The advent of reporter groups in biology provided a means of quantitating immunochemical binding by labeling antibody or antigen and physically separating label incorporated into immune complexes from free label. Although high sensitivity was achieved, quantitative separations were experimentally demanding. Only when it became apparent that reporter groups could provide information, not only about the location of a molecule but also about its microscopic environment, was it possible to design practical non-separation methods. The evolution of early homogenous immunoassays was driven largely by the development of improved detection strategies. The first commercial spin immunoassays, developed by Syva for drug abuse testing during the Vietnam war, were followed by increasingly powerful methods such as immunochemical modulation of enzyme activity, fluorescence, and photo-induced chemiluminescence. Homogeneous methods that quantify analytes at femtomolar concentrations within a few minutes now offer important new opportunities in clinical diagnostics, nucleic acid detection and drug discovery.

  9. Sequential injection immunoassay for environmental measurements.

    PubMed

    Soh, Nobuaki; Tanaka, Mayumi; Hirakawa, Koji; Zhang, RuiQi; Nakajima, Hizuru; Nakano, Koji; Imato, Toshihiko

    2011-01-01

    Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis. PMID:22076332

  10. Multiplex Electrochemical Immunoassay Using Gold Nanoparticle Probes and Immunochromatographic Strips

    SciTech Connect

    Mao, Xun; Baloda, Meenu; Gurung, Anant; Lin, Yuehe; Liu, Guodong

    2008-10-20

    We describe a multiplex electrochemical immunoassay based on the use of gold nanoparticle (Au-NP) probes and immunochromatographic strips (ISs). The approach takes advantage of the speed and low cost of the conventional IS tests and the high sensitivities of the nanoparticle-based electrochemical immunoassays. Rabbit IgG(R-IgG) and human IgM (H-IgM) were used as model targets for the demonstration of the proof of concept. The Au-NPs based sandwich immunoreactions were performed on the IS, and the captured gold nanoparticle labels on the test zones were determined by highly-sensitive stripping voltammetric measurement of the dissolved gold ions (III) with a carbon paste electrode. The detection limits are 1.0 and 1.5 ng/mL with the linear ranges of 2.5-250 ng/mL for quantitative detection of R-IgG and H-IgM, respectively. The total assay time is around 25 minutes. Such multiplex electrochemical immunoassay could be readily highly multiplexed to allow simultaneous parallel detection of numerous proteins and is expected to open new opportunities for protein diagnostics and biosecurity.

  11. APPRAISAL OF PRENATAL ANTI-TOXOPLASMA GONDII (IGG+IGM)- IHA/IGM-ELISA SCREENING IN SINGLE SAMPLES VIA IGG AVIDITY TEST.

    PubMed

    El-Bali, Mohammed; Zaglool, Dina A M; Khodari, Yousif A W; Al-Harthi, Saeed A

    2016-04-01

    Congenital toxoplasmosis is associated with important morbidity and mortality. Since vertical transmission of Toxoplasma gondii can occur in acute cases, antenatal screening for recent infections is vital. Accurate determination of acute toxoplasmosis requires a combination of immunoassays, usually not routinely applied for screening purposes. This study evaluated the anti-T. gondii (IgG+IgM)/IgM prenatal screening procedure by IgG avidity assay. The routine prenatal screening for (IgG+IgM) anti-T. gondii by indirect hemagglutination (IHA) in serum samples was done of 2247 pregnant women who attended two hospitals between 2011 and 2013 revealed 487 (21.7%) positive samples. Examination of IHA-positive sera by IgM and IgG/IgG-avidity concurrent ELISA tests revealed 7 positive and 3 border-line IgM-ELISA titers during the initial check-up of 10 women, who were then followed up at 3-4 week-intervals. Among these, 4 (40%) showed simultaneous high avidity IgG antibodies, indicating distant infection by the parasite, and no anti-T. gondii specific IgG could be detected in follow-up sera of two cases (20%), indicating false IgM initial positive results. Only 4 (40%) women showed simultaneous IgM and low avidity IgG antibodies indicating active infections. Avoidance of an over-diagnosis of acute toxoplasmosis Anti-T. gondii (IgG+IgM)/IgM prenatal screening must be supplemented by a discriminative test like IgG avidity ELISA. PMID:27363056

  12. Automated imatinib immunoassay

    PubMed Central

    Beumer, Jan H.; Kozo, Daniel; Harney, Rebecca L.; Baldasano, Caitlin N.; Jarrah, Justin; Christner, Susan M.; Parise, Robert; Baburina, Irina; Courtney, Jodi B.; Salamone, Salvatore J.

    2014-01-01

    Background Imatinib pharmacokinetic variability and the relationship of trough concentrations with clinical outcomes have been extensively reported. Though physical methods to quantitate imatinib exist, they are not widely available for routine use. An automated homogenous immunoassay for imatinib has been developed, facilitating routine imatinib testing. Methods Imatinib-selective monoclonal antibodies, without substantial cross-reactivity to the N-desmethyl metabolite or N-desmethyl conjugates, were produced. The antibodies were conjugated to 200 nm particles to develop immunoassay reagents on the Beckman Coulter AU480™ analyzer. These reagents were analytically validated using Clinical Laboratory Standards Institute protocols. Method comparison to LC-MS/MS was conducted using 77 plasma samples collected from subjects receiving imatinib. Results The assay requires 4 µL of sample without pre-treatment. The non-linear calibration curve ranges from 0 to 3,000 ng/mL. With automated sample dilution, concentrations of up to 9,000 ng/mL can be quantitated. The AU480 produces the first result in 10 minutes, and up to 400 tests per hour. Repeatability ranged from 2.0 to 6.0% coefficient of variation (CV), and within-laboratory reproducibility ranged from 2.9 to 7.4% CV. Standard curve stability was two weeks and on-board reagent stability was 6 weeks. For clinical samples with imatinib concentrations from 438 – 2,691 ng/mL, method comparison with LC-MS/MS gave a slope of 0.995 with a y-intercept of 24.3 and a correlation coefficient of 0.978. Conclusion The immunoassay is suitable for quantitating imatinib in human plasma, demonstrating good correlation with a physical method. Testing for optimal imatinib exposure can now be performed on routine clinical analyzers. PMID:25551407

  13. Immunoassays in monitoring biotechnological drugs.

    PubMed

    Gygax, D; Botta, L; Ehrat, M; Graf, P; Lefèvre, G; Oroszlan, P; Pfister, C

    1996-08-01

    For the evaluation and interpretation of pharmacokinetic data reliable quantitative determinations are a requirement that can only be met by well-characterized and fully validated analytical methods. To cope with these requirements a method is being established that is based on an integrated and automated fiber-optic biospecific interaction analysis system (FOBIA) for immunoassays. Performance characteristics of this system used in monitoring of recombinant hirudin (CGP 39 393) are presented. Recombinant hirudin is a highly potent and selective inhibitor of human thrombin. Owing to its size and charge, recombinant hirudin is mainly eliminated by glomerular filtration. But only a fraction of the hirudin dose seems to be reabsorbed at the proximal tubule by luminal endocytosis and hydrolyzed by lysosomal enzymes, leaving approximately 50% of the dose to be extracted in the urine. Thus, renal clearance of recombinant hirudin in the absence of renal insufficiency appears to depend primarily on the glomerular filtration rate. During a 3-month i.v. tolerability study in dogs, some of the dogs developed antibodies against recombinant hirudin. The hirudin-antibody complex accumulated in plasma and apparent hirudin plasma concentrations were therefore much higher than expected from single-dose kinetics. Hirudin captured by antibodies showed an extended half-life and the hirudin-antibody complex is still pharmacologically active, as demonstrated by the observed increase in thrombin time. In conclusion, only appropriate analytical methods allow adequate monitoring and pharmacokinetic characterization of biotechnology drugs in biological materials. PMID:8857560

  14. Element-tagged immunoassay with ICP-MS detection: evaluation and comparison to conventional immunoassays

    PubMed Central

    Razumienko, Eva; Ornatsky, Olga; Kinach, Robert; Milyavsky, Michael; Lechman, Eric; Baranov, Vladimir; Winnik, Mitchell A.; Tanner, Scott D.

    2008-01-01

    We have investigated the possibility of using element-tagged antibodies for protein detection and quantification in microplate format using Inductively Coupled Plasma Mass Spectrometry (ICP-MS), and compared the results to conventional immunoassays, such as Enzyme-Linked Immunosorbent Assay (ELISA) and Western blotting. The technique was further employed to detect low levels and measure DNA-binding activity of transcription factor p53 in leukemia cell lysates through its interaction with immobilized oligonucleotides and recognition by element-tagged antibodies. The advantages of ICP-MS detection for routine performance of immunoassays include increased sensitivity, wide dynamic range, minimal interference from complex matrices, and high throughput. Our approach advances the ICP-MS technology and demonstrates its applicability to proteomic studies through the use of antibodies directly labeled with polymer tags bearing multiple atoms of lanthanides. Development of this novel methodology will enable fast and quantitative identification of multiple analytes in a single well. PMID:18456275

  15. A Wash-Free Homogeneous Colorimetric Immunoassay Method

    PubMed Central

    Liu, Huiqiao; Rong, Pengfei; Jia, Hongwei; Yang, Jie; Dong, Bo; Dong, Qiong; Yang, Cejun; Hu, Pengzhi; Wang, Wei; Liu, Haitao; Liu, Dingbin

    2016-01-01

    Rapid and convenient biosensing platforms could be beneficial to timely diagnosis and treatment of diseases in virtually any care settings. Sandwich immunoassays, the most commonly used methods for protein detection, often rely on expensive tags such as enzyme and tedious wash and incubation procedures operated by skilled labor. In this report, we revolutionized traditional sandwich immunoassays by providing a wash-free homogeneous colorimetric immunoassay method without requirement of any separation steps. The proposed strategy was realized by controlling the growth of gold nanoparticles (AuNPs) to mediate the interparticle spacing in the protein-AuNP oligomers. We have demonstrated the successful in vitro detection of cancer biomarker in serum samples from patients with high clinical sensitivity and specificity. PMID:26722373

  16. Mass spectrometric immunoassay

    SciTech Connect

    Nelson, R.W.; Krone, J.R.; Bieber, A.L.; Williams, P.

    1995-04-01

    A new, general method of immunoassay is demonstrated. The approach is based on the microscale immunoaffinity capture of target antigens followed by mass-specific identification and quantitation using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Immunoaffinity capture of antigens effectively overcomes signal suppression effects typically encountered during traditional matrix-assisted laser desorption/ionization analysis of complex biological mixtures while simultaneously concentrating the analyte into a small volume. Sample incubation and processing methods were such that a typical analysis could be performed in less than 1 h while subnanomolar sensitivities were maintained. The technique has been used for the rapid, selective, and quantitative screening of human blood for the presence of myotoxin a, and Mojave toxin from the venoms of the prairie rattlesnake, Crotalus virdis virdis, and the Mojave rattlesnake, Crotalus scutulatus scutulatus. 18 refs., 8 figs.

  17. Lateral Flow Immunoassay.

    PubMed

    Ching, Kathryn H

    2015-01-01

    Lateral flow immunoassays (LFIAs) are a staple in the field of rapid diagnostics. These small handheld devices require no specialized training or equipment to operate, and generate a result within minutes of sample application. They are an ideal format for many types of home test kits, for emergency responders and for food manufacturers and producers looking for a quick evaluation of a given sample. LFIAs rely on high quality monoclonal antibodies that recognize the analyte of interest. As monoclonal antibody technology becomes more accessible to smaller laboratories, there has been increased interest in developing LFIA prototypes for potential commercial manufacture. In this chapter, the basics of designing and building an LFIA prototype are described. PMID:26160571

  18. Immunoassay as an analytical tool in agricultural biotechnology.

    PubMed

    Grothaus, G David; Bandla, Murali; Currier, Thomas; Giroux, Randal; Jenkins, G Ronald; Lipp, Markus; Shan, Guomin; Stave, James W; Pantella, Virginia

    2006-01-01

    Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays. PMID:16915826

  19. Morphological resonances for multicomponent immunoassays

    NASA Astrophysics Data System (ADS)

    Whitten, W. B.; Shapiro, M. J.; Ramsey, J. M.; Bronk, B. V.

    1995-06-01

    An immunoassay technique capable of detecting and identifying a number of species of microorganisms in a single analysis is described. The method uses optical-resonance size discrimination of microspheres to identify antibodies to which stained microorganisms are bound.

  20. Magnetic Beads-based Bioelectrochemical Immunoassay of Polycyclic Aromatic Hydrocarbons

    SciTech Connect

    Lin, Ying-Ying; Liu, Guodong; Wai, Chien M.; Lin, Yuehe

    2007-07-01

    A simple, rapid, and sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) has been developed to detect polycyclic aromatic hydrocarbons (PAHs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PAH-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PAH (HRP-PAH). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreaction among PAH-antibody-coated MBs, PAH analyte, and HRP-PAH. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing 3, 3´, 5, 5´- tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) for electrochemical detection. The voltammetric characteristics of the substrate were investigated, and the reduction peak current of TMB was used to quantify the concentration of PAH. The different parameters, including the amount of HRP-PAH conjugates, the enzyme catalytic reaction time, and the pH of the supporting electrolyte that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 50 pg mL-1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical magnetic immunoassay was successfully evaluated with tap water spiked with PAHs, indicating that this convenient and sensitive technique offers great promise for decentralized environmental applications.

  1. IgM Contributes to Glomerular Injury in FSGS

    PubMed Central

    Strassheim, Derek; Renner, Brandon; Panzer, Sarah; Fuquay, Richard; Kulik, Liudmila; Ljubanović, Danica; Holers, V. Michael

    2013-01-01

    Glomerular IgM and C3 deposits frequently accompany idiopathic FSGS and secondary glomerulosclerosis, but it is unknown whether IgM activates complement, possibly contributing to the pathogenesis of these diseases. We hypothesized that IgM natural antibody binds to neoepitopes exposed in the glomerulus after nonimmune insults, triggering activation of the complement system and further injury. We examined the effects of depleting B cells, using three different strategies, on adriamycin-induced glomerulosclerosis. First, we treated wild-type mice with an anti-murine CD20 antibody, which depletes B cells, before disease induction. Second, we evaluated adriamycin-induced glomerulosclerosis in Jh mice, a strain that lacks mature B cells. Third, we locally depleted peritoneal B cells via hypotonic shock before disease induction. All three strategies reduced deposition of IgM in the glomerulus after administration of adriamycin and attenuated the development of albuminuria. Furthermore, we found that glomerular IgM and C3 were detectable in a subset of patients with FSGS; C3 was present as an activation fragment and colocalized with glomerular IgM, suggesting that glomerular IgM may have bound a cognate ligand. Taken together, these results suggest that IgM activates the complement system within the glomerulus in an animal model of glomerulosclerosis. Strategies that reduce IgM natural antibody or that prevent complement activation may slow the progression of glomerulosclerosis. PMID:23393315

  2. Nanomaterial-based advanced immunoassays.

    PubMed

    Hu, Weihua; Li, Chang Ming

    2011-01-01

    Immunoassay has been the main stream in clinic diagnostics and still attracts extensive research interest in recent years to develop reliable, fast, sensitive, and specific detection methods and platforms to expand its applications in various areas including proteomics, drug discovery, homeland security, food safety, environmental monitoring, and health care. With the dramatic progress in material science, nanotechnology, and bioconjugation techniques, a great diversity of nanomaterials with desirable superior properties have been designed, synthesized, and tailored to facilitate high-performance detections for advanced immunoassays. This paper comprehensively reviews recent advances in nanomaterial-based immunoassay technologies and particularly highlights newly developed strategies associated with interdisciplinary areas for performance enhancement and related mechanisms. The future perspectives of immunosensing technologies are also discussed. PMID:25363746

  3. ELEGANT ENVIRONMENTAL IMMUNOASSAYS

    EPA Science Inventory

    Immunochemical methods are based on selective antibodies directed to a particular target analyte. The specific binding between antibody and analyte can be used for detection and quantitation. Methods such as the enzyme-linked immunosorbent assay (ELISA) can provide a sensitiv...

  4. Fluorescence Immunoassay for Cocaine Detection.

    PubMed

    Nakayama, Hiroshi; Kenjjou, Noriko; Shigetoh, Nobuyuki; Ito, Yuji

    2016-04-01

    A fluorescence immunoassay (FIA) has been developed for the detection of cocaine using norcocaine labeled with merocyanine dye and a monoclonal antibody specific to cocaine. Using this FIA, the detection range for cocaine was between 20.0 and 1700 μg/L with a limit of detection of 20.0 μg/L. Other cocaine derivatives did not interfere significantly with the detection when using this immunoassay technique with cross-reactivity values of less than 20%. Thus this FIA could be considered a useful tool for the detection of cocaine. PMID:26977890

  5. GRBs as Probes of the IGM

    NASA Astrophysics Data System (ADS)

    Cucchiara, Antonino; Totani, Tonomori; Tanvir, Nial

    2016-05-01

    Gamma-ray Bursts (GRBs) are the most powerful explosions known, capable of outshining the rest of gamma-ray sky during their short-lived prompt emission. Their cosmological nature makes them the best tool to explore the final stages in the lives of very massive stars up to the highest redshifts. Furthermore, studying the emission from their low-energy counterparts (optical and infrared) via rapid spectroscopy, we have been able to pin down the exact location of the most distant galaxies as well as placing stringent constraints on their host galaxies and intervening systems at low and high-redshift (e.g. metallicity and neutral hydrogen fraction). In fact, each GRB spectrum contains absorption features imprinted by metals in the host interstellar medium (ISM) as well as the intervening intergalactic medium (IGM) along the line of sight. In this chapter we summarize the progress made using a large dataset of GRB spectra in understanding the nature of both these absorbers and how GRBs can be used to study the early Universe, in particular to measure the neutral hydrogen fraction and the escape fraction of UV photons before and during the epoch of re-ionization.

  6. Protein Adsorption in Microengraving Immunoassays

    PubMed Central

    Song, Qing

    2015-01-01

    Microengraving is a novel immunoassay forcharacterizing multiple protein secretions from single cells. During the immunoassay, characteristic diffusion and kinetic time scales τD and τK determine the time for molecular diffusion of proteins secreted from the activated single lymphocytes and subsequent binding onto the glass slide surface respectively. Our results demonstrate that molecular diffusion plays important roles in the early stage of protein adsorption dynamics which shifts to a kinetic controlled mechanism in the later stage. Similar dynamic pathways are observed for protein adsorption with significantly fast rates and rapid shifts in transport mechanisms when C0* is increased a hundred times from 0.313 to 31.3. Theoretical adsorption isotherms follow the trend of experimentally obtained data. Adsorption isotherms indicate that amount of proteins secreted from individual cells and subsequently captured on a clean glass slide surface increases monotonically with time. Our study directly validates that protein secretion rates can be quantified by the microengraving immunoassay. This will enable us to apply microengraving immunoassays to quantify secretion rates from 104–105 single cells in parallel, screen antigen-specific cells with the highest secretion rate for clonal expansion and quantitatively reveal cellular heterogeneity within a small cell sample. PMID:26501282

  7. Production of IgM rheumatoid factor by normal lymphocytes after stimulation with preparations containing IgM rheumatoid factor from patients with juvenile arthritis.

    PubMed Central

    Nesher, G; Moore, T L; Osborn, T G; Dorner, R W

    1991-01-01

    Preparations containing IgM rheumatoid factor (RF) and hidden IgM RF were isolated from the serum samples of nine patients with juvenile rheumatoid arthritis. Six of these preparations stimulated lymphocytes from normal donors to produce IgG and IgM, of which up to 11% had IgM RF activity. In contrast, the polyclonal activator pokeweed mitogen also stimulated IgM production, but only 1% had IgM RF activity. A relation between the activator and IgM RF or hidden IgM RF is suggested. This is based on the positive correlation between IgM RF concentration in these preparations and their ability to stimulate lymphocytes to produce IgG, IgM, and IgM RF. These data indicate that preparations from patients with juvenile rheumatoid arthritis containing IgM RF and hidden IgM RF are potent stimulants of lymphocytes from normal donors, with specific production of IgM RF. PMID:2015007

  8. IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens.

    PubMed

    Maglione, Paul J; Simchoni, Noa; Black, Samuel; Radigan, Lin; Overbey, Jessica R; Bagiella, Emilia; Bussel, James B; Bossuyt, Xavier; Casanova, Jean-Laurent; Meyts, Isabelle; Cerutti, Andrea; Picard, Capucine; Cunningham-Rundles, Charlotte

    2014-12-01

    IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans. PMID:25320238

  9. Determination of designer drug cross-reactivity on five commercial immunoassay screening kits.

    PubMed

    Regester, Laura E; Chmiel, Jeffrey D; Holler, Justin M; Vorce, Shawn P; Levine, Barry; Bosy, Thomas Z

    2015-03-01

    The detection of new designer drugs is often a difficult issue in forensic urine drug testing as immunoassays are the primary screening methodology for drugs of abuse in many of these laboratories. Cross-reactivity of compounds with immunoassay kits can either aid or complicate the detection of a variety of drug and drug metabolites. For instance, emerging designer drugs that share structural similarities to amphetamines and phencyclidine (PCP) have the potential to cross-react with assays designed to detect these compounds. This study evaluates the cross-reactivity of five commercially available immunoassay reagent kits for 94 designer drugs on a Roche/Hitachi Modular P automated screening instrument. The compounds used in this study are grouped by structural class as follows: 2,5-dimethoxyamphetamines, 2C (2,5-dimethoxyphenethylamines), β-keto amphetamines, substituted amphetamines, piperazines, α-pyrrolidinopropiophenones, tryptamines and PCP analogs. A drug concentration of 100 µg/mL was used to determine cross-reactivity for each assay and resulted in the following positive rates: Microgenics DRI(®) Ecstasy enzyme assay (19%), Microgenics DRI(®) Phencyclidine enzyme assay (20%), Lin-Zhi Methamphetamine enzyme immunoassay (39%), Siemens/Syva(®) EMIT(®)II Plus Amphetamines assay (43%) and CEDIA(®) DAU Amphetamine/Ecstasy assay (57%). Of the 94 designer drugs tested, 14% produced a negative response for all five kits. No designer drug used in this study generated a positive result for all five immunoassay kits. PMID:25492523

  10. Improved buprenorphine immunoassay performance after urine treatment with β-glucuronidase.

    PubMed

    Snyder, Marion L; Darragh, Alicia; Flood, James G; Jones, Jenny; Ropar, Kaitlin; Jarolim, Petr; Melanson, Stacy E F

    2014-01-01

    Buprenorphine (BUP), a semi-synthetic opioid analgesic, is increasingly prescribed for the treatment of chronic pain and opioid dependence. Urine immunoassay screening methods are available for monitoring BUP compliance and misuse; however, these screens may have poor sensitivity or specificity. We evaluated whether the pretreatment of urine with β-glucuronidase (BG) improves the sensitivity and overall accuracy of three BUP enzyme immunoassays when compared with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Urine samples sent to our laboratories for BUP testing (n = 114) were analyzed before and after BG pretreatment by cloned enzyme donor immunoassay (CEDIA), enzyme immunoassay (EIA) and homogenous EIA (HEIA) immunoassays using a common 5 ng/mL cutoff. Total BUP and norbuprenorphine (NBUP) concentrations were measured by LC-MS-MS as the reference method. Urine BG pretreatment improved EIA, HEIA and CEDIA sensitivities from 70, 82 and 94%, respectively, to 97% for each of the three methods, when compared with LC-MS-MS. While the specificity of the EIA and HEIA remained 100% after BG pretreatment, the specificity of the CEDIA decreased from 74 to 67%. Urine pretreatment with BG is recommended to improve sensitivity of the EIA and HEIA BUP screening methods. PMID:24802159

  11. False Positive Lyme Disease IgM Immunoblots in Children.

    PubMed

    Lantos, Paul M; Lipsett, Susan C; Nigrovic, Lise E

    2016-07-01

    In our cross-sectional sample of 7289 serologic tests for Lyme disease, we identified 167 instances of a positive IgM immunoblot but a negative IgG immunoblot test result. Considering that only 71% (95% CI 64%-78%) of patients had Lyme disease, a positive IgM immunoblot alone should be interpreted with caution to avoid over-diagnosis of Lyme disease. PMID:27157898

  12. Comparison of Three Automated Immunoassay Methods for the Determination of Epstein-Barr Virus-Specific Immunoglobulin M ▿

    PubMed Central

    Berth, Mario; Bosmans, Eugene

    2010-01-01

    In this study we compared the performances of three commercially available Epstein-Barr virus (EBV) immunoglobulin M (IgM) assays on highly automated immunoassay platforms: BioPlex 2200 (Bio-Rad Laboratories), Immulite 2000 (Siemens Healthcare Diagnostics), and Liaison (DiaSorin). As a confirmatory method, immunoblotting was performed. The specificity of the three EBV IgM assays was evaluated by testing 293 selected sera from patients with various infectious and noninfectious diseases. After the exclusion of 30 samples, the specificities were 96.2% for Liaison, 98.1% for Immulite, and 97.0% for BioPlex. For evaluation of the sensitivity, samples from 70 consecutive patients with a positive heterophile antibody test were examined, irrespective of clinical or biological findings. After the exclusion of six samples, the sensitivities were 89.1% for Liaison, 84.4% for Immulite, and 89.1% for BioPlex. Finally, in a prospective study performed with 500 samples obtained from consecutive patients and sent in by general practitioners, we also determined Epstein-Barr nuclear antigen IgG and viral capsid antigen IgG in a two-phase approach. Concordance of the EBV serologic status was 96.2% between Liaison and Immulite, 96.4% between Immulite and BioPlex, and 97.8% between BioPlex and Liaison. The three EBV IgM immunoassays that we evaluated have acceptable and comparable performances. PMID:20147496

  13. Multiplex Immunoassay Platforms Based on Shape-Coded Poly(ethylene glycol) Hydrogel Microparticles Incorporating Acrylic Acid

    PubMed Central

    Park, Saemi; Lee, Hyun Jong; Koh, Won-Gun

    2012-01-01

    A suspension protein microarray was developed using shape-coded poly(ethylene glycol) (PEG) hydrogel microparticles for potential applications in multiplex and high-throughput immunoassays. A simple photopatterning process produced various shapes of hydrogel micropatterns that were weakly bound to poly(dimethylsiloxane) (PDMS)-coated substrates. These micropatterns were easily detached from substrates during the washing process and were collected as non-spherical microparticles. Acrylic acids were incorporated into hydrogels, which could covalently immobilize proteins onto their surfaces due to the presence of carboxyl groups. The amount of immobilized protein increased with the amount of acrylic acid due to more available carboxyl groups. Saturation was reached at 25% v/v of acrylic acid. Immunoassays with IgG and IgM immobilized onto hydrogel microparticles were successfully performed with a linear concentration range from 0 to 500 ng/mL of anti-IgG and anti-IgM, respectively. Finally, a mixture of two different shapes of hydrogel microparticles immobilizing IgG (circle) and IgM (square) was prepared and it was demonstrated that simultaneous detection of two different target proteins was possible without cross-talk using same fluorescence indicator because each immunoassay was easily identified by the shapes of hydrogel microparticles. PMID:22969408

  14. Isolation and Characterization of IgM and IgY Antibodies from Plasma of Magellanic Penguins (Spheniscus magellanicus).

    PubMed

    Bizelli, Camila C; Silva, A Sandriana R; da Costa, Jessica D; Vanstreels, Ralph E T; Atzingen, Marina V; Santoro, Marcelo L; Fernandes, Irene; Catão-Dias, José L; Faquim-Mauro, Eliana L

    2015-03-01

    Infectious diseases such as aspergillosis, avian malaria, and viral infections are significant threats to the conservation of penguins, leading to morbidity and mortality of these birds both in captivity and in the wild. The immune response to such infectious diseases is dependent on different mechanisms mediated by cells and soluble components such as antibodies. Antibodies or immunoglobulins are glycoproteins that have many structural and functional features that mediate distinct effector immune functions. Three distinct classes of antibodies have been identified in birds: immunoglobulin A (IgA), immunoglobulin M (IgM), and immunoglobulin Y (IgY). In this study we aim to establish an efficient laboratory method to obtain IgM and IgY antibodies from plasma samples of healthy adult Magellanic penguins (Spheniscus magellanicus). The protocol was developed combining plasma delipidation, sequential precipitation with caprylic acid and ammonium sulfate, and size-exclusion chromatography. The efficiency of the protocol and the identity of the purified IgM and IgY antibodies were confirmed through enzyme-linked immunosorbent assay, Western blotting, one-dimensional and two-dimensional polyacrylamide gel electrophoresis, and lectin binding assay. Structural and physicochemical properties of IgM and IgY from Magellanic penguins were consistent with those of other avian species. This purification protocol will allow for more detailed studies on the humoral immunity of penguins and for the development of high specificity serologic assays to test Magellanic penguins for infectious pathogens. PMID:26292539

  15. Specific Noncompetitive Immunoassay for HT-2 Mycotoxin Detection.

    PubMed

    Arola, Henri O; Tullila, Antti; Kiljunen, Harri; Campbell, Katrina; Siitari, Harri; Nevanen, Tarja K

    2016-02-16

    Here we demonstrate a novel homogeneous one-step immunoassay, utilizing a pair of recombinant antibody antigen-binding fragments (Fab), that is specific for HT-2 toxin and has a positive readout. Advantages over the conventional competitive immunoassay formats such as enzyme-linked immunosorbent assay (ELISA) are the specificity, speed, and simplicity of the assay. Recombinant antibody HT2-10 Fab recognizing both HT-2 and T-2 toxins was developed from a phage display antibody library containing 6 × 10(7) different antibody clones. Specificity of the immunoassay was introduced by an anti-immune complex (IC) antibody binding the primary antibody-HT-2 toxin complex. When the noncompetitive immune complex assay was compared to the traditional competitive assay, an over 10-fold improvement in sensitivity was observed. Although the HT2-10 antibody has 100% cross-reactivity for HT-2 and T-2 toxins, the immune complex assay is highly specific for HT-2 alone. The assay performance with real samples was evaluated using naturally contaminated wheat reference material. The half-maximal effective concentration (EC50) value of the time-resolved fluorescence resonance energy transfer (TR-FRET) assay was 9.6 ng/mL, and the limit of detection (LOD) was 0.38 ng/mL (19 μg/kg). The labeled antibodies can be predried to the assay vials, e.g., microtiter plate wells, and readout is ready in 10 min after the sample application. PMID:26785138

  16. Competitive chemiluminescent anzyme immunoassay for vitamin B12 analysis in human milk.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent discoveries of matrix interferences by haptocorrin (HC) in human milk and serum show that past analyses of vitamin B12 in samples with high HC content might have been inaccurate (Lildballe et al., 2009; Carmel & Agrawal, 2012). We evaluated two competitive enzyme-binding immunoassays for seru...

  17. Application and validation of polybrominated diphenyl ethers immunoassay for environmental and food matrices.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A sensitive magnetic particle enzyme-linked immunoassay (ELISA) was developed to analyze polybrominated diphenyl ethers (PBDEs) in water, milk, fish, chicken and soil samples. The assay is rapid and can be used to analyze fifty samples in about one hour after sample cleanup. The assay has a limit ...

  18. IMMUNOASSAY METHODS FOR MEASURING ATRAZINE AND 3,5,6-TRICHLORO-2-PYRIDINOL IN FOODS

    EPA Science Inventory

    This chapter describes the use of enzyme-linked immunosorbent assay (ELISA) methods for the analysis of two potential environmental contaminants in food sample media, atrazine and 3,5,6-trichloro-2-pyridinol (3,5,6-TCP). Two different immunoassay formats are employed: a magnetic...

  19. Evaluation of two immunoassay procedures for drug testing in hair samples.

    PubMed

    Musshoff, F; Kirschbaum, K M; Graumann, K; Herzfeld, C; Sachs, H; Madea, B

    2012-02-10

    A preliminary initial enzyme-linked immunosorbent assay (LUCIO-Direct ELISA kit) and a preliminary DRI enzyme immunoassay were evaluated for drug detection in head hair with respect to lowered cutoff values recommended in Germany for the control of abstinence in cases of re-granting of drivers' licences. Following drug classes were included: cannabinoids, opiates, cocaine like substances, amphetamine, methamphetamine (and methylenedioxyamphetamines), methadone, and benzodiazepines. 759 analyses were performed using LUCIO-Direct ELISA kits and 936 analyses using DRI enzyme immunoassay tests. Sample size for each drug group and immunoassay test reached from 74 to 178. The LUCIO-Direct ELISA kit revealed a sensitivity of 91% for amphetamine up to 98% for methadone (methamphetamine 92%, cocaine 94%, opiates 94%, benzodiazepines 96%) and values of specificity of 72% for methadone up to 89% for amphetamine and benzodiazepines. The test was not useful for a preliminary screening for tetrahydrocannabinol (sensitivity of 65%) in consideration of a suggested cutoff of 0.02 ng/mg. The DRI enzyme immunoassay test was only useful for morphine and cocaine testing at low recommended new cutoff values (0.1 ng/mg) revealing sensitivities of 94% and 99%, respectively. PMID:21601388

  20. Pholcodine interference in the immunoassay for opiates in urine.

    PubMed

    Svenneby, G; Wedege, E; Karlsen, R L

    1983-01-01

    The excretion in urine after single oral therapeutic doses of morphine derivatives was analysed with radioimmunoassay (RIA) and homogeneous enzyme immunoassay (EMIT) for opiates. In contrast to the rapid excretion of ethylmorphine and codeine, pholcodine showed positive results for opiates 2-6 weeks after intake when the urines were analysed with the RIA-method. When analysed with the EMIT-method, positive results were obtained for pholcodine for approximately 10 days. As pholcodine is a common component in cough mixtures, its prolonged excretion could represent a hazard in interpreting the results from drug analyses of urines. PMID:6347841

  1. Validation of a Microsphere Immunoassay for Serological Leptospirosis Diagnosis in Human Serum by Comparison to the Current Gold Standard

    PubMed Central

    Wynwood, Sarah J.; Burns, Mary-Anne A.; Graham, Glenn C.; Weier, Steven L.; McKay, David B.; Craig, Scott B.

    2015-01-01

    A microsphere immunoassay (MIA) utilising Luminex xMap technology that is capable of determining leptospirosis IgG and IgM independently was developed. The MIA was validated using 200 human samples submitted for routine leptospirosis serology testing. The traditional microscopic agglutination (MAT) method (now 100 years old) suffers from a significant range of technical problems including a dependence on antisera which is difficult to source and produce, false positive reactions due to auto-agglutination and an inability to differentiate between IgG and IgM antibodies. A comparative validation method of the MIA against the MAT was performed and used to determine the ability of the MIA to detect leptospiral antibodies when compared with the MAT. The assay was able to determine samples in the reactive, equivocal and non-reactive ranges when compared to the MAT and was able to differentiate leptospiral IgG antibodies from leptospiral IgM antibodies. The MIA is more sensitive than the MAT and in true infections was able to detect low levels of antibody in the later stages of the acute phase as well as detect higher levels of IgM antibody earlier in the immune phase of the infection. The relatively low cost, high throughput platform and significantly reduced dependency on large volumes of rabbit antisera make this assay worthy of consideration for any microbiological assay that currently uses agglutination assays. PMID:25807009

  2. Intrathecal somatic hypermutation of IgM in multiple sclerosis and neuroinflammation.

    PubMed

    Beltrán, Eduardo; Obermeier, Birgit; Moser, Markus; Coret, Francisco; Simó-Castelló, María; Boscá, Isabel; Pérez-Miralles, Francisco; Villar, Luisa M; Senel, Makbule; Tumani, Hayrettin; Hohlfeld, Reinhard; Casanova, Bonaventura; Dornmair, Klaus

    2014-10-01

    Intrathecal oligoclonal bands of the cerebrospinal fluid are considered the most important immunological biomarkers of multiple sclerosis. They typically consist of clonally expanded IgG antibodies that underwent affinity maturation during sustained stimulation by largely unknown antigens. In addition, ∼40% of patients with multiple sclerosis have oligoclonal bands that consist of expanded IgM antibodies. We investigated the molecular composition of IgM- and IgG-chains from cerebrospinal fluid of 12 patients with multiple sclerosis, seven patients with other neurological diseases, and eight healthy control subjects by high-throughput deep-sequencing and single-cell PCR. Further, we studied the expression of activation-induced cytidine deaminase, the key enzyme for affinity maturation of antibodies, in cerebrospinal fluid samples of 16 patients. From the cerebrospinal fluid of two multiple sclerosis patients we isolated single B cells and investigated the co-expression of antibody chains with activation-induced cytidine deaminase. In striking contrast to IgM-chains from peripheral blood, IgM-chains from cerebrospinal fluid of patients with multiple sclerosis or neuroborreliosis showed a high degree of somatic hypermutation. We found a high content of mutations that caused amino acid exchanges as compared to silent mutations. In addition, more mutations were found in the complementarity determining regions of the IgM-chains, which interact with yet unknown antigens, as compared to framework regions. Both observations provide evidence for antigen-driven affinity maturation. Furthermore, single B cells from the cerebrospinal fluid of patients with multiple sclerosis co-expressed somatically hypermutated IgM-chains and activation-induced cytidine deaminase, an enzyme that is crucial for somatic hypermutation and class switch recombination of antibodies and is normally expressed during activation of B cells in germinal centres. Clonal tracking of particular IgM(+) B

  3. Out-of-Core Hydrodynamic Simulations of the IGM

    NASA Astrophysics Data System (ADS)

    Trac, H.; Pen, U.

    2003-12-01

    Probing the baryons in the intergalactic medium (IGM) through the Lyman alpha forest, the Sunyaev-Zeldovich effect, and the X-ray background is the next important task in cosmology. The evolution of the intergalactic medium is a numerically challenging problem to solve but advancements in hydrodynamic codes and computational techniques now make it tractable to simulate the IGM for the purpose of doing quantitative cosmology. We describe an out-of-core computing paradigm for very high-resolution simulations and a new code designed to handle the high Mach number dynamic range of the IGM. Out-of-core computation refers to the technique of using disk space as virtual memory and transferring data in and out of main memory at high I/O bandwidth. We present some results on the baryon budget and thermodynamic scaling relations from cosmological simulations with 20003 grid cells and 10003 dark matter particles.

  4. IMMUNOASSAYS FOR METAL IONS. (R824029)

    EPA Science Inventory

    Abstract

    Antibodies that recognize chelated forms of metal ions have been used to construct immunoassays for Cd(II), Hg(II), Pb(II), and Ni(II). In this paper, the format of these immunoassays is described and the binding properties of three monoclonal antibodies direc...

  5. Electrokinetic Microstrirring to Enhance Immunoassays

    NASA Astrophysics Data System (ADS)

    Feldman, Hope; Sigurdson, Marin; Meinhart, Carl

    2006-11-01

    Electrokinetic microstirring is used to improve the sensitivity of microfluidic heterogeneous immuno-sensors by enhancing the transport in diffusion-limited reactions. The AC electrokinetic force, Electrothermal Flow, is exploited to create a circular stirring fluid motion, thereby providing more binding opportunities between suspended and wall-immobilized molecules. This process can significantly reduce test times, important for both field-portable biosensors and for lab-based assays. A 2-D numerical simulation model is used to predict the effect of electrothermal flow on a heterogeneous immunoassay resulting from an AC potential applied to two parallel electrodes. The binding is increased by a factor of 7 for an applied voltage of 10 Vrms. The effect was investigated experimentally using a high affinity biotin-streptavidin reaction. Microstirred reaction rates were compared with passive reactions. The measurements show on average an order of magnitude increase in binding between immobilized biotin and fluorescently-labeled streptavidin after 5 minutes. Therefore, this technique shows significant promise for reducing incubation time and enhancing the sensitivity of immunoassays.

  6. Hyper IgM Syndrome with low IgM and thrombocytosis: an unusual case of immunodeficiency.

    PubMed

    Yousef, Ejaz; Arshad Alvi, M

    2016-09-01

    We report a 5 years old male child with low serum IgG, IgA and IgM levels, who presented with recurrent perianal and oral ulcers, intermittent fever, and protracted diarrhea. Despite the lack of typical respiratory symptoms, low serum IgM level and persistent thrombocytosis, an X-linked hyper-IgM syndrome (X-HIGM) was considered. Laboratory investigations revealed a diagnosis of hyper-IgM syndrome caused by CD40L deficiency. PMID:27608476

  7. Impact of IgM Antibodies on Cross-Protection against Pneumococcal Serogroups 6 and 19 after Immunization with 7-Valent Pneumococcal Conjugate Vaccine in Children.

    PubMed

    Cho, Hye-Kyung; Park, In Ho; Burton, Robert L; Kim, Kyung-Hyo

    2016-06-01

    Although it is well known that pneumococcal conjugate vaccines provide cross-protection against some vaccine-related serotypes, these mechanisms are still unclear. This study was performed to investigate the role of cross-protective IgM antibodies against vaccine-related serotypes 6A, 6C, and 19A induced in children aged 12-23 months after immunization with 7-valent pneumococcal conjugate vaccine (PCV7). We obtained serum samples from 18 Korean children aged 12-23 months after a PCV7 booster immunization. The serum IgG and IgM concentrations of serotypes 6B and 19F were measured by enzyme-linked immunosorbent assay (ELISA) in serum. The opsonic indices (OIs) against vaccine serotypes 6B and 19F and vaccine-related serotypes 6A, 6C, and 19A were determined by an opsonophagocytic killing assay (OPA) in IgM-depleted and control serum. Both IgG and IgM antibodies in ELISA and opsonic indices in OPA against serotypes 6B and 19F were demonstrated in the immune serum. IgM depletion decreased the OIs against vaccine serotypes 6B (geometric means of OIs (GMIs) of 3,009 vs. 1,396, 38% reduction) and 19F (1,117 vs. 750, 36% reduction). In addition, IgM depletion markedly decreased the OIs against vaccine-related serotypes 6A (GMIs of 961 vs. 329, 70% reduction), 6C (432 vs. 185, 72% reduction), and 19A (301 vs. 166, 58% reduction). The booster immunization PCV7 induced protective antibodies in the form of both IgG and IgM isotypes. IgM antibodies contributed to eliciting cross-protection against vaccine-related serotypes as well as against vaccine serotypes. PMID:27247505

  8. Impact of IgM Antibodies on Cross-Protection against Pneumococcal Serogroups 6 and 19 after Immunization with 7-Valent Pneumococcal Conjugate Vaccine in Children

    PubMed Central

    Burton, Robert L.

    2016-01-01

    Although it is well known that pneumococcal conjugate vaccines provide cross-protection against some vaccine-related serotypes, these mechanisms are still unclear. This study was performed to investigate the role of cross-protective IgM antibodies against vaccine-related serotypes 6A, 6C, and 19A induced in children aged 12-23 months after immunization with 7-valent pneumococcal conjugate vaccine (PCV7). We obtained serum samples from 18 Korean children aged 12-23 months after a PCV7 booster immunization. The serum IgG and IgM concentrations of serotypes 6B and 19F were measured by enzyme-linked immunosorbent assay (ELISA) in serum. The opsonic indices (OIs) against vaccine serotypes 6B and 19F and vaccine-related serotypes 6A, 6C, and 19A were determined by an opsonophagocytic killing assay (OPA) in IgM-depleted and control serum. Both IgG and IgM antibodies in ELISA and opsonic indices in OPA against serotypes 6B and 19F were demonstrated in the immune serum. IgM depletion decreased the OIs against vaccine serotypes 6B (geometric means of OIs (GMIs) of 3,009 vs. 1,396, 38% reduction) and 19F (1,117 vs. 750, 36% reduction). In addition, IgM depletion markedly decreased the OIs against vaccine-related serotypes 6A (GMIs of 961 vs. 329, 70% reduction), 6C (432 vs. 185, 72% reduction), and 19A (301 vs. 166, 58% reduction). The booster immunization PCV7 induced protective antibodies in the form of both IgG and IgM isotypes. IgM antibodies contributed to eliciting cross-protection against vaccine-related serotypes as well as against vaccine serotypes. PMID:27247505

  9. Novel immunoassay formats for integrated microfluidic circuits: diffusion immunoassays (DIA)

    NASA Astrophysics Data System (ADS)

    Weigl, Bernhard H.; Hatch, Anson; Kamholz, Andrew E.; Yager, Paul

    2000-03-01

    Novel designs of integrated fluidic microchips allow separations, chemical reactions, and calibration-free analytical measurements to be performed directly in very small quantities of complex samples such as whole blood and contaminated environmental samples. This technology lends itself to applications such as clinical diagnostics, including tumor marker screening, and environmental sensing in remote locations. Lab-on-a-Chip based systems offer many *advantages over traditional analytical devices: They consume extremely low volumes of both samples and reagents. Each chip is inexpensive and small. The sampling-to-result time is extremely short. They perform all analytical functions, including sampling, sample pretreatment, separation, dilution, and mixing steps, chemical reactions, and detection in an integrated microfluidic circuit. Lab-on-a-Chip systems enable the design of small, portable, rugged, low-cost, easy to use, yet extremely versatile and capable diagnostic instruments. In addition, fluids flowing in microchannels exhibit unique characteristics ('microfluidics'), which allow the design of analytical devices and assay formats that would not function on a macroscale. Existing Lab-on-a-chip technologies work very well for highly predictable and homogeneous samples common in genetic testing and drug discovery processes. One of the biggest challenges for current Labs-on-a-chip, however, is to perform analysis in the presence of the complexity and heterogeneity of actual samples such as whole blood or contaminated environmental samples. Micronics has developed a variety of Lab-on-a-Chip assays that can overcome those shortcomings. We will now present various types of novel Lab- on-a-Chip-based immunoassays, including the so-called Diffusion Immunoassays (DIA) that are based on the competitive laminar diffusion of analyte molecules and tracer molecules into a region of the chip containing antibodies that target the analyte molecules. Advantages of this

  10. Immunoassay based water quality analysis: A new tool for drinking water supply management

    SciTech Connect

    Kostyshyn, C.R.; Brown, W.; Hervey, E.; Hull, C.

    1996-11-01

    The recent availability of enzyme-linked immunosorbent assay (ELISA) tests for the analysis of organic environmental contaminants provides drinking water utility managers and operators with a new tool for managing treatment operations and monitoring source watersheds. Immunoassay technology permits rapid, inexpensive and accurate in-plant testing of many SDWA regulated organic contaminants at concentrations well below established MCL`s. Analytical testing which would not be practicable due to the high cost or long turnaround time limitations of conventional testing methods is now being performed using immunoassay based analysis. Water quality data generated using immunoassay based methods are being utilized by drinking water utilities as an integral part of source watershed management programs, process operations optimization efforts, pro-active raw and finished water testing programs, and flood and incident response management.

  11. Mycobacterium tuberculosis Lipolytic Enzymes as Potential Biomarkers for the Diagnosis of Active Tuberculosis

    PubMed Central

    Brust, Belinda; Lecoufle, Mélanie; Tuaillon, Edouard; Dedieu, Luc; Canaan, Stéphane; Valverde, Viviane; Kremer, Laurent

    2011-01-01

    Background New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. Methods Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. Results A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. Conclusion These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high

  12. [X-linked hyper-IGM syndrome associated to sclerosing cholangitis and gallbladder neoplasm: clinical case].

    PubMed

    Rodríguez, Cristián; Carrión, Flavio; Marinovic, María Angélica; Chávez, Eduardo; Preisler, Jessica; Pooley, Francisco; Futatani, Takeshi; Ochs, Hans D

    2003-03-01

    We report a 11 years old male diagnosed as a X-linked hyper-IgM syndrome that presented with recurrent infections and sclerosing cholangitis and later developed a gallbladder cancer. Immunological evaluation showed decreased levels of serum IgG and IgA with elevated levels of IgM. Study of CD40 ligand expression on mitogen activated peripheral blood mononuclear cells revealed total absence of this marker on T lymphocytes. Molecular analysis detected, in the patient and his mother, a nonsense mutation in exon 1 of the transmembrane segment of the CD40 ligand. He also presented elevation of alkaline phosphatases and mild elevation of liver enzymes. Liver biopsy demonstrated the presence of idiopathic sclerosing cholangitis. The patient was started on monthly IVIG therapy at 400 mg/kg, as well as ursodeoxycholic acid and vitamin E, with normalization of his IgG and IgM levels a decrease in the incidence of infections and normalization of liver function. Three years after diagnosis, we detected the presence of polyps inside the gallbladder that were reported at biopsy as adenocarcinoma. He underwent hepatic bisegmentectomy (VI B-V) and local lymphadenectomy. PMID:12790080

  13. Preparation of horseradish peroxidase hydrazide and its use in immunoassay.

    PubMed

    Shrivastav, Tulsidas G

    2003-01-01

    Preparation of horseradish peroxidase (HRP) hydrazide that is HRP linked to adipic acid dihydrazide (HRP-ADH) and its use in enzyme immunoassay (EIA) is described. In this new strategy, horseradish peroxidase was conjugated to adipic acid dihydrazide using a carbodiimide coupling method. The resulting HRP-ADH was then coupled to cortisol-21-hemisuccinate (Cortisol-21-HS) to prepare enzyme conjugate. The prepared cortisol-21-HS coupled ADH-HRP (Cortisol-21-HS-ADH-HRP) enzyme conjugate was used for the development of an enzyme linked immunosorbent assay (ELISA) for direct estimation of cortisol. To the cortisol antibody coated microtiter wells, standard or serum samples (50 microL), along with cortisol-21-HS-ADH-HRP enzyme conjugate (100 microL) were incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using tetramethyl benzidine/hydrogen peroxide (TMB/H2O2) as substrate. The sensitivity of the assay was 0.05 microg/dL and the analytical recovery ranged from 92.9 to 101.7%. PMID:12953974

  14. Development of sub-micron patterned carbon electrodes for immunoassays.

    PubMed

    Dontha, N; Nowall, W B; Kuhr, W G

    1999-02-01

    Sub-micron sized domains of a carbon surface are derivatized with antibodies using biotin/avidin technology. These sites are spatially-segregated from, and directly adjacent to, electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains are kept to a minimum (e.g. less than a micron) to maintain the high sensitivity required for the measurement of enzyme-linked cofactors in an enzyme-linked immunoassay (ELISA). This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This allows the construction of microscopic arrays of active ELISA sites on a carbon substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators; thus maximizing sensitivity and detection of the enzyme mediator. The carbon electrode surface is characterized with respect to its chemical structure and electron transfer properties following each step of the antibody immobilization process. The characterization of specific modifications of micron regions of the carbon surface requires analytical methodology that has both high spatial resolution and sensitivity. We have used fluorescence microscopy with a cooled CCD imaging system to visualize the spatial distribution of enzyme immobilization sites (indicated by fluorescence from Texas-Red labeled antibody) across the carbon surface. The viability of the enzyme attached to the surface in this manner was demonstrated by imaging the distribution of an insoluble, fluorescent product. PMID:10698570

  15. The development of immunoassays for detection of chemical warfare agents

    SciTech Connect

    Lenz, D.E.

    1995-06-01

    With the advent of enzyme linked immunoabsorbant assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were reported as capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detecting soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition, these antibodies were highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies reported an extension of the level of sensitivity to -80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These antibodies offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances.

  16. [Laser-time-resolved fluorescence spectroscopy in immunoassays].

    PubMed

    Pan, L; Du, J; Xie, W; Du, Q; Yun, Q

    2000-06-01

    This paper described a laser-excited time-resolved fluoroimmunoassay set. It made lanthanide ion to couple the anhydrde of diethylenetriaminepentaacetic acid (DTPAA) for labeling antibodies. The experiment used polystyrene tap coated with HCV antigen as the solid phase and a chelate of the rare earth metal europium as fluorescent label. A nitrogen laser beam was used to excite the Eu3- chelates and after 60 microseconds delay time, the emission fluorescence was measured. Background fluorescence of short lifetimes caused by serum components and Raman scattering can be eliminated by set the delay time. In the system condition, fluorescent spectra and fluorescent lifetimes of Eu3+ beta-naphthoyltrifluroacetone (NTA) chelates were measured. The fluorescent lifetime value is 650 microseconds. The maximum emission wavelength is 613 nm. The linear range of europium ion concentration is 1 x 10(-7)-1 x 10(-11) g.mL-1 and the detection limit is 1 x 10(-13) g.mL-1. The relative standard deviation of determination (n = 12) for samples at 0.01 ng.mL-1 magnitude is 6.4%. Laser-TRFIA was also found to be suitable for diagnosis of HCV. The sensitivity and specificity were comparable to enzyme immunoassay. The result was obtained with laser-TRFIA for 29 human correlated well with enzyme immunoassay. PMID:12958930

  17. Inter-laboratory Variability of Current Immunoassay Methods for Tacrolimus among Japanese Hospitals.

    PubMed

    Miura, Masatomo; Masuda, Satohiro; Egawa, Hiroto; Yuzawa, Kenji; Matsubara, Kazuo

    2016-08-01

    The aim of this study was to assess inter-hospital laboratory variability (coefficient of variation; CV) of immunoassay methods for tacrolimus and the comparability of control samples and results obtained by immunoassay measurements. One hundred seven hospital laboratories routinely performing therapeutic drug monitoring (TDM) of tacrolimus participated in the study. Thirteen spiked samples with known tacrolimus concentrations in the range of 0-26.0 ng/mL were prepared. Each spiked sample was analyzed according to the manufacturer's instructions using an affinity column-mediated immunoassay (ACMIA) on a Dimension(®) analyzer, the enzyme multiplied immunoassay technique (EMIT) on a Viva-E(®) analyzer, a chemiluminescent enzyme immunoassay (CLIA) on the Architect(®) system, and the electro-chemiluminescence immunoassay (ECLIA) on a cobas(®) analyzer. The 20% coefficient of variation values for the CLIA, ACMIA, EMIT, and ECLIA assays in the hospital laboratories were 1.82, 5.36, 4.59, and 0.89 ng/mL, respectively. CLIA and ECLIA had positive biases at concentrations of tacrolimus above 12 ng/mL relative to the spiked concentration, whereas the assay bias for ACMIA tended to be more negative at concentrations of tacrolimus above 6 ng/mL. EMIT had positive biases over the wide concentration range of 0.0-26.0 ng/mL (mean of mean errors 1.224). CLIA and ECLIA provided adequate precision at the target tacrolimus concentration of 3.0 ng/mL, whereas ACMIA and EMIT were unable to respond to target concentrations between 3.0 and 5.0 ng/mL for renal transplant recipients. Appropriate assessment of tacrolimus concentration by an assay having higher sensitivity, precision, and accuracy is necessary to ensure long-term survival of transplant recipients receiving tacrolimus. PMID:27263473

  18. A reassessment of IgM memory subsets in humans

    PubMed Central

    Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M.; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K.; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-01-01

    From paired blood and spleen samples from three adult donors we performed high-throughput V-h sequencing of human B-cell subsets defined by IgD and CD27 expression: IgD+CD27+ (“MZ”), IgD−CD27+(“memory”, including IgM (“IgM-only”), IgG and IgA) and IgD−CD27− cells (“double-negative”, including IgM, IgG and IgA). 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for Vh-gene mutation, H-CDR3-length, and Vh/Jh usage, comparing these different characteristics with all sequences from their subset of origin, for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency, and lower Vh4 and higher Jh6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (between MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The “IgM-only” subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27+ switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells, and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

  19. A Reassessment of IgM Memory Subsets in Humans.

    PubMed

    Bagnara, Davide; Squillario, Margherita; Kipling, David; Mora, Thierry; Walczak, Aleksandra M; Da Silva, Lucie; Weller, Sandra; Dunn-Walters, Deborah K; Weill, Jean-Claude; Reynaud, Claude-Agnès

    2015-10-15

    From paired blood and spleen samples from three adult donors, we performed high-throughput VH sequencing of human B cell subsets defined by IgD and CD27 expression: IgD(+)CD27(+) ("marginal zone [MZ]"), IgD(-)CD27(+) ("memory," including IgM ["IgM-only"], IgG and IgA) and IgD(-)CD27(-) cells ("double-negative," including IgM, IgG, and IgA). A total of 91,294 unique sequences clustered in 42,670 clones, revealing major clonal expansions in each of these subsets. Among these clones, we further analyzed those shared sequences from different subsets or tissues for VH gene mutation, H-CDR3-length, and VH/JH usage, comparing these different characteristics with all sequences from their subset of origin for which these parameters constitute a distinct signature. The IgM-only repertoire profile differed notably from that of MZ B cells by a higher mutation frequency and lower VH4 and higher JH6 gene usage. Strikingly, IgM sequences from clones shared between the MZ and the memory IgG/IgA compartments showed a mutation and repertoire profile of IgM-only and not of MZ B cells. Similarly, all IgM clonal relationships (among MZ, IgM-only, and double-negative compartments) involved sequences with the characteristics of IgM-only B cells. Finally, clonal relationships between tissues suggested distinct recirculation characteristics between MZ and switched B cells. The "IgM-only" subset (including cells with its repertoire signature but higher IgD or lower CD27 expression levels) thus appear as the only subset showing precursor-product relationships with CD27(+) switched memory B cells, indicating that they represent germinal center-derived IgM memory B cells and that IgM memory and MZ B cells constitute two distinct entities. PMID:26355154

  20. [FT4 immunoassay interference : A case report].

    PubMed

    Chaabouni, Khansa; Hargafi, Khaoula; Elleuch, Aida; Messedi, Mariem; Turki, Mouna; Lahyani, Amina; Ayedi, Fatma

    2015-04-01

    Measurement of thyrotropin and free thyroxin made using immunoassays are usually needed in clinical endocrinology. Here, we report a case of a patient with type 2 diabetes who presented a weight loss. To eliminate hyperthyroidism, thyroid function tests were performed. Free thyroxin (FT4) was decreased using two automated immunoassays TOSOH AIA 1800 and Roche ELECSYS 2010, with a normal thyrotropin value. Thyroid function tests repeated a month later were normal. The patient's history revealed contact with sheep, which may partly explain the interference. Investigations into the patient's serum were carried out using both the PEG test and dilution test. Interference factors were probably antibodies. Despite progress in immunoassays, we should be aware of interference occurrence since it can lead to false results, unnecessary investigations and incorrect treatment. Thus, simple tests must be carried out as if interference in immunoassays were suspected. Dilutions and PEG tests are generally performed as first line investigations. PMID:26375746

  1. A fluorometric enzyme immunoassay for follitropin and lutropin.

    PubMed

    Huguet, J; Bonnin, M R; Guillén, E; Navarro, M A

    1991-09-01

    The analytical performance of the Stratus (Baxter) automatic analyser for human lutropin and follitropin determination was evaluated and compared with that of an immunoradiometric assay. Within-run and between-run imprecision were lower than those of the immunoradiometric assay. No significant differences were obtained in the parallelism study. A good and significant correlation was obtained between both methods. Results obtained for both methods were not interchangeable. The Stratus automatic analyser can replace radioisotopic methods, thus eliminating radioactive hazards. PMID:1760486

  2. Impact of anti-PEG IgM antibodies on the pharmacokinetics of pegylated asparaginase preparations in mice.

    PubMed

    Poppenborg, Sabine M; Wittmann, Julia; Walther, Wolfgang; Brandenburg, Gunda; Krähmer, Ralf; Baumgart, Joachim; Leenders, Frank

    2016-08-25

    The potential impact of pre-existing anti-PEG antibodies on the asparaginase activity kinetics of two pegylated l-asparaginase preparations - pegylated recombinant l-asparaginase (PEG-rASNase MC0609) and pegaspargase (pegylated Escherichia colil-asparaginase) - was investigated in immune competent, naïve B6D2F1-hybrid mice. To generate anti-PEG antibodies, mice were pre-sensitised by repeated injections of 40kDa PEG-Diol without being conjugated to a carrier. Successful PEG-Diol pre-sensitisation was verified by analysis of anti-PEG antibody titers in serum. 88-100% of animals developed PEG-specific anti-PEG IgM antibodies after PEG-Diol pre-sensitisation. All animals positive for anti-PEG IgM antibodies and control animals (without prior PEG-Diol pre-sensitisation) were treated once with PEG-rASNase MC0609 or pegaspargase, and asparaginase enzyme activity levels and immunogenicity of both preparations were analysed. Known serum asparaginase activity profiles were measured after treatment with PEG-rASNase MC0609 or pegaspargase in all treatment groups. No rapid decrease of asparaginase activity was observed - irrespective of successful PEG-Diol pre-sensitisation and presence of acquired anti-drug-IgG and/or anti-PEG IgM antibodies. In conclusion, the pharmacokinetics of pegylated l-asparaginase was unaffected by the presence of pre-existing anti-PEG IgM antibodies in immune competent B6D2F1-hybrid mice Probably the titre or affinity of these anti-PEG IgM antibodies were too low to influence the pharmacokinetics of PEG-rASNase MC0609 or pegaspargase or anti-PEG IgM antibodies bound to PEG-ASNase without neutralising capabilities. Thus, early loss of asparaginase activity as observed in serum of ALL patients is a complex process and cannot be explained solely by the existence of pre-existing anti-PEG antibodies. PMID:27292820

  3. IGM Heating and AGN activity in Fossil Galaxy Groups

    NASA Astrophysics Data System (ADS)

    Miraghaei, H.; Khosroshahi, H. G.; Klöckner, H.-R.; Ponman, T. J.; Jetha, N. N.; Raychaudhury, S.

    2014-07-01

    Fossil galaxy groups are energetically and morphologically ideal environments to study the intergalactic medium (IGM) heating, because their inter-galactic gas is undisturbed due to the lack of recent group scale mergers. We study the role of active galactic nuclei (AGN) in heating the IGM in a sample of five fossil galaxy groups by employing properties at 610 MHz and 1.4 GHz. We find that two of the dominant galaxies in fossil groups, ESO 3060170 and RX J1416.4+2315, are associated with the radio lobes. We evaluate the PdV work of the radio lobes and their corresponding heating power and compare to the X-ray emission loss within cooling radius. Our results show that the power due to mechanical heating is not sufficiently high to suppress the cooling.

  4. Fast and Sensitive Detection of Enteropathogenic Yersinia by Immunoassays

    PubMed Central

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe

    2014-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 103 CFU/ml to 8.8 × 104 CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 105 CFU/ml to 106 CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  5. Fast and sensitive detection of enteropathogenic Yersinia by immunoassays.

    PubMed

    Laporte, Jérôme; Savin, Cyril; Lamourette, Patricia; Devilliers, Karine; Volland, Hervé; Carniel, Elisabeth; Créminon, Christophe; Simon, Stéphanie

    2015-01-01

    Yersinia enterocolitica and Yersinia pseudotuberculosis, the two Yersinia species that are enteropathogenic for humans, are distributed worldwide and frequently cause diarrhea in inhabitants of temperate and cold countries. Y. enterocolitica is a major cause of foodborne disease resulting from consumption of contaminated pork meat and is further associated with substantial economic cost. However, investigation of enteropathogenic Yersinia species is infrequently performed routinely in clinical laboratories because of their specific growth characteristics, which make difficult their isolation from stool samples. Moreover, current isolation procedures are time-consuming and expensive, thus leading to underestimates of the incidence of enteric yersiniosis, inappropriate prescriptions of antibiotic treatments, and unnecessary appendectomies. The main objective of the study was to develop fast, sensitive, specific, and easy-to-use immunoassays, useful for both human and veterinary diagnosis. Monoclonal antibodies (MAbs) directed against Y. enterocolitica bioserotypes 2/O:9 and 4/O:3 and Y. pseudotuberculosis serotypes I and III were produced. Pairs of MAbs were selected by testing their specificity and affinity for enteropathogenic Yersinia and other commonly found enterobacteria. Pairs of MAbs were selected to develop highly sensitive enzyme immunoassays (EIAs) and lateral flow immunoassays (LFIs or dipsticks) convenient for the purpose of rapid diagnosis. The limit of detection of the EIAs ranged from 3.2 × 10(3) CFU/ml to 8.8 × 10(4) CFU/ml for pathogenic serotypes I and III of Y. pseudotuberculosis and pathogenic bioserotypes 2/O:9 and 4/O:3 of Y. enterocolitica and for the LFIs ranged from 10(5) CFU/ml to 10(6) CFU/ml. A similar limit of detection was observed for artificially contaminated human feces. PMID:25355759

  6. Smart CuS Nanoparticles as Peroxidase Mimetics for the Design of Novel Label-Free Chemiluminescent Immunoassay.

    PubMed

    Yang, Zhanjun; Cao, Yue; Li, Juan; Lu, Mimi; Jiang, Zhikang; Hu, Xiaoya

    2016-05-18

    In the present work, a novel label-free chemiluminescent (CL) immunoassay method was designed by employing smart CuS nanoparticles (CuSNPs) as peroxidase mimetics. The CuSNPs were synthesized through a simple coprecipitation method, and showed high catalytic activity and stability. This efficient label-free CL immunoassay could be easily achieved through a simple strategy. First, CuSNPs dispersed in chitosan were modified on the epoxy-functionalized glass slide to form a solid CL signal interface. Streptavidin was then used to functionalize CuSNPs to capture the biotinylated antibody, further producing a sensing interface. After online incubation with antigen molecules, the formed antibody-antigen complex on the biosensing substrate could prevent the diffusion channel of CL substrate toward the signal interface, and restrained the mimic enzyme-catalyzed CL reaction, finally resulting in the decrease of CL signals of the assay system. Compared to the label-based CL immunoassay, the proposed label-free assay mode is more simple, cheap and fast. Using a model analyte alpha-fetoprotein, the label-free CL immunoassay method had a linear range of 0.1-60 ng/mL and a low detection limit of 0.07 ng/mL. Moreover, the peroxidase mimetic-based label-free CL immunoassay system showed good specificity, acceptable repeatability, and good accuracy. The study provided a promising strategy for the development of highly efficient label-free CL immunoassay system. PMID:27137349

  7. Evaluation of commercially available diagnostic tests for the detection of dengue virus NS1 antigen and anti-dengue virus IgM antibody.

    PubMed

    Hunsperger, Elizabeth A; Yoksan, Sutee; Buchy, Philippe; Nguyen, Vinh Chau; Sekaran, Shamala Devi; Enria, Delia A; Vazquez, Susana; Cartozian, Elizabeth; Pelegrino, Jose L; Artsob, Harvey; Guzman, Maria G; Olliaro, Piero; Zwang, Julien; Guillerm, Martine; Kliks, Susie; Halstead, Scott; Peeling, Rosanna W; Margolis, Harold S

    2014-10-01

    Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60-75% and specificity 71-80%; NS1 RDT sensitivity was 38-71% and specificity 76-80%; the IgM anti-DENV RDTs sensitivity was 30-96%, with a specificity of 86-92%, and IgM anti-DENV ELISA sensitivity was 96-98% and specificity 78-91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88-94%. PMID:25330157

  8. Evaluation of Commercially Available Diagnostic Tests for the Detection of Dengue Virus NS1 Antigen and Anti-Dengue Virus IgM Antibody

    PubMed Central

    Hunsperger, Elizabeth A.; Yoksan, Sutee; Buchy, Philippe; Nguyen, Vinh Chau; Sekaran, Shamala Devi; Enria, Delia A.; Vazquez, Susana; Cartozian, Elizabeth; Pelegrino, Jose L.; Artsob, Harvey; Guzman, Maria G.; Olliaro, Piero; Zwang, Julien; Guillerm, Martine; Kliks, Susie; Halstead, Scott; Peeling, Rosanna W.; Margolis, Harold S.

    2014-01-01

    Commercially available diagnostic test kits for detection of dengue virus (DENV) non-structural protein 1 (NS1) and anti-DENV IgM were evaluated for their sensitivity and specificity and other performance characteristics by a diagnostic laboratory network developed by World Health Organization (WHO), the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Pediatric Dengue Vaccine Initiative (PDVI). Each network laboratory contributed characterized serum specimens for the panels used in the evaluation. Microplate enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT formats) were represented by the kits. Each ELISA was evaluated by 2 laboratories and RDTs were evaluated by at least 3 laboratories. The reference tests for IgM anti-DENV were laboratory developed assays produced by the Armed Forces Research Institute for Medical Science (AFRIMS) and the Centers for Disease Control and Prevention (CDC), and the NS1 reference test was reverse transcriptase polymerase chain reaction (RT-PCR). Results were analyzed to determine sensitivity, specificity, inter-laboratory and inter-reader agreement, lot-to-lot variation and ease-of-use. NS1 ELISA sensitivity was 60–75% and specificity 71–80%; NS1 RDT sensitivity was 38–71% and specificity 76–80%; the IgM anti-DENV RDTs sensitivity was 30–96%, with a specificity of 86–92%, and IgM anti-DENV ELISA sensitivity was 96–98% and specificity 78–91%. NS1 tests were generally more sensitive in specimens from the acute phase of dengue and in primary DENV infection, whereas IgM anti-DENV tests were less sensitive in secondary DENV infections. The reproducibility of the NS1 RDTs ranged from 92-99% and the IgM anti-DENV RDTs from 88–94%. PMID:25330157

  9. IgM rheumatoid factor (RF), IgA RF, IgE RF, and IgG RF detected by ELISA in rheumatoid arthritis.

    PubMed Central

    Gioud-Paquet, M; Auvinet, M; Raffin, T; Girard, P; Bouvier, M; Lejeune, E; Monier, J C

    1987-01-01

    One hundred patients with rheumatoid arthritis (RA), of whom 73 were seropositive by latex or Waaler-Rose (WR) assays, or both, 100 healthy subjects, and 102 diseased controls (22 patients with systemic lupus erythematosus (SLE) and 80 with bronchial asthma) were evaluated for the presence of IgM rheumatoid factor (RF), IgA RF, IgE RF, and IgG RF by an enzyme linked immunosorbent assay (ELISA). Ninety two per cent, 65%, 68%, and 66% of the patients with RA were found to be positive for IgM, IgA, IgE, and IgG respectively. A positive correlation existed between the levels of IgM RF and IgA RF on the one hand and disease activity on the other, and the levels of IgM RF and IgA RF correlated with the levels of circulating immune complexes as measured by a C1q binding assay. The presence of extra-articular features also correlated positively with the levels of IgA RF and IgE RF. Five out of six patients with Sjögren's syndrome had very high levels of IgA RF. Of 47 patients typed for HLA-DR, DR1 and DR2 were significantly more frequent in those with the highest levels of IgM RF. Conversely, DR3 was associated with low levels or absence of IgA RF and IgE RF. These results suggest that immune response genes may regulate the level of different RF isotypes. The frequencies of IgM, IgA, IgE, and IgG RF were 59%, 36%, 9%, and 27% respectively in SLE and 25%, 2.5%, 70%, and 59% in bronchial asthma. PMID:3813676

  10. A Rapid, Multiplexed, High-Throughput Flow-Through Membrane Immunoassay: A Convenient Alternative to ELISA

    PubMed Central

    Ramachandran, Sujatha; Singhal, Mitra; McKenzie, Katherine G.; Osborn, Jennifer L.; Arjyal, Amit; Dongol, Sabina; Baker, Stephen G.; Basnyat, Buddha; Farrar, Jeremy; Dolecek, Christiane; Domingo, Gonzalo J.; Yager, Paul; Lutz, Barry

    2013-01-01

    This paper describes a rapid, high-throughput flow-through membrane immunoassay (FMIA) platform. A nitrocellulose membrane was spotted in an array format with multiple capture and control reagents for each sample detection area, and assay steps were carried out by sequential aspiration of sample and reagents through each detection area using a 96-well vacuum manifold. The FMIA provides an alternate assay format with several advantages over ELISA. The high surface area of the membrane permits high label concentration using gold labels, and the small pores and vacuum control provide rapid diffusion to reduce total assay time to ~30 min. All reagents used in the FMIA are compatible with dry storage without refrigeration. The results appear as colored spots on the membrane that can be quantified using a flatbed scanner. We demonstrate the platform for detection of IgM specific to lipopolysaccharides (LPS) derived from Salmonella Typhi. The FMIA format provides analytical results comparable to ELISA in less time, provides integrated assay controls, and allows compensation for specimen-to-specimen variability in background, which is a particular challenge for IgM assays. PMID:26835678

  11. Sensitive chemiluminescent immunoassay of triclopyr by digital image analysis.

    PubMed

    Díaz, Aurora N; Sánchez, Francisco G; Baro, Enrique N; Díaz, Ana F G; Aguilar, Alfonso; Algarra, Manuel

    2012-08-15

    An image based detection of chemiluminescence enzyme-linked immunosorbent assay (CL-ELISA) for the quantification of triclopyr has been developed. The immunoassay was an indirect competitive immunoassay with an anti-rabbit secondary antibody conjugated to horseradish peroxidase (HRP). Chemiluminescence was produced by the luminol/H(2)O(2)/HRP reaction, detected by a monochrome video CCD camera and digitized with an Imagraph IC-PCI frame grabber using a custom program developed in C(++) (Microsoft Visual C(++) 6.0). Two main improvements are reported in the data processing software: the implementation of a circular mesh covering the perimeter of each well, eliminating diffuse light from the neighboring wells, and the use of volume (the integration of light intensity of all pixels that define a well) as an analytical signal instead of CL intensity or area (as usual in commercial plate readers) to improve precision for normalization of the total light output. The standard curve was produced for 0.01-10 ng/L triclopyr. The limit of detection was 0.8 ng/L and the variation coefficient was 3.07% (n=10, P=0.05). PMID:22841045

  12. Detection of the Antimicrobial Triclosan in Environmental Samples by Immunoassay.

    PubMed

    Ahn, Ki Chang; Ranganathan, Anupama; Bever, Candace S; Hwang, Sung Hee; Holland, Erika B; Morisseau, Kevin; Pessah, Isaac N; Hammock, Bruce D; Gee, Shirley J

    2016-04-01

    A sensitive, competitive enzyme-linked immunosorbent assay (ELISA) for the detection of the antimicrobial triclosan (TCS; 2,4,4'-trichloro-2'-hydroxydiphenyl ether) was developed. Novel immunizing haptens were synthesized by derivatizing at the 4-Cl position of the TCS molecule. Compounds derived from substitutions at 4'-Cl and that replaced the 2'-OH with a Cl atom were designed as unique coating antigen haptens. Polyclonal rabbit antisera were screened against the coating antigen library to identify combinations of immunoreagents resulting in the most sensitive assays. The most sensitive assay identified was one utilizing antiserum no. 1155 and a heterologous competitive hapten, where the 2'-OH group was substituted with a Cl atom. An IC50 value and the detection range for TCS in assay buffer were 1.19 and 0.21-6.71 μg/L, respectively. The assay was selective for TCS, providing low cross-reactivity (<5%) to the major metabolites of TCS and to brominated diphenyl ether-47. A second assay utilizing a competitive hapten containing Br instead of Cl substitutions was broadly selective for both brominated and chlorinated diphenylethers. Using the most sensitive assay combination, we measured TCS concentrations in water samples following dilution. Biosolid samples were analyzed following the dilution of a simple solvent extract. The immunoassay results were similar to those determined by LC-MS/MS. This immunoassay can be used as a rapid and convenient tool to screen for human and environmental exposure. PMID:26937944

  13. Multiplex Detection of Plant Pathogens Using a Microsphere Immunoassay Technology

    PubMed Central

    Charlermroj, Ratthaphol; Himananto, Orawan; Seepiban, Channarong; Kumpoosiri, Mallika; Warin, Nuchnard; Oplatowska, Michalina; Gajanandana, Oraprapai; Grant, Irene R.; Karoonuthaisiri, Nitsara; Elliott, Christopher T.

    2013-01-01

    Plant pathogens are a serious problem for seed export, plant disease control and plant quarantine. Rapid and accurate screening tests are urgently required to protect and prevent plant diseases spreading worldwide. A novel multiplex detection method was developed based on microsphere immunoassays to simultaneously detect four important plant pathogens: a fruit blotch bacterium Acidovorax avenae subsp. citrulli (Aac), chilli vein-banding mottle virus (CVbMV, potyvirus), watermelon silver mottle virus (WSMoV, tospovirus serogroup IV) and melon yellow spot virus (MYSV, tospovirus). An antibody for each plant pathogen was linked on a fluorescence-coded magnetic microsphere set which was used to capture corresponding pathogen. The presence of pathogens was detected by R-phycoerythrin (RPE)-labeled antibodies specific to the pathogens. The assay conditions were optimized by identifying appropriate antibody pairs, blocking buffer, concentration of RPE-labeled antibodies and assay time. Once conditions were optimized, the assay was able to detect all four plant pathogens precisely and accurately with substantially higher sensitivity than enzyme-linked immunosorbent assay (ELISA) when spiked in buffer and in healthy watermelon leaf extract. The assay time of the microsphere immunoassay (1 hour) was much shorter than that of ELISA (4 hours). This system was also shown to be capable of detecting the pathogens in naturally infected plant samples and is a major advancement in plant pathogen detection. PMID:23638044

  14. Progress toward multiplexed sample-to-result detection in low resource settings using microfluidic immunoassay cards.

    PubMed

    Lafleur, Lisa; Stevens, Dean; McKenzie, Katherine; Ramachandran, Sujatha; Spicar-Mihalic, Paolo; Singhal, Mitra; Arjyal, Amit; Osborn, Jennifer; Kauffman, Peter; Yager, Paul; Lutz, Barry

    2012-03-21

    In many low resource settings multiple diseases are endemic. There is a need for appropriate multi-analyte diagnostics capable of differentiating between diseases that cause similar clinical symptoms. The work presented here was part of a larger effort to develop a microfluidic point-of-care system, the DxBox, for sample-to-result differential diagnosis of infections that present with high rapid-onset fever. Here we describe a platform that detects disease-specific antigens and IgM antibodies. The disposable microfluidic cards are based on a flow-through membrane immunoassay carried out on porous nitrocellulose, which provides rapid diffusion for short assay times and a high surface area for visual detection of colored assay spots. Fluid motion and on-card valves were driven by a pneumatic system and we present designs for using pneumatic control to carry out assay functions. Pneumatic actuation, while having the potential advantage of inexpensive and robust hardware, introduced bubbles that interfered with fluidic control and affected assay results. The cards performed all sample preparation steps including plasma filtration from whole blood, sample and reagent aliquoting for the two parallel assays, sample dilution, and IgG removal for the IgM assays. We demonstrated the system for detection of the malarial pfHRPII antigen (spiked) and IgM antibodies to Salmonella Typhi LPS (patient plasma samples). All reagents were stored on card in dry form; only the sample and buffer were required to run the tests. Here we detail the development of this platform and discuss its strengths and weaknesses. PMID:22311085

  15. EVALUATION OF IMMUNOASSAY METHODS FOR DETERMINATION OF 3,5,6-TRICHLORO-2-PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    EPA Science Inventory

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by the RaPID (TM) commercial immunoassay testing ...

  16. Development of a multiplexed bead-based immunoassay for the simultaneous detection of antibodies to 17 pneumococcal proteins.

    PubMed

    Shoma, S; Verkaik, N J; de Vogel, C P; Hermans, P W M; van Selm, S; Mitchell, T J; van Roosmalen, M; Hossain, S; Rahman, M; Endtz, H Ph; van Wamel, W J B; van Belkum, A

    2011-04-01

    Presently, several pneumococcal proteins are being evaluated as potential vaccine candidates. Here, we gather novel insights in the immunogenicity of PLY, PsaA, PspA, PspC, NanA, Hyl, PpmA, SlrA, Eno, IgA1-protease, PdBD, BVH-3, SP1003, SP1633, SP1651, SP0189 and SP0376. We developed a multiplex bead-based immunoassay (xMAP(®) Technology, Luminex Corporation) to simultaneously quantify antibodies against these 17 pneumococcal proteins in serum. The median fluorescence intensity (MFI) values obtained for human pooled serum with the multiplex assay were between 82% and 111% (median 94%) of those obtained with the singleplex assays. For IgG, the coefficient of variation (CV) in serum ranged from 2% to 9%, for IgA, the CV ranged from 3% to 14% and for IgM, the CV ranged from 11% to 15%. Using this immunoassay, we showed that anti-pneumococcal antibody levels exhibited extensive inter-individual variability in young children suffering from invasive pneumococcal disease. All proteins, including the proteins with, as yet, unknown function, were immunogenic. In conclusion, the multiplex Streptococcus pneumoniae immunoassay based on proteins is reproducible. This assay can be used to monitor anti-S. pneumoniae antibody responses in a material- and time-saving manner. PMID:21086008

  17. A natural IgM antibody does inhibit polyclonal and antigen-specific IgM but not IgG B-cell responses.

    PubMed

    Kiss, K; Uher, F; Gergely, J

    1994-03-01

    Since a B-cell growth-inhibitory natural IgM antibody was identified in the culture supernatants of LPS-stimulated murine splenic B lymphocytes [11], attempts have been made to define other possible functional role(s) of this antibody. Here we show that this regulatory IgM is able to inhibit not only the proliferation of splenic B cells, but also their IgM secretion during LPS-induced polyclonal, as well as antigen (FITC-KLH)-specific antibody responses. In contrast, IgG1 production of hapten (FITC)-specific B cells neither during restimulation with LPS nor in the presence of carrier-specific T lymphocytes in vitro was affected by regulatory IgM. Therefore, whereas newly emerging naive B cells are highly susceptible, IgG-secreting B cells appear to be completely resistant to inactivation by the regulatory IgM autoantibody. PMID:7518418

  18. Development of an Ultrasensitive Immunoassay for Detecting Tartrazine

    PubMed Central

    Li, Zhuokun; Song, Shanshan; Xu, Liguang; Kuang, Hua; Guo, Shidong; Xu, Chuanlai

    2013-01-01

    We have developed an ultrasensitive indirect competitive enzyme-linked immunosorbent assay for the determination of tartrazine. Two carboxylated analogues of tartrazine with different spacer lengths, and one derivative from commercial tartrazine after a little chemical modification, were synthesized as haptens in order to produce antibodies specific to tartrazine. The effect of sulfonic acid groups on the hapten structure of tartrazine was also studied carefully for the first time. A most specific monoclonal antibody against tartrazine was created and exhibited an IC50 value of 0.105 ng/mL and a limit of detection of 0.014 ng/mL, with no cross-reactivity to other structurally-related pigments. The established immunoassay was applied to the determination of tartrazine in fortified samples of orange juice and in real positive samples of carbonated beverages. PMID:23799494

  19. New developments in particle-based tests and immunoassays.

    PubMed

    Bangs, L B

    1990-09-01

    Latex agglutination tests were invented in 1957. Thirty years later, new tests are still being devised and applied to new analytes. Reproducibility and readability continue to improve. Qualitative tests have now evolved to quantitative particle immunoassays: agglutination is detected by spectrophotometers or nephelometers, in tubes or 96-well plates. These same particles are now also being used in particle capture ELIST and ELISA (enzyme linked immunosorbent tests and assays) where particles are caught upon a filter and act as supports for sandwich tests (those "+/-" or "blue-dot" tests). These also can be quantified, as in the Abbott IM x assay system. Dyed microspheres now function as the color tags in over-the-counter sandwich-type pregnancy tests. In the future, results from assays using this technology could be read on reflectometers (strip readers). Currently, magnetic particles are used in solid phase radioimmunoassays and DNA probes. PMID:10148953

  20. Nanoparticle-based immunosensors and immunoassays for aflatoxins.

    PubMed

    Wang, Xu; Niessner, Reinhard; Tang, Dianping; Knopp, Dietmar

    2016-03-17

    Aflatoxins are naturally existing mycotoxins produced mainly by Aspergillus flavus and Aspergillus parasiticus, present in a wide range of food and feed products. Because of their extremely high toxicity and carcinogenicity, strict control of maximum residue levels of aflatoxins in foodstuff is set by many countries. In daily routine, different chromatographic methods are used almost exclusively. As supplement, in several companies enzyme immunoassay-based sample testing as primary screening is performed. Recently, nanomaterials such as noble metal nanoparticles, magnetic particles, carbon nanomaterials, quantum dots, and silica nanomaterials are increasingly utilized for aflatoxin determination to improve the sensitivity and simplify the detection. They are employed either as supports for the immobilization of biomolecules or as electroactive or optical labels for signal transduction and amplification. Several nanoparticle-based electrochemical, piezoelectric, optical, and immunodipstick assays for aflatoxins have been developed. In this review, we summarize these recent advances and illustrate novel concepts and promising applications in the field of food safety. PMID:26920768

  1. A redox-mediated chromogenic reaction and application in immunoassay.

    PubMed

    Yu, Ru-Jia; Ma, Wei; Peng, Mao-Pan; Bai, Zhi-Shan; Long, Yi-Tao

    2016-08-31

    A novel redox-mediated chromogenic reaction was demonstrated based on the reaction between HAuCl4 and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which generate various color responses from red to green in the resulting solutions. Various redox substance could be used to mediate the reaction and trigger a distinct color response. We established a sensitive hydrogen peroxide colorimetric sensor based on the redox-mediated chromogenic reaction and depicted the application both in detection of enzyme and in an immunoassay. Combining the traditional chromogenic reagent with gold nanoparticles, our assay has the advantage in short response time (within three minutes), high sensitivity (10(-12) g mL(-1) for HBsAg) and stability. PMID:27506364

  2. The development of immunoassays for detection of chemical warfare agents

    SciTech Connect

    Lenz, D.E.; Brimfield, A.A.; Cook, L.

    1996-10-01

    With the advent of enzyme linked immunoabsorbent assays (ELISA) and monoclonal antibodies in the last two decades, there has been considerable effort devoted to the development of antibodies to detect and quantify low molecular weight toxic substances in environmental or biological fluids. Polyclonal antibodies against paraoxon (the toxic metabolite of parathion) were capable of detecting paraoxon in body fluids at a level of 10{sup -9} M ({approximately}260 pg/mL) when used in a competitive inhibition enzyme immunoassay (CIEIA). Monoclonal antibodies developed against a structural analogue of the chemical warfare agent soman were capable of detection soman in buffer solutions at a level of 10{sup -6} M ({approximately}180 ng/mL). In addition these antibodies were found to be highly specific for soman even in the presence of its major hydrolysis product. Subsequent studies with antisoman monoclonal antibodies extended the level of sensitivity to {approximately}80 ng/mL. Furthermore these antibodies did not cross react with other chemical warfare nerve agents such as sarin or tabun. In all cases, the time for a confirmatory test was two hours or less. Immunoassays for T-2 micotoxins have also been reported with a minimal detection range of 2 pg/assay to 50 ng/assay for the polyclonal and monoclonal T-2 antibodies respectively. These reagents offer a sensitive, rapid and low cost approach to the diagnosis or detection of the presence of toxic chemical substances. More recent efforts have focussed on developing antibodies specific for sulfur mustard a highly reactive vesicating agent.

  3. Ca2+-Regulated Photoproteins: Effective Immunoassay Reporters

    PubMed Central

    Frank, Ludmila A.

    2010-01-01

    Ca2+-regulated photoproteins of luminous marine coelenterates are of interest and a challenge for researchers as a unique bioluminescent system and as a promising analytical instrument for both in vivo and in vitro applications. The proteins are comprehensively studied as to biochemical properties, tertiary structures, bioluminescence mechanism, etc. This knowledge, along with available recombinant proteins serves the basis for development of unique bioluminescent detection systems that are “self-contained”, triggerable, fast, highly sensitive, and non-hazardous. In the paper, we focus on the use of photoproteins as reporters in binding assays based on immunological recognition element—bioluminescent immunoassay and hybridization immunoassay, their advantages and prospects. PMID:22163526

  4. ABO (histo) blood group phenotype development and human reproduction as they relate to ancestral IgM formation: A hypothesis.

    PubMed

    Arend, Peter

    2016-01-01

    The formation of a histo (blood) group) ABO phenotype and the exclusion of an autoreactive IgM or isoagglutinin activity arise apparently in identical glycosylation of complementary domains on cell surfaces and plasma proteins. The fundamental O-glycan emptiness of the circulating IgM, which during the neonatal amino acid sequencing of the variable regions is exerting germline-specific O-GalNAc glycan-reactive serine/threonine residues that in the plasma of the adult human blood group O individuals apparently remain associated with the open glycosidic sites on the ABOH convertible red cell surface, must raise suggestions on a transient expression of developmental glycans, which have been "lost" over the course of maturation. In fact, while the mammalian non-somatic, embryogenic stem cell (ESC)- germ cell (GC) transformation is characterized by a transient and genetically as-yet-undefined trans-species-functional O-GalNAc glycan expression, in the C57BL/10 mouse such expression was potentially identified in growth-dependent, blood group A-like GalNAc glycan-bearing, ovarian glycolipids complementary with the syngeneic anti-A reactive IgM, which does not appear in early ovariectomized animals. This non-somatically encoded, polyreactive, ancestral IgM molecule has not undergone clonal selection and does primarily not differentiate between self and non-self and might, due to amino acid hydroxyl groups, highly suggest substrate competition with subsequent O-glycosylations in ongoing ESC-GC transformations and affecting GC maturation. However, the membrane-bound somatic N/O-glycotransferases, which initiate, after formation of the zygote, the complex construction of the human ABO phenotypes in the trans cisternae of the Golgi apparatus, are associated and/or completed with soluble enzyme versions exerting identical specificities in plasma and likely competing vice versa by glycosylation of neonatal IgM amino acids, where they suggest to accomplish the clearance of anti

  5. Highly sensitive immunoassay based on immunogold-silver amplification and inductively coupled plasma mass spectrometric detection.

    PubMed

    Liu, Rui; Liu, Xing; Tang, Yurong; Wu, Li; Hou, Xiandeng; Lv, Yi

    2011-03-15

    In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL(-1) with a limit of detection (LOD, 3σ) of 0.03 ng mL(-1) (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay. PMID:21348438

  6. Development and application of recombinant antibody-based immunoassays to tetraconazole residue analysis in fruit juices.

    PubMed

    Plana, Emma; Moreno, Maria-José; Montoya, Ángel; Manclús, Juan J

    2014-01-15

    Tetraconazole is currently used as a fungicide in fruit and vegetables. The aim of this work was the development of immunochemical techniques based on recombinant antibodies for the screening of tetraconazole residues in fruit juices. Recombinant antibodies were produced from a hybridoma cell line secreting a monoclonal antibody specific for tetraconazole and from lymphocytes of mice hyperimmunised with tetraconazole haptens conjugated to bovine serum albumin. From these antibodies, enzyme-linked immunosorbent assays in the conjugate-coated format were developed, which were able to detect tetraconazole standards down to 1ng/mL. From recovery studies with spiked samples, these immunoassays determined tetraconazole in orange and apple juices with acceptable reproducibility (coefficients of variation below 25%) and recoveries (ranging from 78% to 145%) for a screening technique. The analytical performance of RAb-based immunoassays was fairly similar to that of the MAb-based immunoassays. Due to their simplicity and high sample throughput, the developed recombinant-based immunoassays can be valuable analytical tools for the screening of tetraconazole residues in fruit juices at regulatory levels. PMID:24054232

  7. A novel immunosensor for detecting toxoplasma gondii-specific IgM based on goldmag nanoparticles and graphene sheets.

    PubMed

    Jiang, Shuting; Hua, Erhui; Liang, Mo; Liu, Bei; Xie, Guoming

    2013-01-01

    A novel electrochemical immunosensor for detecting toxoplasma gondii-specific IgM (Tg-IgM) was constructed based on goldmag (Au-Fe(3)O(4)) nanoparticles and graphene sheets (GS). Thionine (Thi), as a mediator, was first electropolymerized on a nafion-GS (Nf-GS) modified electrode. Subsequently, gold nanoparticles (AuNPs) were attached onto the poly-thionine film through π-stacking interactions, and then were used to immobilize toxoplasma gondii antigen (Tg-Ag) for immunosensor fabrication. A sandwich-type immunoassay for Tg-IgM was performed using Au-Fe(3)O(4) labeled anti-IgM-horseradish peroxidase (HRP) as trace label. Electrochemical detection was carried out in the presence of H(2)O(2) as HRP substrate. Using Au-Fe(3)O(4) provided a simple, non-chemical damaging method for regeneration, and enhanced the HRP reduction ability toward H(2)O(2). The AuNPs/Thi/Nf-GS nanocomposite also had good conductivity and biocompatibility, which effectively improved the immunosensor sensitivity. Under optimal conditions, the immunosensor can detect Tg-IgM in two linear ranges from 0.0375 to 1.2 AU mL(-1) and from 2.0 to 18 AU mL(-1) with a detection limit of 0.016 AU mL(-1) (S/N=3). The immunosensor exhibited good reproducibility, stability, and selectivity as well. PMID:23010058

  8. Role of natural and immune IgM antibodies in immune responses.

    PubMed

    Boes, M

    2000-12-01

    IgM antibodies constitute the major component of the natural antibodies and is also the first class of antibodies produced during a primary antibody response. The IgM-type antibodies differ from other classes of antibodies in that they are predominantly produced by B1 cells, in the absence of apparent stimulation by specific antigens. In addition, IgM antibodies are mostly encoded by germline V gene segments and have low affinities but broad specificites to both foreign and self structures. New developments regarding the function of both immune IgM antibodies and natural IgM antibodies will be examined here. PMID:11451419

  9. Bicentric evaluation of Access Toxo immunoglobulin M (IgM) and IgG assays and IMx toxo IgM and IgG assays and comparison with Platelia Toxo IgM and IgG assays.

    PubMed Central

    Decoster, A; Lecolier, B

    1996-01-01

    The recent Access immunoanalysis system (Sanofi Diagnostics Pasteur) for the serological diagnosis of toxoplasmosis was compared with the Abbott Toxo IMx EIA system, taking the Platelia Toxo immunoglobulin G (IgG) and Platelia Toxo IgM systems as references and using as confirmation methods an indirect fluorescence assay or a dye test for IgG and an immunosorbent agglutination assay (ISAGA) for IgM. A total of 1,461 serum samples were studied, of which 128 were collected from 42 recently seroconverted patients. Sensitivity and specificity rates of the Access system were 97.7 and 99.5%, respectively, for IgM and 98.6 and 100%, respectively, for IgG. Sensitivity and specificity rates of the Abbott IMx EIA system were 91 and 100%, respectively, for IgM and 92.5 and 100%, respectively, for IgG. The Access Toxo IgG and IgM EIA systems were found to be more sensitive than the Abbott Toxo IgG and IgM IMx EIA systems. PMID:8784554

  10. Chemiluminescence Resonance Energy Transfer Competitive Immunoassay Employing Hapten-Functionalized Quantum Dots for the Detection of Sulfamethazine.

    PubMed

    Ma, Mingfang; Wen, Kai; Beier, Ross C; Eremin, Sergei A; Li, Chenglong; Zhang, Suxia; Shen, Jianzhong; Wang, Zhanhui

    2016-07-20

    We describe a new strategy for using chemiluminescence resonance energy transfer (CRET) by employing hapten-functionalized quantum dots (QDs) in a competitive immunoassay for detection of sulfamethazine (SMZ). Core/multishell QDs were synthesized and modified with phospholipid-PEG. The modified QDs were functionalized with the hapten 4-(4-aminophenyl-sulfonamido)butanoic acid. The CRET-based immunoassay exhibited a limit of detection for SMZ of 9 pg mL(-1), which is >4 orders of magnitude better than a homogeneous fluorescence polarization immunoassay and is 2 orders of magnitude better than a heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach for detection of chemical contaminants. PMID:27362827

  11. In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis.

    PubMed

    Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C; Velineni, Sridhar; Timoney, John F; Natarajaseenivasan, Kalimuthusamy

    2016-01-01

    Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. PMID:26607308

  12. In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis

    PubMed Central

    Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C.; Velineni, Sridhar; Timoney, John F.

    2015-01-01

    Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. PMID:26607308

  13. [Role of line immunoassay in the diagnosis of early HIV infection: a diagnostic case].

    PubMed

    Soylar, Muhammed; Altuğlu, Imre; Sertöz, Rüçhan; Gökengin, Deniz

    2013-07-01

    Combined p24 antigen-HIV antibody fourth-generation assays that identify most of the early HIV infections have been used extensively worldwide for several years. This poses challenges for the traditional algorithm of line immunoassay (LIA) confirmation. LIA tests are useful methods with their high specificity and their ability to differentiate HIV-1 from HIV-2, but they are reactive days after the fourth generation enzyme immunoassays. With acute HIV infection, high levels of infectious virus are detectable in serum and genital secretions. The rate of transmission during acute HIV infection is higher than the established HIV infection, for this reason, new HIV testing strategies need to focus on sensitivity, especially for this highly contagious phase immediately after infection. Serum sample of a patient sent to Ege University Hospital Clinical Virology Laboratory was repeatedly reactive with low signal/cutoff ratios with two different commercial fourth generation enzyme immunoassays (Architect HIV Ag/Ab Combo Reagent Kit, Abbott, Germany and Vidas HIV Duo Quick, Biomerieux, France). The sample was non-reactive with the LIA (INNO-LIA HIV I/II Score, Innogenetics, Belgium) and HIV RNA (RealTime HIV-I Amplification Reagent Kit, Abbott, USA) result was positive (4.1 x 10(5) copies/ml). With the presentation of this case, the role of LIA in the diagnosis of early HIV infection and its place in test algorithms were questioned. PMID:23971936

  14. The contribution of naturally occurring IgM antibodies, IgM cross-reactivity and complement dependency in murine humoral responses to pneumococcal capsular polysaccharides.

    PubMed

    Jones, Hannah E; Taylor, Philip R; McGreal, Eamon; Zamze, Susanne; Wong, Simon Y C

    2009-09-25

    Immunogenicity of 12 capsular polysaccharides (CPS) from Streptococcus pneumoniae did not correlate with pre-existing levels of natural IgM anti-CPS antibodies in mice. Immunization of mice with individual CPS, with the exception of type 14 (the only neutral CPS tested), increased serum IgM that also bound other CPS serotypes independent of structural similarity or commonly known contaminants. Surprisingly only IgM response to type 4 (which has a small immunodominant epitope) was dependent on either complement C3 or complement receptors CD35/CD21. IgG anti-CPS responses were infrequently induced, but critically dependent on complement. Our results have clarified the role of complement in the induction of IgM and IgG anti-CPS antibody responses in mice and have implications for CPS vaccine development. PMID:19660585

  15. Thrombocytopenia in Malaria with Immunoglobulin (IgM) Changes

    PubMed Central

    Beale, P. J.; Cormack, J. D.; Oldrey, T. B. N.

    1972-01-01

    Of 33 cases of naturally occurring human malaria 32 were found to have significant thrombocytopenia. Only one patient showed signs of bleeding. The lowest platelet levels were found between the day of diagnosis and the fourth day of treatment. Thereafter they returned to normal values. No other factors could be found to correlate with the presence or depth of thrombocytopenia, and no evidence of intravascular coagulation was found in any case. A rise in the immunoglobulin IgM was found in all 13 cases in which it was estimated. Since thrombocytopenia can occur independently of intravascular coagulation the latter should be diagnosed and heparin given only after clotting factors have been shown to be depleted. ImagesFIG. 3 PMID:5008661

  16. Investigation Of The Diffuse IGM By Cross-Correlation Studies

    SciTech Connect

    Farnsworth, Damon; Brown, Shea; Rudnick, Lawrence

    2009-12-18

    We present results from the first cross-correlation search for the synchrotron component of the diffuse intergalactic medium (IGM) in filamentary large scale structure (LSS). We used the low resolution (36') Bonn survey at 21cm, with the infrared 2MASS catalog as a tracer of the LSS. Synchrotron emission likely results from LSS formation shocks and feedback from AGN and galactic winds [2]. We determined 3{sigma} upper limits to the diffuse emission in units of flux per galaxy; these correspond to filament equipartition magnetic fields as low as 0.2 {mu}G. The detection threshold for the average (peak) filament brightness is 1 (7) mK for 0.03

  17. Automated homogeneous immunoassay analysis of cotinine in urine.

    PubMed

    Niedbala, R Sam; Haley, Nancy; Kardos, Stephanie; Kardos, Keith

    2002-04-01

    A study was conducted to evaluate the performance comparison of a homogeneous enzyme immunoassay (EIA) designed to detect cotinine in urine and carbon monoxide (CO) breath measurements to determine smoking status. The clinical comparison was done using urine and breath specimens from 218 volunteers. Urine samples were analyzed by immunoassay and confirmed by gas chromatography-mass spectrometry (GC-MS). Breath carbon monoxide was determined by a commercial analyzer. Using cutoffs of 10 ppm for CO and 500 ng/mL for urinary cotinine, the relative sensitivity/specificity was 93.6%/74.0%. The positive predictive value was 86.8%, and the negative predictive value was 86.5%. However, comparison of the EIA to GC-MS showed a sensitivity/specificity of 96.2%/98.4% and a positive predictive value of 99.3%. The EIA was also evaluated non-clinically for precision, stability, recovery, and interferences. In addition, the non-clinical evaluation demonstrated coefficients of variation from 0.37 to 1.09% across cotinine concentrations ranging from 0 to 5000 ng/mL. The assay was found to be highly specific for cotinine and cross-reacted to a limited degree with 3-hydroxycotinine. Finally, multiple freeze-thaw cycles of urines containing cotinine showed no degradation of the drug in the specimen when tested in the EIA. Thus, the EIA tested is a rapid, lab-based test that can reliably determine cotinine levels and their relation to smoking status. PMID:11991533

  18. Bipolar disorder course, impaired glucose metabolism and antioxidant enzymes activities: A preliminary report.

    PubMed

    Mansur, Rodrigo B; Rizzo, Lucas B; Santos, Camila M; Asevedo, Elson; Cunha, Graccielle R; Noto, Mariane N; Pedrini, Mariana; Zeni-Graiff, Maiara; Gouvea, Eduardo S; Cordeiro, Quirino; Reininghaus, Eva Z; McIntyre, Roger S; Brietzke, Elisa

    2016-09-01

    This study aimed to examine the role of oxidative stress in bipolar disorder (BD) by evaluating the relationship among antioxidant enzymes activities, impaired glucose metabolism (IGM) and illness course. We measured the activities of plasma superoxide dismutase (SOD) and glutathione peroxidase (GPx) in individuals with BD (N = 55) and healthy controls (N = 28). Information related to current and past psychiatric/medical history, as well as prescription of any pharmacological treatments was captured. Impaired glucose metabolism was operationalized as pre-diabetes or type 2 diabetes mellitus. Our results showed that, after adjustment for age, gender, alcohol use, smoking and current medication, both BD (p < 0.001) and IGM (p = 0.019) were associated with increased GPx activity, whereas only BD was associated with decreased SOD activity (p = 0.008). We also observed an interaction between BD and IGM on SOD activity (p = 0.017), whereas the difference between BD and controls was only significant in individuals with IGM (p = 0.009). IGM, GPx and SOD activity were independently associated with variables of illness course. Moreover, IGM moderated the association between SOD activity and number of mood episodes (p < 0.001), as a positive correlation between SOD activity and mood episodes was observed only in participants with IGM. In conclusion, BD and IGM are associated with independent and synergistic effects on markers of oxidative stress. The foregoing observations suggest that the heterogeneity observed in previous studies evaluating antioxidant enzymes in BD may be a function of concurrent IGM; and that imbalances in the oxidative system may subserve the association between BD and IGM, as well as its relationship with illness course. PMID:27281261

  19. Low molecular weight IgM in selective IgA deficiency.

    PubMed Central

    Kwitko, A O; Roberts-Thomson, P J; Shearman, D J

    1982-01-01

    Thirty-nine persons with selective IgA deficiency were studied. These comprised 27 subjects found by population screening and 12 by other means. Low molecular weight (LMW) serum IgM was sought in 28 of the 39 persons. Nine of the 28 (32%) had LMW IgM detectable by a sensitive gel filtration technique. Of 17 patients discovered by screening, five (29%) had LMW IgM. In the nine positive persons, LMW IgM constituted up to 17% of the total serum IgM concentration. Eight of the nine IgA deficient persons with LMW IgM, had clinical disease while associated disease in the entire IgA deficient population was less frequent. Serum immune complexes were demonstrated in five of seven subjects with LMW IgM using a C1q-dependent radioimmunoassay; four of these had immune complex associated disorders, three with polyarthritis and one with glomerulonephritis. Because circulating immune complexes are frequently detected in IgA deficient persons without disease, it is proposed that the presence of LMW serum IgM in IgA deficiency may be associated with disease due to the formation of specific pathogenic immune complexes. PMID:7172505

  20. Theoretical limitations of quantification for noncompetitive sandwich immunoassays.

    PubMed

    Woolley, Christine F; Hayes, Mark A; Mahanti, Prasun; Douglass Gilman, S; Taylor, Tom

    2015-11-01

    Immunoassays exploit the highly selective interaction between antibodies and antigens to provide a vital method for biomolecule detection at low concentrations. Developers and practitioners of immunoassays have long known that non-specific binding often restricts immunoassay limits of quantification (LOQs). Aside from non-specific binding, most efforts by analytical chemists to reduce the LOQ for these techniques have focused on improving the signal amplification methods and minimizing the limitations of the detection system. However, with detection technology now capable of sensing single-fluorescence molecules, this approach is unlikely to lead to dramatic improvements in the future. Here, fundamental interactions based on the law of mass action are analytically connected to signal generation, replacing the four- and five-parameter fittings commercially used to approximate sigmoidal immunoassay curves and allowing quantitative consideration of non-specific binding and statistical limitations in order to understand the ultimate detection capabilities of immunoassays. The restrictions imposed on limits of quantification by instrumental noise, non-specific binding, and counting statistics are discussed based on equilibrium relations for a sandwich immunoassay. Understanding the maximal capabilities of immunoassays for each of these regimes can greatly assist in the development and evaluation of immunoassay platforms. While many studies suggest that single molecule detection is possible through immunoassay techniques, here, it is demonstrated that the fundamental limit of quantification (precision of 10 % or better) for an immunoassay is approximately 131 molecules and this limit is based on fundamental and unavoidable statistical limitations. PMID:26342315

  1. The Long Elusive IgM Fc Receptor, FcμR

    PubMed Central

    Kubagawa, Hiromi; Oka, Satoshi; Kubagawa, Yoshiki; Torii, Ikuko; Takayama, Eiji; Kang, Dong-Won; Jones, Dewitt; Nishida, Naonori; Miyawaki, Toshio; Bertoli, Luigi F.; Sanders, Sheila K.; Honjo, Kazuhito

    2014-01-01

    IgM exists as both a monomer on the surface of B cells and a pentamer secreted by plasma cells. Both preimmune “natural” and antigen-induced “immune” IgM antibodies are important for protective immunity and for immune regulation of autoimmune processes by recognizing pathogens and self-antigens. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed but fail to fully account for the IgM-mediated protection and regulation. A major reason for this deficit in our understanding of IgM function seems to be lack of data on a long elusive Fc receptor for IgM (FcμR). We have recently identified a bona fide FcμR in both humans and mice. In this article we briefly review what we have learned so far about FcμR. PMID:24793544

  2. Multiplexed Microsphere Suspension Array-Based Immunoassays.

    PubMed

    Lin, Andrew; Salvador, Alexandra; Carter, J Mark

    2015-01-01

    ELISA is an extremely powerful tool to detect analytes because of its sensitivity, selectivity, reproducibility and ease of use. Here we describe sandwich immunoassays performed in suspension on spectrally unique microspheres developed by Luminex. Luminex assays offer the benefit of multiplex analysis of large numbers of analytes in a single reaction. Because the microspheres are spectrally unique, many microspheres, each attached to various antibodies, can be added to a single sample. Luminex instruments can distinguish each microsphere and detect the intensity of a reporter signal for each microsphere. Results are reported in Median Fluorescent Intensities for each analyte. Luminex assays can be used to detect up to 500 analytes in a high-throughput format. Luminex refers to this technology as xMAP(®). Here we describe a routine protocol for a Luminex immunoassay. Other Luminex assays would have to be optimized for specific conditions according to their use. PMID:26160569

  3. Prevalence of Legionella-specific IgG and IgM antibody in a dental clinic population.

    PubMed

    Fotos, P G; Westfall, H N; Snyder, I S; Miller, R W; Mutchler, B M

    1985-12-01

    This study was undertaken to determine the frequency of Legionella infection in a dental clinic setting. Serum samples from 270 dental clinic personnel were evaluated using an enzyme-linked immunosorbent assay to detect Legionella-specific IgM and IgG antibodies. The pooled-species whole-cell-antigen preparation used in these assays was derived from six Legionella pneumophila strains and one strain each from Legionella bozemanii and Legionella micdadei. Significant levels of IgG and IgM antibodies were found in 20% and 16%, respectively, of the samples. This compares with 8% and 10%, respectively, for a randomly selected non-clinical group from the region (P less than 0.005). Samples from clinic personnel with significant IgG titers (greater than 1:128) were also evaluated for activity to each of the eight single-species antigens, with the following results: L. pneumophila, 45% (combined six strains); L. micdadei, 37%; and L. bozemanii, 18%. Comparing individuals' "years spent in the clinic environment" with the incidence of significant antibody levels strongly suggests that the risk of Legionella infection increases proportionately with increased clinic exposure time (P less than 0.05). Analysis of these data implies that Legionella may be present in the dental clinic environment, thus creating an increased risk for clinical personnel or patients. PMID:3865949

  4. Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays.

    PubMed

    Chen, Hui; Hagström, Anna E V; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C; Atmar, Robert L; Willson, Richard C

    2016-01-01

    In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 10(6) virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings. PMID:27075635

  5. Flotation Immunoassay: Masking the Signal from Free Reporters in Sandwich Immunoassays

    PubMed Central

    Chen, Hui; Hagström, Anna E. V.; Kim, Jinsu; Garvey, Gavin; Paterson, Andrew; Ruiz-Ruiz, Federico; Raja, Balakrishnan; Strych, Ulrich; Rito-Palomares, Marco; Kourentzi, Katerina; Conrad, Jacinta C.; Atmar, Robert L.; Willson, Richard C.

    2016-01-01

    In this work, we demonstrate that signal-masking reagents together with appropriate capture antibody carriers can eliminate the washing steps in sandwich immunoassays. A flotation immunoassay (FI) platform was developed with horseradish peroxidase chemiluminescence as the reporter system, the dye Brilliant Blue FCF as the signal-masking reagent, and buoyant silica micro-bubbles as the capture antibody carriers. Only reporters captured on micro-bubbles float above the dye and become visible in an analyte-dependent manner. These FIs are capable of detecting proteins down to attomole levels and as few as 106 virus particles. This signal-masking strategy represents a novel approach to simple, sensitive and quantitative immunoassays in both laboratory and point-of-care settings. PMID:27075635

  6. Enzyme immunoassay by dynamic enhanced vibrational spectroscopy of the enzyme reaction product

    NASA Astrophysics Data System (ADS)

    Zhao, Haiying; Dou, Xiaoming

    2005-01-01

    This paper reports a kind of application of surface-enhanced Raman scattering (SERS) to immunology. In the proposed system, antibody immobilized on a solid substrate reacts with antigen, which binds with another antibody labeled with peroxidase. If this immunocomplex is subjected to reaction with o-phenylenediamine and hydrogenperoxide at 37°C, azoaniline is generated. This azo compound is adsorbed on a silver colloid and only the azo compound gives a strong surface-enhanced resonance Raman (SERRS) spectrum. A linear relationship was observed between the peak intensity of the N=N stretching band and the concentration of antigen, revealing that one can determine the concentration of antigen by the SERRS measurement of the reaction product.

  7. Age‐related aspects of human IgM+ B cell heterogeneity

    PubMed Central

    Martin, Victoria; Wu, Yu‐Chang; Kipling, David

    2015-01-01

    The CD27+IgD+ B cell population, known as IgM memory, reduces with age. It is thought that this population is responsible for pneumococcal polysaccharide T‐independent responses, and that the age‐related reduction might be partially responsible for the increased susceptibility of older people to bacterial pathogens. There are other IgM+ B cell populations that do not express IgD. We compared the different IgM populations using high‐throughput sequencing of the immunoglobulin (Ig) gene repertoire and multidimensional cell phenotyping and found that the different populations of IgM cells, defined by CD27 and IgD expression, have repertoire differences. Some of these differences are likely indicative of different selection pressures in an immune response, although the older individuals were found to have a changed repertoire in naive B cells, which may contribute to some of the changes seen in memory cells. In addition, even within the CD27+IgD+ IgM memory population there are multiple cell types. We show that the level of IgM expression varies substantially and hypothesize that this distinguishes between T‐dependent and T‐independent types of IgM memory cells. Significant age‐related changes in the relative proportions of these populations may exacerbate the reduction in T‐independent responders in old age. PMID:26152370

  8. Magnetic-bead-based sub-femtomolar immunoassay using resonant Raman scattering signals of ZnS nanoparticles.

    PubMed

    Ding, Yadan; Cong, Tie; Chu, Xueying; Jia, Yan; Hong, Xia; Liu, Yichun

    2016-07-01

    Highly sensitive, specific, and selective immunoassays are of great significance for not only clinical diagnostics but also food safety, environmental monitoring, and so on. Enzyme-linked immunosorbent assays and fluorescence-based and electrochemical immunoassays are important intensively investigated immunoassay techniques. However, they might suffer from low sensitivity or false-positive results. In this work, a simple, reliable, and ultrasensitive magnetic-bead-based immunoassay was performed using biofunctionalized ZnS semiconductor nanocrystals as resonant Raman probes. The resonant Raman scattering of ZnS nanocrystals displays evenly spaced multi-phonon resonant Raman lines with narrow bandwidths and has strong resistance to environmental variation due to the nature of the electron-phonon interaction, thus rendering reliable signal readout in the immunoassays. The superparamagnetic Fe3O4 nanoparticles facilitated greatly the separation, purification, and concentration processes. It is beneficial for both reducing the labor intensity and amplifying the detection signals. The immobilization of antibodies on the surface of magnetic beads, the preparation of resonant Raman probes, and the immunological recognition between the antibody and analyte all occurred in the liquid phase, which minimized the diffusion barriers and boundary layer constraints. All these factors contributed to the ultralow detection limit of human IgG, which was determined to be about 0.5 fM (∼0.08 pg/ml). It is nearly the highest sensitivity obtained for IgG detection. This work shall facilitate the design of nanoplatforms for ultrasensitive detections of proteins, DNAs, bacteria, explosives, and so on. Graphical abstract An ultrasensitive magnetic-bead-based immunoassay was performed using multi-phonon resonant Raman lines of ZnS nanoparticles as detection signals. PMID:27173389

  9. Interaction between the IGM and a dwarf galaxy

    NASA Astrophysics Data System (ADS)

    Lora, V.; Raga, A. C.; Grebel, E. K.

    2015-04-01

    Dwarf Galaxies are the most common objects in the Universe and are believed to contain large amounts of dark matter. There are mainly three morphologic types of dwarf galaxies: dwarf ellipticals, dwarf spheroidals and dwarf irregulars. Dwarf irregular galaxies are particularly interesting in dwarf galaxy evolution, since dwarf spheroidal predecessors could have been very similar to them. Therefore, a mechanism linked to gas-loss in dwarf irregulars should be observed, i.e. ram pressure stripping. In this paper, we study the interaction between the ISM of a dwarf galaxy and a flowing IGM. We derive the weak-shock, plasmon solution corresponding to the balance between the post-bow shock pressure and the pressure of the stratified ISM (which we assume follows the fixed stratification of a gravitationally dominant dark matter halo). We compare our model with previously published numerical simulations and with the observed shape of the HI cloud around the Ho II and Pegasus dwarf irregular galaxies. We show that such a comparison provides a straightforward way for estimating the Mach number of the impinging flow.

  10. Sunitinib-associated pseudothrombocytopenia induced by IgM antibody.

    PubMed

    Albersen, Arjan; Porcelijn, Leendert; Schilders, Joyce; Zuetenhorst, Hanneke; Njo, Tjin; Hamberg, Paul

    2013-01-01

    Thrombocytopenia is a well-documented adverse reaction of sunitinib. Thrombocytopenia was observed in a patient with metastatic renal clear-cell carcinoma undergoing sunitinib treatment. Platelet count in an ethylenediaminetetraacetic acid (EDTA) sample was 19 × 10(9)/l. To exclude pseudothrombocytopenia (PTCP), a platelet count in citrate-anticoagulated blood was performed, showing a platelet count of 6 × 10(9)/l. Due to the apparent thrombocytopenia, the patient received platelet concentrates. Subsequent analyses revealed PTCP whereby platelet clumping was most abundant in citrate - followed by EDTA- and heparin-anticoagulated blood samples. This effect was partially reversed after placing blood samples at 37°C. The IgM antiplatelet autoantibodies responsible for in vitro agglutination are temperature and multianticoagulant dependent and did not react to amikacin pre-supplementation. Remarkably, the antibody revealed specificity to platelet antigens other than GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIV, and GPV. After 16 days of discontinuing sunitinib, no PTCP and no platelet reactive antibodies could be detected. We report a case of PTCP with clear time-relation with sunitinib, strongly suggesting the mechanism to be sunitinib dependent. Since this finding has not been described before, non-recognition of PTCP during sunitinib treatment might lead to dose reduction or unwarranted therapy. PMID:23066976

  11. Development and preliminary validation of an antibody filtration-assisted single-dilution chemiluminometric immunoassay for potency testing of Piscirickettsia salmonis vaccines.

    PubMed

    Wilda, Maximiliano; Lavoria, María Ángeles; Giráldez, Adrián; Franco-Mahecha, Olga Lucía; Mansilla, Florencia; Érguiz, Matías; Iglesias, Marcela Elvira; Capozzo, Alejandra Victoria

    2012-11-01

    Challenge with live pathogens could be substituted by serology for many veterinary diseases, however little progress has been made in the development of alternative batch vaccine potency tests for fish. This study reports the development and preliminary validation of a single-dilution filtration-assisted chemiluminometric immunoassay (SD FAL-ELISA) applied to measure anti Piscirickettsia salmonis IgM in individual or pooled serum and mucus samples. The assay was set up to test a single-dilution of the sample. Serum SD FAL-ELISA yielded a sensitivity of 90% and a specificity of 96%. SD FAL-ELISA was applied to evaluate pooled and individual samples from P. salmonis challenge assessments. Relative-light units values (RLU) obtained by SD FAL-ELISA were proportional to antibody levels in serum. RLU values obtained from pooled and individual serum samples increased with the observed relative percent survival (RPS) values, indicating a correlation between protection and specific IgM levels. Results obtained for specific IgM in mucus samples was not related to the RPS, but discriminated the vaccine that yielded high RPS (86.4%) from the others (40.9 and 54.5%). This is the first report on the development of an indirect high-throughput serological assessment for P. salmonis vaccine potency testing using both pooled or individual serum and cutaneous mucus samples. PMID:23040097

  12. IgM Promotes the Clearance of Small Particles and Apoptotic Microparticles by Macrophages

    PubMed Central

    Litvack, Michael L.; Post, Martin; Palaniyar, Nades

    2011-01-01

    Background Antibodies are often involved in enhancing particle clearance by macrophages. Although the mechanisms of antibody-dependent phagocytosis have been studied for IgG in greater detail, very little is known about IgM-mediated clearance. It has been generally considered that IgM does not support phagocytosis. Recent studies indicate that natural IgM is important to clear microbes and other bioparticles, and that shape is critical to particle uptake by macrophages; however, the relevance of IgM and particle size in their clearance remains unclear. Here we show that IgM has a size-dependent effect on clearance. Methodology/Principal Findings We used antibody-opsonized sheep red blood cells, different size beads and apoptotic cells to determine the effect of human and mouse IgM on phagocytosis by mouse alveolar macrophages. Our microscopy (light, epifluorescence, confocal) and flow cytometry data show that IgM greatly enhances the clearance of small particles (about 1–2 micron) by these macrophages. There is an inverse relationship between IgM-mediated clearance by macrophages and the particle size; however, macrophages bind and internalize many different size particles coated with IgG. We also show that IgM avidly binds to small size late apoptotic cells or bodies (2–5 micron) and apoptotic microparticles (<2 µm) released from dying cells. IgM also promotes the binding and uptake of microparticle-coated beads. Conclusions/Significance Therefore, while the shape of the particles is important for non-opsonized particle uptake, the particle size matters for antibody-mediated clearance by macrophages. IgM particularly promotes the clearance of small size particles. This finding may have wider implications in IgM-mediated clearing of antigens, microbial pathogens and dying cells by the host. PMID:21448268

  13. Transcriptional Heterogeneity of IgM+ Cells in Rainbow Trout (Oncorhynchus mykiss) Tissues

    PubMed Central

    Abós, Beatriz; Castro, Rosario; Pignatelli, Jaime; Luque, Alfonso; González, Lucia; Tafalla, Carolina

    2013-01-01

    Two major classes of B lymphocytes have been described to date in rainbow trout: IgM+ and IgT+ cells. IgM+ cells are mainly localized in the spleen, peripheral blood and kidney but are also found in other tissues. However, differences among IgM+ cell populations attending to its location are poorly defined in fish. Thus, the aim of this work was to characterize the expression of different immune molecules such as chemokine receptors, Toll-like receptors (TLRs) and transcription factors on sorted IgM+ lymphocytes from different rainbow trout tissues. IgM+ populations from blood, spleen, kidney, gills, intestine and liver were isolated by cell sorting and the constitutive levels of transcription of these genes evaluated by real-time PCR. To further characterize B cells, we identified an MS4A sequence. In humans, the MS4A family includes several genes with immune functions, such as the B cell marker CD20 or FcRβ. Subsequently, we have also evaluated the mRNA levels of this MS4A gene in the different IgM+ populations. The relevant differences in transcriptional patterns observed for each of these IgM+ populations analyzed, point to the presence of functionally different tissue-specific B cell populations in rainbow trout. The data shown provides a pattern of genes transcribed in IgM+ B cells not previously revealed in teleost fish. Furthermore, the constitutive expression of all the TLR genes analyzed in IgM+ cells suggests an important role for these cells in innate immunity. PMID:24324826

  14. Expression and glycoengineering of functionally active heteromultimeric IgM in plants

    PubMed Central

    Loos, Andreas; Gruber, Clemens; Altmann, Friedrich; Mehofer, Ulrich; Hensel, Frank; Grandits, Melanie; Oostenbrink, Chris; Stadlmayr, Gerhard; Furtmüller, Paul G.; Steinkellner, Herta

    2014-01-01

    IgM antibodies are an important player of the human’s innate defense mechanisms and increasingly have gained interest as therapeutics. Although the expression of IgM antibodies in mammalian cell culture is established, this approach remains costly and alternative methods have not been developed yet. Plants have a proven record for the production of therapeutically relevant recombinant proteins. However, whether they are able to express proteins like IgM antibodies, which range among the most complex human proteins, remains unknown so far. Here we report the in planta generation of the functionally active monoclonal antitumor IgM PAT-SM6 (SM6). SM6 efficiently accumulates in plant leaves and assembles correctly into heterooligomers (pentamers and hexamers). Detailed glycosylation analysis exhibited complex and oligomannosidic N-glycans in a site-specific manner on human-serum IgM and on plant- and human-cell-line–produced SM6. Moreover, extensive in planta glycoengineering allowed the generation of SM6 decorated with sialylated human-type oligosaccharides, comparable to plasma-derived IgM. A glycosylated model of pentameric IgM exhibits different accessibility of the glycosylation sites, explaining site-specific glycosylation. Biochemical and biophysical properties and importantly biological activities of plant-derived SM6 glycoforms are comparable to the human-cell–derived counterparts. The in planta generation of one of the most complex human proteins opens new pathways toward the production of difficult-to-express proteins for pharmaceutical applications. Moreover, the generation of IgMs with a controlled glycosylation pattern allows the study of the so far unknown contribution of sugar moieties to the function of IgMs. PMID:24706782

  15. Cytomegalovirus IgM Seroprevalence among Women of Reproductive Age in the United States

    PubMed Central

    Wang, Chengbin; Dollard, Sheila C.; Amin, Minal M.; Bialek, Stephanie R.

    2016-01-01

    Cytomegalovirus (CMV) IgM indicates recent active CMV infection. CMV IgM seroprevalence is a useful marker for prevalence of transmission. Using data from the National Health and Nutrition Examination Survey (NHANES) III 1988–1994, we present estimates of CMV IgM prevalence by race/ethnicity, provide a comparison of IgM seroprevalence among all women and among CMV IgG positive women, and explore factors possibly associated with IgM seroprevalence, including socioeconomic status and exposure to young children. There was no difference in IgM seroprevalence by race/ethnicity among all women (3.1%, 2.2%, and 1.6% for non-Hispanic white, non-Hispanic black and Mexican American, respectively; P = 0.11). CMV IgM seroprevalence decreased significantly with increasing age in non-Hispanic black women (P<0.001 for trend) and marginally among Mexican American women (P = 0.07), while no apparent trend with age was seen in non-Hispanic white women (P = 0.99). Among 4001 IgG+ women, 118 were IgM+, resulting in 4.9% IgM seroprevalence. In IgG+ women, IgM seroprevalence varied significantly by age (5.3%, 7.3%, and 3.7% for women of 12–19, 20–29, and 30–49 years; P = 0.04) and race/ethnicity (6.1%, 2.7%, and 2.0% for non-Hispanic white, non-Hispanic black, and Mexican American; P<0.001). The factors reported associated with IgG seroprevalence were not associated with IgM seroprevalence. The patterns of CMV IgM seroprevalence by age, race/ethnicity, and IgG serostatus may help understanding the epidemiology of congenital CMV infection as a consequence of vertical transmission and are useful for identifying target populations for intervention to reduce CMV transmission. PMID:26990759

  16. Production of anti-idiotype antibodies for deoxynivalenol and their evaluation with three immunoassay platforms.

    PubMed

    Maragos, C M

    2014-05-01

    Immunoassays for deoxynivalenol (DON) that involve binding to DON-specific antibodies have been widely developed. In such assays, the responses of samples are generally compared with calibration curves generated by using DON in competition with labeled reagents such as enzymatic or fluorescent conjugates of the toxin. However, materials that mimic the toxin can also be used, provided that they compete effectively with the labeled reagents for the DON-specific antibodies. Examples include certain types of anti-idiotype antibodies, obtained by the immunization of animals with toxin-specific antibodies. In the present work, anti-idiotype antibodies were developed which mimicked DON in the ability to bind to a DON-specific monoclonal antibody (Mab). Fab fragments of the Mab (Ab1) were used to immunize rabbits. Sera were screened by competitive direct enzyme linked immunosorbent assay (CD-ELISA) for the presence of anti-idiotype antibodies (Ab2). In order to determine the most effective screening format and also the potential efficacy in various forms of biosensors, the sera were further evaluated in biolayer interferometry (BLI) and fluorescence polarization immunoassay (FPIA) formats. All three formats were used to demonstrate the presence of anti-idiotypes capable of binding to the paratope of the DON antibody (subtypes Ab2β or Ab2γ). Such materials have the potential to replace DON as calibrants in immunoassays for this toxin. PMID:24526340

  17. Solid-phase competitive and sandwich-type erythro-immunoassays for human chorionic gonadotropin.

    PubMed

    Gupta, S K; Guesdon, J L; Avrameas, S; Talwar, G P

    1985-06-25

    A simple '1-step' competitive erythro-immunoassay for human chorionic gonadotropin (hCG) employing V-shaped well microtitration plates coated with monoclonal anti-beta-hCG antibody has been described. hCG of the test sample competes with the antigen-coupled sheep erythrocytes for binding to the antibody on the solid surface. The assay is able to detect up to 31.25 ng hCG/ml. A higher sensitivity enabling detection up to 0.25 ng hCG/ml is attained by the sandwich erythro-immunoassay using a chimera antibody prepared by coupling monoclonal anti-alpha-hCG antibody to an affinity-purified polyclonal antibody specific for sheep erythrocytes. This assay is amenable to the qualitative as well as quantitative use as described. The urinary components do not interfere in the assay. Results obtained by this assay on 47 human urine samples correlated well with the values obtained by '2-step' sandwich enzyme immunoassay and radioimmunoassay. PMID:2409175

  18. Validation of a new homogeneous immunoassay for the detection of carisoprodol in urine.

    PubMed

    Wang, Guohong; Huynh, Kim; Barhate, Rekha; Rodrigues, Warren; Moore, Christine; Coulter, Cynthia; Vincent, Michael; Soares, James

    2011-03-01

    The objective of this project was to validate a new high-throughput homogeneous enzyme immunoassay (HEIA) for the rapid detection of carisoprodol in human urine. Carisoprodol (Soma(®)) and meprobamate are widely prescribed as musculoskeletal pain relief drugs and are listed as one of the 10 most frequently identified drugs associated with DUI cases. Carisoprodol has a short elimination half-life of 1-3 h; however, its major active metabolite, meprobamate, has a longer elimination half-life of 6-17 h. As a result, it is important for an immunoassay to cross-react with both compounds. The advantage of this new assay is that cutoff concentrations can be adjusted between 100 and 500 ng/mL. The reportable range was 25 to 1000 ng/mL for carisoprodol and 50 to 10,000 ng/mL for meprobamate. The intraday coefficient of variation (% CV) for the semi-quantitative assay was less than 1%. The homogeneous assay was validated with a total of 86 urine samples previously analyzed by liquid chromatography-tandem mass spectrometry with carisoprodol concentrations ranging from 50 to 10,000 ng/mL. The accuracy was found to be 100% when immunoassay cutoff concentrations of carisoprodol and meprobamate were set at 100 and 1000 ng/mL, respectively. PMID:21396230

  19. Development of an incurred cornbread model for gluten detection by immunoassays.

    PubMed

    Sharma, Girdhari M; Khuda, Sefat E; Pereira, Marion; Slate, Andrew; Jackson, Lauren S; Pardo, Christopher; Williams, Kristina M; Whitaker, Thomas B

    2013-12-11

    Gluten that is present in food as a result of cross-contact or misbranding can cause severe health concerns to wheat-allergic and celiac patients. Immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and lateral flow device (LFD), are commonly used to detect gluten traces in foods. However, the performance of immunoassays can be affected by non-assay-related factors, such as food matrix and processing conditions. Gluten (0-500 ppm) and wheat flour (20-1000 ppm) incurred cornbread was prepared at different incurred levels and baking conditions (204.4 °C for 20, 27, and 34 min) to study the accuracy and precision of gluten measurement by seven immunoassay kits (three LFD and four ELISA kits). The stability and immunoreactivity of gluten proteins, as measured by western blot using three different antibodies, were not adversely affected by the baking conditions. However, the gluten recovery varied depending upon the ELISA kit and the gluten source used to make the incurred cornbread, affecting the accuracy of gluten quantification (BioKits, 9-77%; Morinaga, 91-137%; R-Biopharm, 61-108%; and Romer Labs, 113-190%). Gluten recovery was reduced with increased baking time for most ELISA kits analyzed. Both the sampling and analytical variance increased with an increase in the gluten incurred level. The predicted analytical coefficient of variation associated with all ELISA kits was below 12% for all incurred levels, indicative of good analytical precision. PMID:24245605

  20. COMPARISON OF IMMUNOASSAY AND GAS CHROMATOGRAPHY/MASS SPECTROMETRY METHODS FOR MEASURING 3,5,6-TRICHLORO-2PYRIDINOL IN MULTIPLE SAMPLE MEDIA

    EPA Science Inventory

    Two enzyme-linked immunosorbent assay (ELISA) methods were evaluated for the determination of 3,5,6-trichloro-2-pyridinol (3,5,6-TCP) in multiple sample media (dust, soil, food, and urine). The dust and soil samples were analyzed by a commercial RaPID immunoassay testing kit. ...

  1. Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor.

    PubMed

    Kerishnan, Jesinda P; Gopinath, Subash C B; Kai, Sia Bik; Tang, Thean-Hock; Ng, Helen Lee-Ching; Rahman, Zainal Ariff Abdul; Hashim, Uda; Chen, Yeng

    2016-01-01

    The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5(+)/6(+)) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients. PMID:27279791

  2. Detection of IgM antibrucella antibody in the absence of IgGs: a challenge for the clinical interpretation of brucella serology.

    PubMed

    Solís García Del Pozo, Julián; Lorente Ortuño, Santiago; Navarro, Elena; Solera, Javier

    2014-12-01

    The use of enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgM antibodies antibrucella has become widespread in the diagnosis of human brucellosis. IgM anti-Brucella antibodies are indicative of acute infection. Between 2009-2013, 5307 patients were evaluated for serologic diagnosis at the Microbiology Laboratory of the Albacete General Hospital. A ELISA IgM-positive, IgG-negative anti-Brucella antibody serology pattern was detected in 17 of those patients. Epidemiology data, symptoms, laboratory data, treatment and outcome from these patients were reviewed. Sixteen patients presented with musculoskeletal pain, fatigue and/or fever and 1 was asymptomatic. Five patients received treatment with doxycycline combined with rifampin, gentamycin or streptomycin during 6-12 weeks, with no improvement. None of the 17 patients were finally diagnosed with brucellosis. Our results indicate that anti-Brucella IgM positive serology, per se, is not enough to diagnose acute brucellosis and other methods should be used for confirmation. Brucella serology data should be interpreted taking into account the patient's clinical history and epidemiological context. PMID:25474572

  3. Detection of Human Papillomavirus 16-Specific IgG and IgM Antibodies in Patient Sera: A Potential Indicator of Oral Squamous Cell Carcinoma Risk Factor

    PubMed Central

    Kerishnan, Jesinda P.; Gopinath, Subash C.B.; Kai, Sia Bik; Tang, Thean-Hock; Ng, Helen Lee-Ching; Rahman, Zainal Ariff Abdul; Hashim, Uda; Chen, Yeng

    2016-01-01

    The association between human papillomavirus type 16 (HPV16) and oral cancer has been widely reported. However, detecting anti-HPV antibodies in patient sera to determine risk for oral squamous cell carcinoma (OSCC) has not been well studied. In the present investigation, a total of 206 OSCC serum samples from the Malaysian Oral Cancer Database & Tissue Bank System, with 134 control serum samples, were analyzed by enzyme-linked immunosorbant assay (ELISA) to detect HPV16-specific IgG and IgM antibodies. In addition, nested PCR analysis using comprehensive consensus primers (PGMY09/11 and GP5+/6+) was used to confirm the presence of HPV. Furthermore, we have evaluated the association of various additional causal factors (e.g., smoking, alcohol consumption, and betel quid chewing) in HPV-infected OSCC patients. Statistical analysis of the Malaysian population indicated that OSCC was more prevalent in female Indian patients that practices betel quid chewing. ELISA revealed that HPV16 IgG, which demonstrates past exposure, could be detected in 197 (95.6%) OSCC patients and HPV16-specific IgM was found in a total of 42 (20.4%) OSCC patients, indicating current exposure. Taken together, our study suggest that HPV infection may play a significant role in OSCC (OR: 13.6; 95% CI: 3.89-47.51) and HPV16-specific IgG and IgM antibodies could represent a significant indicator of risk factors in OSCC patients. PMID:27279791

  4. Multiplexed In-cell Immunoassay for Same-sample Protein Expression Profiling

    PubMed Central

    Shang, Jing; Zrazhevskiy, Pavel; Postupna, Nadia; Keene, C. Dirk; Montine, Thomas J.; Gao, Xiaohu

    2015-01-01

    In-cell immunoassays have become a valuable tool for protein expression analysis complementary to established assay formats. However, comprehensive molecular characterization of individual specimens has proven challenging and impractical due to, in part, a singleplex nature of reporter enzymes and technical complexity of alternative assay formats. Herein, we describe a simple and robust methodology for multiplexed protein expression profiling on the same intact specimen, employing a well-characterized enzyme alkaline phosphatase for accurate quantification of all targets of interest, while overcoming fundamental limitations of enzyme-based techniques by implementing the DNA-programmed release mechanism for segregation of sub-sets of target-bound reporters. In essence, this methodology converts same-sample multi-target labeling into a set of isolated singleplex measurements performed in a parallel self-consistent fashion. For a proof-of-principle, multiplexed detection of three model proteins was demonstrated on cultured HeLa cells, and two clinically-relevant markers of dementia, β-amyloid and PHF-tau, were profiled in formalin-fixed paraffin embedded brain tissue sections, uncovering correlated increase in abundance of both markers in the “Alzheimer’s disease” cohort. Featuring an analytically powerful yet technically simple and robust methodology, multiplexed in-cell immunoassay is expected to enable insightful same-sample protein profiling studies and become broadly adopted in biomedical research and clinical diagnostics. PMID:26328896

  5. Analysis of drugs in human tissues by supercritical fluid extraction/immunoassay

    NASA Astrophysics Data System (ADS)

    Furton, Kenneth G.; Sabucedo, Alberta; Rein, Joseph; Hearn, W. L.

    1997-02-01

    A rapid, readily automated method has been developed for the quantitative analysis of phenobarbital from human liver tissues based on supercritical carbon dioxide extraction followed by fluorescence enzyme immunoassay. The method developed significantly reduces sample handling and utilizes the entire liver homogenate. The current method yields comparable recoveries and precision and does not require the use of an internal standard, although traditional GC/MS confirmation can still be performed on sample extracts. Additionally, the proposed method uses non-toxic, inexpensive carbon dioxide, thus eliminating the use of halogenated organic solvents.

  6. Development of SERS substrates for immunoassay applications

    NASA Astrophysics Data System (ADS)

    Celik, Okkes; Kahraman, Mehmet

    2016-03-01

    Surface-enhanced Raman scattering (SERS) is an emerging technique for the detection and identification of biological structures. SERS is based on immunoassay methods are mostly used for the specific detection and identification of bacteria. In this study, SERS substrates are developed with deposition of synthesized spherical 13 nm gold nanoparticles (AuNPs) and 50 nm silver nanoparticles (AgNPs) on regular glass slides with convective assembly method for SERS based immunoassay for the detection and identification of bacteria. The synthesized NPs are characterized by UV-vis absorption spectroscopy, dynamic light scattering (DLS) and atomic force microscopy (AFM). Colloidal suspensions are concentrated by centrifugation to obtain thin films by the deposition of NPs on a regular glass slide with the convective assembly. The experimental parameters for the convective assembly are optimized by changing of NP concentration, stage velocity and NPs volume dropped between two glass slides. Structural characterization of thin films is performed by AFM and SEM. SERS is also used for the optical characterization of the prepared thin films of NPs. In this study, 4- aminothiophenol (4-ATP) is used as probe molecules to evaluate SERS activity of the thin films depending on the type and concentration of NPs. The results demonstrate that, SERS performances of the thin films are dependent on not only the type of NPs but also it depends on the concentration of NPs which forms thin films. The thin film having highest SERS activity could be used for the SERS-based immunoassays for the detection and identification of bacteria.

  7. Magnetic Electrochemical Immunoassays with Quantum Dot Labels for Detection of Phosphorylated Acetylcholinesterase in Plasma

    SciTech Connect

    Wang, Hua; Wang, Jun; Timchalk, Charles; Lin, Yuehe

    2008-11-01

    A new magnetic electrochemical immunoassay has been developed as a tool for biomonitoring exposures to organophosphate (OP) compounds, e.g., insecticides and chemical nerve agents, by directly detecting organophosphorylated acetylcholinesterase (OP-AChE). This immunoassay uniquely incorporates highly efficient magnetic separation with ultrasensitive square wave voltammetry (SWV) analysis with quantum dots (QDs) as labels. A pair of antibodies was used to achieve the specific recognition of OP-AChE that was prepared with paraoxon as an OP model agent. Antiphosphoserine polyclonal antibodies were anchored on amorphous magnetic particles preferably chosen to capture OP-AChE from the sample matrixes by binding their phosphoserine moieties that were exposed through unfolding the protein adducts. This was validated by electrochemical examinations and enzyme-linked immunosorbent assays. Furthermore, antihuman AChE monoclonal antibodies were labeled with cadmium-source QDs to selectively recognize the captured OP-AChE, as characterized by transmission electron microscopy. The subsequent electrochemical SWV analysis of the cadmium component released by acid from the coupled QDs was conducted on disposable screen-printed electrodes. Experimental results indicated that the SWV-based immunoassays could yield a linear response over a broad concentration range of 0.3-300 ng/mL OP-AChE in human plasma with a detection limit of 0.15 ng/mL. Such a novel electrochemical immunoassay holds great promise as a simple, selective, sensitive, and field-deployable tool for the effective biomonitoring and diagnosis of potential exposures to nerve agents and pesticides.

  8. A nonenzymatic optical immunoassay strategy for detection of Salmonella infection based on blue silica nanoparticles.

    PubMed

    Sun, Qian; Zhao, Guangying; Dou, Wenchao

    2015-10-22

    A novel nonenzymatic optical immunoassay strategy was for the first time designed and utilized for sensitive detection of antibody to Salmonella pullorum and Salmonella gallinarum (S. pullorum and S. gallinarum) in serum. The optical immunoassay strategy was based on blue silica nanoparticles (Blue-SiNps) and magnetic beads (MB). To construct such an optical immunoassay system, the Blue-SiNPs were first synthesized by inverse microemulsion method, characterized by SEM, Zeta potential and FTIR. Two nanostructures including Blue-SiNPs and MB were both functionalized with antibody against S. pullorum and S. gallinarum (anti-PG) without using enzyme labeled antibody. Anti-PG functionalized blue silica nanoparticles (IgG-Blue-SiNps) were used as signal transduction labels, while anti-PG functionalized magnetic beads (IgG-MB) were selected to separate and enrich the final sandwich immune complexes. In the process of detecting negative serum, a sandwich immunocomplex is formed between the IgG-MB and IgG-Blue-SiNPs. With the separation of the immunocomplex using an external magnetic field, the final plaque displayed bright blue color. While in the detection of infected serum, IgG-MB and anti-PG formed sandwich immunocomplexes, IgG-Blue-SiNPs were unable to bind to the limited sites of the antigen, and a light brown plaque was displayed in the bottom of microplate well. Stable results were obtained with an incubation time of 60 min at room temperature, and different colors corresponding to different results can be directly detected with naked eye. The reaction of IgG-Blue-SiNPs with S. pullorum was inhibited by 1:100 dilution of positive chicken serum. Such a simple immunoassay holds great potential as sensitive, selective and point-of-care (POC) tool for diagnosis of other biological molecules. PMID:26526916

  9. Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

    PubMed

    Augustine, Swinburne A J; Simmons, Kaneatra J; Eason, Tarsha N; Griffin, Shannon M; Curioso, Clarissa L; Wymer, Larry J; Fout, G Shay; Grimm, Ann C; Oshima, Kevin H; Dufour, Al

    2015-10-01

    There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H

  10. Age-Related Decline in Natural IgM Function: Diversification and Selection of the B-1a Cell Pool with Age.

    PubMed

    Holodick, Nichol E; Vizconde, Teresa; Hopkins, Thomas J; Rothstein, Thomas L

    2016-05-15

    Streptococcus pneumoniae is the most common cause of pneumonia, which claims the lives of people over the age of 65 y seven times more frequently than those aged 5-49 y. B-1a cells provide immediate and essential protection from S. pneumoniae through production of natural Ig, which has minimal insertion of N-region additions added by the enzyme TdT. In experiments with SCID mice infected with S. pneumoniae, we found passive transfer of IgG-depleted serum from aged (18-24 mo old) mice had no effect whereas IgG-depleted serum from young (3 mo old) mice was protective. This suggests protective natural IgM changes with age. Using single cell PCR we found N-region addition, which is initially low in fetal-derived B-1a cell IgM developing in the absence of TdT, increased in 7- to 24-mo-old mice as compared with 3-mo-old mice. To determine the mechanism responsible for the age related change in B-1a cell IgM, we established a mixed chimera system in which mice were reconstituted with allotype-marked mature peritoneal B-1a cells and adult bone marrow cells. We demonstrated even in the presence of mature peritoneal B-1a cells, adult bone marrow contributed to the mature B-1a cell pool. More importantly, using this system we found over a 10-mo-period peritoneal B-1a cell IgM changed, showing the number of cells lacking N-region additions at both junctions fell from 49 to 29% of sequences. These results strongly suggest selection-induced skewing alters B-1a cell-derived natural Ab, which may in turn be responsible for the loss of natural IgM-mediated protection against pneumococcal infection. PMID:27183643

  11. Nanomaterial Labels in Electrochemical Immunosensors and Immunoassays

    SciTech Connect

    Liu, Guodong; Lin, Yuehe

    2007-12-15

    This article reviews recent advances in nanomaterial labels in electrochemical immunosensors and immunoassays. Various nanomaterial labels are discussed, including colloidal gold/silver, semiconductor nanoparticles, and markers loaded nanocarriers (carbon nanotubes, apoferritin, silica nanoparticles, and liposome beads). The enormous signal enhancement associated with the use of nanomaterial labels and with the formation of nanomaterial–antibody-antigen assemblies provides the basis for ultrasensitive electrochemical detection of disease-related protein biomarkers, biothreat agents, or infectious agents. In general, all endeavors cited here are geared to achieve one or more of the following goals: signal amplification by several orders of magnitude, lower detection limits, and detecting multiple targets.

  12. Fluorescent immunoassay visualization of sorbed pollutants

    SciTech Connect

    Moore, W.K.; Mossman, D.J.; Schwab, A.P.; Feldbush, T.L.

    1994-12-31

    Current methods of detecting sorbed soil pollutants require that the contaminant be extracted from the soil. In an effort to make detection simpler and safer, standard fluorescent immunoassay techniques are being modified to allow fluorescent tags on the pollutant to be viewed and photographed with epifluorescent microscopy. Initial research focuses on detecting chlorinated benzenes on various soil types and developing a technique for tagging these pollutants with appropriate antibodies. This should lead to detection in actual soil cores and a better understanding of how contaminants progress through different soils.

  13. Gliadin Detection in Food by Immunoassay

    NASA Astrophysics Data System (ADS)

    Grant, Gordon; Sporns, Peter; Hsieh, Y.-H. Peggy

    Immunoassays are very sensitive and efficient tests that are commonly used to identify a specific protein. Examples of applications in the food industry include identification of proteins expressed in genetically modified foods, allergens, or proteins associated with a disease, including celiac disease. This genetic disease is associated with Europeans and affects about one in every 200 people in North America. These individuals react immunologically to wheat proteins, and consequently their own immune systems attack and damage their intestines. This disease can be managed if wheat proteins, specifically "gliadins," are avoided in foods.

  14. Industrial Fungal Enzymes: An Occupational Allergen Perspective

    PubMed Central

    Green, Brett J.; Beezhold, Donald H.

    2011-01-01

    Occupational exposure to high-molecular-weight allergens is a risk factor for the development and pathogenesis of IgE-mediated respiratory disease. In some occupational environments, workers are at an increased risk of exposure to fungal enzymes used in industrial production. Fungal enzymes have been associated with adverse health effects in the work place, in particular in baking occupations. Exposure-response relationships have been demonstrated, and atopic workers directly handling fungal enzymes are at an increased risk for IgE-mediated disease and occupational asthma. The utilization of new and emerging fungal enzymes in industrial production will present new occupational exposures. The production of antibody-based immunoassays is necessary for the assessment of occupational exposure and the development of threshold limit values. Allergen avoidance strategies including personal protective equipment, engineering controls, protein encapsulation, and reduction of airborne enzyme concentrations are required to mitigate occupational exposure to fungal enzymes. PMID:21747869

  15. Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays

    PubMed Central

    Vollmer, Tanja; Diekmann, Juergen; Eberhardt, Matthias; Knabbe, Cornelius; Dreier, Jens

    2016-01-01

    Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes. PMID:27556482

  16. Monitoring of Anti-Hepatitis E Virus Antibody Seroconversion in Asymptomatically Infected Blood Donors: Systematic Comparison of Nine Commercial Anti-HEV IgM and IgG Assays.

    PubMed

    Vollmer, Tanja; Diekmann, Juergen; Eberhardt, Matthias; Knabbe, Cornelius; Dreier, Jens

    2016-01-01

    Diagnosis of hepatitis E virus (HEV) is usually determined serologically by detection of the presence of immunoglobulin (Ig)M antibodies or rising anti-HEV IgG titers. However, serological assays have demonstrated a significant variation in their sensitivities and specificities. In this study, we present the systematic comparison of different immunological anti-HEV assays using complete seroconversion panels of 10 virologically confirmed HEV genotype 3 infected individuals. Assay sensitivities were further evaluated by testing serially diluted World Health Organization (WHO) reference reagent for hepatitis E virus antibody and one patient sample infected with HEV genotype 3. Anti-HEV IgM and IgG antibody presence was determined using the immunological assays Wantai HEV IgM/IgG enzyme-linked immunosorbent assay (ELISA) (Sanbio, Uden, The Netherlands), recomWell HEV IgM/IgG (Mikrogen, Neuried, Germany), HEV IgM ELISA 3.0, HEV ELISA, HEV ELISA 4.0, Assure HEV IgM Rapid Test (all MP Biomedicals Europe, Illkirch Cedex, France) and Anti-HEV ELISA (IgM/IgG, Euroimmun, Lübeck, Germany). The assays showed differences regarding their analytical and diagnostic sensitivities, with anti-HEV IgM assays (n = 5) being more divergent compared to anti-HEV IgG (n = 4) assays in this study. Considerable variations were observed particularly for the detection period of IgM antibodies. This is the first study systematically characterizing serologic assays on the basis of seroconversion panels, providing sample conformity for a conclusive comparison. Future studies should include the assay comparison covering the four different genotypes. PMID:27556482

  17. Circulating IgM Requires Plasma Membrane Disruption to Bind Apoptotic and Non-Apoptotic Nucleated Cells and Erythrocytes

    PubMed Central

    Hesketh, Emily E.; Dransfield, Ian; Kluth, David C.; Hughes, Jeremy

    2015-01-01

    Autoimmunity is associated with defective phagocytic clearance of apoptotic cells. IgM deficient mice exhibit an autoimmune phenotype consistent with a role for circulating IgM antibodies in apoptotic cell clearance. We have extensively characterised IgM binding to non-apoptotic and apoptotic mouse thymocytes and human Jurkat cells using flow cytometry, confocal imaging and electron microscopy. We demonstrate strong specific IgM binding to a subset of Annexin-V (AnnV)+PI (Propidium Iodide)+ apoptotic cells with disrupted cell membranes. Electron microscopy studies indicated that IgM+AnnV+PI+ apoptotic cells exhibited morphologically advanced apoptosis with marked plasma membrane disruption compared to IgM-AnnV+PI+ apoptotic cells, suggesting that access to intracellular epitopes is required for IgM to bind. Strong and comparable binding of IgM to permeabilised non-apoptotic and apoptotic cells suggests that IgM bound epitopes are 'apoptosis independent' such that IgM may bind any cell with profound disruption of cell plasma membrane integrity. In addition, permeabilised erythrocytes exhibited significant IgM binding thus supporting the importance of cell membrane epitopes. These data suggest that IgM may recognize and tag damaged nucleated cells or erythrocytes that exhibit significant cell membrane disruption. The role of IgM in vivo in conditions characterized by severe cell damage such as ischemic injury, sepsis and thrombotic microangiopathies merits further exploration. PMID:26121639

  18. Sensitive, Fast, and Specific Immunoassays for Methyltestosterone Detection

    PubMed Central

    Kong, Na; Song, Shanshan; Peng, Juan; Liu, Liqiang; Kuang, Hua; Xu, Chuanlai

    2015-01-01

    An indirect competitive enzyme-linked immunosorbent assay (icELISA) and an immunochromatographic strip assay using a highly specific monoclonal antibody, were developed to detect methyltestosterone (MT) residues in animal feed. The optimized icELISA had a half-inhibition concentration value of 0.26 ng/mL and a limit of detection value of 0.045 ng/mL. There was no cross-reactivity with eight analogues, revealing high specificity for MT. Based on icELISA results, the recovery rate of MT in animal feed was 82.4%–100.6%. The results were in accordance with those obtained by gas chromatography-mass spectrometry. The developed immunochromatographic strip assay, as the first report for MT detection, had a visual cut-off value of 1 ng/mL in PBS, 2.5 ng/g in fish feed, and 2.5 ng/g in pig feed. Therefore, these immunoassays are useful and fast tools for MT residue detection in animal feed. PMID:25938198

  19. Sensitive giant magnetoresistive-based immunoassay for multiplex mycotoxin detection.

    PubMed

    Mak, Andy C; Osterfeld, Sebastian J; Yu, Heng; Wang, Shan X; Davis, Ronald W; Jejelowo, Olufisayo A; Pourmand, Nader

    2010-03-15

    Rapid and multiplexed measurement is vital in the detection of food-borne pathogens. While highly specific and sensitive, traditional immunochemical assays such as enzyme-linked immunosorbent assays (ELISAs) often require expensive read-out equipment (e.g. fluorescent labels) and lack the capability of multiplex detection. By combining the superior specificity of immunoassays with the sensitivity and simplicity of magnetic detection, we have developed a novel multiplex magnetic nanotag-based detection platform for mycotoxins that functions on a sub-picomolar concentration level. Unlike fluorescent labels, magnetic nanotags (MNTs) can be detected with inexpensive giant magnetoresistive (GMR) sensors such as spin-valve sensors. In the system presented here, each spin-valve sensor has an active area of 90 microm x 90 microm, arranged in an 8 x 8 array. Sample is added to the antibody-immobilized sensor array prior to the addition of the biotinylated detection antibody. The sensor response is recorded in real time upon the addition of streptavidin-linked MNTs on the chip. Here we demonstrate the simultaneous detection of multiple mycotoxins (aflatoxins B(1), zearalenone and HT-2) and show that a detection limit of 50 pg/mL can be achieved. PMID:20047828

  20. IL-1beta expression in IgM monoclonal gammopathy and its relationship to multiple myeloma.

    PubMed

    Donovan, K A; Lacy, M Q; Gertz, M A; Lust, J A

    2002-03-01

    We have shown that IL-1beta is not detectable in normal plasma cells but is produced by plasma cells from virtually all patients with multiple myeloma (MM). To extend our earlier work, IL-1beta expression was determined in 13 newly diagnosed patients with IgM monoclonal gammopathy. Eleven patients with Waldenstrom's macroglobulinemia (WM) and two patients with IgM MM were investigated for IL-1beta expression by in situ hybridization (ISH). All patients with WM had bone marrow biopsies consistent with the diagnosis, an IgM M-protein in the serum, and subsequently required chemotherapy. Seven of 11 patients with WM had an M-protein >3 g/dl and five patients had bone surveys performed that were negative for osteolytic disease. Two patients were diagnosed with IgM MM because of the presence of significant osteolytic disease on a metastatic bone survey. ISH for kappa, lambda, and IL-1beta expression was performed on bone marrow aspirates from each of the 13 patients. None of the neoplastic cells from the 11 patients with WM showed detectable IL-1beta expression by ISH. However, the neoplastic cells from both patients with IgM MM expressed IL-1beta mRNA at high levels. This aberrant IL-1beta production may explain the presence of bone lesions in the patients with IgM MM. PMID:11896542

  1. Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis.

    PubMed

    Wang, Hongsheng; Coligan, John E; Morse, Herbert C

    2016-01-01

    Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. PMID:27014278

  2. Physiological IgM Class Catalytic Antibodies Selective for Transthyretin Amyloid*

    PubMed Central

    Planque, Stephanie A.; Nishiyama, Yasuhiro; Hara, Mariko; Sonoda, Sari; Murphy, Sarah K.; Watanabe, Kenji; Mitsuda, Yukie; Brown, Eric L.; Massey, Richard J.; Primmer, Stanley R.; O'Nuallain, Brian; Paul, Sudhir

    2014-01-01

    Peptide bond-hydrolyzing catalytic antibodies (catabodies) could degrade toxic proteins, but acquired immunity principles have not provided evidence for beneficial catabodies. Transthyretin (TTR) forms misfolded β-sheet aggregates responsible for age-associated amyloidosis. We describe nucleophilic catabodies from healthy humans without amyloidosis that degraded misfolded TTR (misTTR) without reactivity to the physiological tetrameric TTR (phyTTR). IgM class B cell receptors specifically recognized the electrophilic analog of misTTR but not phyTTR. IgM but not IgG class antibodies hydrolyzed the particulate and soluble misTTR species. No misTTR-IgM binding was detected. The IgMs accounted for essentially all of the misTTR hydrolytic activity of unfractionated human serum. The IgMs did not degrade non-amyloidogenic, non-superantigenic proteins. Individual monoclonal IgMs (mIgMs) expressed variable misTTR hydrolytic rates and differing oligoreactivity directed to amyloid β peptide and microbial superantigen proteins. A subset of the mIgMs was monoreactive for misTTR. Excess misTTR was dissolved by a hydrolytic mIgM. The studies reveal a novel antibody property, the innate ability of IgMs to selectively degrade and dissolve toxic misTTR species as a first line immune function. PMID:24648510

  3. Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis

    PubMed Central

    Wang, Hongsheng; Coligan, John E.; Morse, Herbert C.

    2016-01-01

    Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions. PMID:27014278

  4. The role of IgM rheumatoid factor in experimental immune vasculitis.

    PubMed Central

    Floyd, M; Tesar, J T

    1979-01-01

    The effect of IgM rhematoid factor (RF) on reversepassive cutaneous Arthus reaction in rats was studied. The RF was obtained from the serum cryoglobulin of a patient with symptoms of purpura, arthralgia and digital gangrene. The cryoglobulins was of IgG-IgM type and when given i.v it induced a prompt hypocomplementaemia in experimental animals. The purified RF also induced low serum complement levels when injected i.v. along with complexes of non-complement-fixing, aggregated IgG. A reverse passive Arthus reaction was induced by intradermal injection of IgG anti-bovine serum albumin (BSA), followed by an i.v. dose of antigen (Ag). The cutaneous inflammatory reaction was aggravated by simultaneous administration of IgM RF intradermally, but not by IgM without antibody (Ab) properties. Intradermal injection of low concentrations of non-complement-fixing IgG anti-BSA, along with normal human IgM, followed by i.v. injection of BSA, resulted in a complete lack of cutaneous inflammation. At higher Ab concentrations there was only a mild inflammation. However, when IgM RF was substituted for normal IgM and injected with non-complement-fixing anti-BSA, an effective reverse passive cutaneous Arthus reaction and vasculitis was induced. The inflammatory response was greatly suppressed by decomplementation of animals by cobra venom factor. This study provides evidence favouring an inflammatory, complement-dependent role for RF in vasculitis. PMID:157238

  5. Platelet antibodies of the IgM class in immune thrombocytopenic purpura

    SciTech Connect

    Cines, D.B.; Wilson, S.B.; Tomaski, A.; Schreiber, A.D.

    1985-04-01

    The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, the authors studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. They observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP. They obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3. The authors demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP.

  6. Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay

    PubMed Central

    Wynwood, S. J.; Burns, M. A.; Graham, G. C.; Weier, S. L.; McKay, D. B.; Craig, S. B.

    2016-01-01

    Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations. PMID:26835139

  7. Serological diagnosis of Leptospirosis in bovine serum samples using a microsphere immunoassay.

    PubMed

    Wynwood, S J; Burns, M A; Graham, G C; Weier, S L; McKay, D B; Craig, S B

    2016-01-01

    Leptospirosis causes significant economic loss within the cattle industry worldwide. Current diagnostic methods are generally inadequate for dealing with large numbers of samples, are outdated, and provide little useful diagnostic and epidemiological information. This aim of this study was to apply a microsphere immunoassay (MIA), utilising Luminex xMap technology, to 200 bovine serum samples to determine this method's usefulness in leptospirosis diagnosis in comparison with the current gold standard, the microscopic agglutination test (MAT). Although MAT is the most widely used laboratory test for the diagnosis of leptospirosis, its reliance on live cultures, subjective interpretation of results and an inability to differentiate between antibody classes, suggest MAT is no longer the best method for the diagnosis of leptospirosis. The results presented in this paper show that MIA was able to determine reactive from non-reactive samples when compared with MAT, and was able to differentiate IgG and IgM classes of antibody. The results suggest increased sensitivity in MIA and the ability to multiplex up to 500 antigens at one time allows for significant improvements in cost-effectiveness as well as a reduced dependency on live cultures. The relatively low cost, high throughput platform and differentiation of antibody class, as shown in previous research, make this assay worthy of consideration for the diagnosis of leptospirosis in small-scale or large-scale bovine populations. PMID:26835139

  8. Microarray-integrated optoelectrofluidic immunoassay system.

    PubMed

    Han, Dongsik; Park, Je-Kyun

    2016-05-01

    A microarray-based analytical platform has been utilized as a powerful tool in biological assay fields. However, an analyte depletion problem due to the slow mass transport based on molecular diffusion causes low reaction efficiency, resulting in a limitation for practical applications. This paper presents a novel method to improve the efficiency of microarray-based immunoassay via an optically induced electrokinetic phenomenon by integrating an optoelectrofluidic device with a conventional glass slide-based microarray format. A sample droplet was loaded between the microarray slide and the optoelectrofluidic device on which a photoconductive layer was deposited. Under the application of an AC voltage, optically induced AC electroosmotic flows caused by a microarray-patterned light actively enhanced the mass transport of target molecules at the multiple assay spots of the microarray simultaneously, which reduced tedious reaction time from more than 30 min to 10 min. Based on this enhancing effect, a heterogeneous immunoassay with a tiny volume of sample (5 μl) was successfully performed in the microarray-integrated optoelectrofluidic system using immunoglobulin G (IgG) and anti-IgG, resulting in improved efficiency compared to the static environment. Furthermore, the application of multiplex assays was also demonstrated by multiple protein detection. PMID:27190571

  9. Fluorimetric Immunoassay for Multianalysis of Aflatoxins

    PubMed Central

    2013-01-01

    A sensitive fluorimetric ELISA was developed for the analysis of aflatoxins. The assay was performed in a 384 microwell plate, wherein high specificity monoclonal antibody against AFM1 (mAb-AFM1) was used as capture antibody and FITC conjugated secondary antibody was used for detection and quantification of the analyte. The linear range of the immunoassay was found to be 6.25–50 pg/mL. AFM1 as low as 1 pg/mL was detected by this method with assay volume 40 μL. The multi-analysis of different aflatoxins was also investigated in the microwell plate, based on the cross-reactivity (CR) approach. Real milk samples were tested along with certified reference material by standard addition method and recovery analysis was done. The mAb-AFM1 showed 23.2% CR with AFB1, 50% CR with respect to AFM2, and least CR towards AFG1 (<1%). Furthermore, mixture analysis of AFM2 and AFB1 was carried out at specific concentrations of AFM1. The advantages of this developed immunoassay are high sensitivity, high throughput, multianalyte detection, versatility, and ease of handling. PMID:24000318

  10. [A case of multifocal motor neuropathy with IgM lambda anti-GM1 antibody and IgM kappa paraprotein reacting exclusively with GM2].

    PubMed

    Arai, Motomi; Kusunoki, Susumu

    2009-01-01

    A 57-year-old previously healthy woman visited our clinic complaining of frequent muscle cramps and progressive weakness in the hands and fingers for 3 years. On examination, cranial nerves were unremarkable. There were moderate weakness and mild muscle wasting with fasciculation in the left thumb flexor and interossei on both sides. Tendon reflexes were hypoactive. There were no pathologic reflexes or sensory deficit. The cerebrospinal fluid was unremarkable. Nerve conduction studies demonstrated conduction block in the right ulnar nerve. Compound muscle action potential in the left median nerve was low-normal. Distal motor latencies, motor and sensory nerve conduction velocities were normal in all nerves tested. A diagnosis of multifocal motor neuropathy was made. Two courses of intravenous immunoglobulin infusion gave no beneficial effects. The patient had IgM kappa monoclonal gammopathy of undetermined significance. Her serum IgM reacted with GM2, GM1, and GA1 but not with GD1a, GD1b, GD3, GalNAc-GD1a, GT1b, GQ1b, galactocerebroside, or sulfated glucuronyl paragloboside. IgM kappa paraprotein reacted exclusively with GM2. Only IgM lambda bound to GM1 and GA1, suggesting the possibility that another paraprotein, though undetectable by immunoelectrohoresis, had a reactivity with GM1 and GA1. This case showed previously unreported antigenic specificity of paraproteins in cases of MMN. PMID:19348179

  11. Enzyme markers

    MedlinePlus

    ... or defects passed down through families (inherited) can affect how enzymes work. Some enzymes are affected by several genes. Test results are usually reported as a percentage of normal enzyme activity.

  12. Demonstration of immunoglobulin M class antibodies to Toxoplasma gondii antigenic component p35000 by enzyme-linked antigen immunosorbent assay.

    PubMed Central

    Lindenschmidt, E G

    1986-01-01

    On the basis that 89% of 48 acute-phase toxoplasmosis patients showed immunoglobulin M (IgM) class antibodies to the 35,000-molecular-weight antigenic component (p35000) of Toxoplasma gondii, as demonstrated by IgM immunoblotting, the antigen was purified by sucrose gradient centrifugation and enzyme labeled for use in an enzyme-linked antigen immunosorbent assay (ELA) for the demonstration of IgM class antibodies to the p35000 component. The ELA showed a specificity of 96% with 139 serum specimens at a serum dilution of only 1:5. The test serologically detected 73 symptomatic acute-phase toxoplasmosis patients; 64 were positive in the 19S IgM indirect immunofluorescent-antibody test, and 9 were negative, although they showed IgM antibodies to p35000, as demonstrated by IgM immunoblotting. Also, the ELA turned out to be independent of IgM rheumatoid factors in six acute-phase toxoplasmosis serum specimens. PMID:3536996

  13. An on-bacterium flow cytometric immunoassay for protein quantification.

    PubMed

    Lan, Wen-Jun; Lan, Wei; Wang, Hai-Yan; Yan, Lei; Wang, Zhe-Li

    2013-09-01

    The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was <10%. The comparison between this immunoassay and a polystyrene bead-based flow cytometric immunoassay showed a correlation coefficient of 0.998 for serum CEA or 0.996 for serum CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein. PMID:23739299

  14. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    NASA Astrophysics Data System (ADS)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-04-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  15. Site-Specific N-Glycosylation of Recombinant Pentameric and Hexameric Human IgM

    NASA Astrophysics Data System (ADS)

    Moh, Edward S. X.; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.

    2016-07-01

    Glycosylation is known to play an important role in IgG antibody structure and function. Polymeric IgM, the largest known antibody in humans, displays five potential N-glycosylation sites on each heavy chain monomer. IgM can exist as a pentamer with a connecting singly N-glycosylated J-chain (with a total of 51 glycosylation sites) or as a hexamer (60 glycosylation sites). In this study, the N-glycosylation of recombinant pentameric and hexameric IgM produced by the same human cell type and culture conditions was site-specifically profiled by RP-LC-CID/ETD-MS/MS using HILIC-enriched tryptic and GluC glycopeptides. The occupancy of all putative N-glycosylation sites on the pentameric and hexameric IgM were able to be determined. Distinct glycosylation differences were observed between each of the five N-linked sites on the IgM heavy chains. While Asn171, Asn332, and Asn395 all had predominantly complex type glycans, differences in glycan branching and sialylation were observed between the sites. Asn563, a high mannose-rich glycosylation site that locates in the center of the IgM polymer, was only approximately 60% occupied in both the pentameric and hexameric IgM forms, with a difference in relative abundance of the glycan structures between the pentamer and hexamer. This study highlights the information obtained by characterization of the site-heterogeneity of a highly glycosylated protein of high molecular mass with quaternary structure, revealing differences that would not be seen by global glycan or deglycosylated peptide profiling.

  16. Differentiation of Classical Swine Fever Virus Infection from CP7_E2alf Marker Vaccination by a Multiplex Microsphere Immunoassay

    PubMed Central

    Xia, Hongyan; Harimoorthy, Rajiv; Vijayaraghavan, Balaje; Blome, Sandra; Widén, Frederik; Beer, Martin; Belák, Sándor

    2014-01-01

    Classical swine fever (CSF) is a highly contagious viral disease of pigs that has a tremendous socioeconomic impact. Vaccines are available for disease control. However, most industrialized countries are implementing stamping-out strategies to eliminate the disease and avoid trade restrictions. These restrictions can be avoided through the use of marker vaccines such as CP7_E2alf. Marker vaccines have to be accompanied by reliable and robust discriminatory assays. In this context, a multiplex microsphere immunoassay for serological differentiation of infected from vaccinated animals (DIVA) was developed to distinguish CSF virus (CSFV)-infected animals from CP7_E2alf-vaccinated animals. To this end, three viral proteins, namely, CSFV E2, CSFV Erns, and bovine viral diarrhea virus (BVDV) E2, were produced in insect cells using a baculovirus expression system; they were used as antigens in a microsphere immunoassay, which was further evaluated by testing a large panel of pig sera and compared to a well-characterized commercial CSFV E2 antibody enzyme-linked immunosorbent assays (ELISAs) and a test version of an improved CSFV Erns antibody ELISA. Under a cutoff median fluorescence intensity value of 5,522, the multiplex microsphere immunoassay had a sensitivity of 98.5% and a specificity of 98.9% for the detection of antibodies against CSFV E2. The microsphere immunoassay and the CSFV Erns ELISA gave the same results for 155 out of 187 samples (82.8%) for the presence of CSFV Erns antibodies. This novel multiplex immunoassay is a valuable tool for measuring and differentiating immune responses to vaccination and/or infection in animals. PMID:25378351

  17. Self-concentrating buoyant glass microbubbles for high sensitivity immunoassays.

    PubMed

    Juang, Duane S; Hsu, Chia-Hsien

    2016-02-01

    Here, we report the novel application of a material with self-concentrating properties for enhancing the sensitivity of immunoassays. Termed as glass microbubbles, they are antibody functionalized buoyant hollow glass microspheres that simultaneously float and concentrate into a dense monolayer when dispensed in a liquid droplet. This self-concentrating charactaristic of the microbubbles allow for autonomous signal localization, which translates to a higher sensitivity compared to other microparticle-based immunoassays. We then demonstrated a "microbubble array" platform consisting of the glass microbubbles floating in a microfluidic liquid hemisphere array for performing multiplex immunoassays. PMID:26620967

  18. Immunoassay procedures for fiber optic sensors

    NASA Astrophysics Data System (ADS)

    Ligler, Frances S.

    1988-04-01

    There is an increasing need for the development of an ultrasensitive immunoassay for use with fiber optic sensors. These detection systems can be used for such applications as disease diagnosis, detection of chemical and biological warfare agents or drugs of abuse, pollution control, therapeutic monitoring, and explosive detection. This specific program is designed to produce generic chemistries for use with existing fiber optic-based sensors to detect pathogens of particular threat to Army personnel as determined by USAMRIID. The detection system under development involves the attachment of antibodies to an optical fiber at high density. In addition, the immobilization must be achieved in a way which retains the antibody's ability to bind antigen. The functionality of the antibody will be tested through the binding of a labelled antigen. In the future, this assay could incorporate the antibodies developed by the Army for pathogens of particularly military concern.

  19. Nanoparticles for Enhanced Sensitivity in Electrochemical Immunoassays

    SciTech Connect

    Lin, Yuehe; Wang, Jun; Wang, Hua; Wu, Hong; Tang, Zhiwen

    2008-10-12

    In this manuscript, we report on electrochemical biosensors based on various nanoparticles (NPs) as labels for sensitive detection of protein biomarkers. We used silica nanoparticle as a carrier to loading a large amount of electroactive species such as poly(guanine) for sensitive immunoassay of tumor necrosis factor-alpha (TNF-a). We took the advantages of the unique hollow structure and reconstruction properties of apoferritin to prepare Cd3(PO4)2 nanoparticles as labels for sensitive assay of TNF-a. A novel immunochromatographic/electro-chemical biosensor based on quantum dots as labels has also been developed for rapid and sensitive detection of prostate-specific antigen (PSA) in human serum. These biosensors are quite sensitive with the detection limit at pM level and these approaches based on nanoparticle labels offer a new avenue for sensitive detection of protein biomarkers.

  20. Monoclonal Antibodies Specific for Immunorecessive Epitopes of Glucuronoxylomannan, the Major Capsular Polysaccharide of Cryptococcus neoformans, Reduce Serotype Bias in an Immunoassay for Cryptococcal Antigen▿

    PubMed Central

    Percival, Ann; Thorkildson, Peter; Kozel, Thomas R.

    2011-01-01

    Immunoassay for detection of glucuronoxylomannan (GXM), the major capsular polysaccharide of Cryptococcus neoformans, is an important tool for diagnosis of cryptococcosis. However, immunoassays that are based solely or in part on detection with polyclonal antibodies may show serotype bias in detection of GXM, particularly limited sensitivity for serotype C. In this study, we describe detection of GXM in an antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) that used a cocktail of two monoclonal antibodies (MAbs). MAb F12D2 was previously produced by immunization with GXM that had been treated to remove O-acetyl groups, a major source of serotype specificity. MAb F12D2 has a high degree of reactivity with GXM of serotypes A, B, C, and D, but the reactivity with serotype D was less than was found with other MAbs. MAb 339 is highly reactive with GXM of serotypes A and D. Use of a combination of the two MAbs produced an immunoassay that had the best properties of both MAbs, including good reactivity with serotype C, which is an emerging threat in sub-Saharan Africa. These results suggest that next-generation immunoassays for diagnosis of cryptococcosis may be formulated by (i) use of immunization and hybridoma screening strategies that are designed to prospectively meet the needs of immunoassay performance and (ii) careful selection of MAbs that span the expected polysaccharide serotypes in the subject patient population. PMID:21697342

  1. "Smart" mobile affinity matrix for microfluidic immunoassays.

    PubMed

    Malmstadt, Noah; Hoffman, Allan S; Stayton, Patrick S

    2004-08-01

    There is a current need for simple methods for immobilizing biomolecules within microfluidic channels. Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements. Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide)(PNIPAAm) and co-modified with biotinylated poly(ethylene glycol)(PEG). PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST)( approximately 28 degrees C in the solutions used here). This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate)(PET) microchannels. Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads. These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus. The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration. The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process. This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules. Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels. PMID:15269814

  2. A Monoclonal IgM Protein with Antibody-like Activity for Human Albumin

    PubMed Central

    Hauptman, Stephen; Tomasi, Thomas B.

    1974-01-01

    The serum of a patient (L'ec) with an IgM lambda monoclonal protein was noted to bind albumin on immunoelectrophoresis. Analytical ultracentrifugation of the L'ec serum demonstrated 23S and 12S peaks, but no 4S (albumin) boundary. Immunologically identical 20S and 9S IgM proteins were isolated from the serum and the addition in vitro of either the patient's albumin or albumin isolated from normal serum was shown to reconstitute the 23S and 12S boundaries. The binding of high molecular weight IgM to albumin was demonstated by Sephadex G200 chromatography with 125I-labeled albumin and isolated IgM. Immunoelectrophoresis of the L'ec IgM developed with aggregated albumin (reverse immunoelectrophoresis) also demonstrated the binding of albumin to IgM. That all of the patient's IgM complexed with albumin was shown by affinity chromatography employing an aggregated albumin-immunoadsorbent column. Binding was shown to be of the noncovalent type by polyacrylamide gel electrophoresis in 8 M urea. With hot trypsin proteolysis, Fabμ and Fcμ5 fragments were isolated, and monomer albumin was shown to complex only with the Fabμ fragment by both analytical ultracentrifugation and molecular sieve chromatogaphy employing 125I-labeled Fab fragments. 1 mol of Fabμ fragment bound 1 mol of monomer albumin. Polymers of human albumin, produced by heat aggregation, precipitated with the isolated L'ec protein on gel diffusion analysis and, when coated on sheep red blood cells, gave a hemagglutination titer greater than 1 million with the whole L'ec serum. 50 additional monoclonal IgM, 33 IgA, and 80 IgG sera failed to show precipitation or hemagglutination with aggregated albumin. Native monomer albumin inhibited precipitation only at high concentrations (> 50 mg/ml); dimer albumin or fragments of albumin produced by trypsin digestion inhibited at low concentrations (0.4 mg/ml). No reactivity occurred with the albumin of five other mammalian species, including bovine. The L'ec protein

  3. West Nile virus IgM and IgG antibodies three years post- infection

    PubMed Central

    Papa, A; Anastasiadou, A; Delianidou, M

    2015-01-01

    Background: West Nile virus (WNV) causes to humans a variety of symptoms, from asymptomatic infection to severe neuroinvasive disease. In a previous study, it was shown that WNV IgM antibodies persisted in three of 26 (12%) patients, nine months after onset of the symptoms. The aim of the present study was to test 10 of these patients, three years post-infection for probable persistence of IgM antibodies and to investigate their IgG antibody patterns. Material and Methods: In summer 2013 serum samples were collected from 10 persons who were infected with WNV in 2010; 6 of them had a neuroinvasive disease. The three persons with detectable WNV IgM antibodies, nine months after onset of the symptoms, were included in the study. All samples were tested by ELISA in parallel with their stored paired samples taken in 2011. The positive results were confirmed by neutralization test. Results: WNV IgM antibodies were still detectable in the three persons, while high levels of WNV IgG and neutralizing antibodies were present in nine of the 10 persons, regardless the involvement of the nervous system. Conclusions: WNV IgM antibodies persist for more than three years in 12% of patients with WNV infection, while WNV IgG antibodies persist and even increase their levels, regardless the involvement of the nervous system, suggesting that the immune response in the symptomatic WNV infections is strong and long-lasting. Hippokratia 2015, 19 (1): 34-36. PMID:26435644

  4. Distribution of radiolabeled human and mouse monoclonal IgM antibodies in murine models

    SciTech Connect

    Halpern, S.E.; Hagan, P.L.; Chen, A.; Birdwell, C.R.; Bartholomew, R.M.; Burnett, K.G.; David, G.S.; Poggenburg, K.; Merchant, B.; Carlo, D.J.

    1988-10-01

    The distribution and kinetics of six human and one murine monoclonal IgM antibodies (MoAb) were studied in BALB/c mice. Labeling was with /sup 111/In, /sup 75/Se, and /sup 125/I. The monomers and pentamers of certain MoAbs were studied. Human distribution studies were also performed. The serum containing (/sup 111/In)MoAb was obtained from one of the patients 24 hr after administration and injected into mice which were then killed and assayed for /sup 111/In distribution. In general, the (/sup 75/Se) and (/sup 111/In)MoAbs had distribution and kinetic patterns that were similar while the /sup 125/I-labeled MoAbs dehalogenated after 4 hr. Monomers and pentamers had highly similar distributions suggesting that the distribution of IgMs may be based on factors other than molecular size. The murine IgM showed a somewhat different distribution in mice than did human IgMs. Serum from the patient containing (/sup 111/In)MoAb had a distribution in mice similar to that of the patient with high liver and gastrointestinal uptake. The human imaging indicates that it is possible to target tumor with human IgM MoAbs, but significant problems remain in regard to their clinical use.

  5. Further insights into the anti-PF4/heparin IgM immune response.

    PubMed

    Krauel, Krystin; Schulze, Annika; Jouni, Rabie; Hackbarth, Christine; Hietkamp, Bernhard; Selleng, Sixten; Koster, Andreas; Jensch, Inga; van der Linde, Julia; Schwertz, Hansjörg; Bakchoul, Tamam; Hundt, Matthias; Greinacher, Andreas

    2016-04-01

    Anti-platelet factor 4 (PF4)/heparin antibodies are not only the cause of heparin-induced thrombocytopenia but might also play a role in the antibacterial host defence. Recently, marginal zone (MZ) B cells were identified to be crucial for anti-PF4/heparin IgG antibody production in mice. Combining human studies and a murine model of polymicrobial sepsis we further characterised the far less investigated anti-PF4/heparin IgM immune response. We detected anti-PF4/heparin IgM antibodies in the sera of paediatric patients < 6 months of age after cardiac surgery and in sera of splenectomised mice subjected to polymicrobial sepsis. In addition, PF4/heparin-specific IgM B cells were not only found in murine spleen, but also in peritoneum and bone marrow upon in vitro stimulation. Together, this indicates involvement of additional B cell populations, as MZ B cells are not fully developed in humans until the second year of life and are restricted to the spleen in mice. Moreover, PF4/heparin-specific B cells were detected in human cord blood upon in vitro stimulation and PF4-/- mice produced anti-PF4/heparin IgM antibodies after polymicrobial sepsis. In conclusion, the anti-PF4/heparin IgM response is a potential innate immune reaction driven by a B cell population distinct from MZ B cells. PMID:26467272

  6. Influenza Virus-Specific Neutralizing IgM Antibodies Persist for a Lifetime

    PubMed Central

    Skountzou, Ioanna; Satyabhama, Lakshmipriyadarshini; Stavropoulou, Anastasia; Ashraf, Zuhha; Esser, E. Stein; Vassilieva, Elena; Koutsonanos, Dimitrios; Compans, Richard

    2014-01-01

    Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly defined in vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50× 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+ plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did. PMID:25165027

  7. Regulation of the immune response by natural IgM: lessons from warm autoimmune hemolytic anemia.

    PubMed

    Stahl, Dorothea; Sibrowski, Walter

    2003-01-01

    Natural autoantibodies are immunoglobulins of isotypes IgM, IgG and IgA that are present under physiological conditions and that are directed toward self-antigens. Repertoires of self-reactive antibodies have been analysed intensively during the last decade and have been shown to be altered in a variety of autoimmune disorders, immunodeficiency syndromes and lymphoproliferative diseases. Immunoglobulin interactions via variable regions of antibody molecules account significantly for the functional integrity of natural self-reactive antibody repertoires. Recent data indicate that natural immunoglobulins of the isotype IgM might prove particularly useful for the control of antibody-mediated autoimmune diseases under certain conditions. Warm autoimmune hemolytic anemia (WAIHA), an IgG-mediated autoimmune disorder, turns out to be a clinically relevant in vivo model to analyse the impact of autologous IgM on the development of IgG-mediated autoimmunity in humans. We here summarize current knowledge on the role of autologous IgM for regulating self-reactivity. Since natural self-reactive antibodies are critical for the regulation not only of auto- but also of alloimmune responses, as they occur for example in the setting of organ transplantation, regulation of immune homeostasis by pools of human normal IgM might be an interesting therapeutic target of broad interest for clinical medicine. PMID:12871191

  8. The old but new IgM Fc receptor (FcμR).

    PubMed

    Kubagawa, Hiromi; Kubagawa, Yoshiki; Jones, Dewitt; Nasti, Tahseen H; Walter, Mark R; Honjo, Kazuhito

    2014-01-01

    IgM is the first Ig isotype to appear during phylogeny, ontogeny and the immune response. The importance of both pre-immune "natural" and antigen-induced "immune" IgM antibodies in immune responses to pathogens and self-antigens has been established by studies of mutant mice deficient in IgM secretion. Effector proteins interacting with the Fc portion of IgM, such as complement and complement receptors, have thus far been proposed, but fail to fully account for the IgM-mediated immune protection and regulation of immune responses. Particularly, the role of the Fc receptor for IgM (FcμR) in such effector functions has not been explored until recently. We have identified an authentic FcμR in humans using a functional cloning strategy and subsequently in mice by RT-PCR and describe here its salient features and the immunological consequences of FcμR deficiency in mice. Since the FcμR we cloned was identical to Toso or Fas inhibitory molecule 3 (FAIM3), there have been spirited debates regarding the real function of FcμR/Toso/FAIM3 and we will also comment on this topic. PMID:25116093

  9. β-D-glucosidase assisted gold dissolution as non-optical and quantifiable detection technique for immunoassays.

    PubMed

    Koehler, F M; Raso, R A; Grass, R N; Stark, W J

    2013-12-01

    Immunoassays are used for detecting protein targets for various applications. Here, a modification of immunoassays to allow a purely electrical detection of the target protein concentration is shown. The modification comprises a β-D-glucosidase as reporter enzyme and a cyanogenic glycoside as substrate. The enzymatic reaction produces cyanide in small quantities. For electrical detection of the cyanide, a novel sensor is developed, based on a gold micro wire. The cyanide dissolves the gold wire and changes the electrical resistance of the wire. Monitoring the resistance change allows a quantitative measurement of the target human C-reactive protein (an inflammatory marker) in blood plasma in the physiological relevant concentration range. PMID:23670861

  10. Identification of the minimal binding region of a Plasmodium falciparum IgM binding PfEMP1 domain

    PubMed Central

    Semblat, Jean-Philippe; Ghumra, Ashfaq; Czajkowsky, Daniel M.; Wallis, Russell; Mitchell, Daniel A.; Raza, Ahmed; Rowe, J.Alexandra

    2015-01-01

    Binding of host immunoglobulin is a common immune evasion mechanism demonstrated by microbial pathogens. Previous work showed that the malaria parasite Plasmodium falciparum binds the Fc-region of human IgM molecules, resulting in a coating of IgM on the surface of infected erythrocytes. IgM binding is a property of P. falciparum strains showing virulence-related phenotypes such as erythrocyte rosetting. The parasite ligands for IgM binding are members of the diverse P. falciparum Erythrocyte Membrane Protein One (PfEMP1) family. However, little is known about the amino acid sequence requirements for IgM binding. Here we studied an IgM binding domain from a rosette-mediating PfEMP1 variant, DBL4ζ of TM284var1, and found that the minimal IgM binding region mapped to the central region of the DBL domain, comprising all of subdomain 2 and adjoining parts of subdomains 1 and 3. Site-directed mutagenesis of charged amino acids within subdomain 2, predicted by molecular modelling to form the IgM binding site, showed no marked effect on IgM binding properties. Overall, this study identifies the minimal IgM binding region of a PfEMP1 domain, and indicates that the existing homology model of PfEMP1-IgM interaction is incorrect. Further work is needed to identify the specific interaction site for IgM within the minimal binding region of PfEMP1. PMID:26094597

  11. New 21 cm Power Spectrum Upper Limits From PAPER II: Constraints on IGM Properties at z = 7.7

    NASA Astrophysics Data System (ADS)

    Pober, Jonathan; Ali, Zaki; Parsons, Aaron; Paper Team

    2015-01-01

    Using a simulation-based framework, we interpret the power spectrum measurements from PAPER of Ali et al. in the context of IGM physics at z = 7.7. A cold IGM will result in strong 21 cm absorption relative to the CMB and leads to a 21 cm fluctuation power spectrum that can exceed 3000 mK^2. The new PAPER measurements allow us to rule out extreme cold IGM models, placing a lower limit on the physical temperature of the IGM. We also compare this limit with a calculation for the predicted heating from the currently observed galaxy population at z = 8.

  12. Development of immunoassays for human urokinase

    NASA Technical Reports Server (NTRS)

    Atassi, M. Zouhair

    1988-01-01

    Radioimmune assays (RIA) and enzyme linked immune assays for measurement of pro-urokinase and the two active forms of the enzyme were developed. Polyclonal and monoclonal antibodies, with desired specificities against preselected synthetic regions of urokinase (UK), were obtained by immunization with the respective synthetic peptides and used to develop RIA for zymogen and the two activated forms of UK.

  13. Detection of narcotics with an immunoassay film badge

    SciTech Connect

    Lukens, H.R.

    1993-12-31

    Efficient personnel performance, a major requirement for a safe nuclear industry, is jeopardized where personnel use narcotics. However, detection of narcotics at nuclear plants is a challenge. The unique specificity and sensitivity of an immunoassay has been implemented in the form of a small, dry immunoassay film badge (IFB) for the detection of vapors emitted by narcotics. The device is suitable as an area monitor, and its characteristics are suitable for use as a breath monitor for the detection of drug use.

  14. IgM–IgG cryoglobulinaemia with IgM paraprotein component

    PubMed Central

    Klein, F.; van Rood, J. J.; van Furth, R.; Radema, H.

    1968-01-01

    Four patients with mixed IgM–IgG cryoglobulinaemia are described. Clinically they all had some features of an autoimmune disease, while two of them had a lympho-epithelial tumour in the parotid gland. The mixed cryoglobulins of all patients contained an IgM paraprotein with the properties of a rheumatoid factor. They can be regarded as cryoprecipitates of a rheumatoid factor with autologous IgG. In one case the parotid tumour, and not the bone marrow, produced the IgM paraprotein. The clinical significance of the cryoglobulins is discussed. The IgM paraproteins with rheumatoid factor activity may be an expression of an underlying abnormality of the immunological system of these patients. ImagesFig. 1Fig. 2Fig. 4Fig. 5Fig. 6Fig. 7 PMID:5701952

  15. Diagnostic and prognostic significance of the IgM antibody to the Hepatitis delta virus

    SciTech Connect

    Farci, P.; Gerin, J.L.; Aragona, M.; Lindsey, I.; Crivelli, O.; Balestrieri, A.; Smedile, A.; Thomas, H.C.; Rizzetto, M.

    1986-03-21

    The IgM class antibody to the hepatitis delta virus (HDV) was determined in different clinical categories of hepatitis B surface antigen carriers infected by the HDV (positive in the test for total antibody to HDV). The IgM antibody was found at high titers in each 70 patients with inflammatory liver disease and at a low titer in one six patients with inactive cirrhosis; it was not found in eight carriers with normal liver histology. Testing for Igm antibody to HDV distinguishes hepatitis B surface antigen carriers who have underlying inflammatory HDV liver disease from those with past HDV infection and provides prognostic information on the course of chronic HDV hepatitis.

  16. Comparison of immunoassay screening tests and LC-MS-MS for urine detection of benzodiazepines and their metabolites: results of a national proficiency test.

    PubMed

    Bertol, Elisabetta; Vaiano, Fabio; Borsotti, Maurizio; Quercioli, Massimo; Mari, Francesco

    2013-01-01

    For most diverse purposes, different immunoassay (IA) screening methods are usually used to detect benzodiazepines and their metabolites in urine. In this study, we compared the main IAs used in forensic toxicology (Cloned Enzyme Donor Immunoassay, CEDIA®; Enzyme-Multiplied Immunoassay Technique, EMIT®; Fluorescent Polarization ImmunoAssay, FPIA®; Kinetic Interaction of Microparticles in Solution, KIMS® and Immunochromatographic Techniques, IMC) with liquid chromatography-tandem mass spectrometry (LC-MS-MS). Twelve urine specimens were analyzed by 178 laboratories in Italy that participated in a National Proficiency Test, providing both qualitative and semi-quantitative results. Each IA was evaluated by the parameters: true positive, true negative, false positive (FP), false negative (FN), sensitivity (SENS), specificity (SPEC), positive predictive value, negative predictive value (NPV) and accuracy. SPEC was affected by a high FP rate for all IAs. The lowest SENS and NPV were provided by FPIA due to a high number of FN cases. Comparing IA semi-quantitative data with LC-MS-MS results, an overestimation of benzodiazepine amount is noted. This paper draws attention to the problem of the careless use of IA tests for forensic purposes as they may provide FP and/or FN results that can lead to errors of great severity. PMID:23943436

  17. Reinvestigating the Role of IgM in Rabies Virus Postexposure Vaccination

    PubMed Central

    Dorfmeier, Corin L.; Shen, Shixue; Tzvetkov, Evgeni P.

    2013-01-01

    B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM−/−) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM−/− mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM−/− mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM−/− mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine. PMID:23760250

  18. Reinvestigating the role of IgM in rabies virus postexposure vaccination.

    PubMed

    Dorfmeier, Corin L; Shen, Shixue; Tzvetkov, Evgeni P; McGettigan, James P

    2013-08-01

    B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM(-/-)) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM(-/-) mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM(-/-) mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM(-/-) mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine. PMID:23760250

  19. Suppression of IgM Proteolysis by Conformational Stabilization Through Excipients

    PubMed Central

    Mueller, Monika; Loh, Maybelle Q. T.; Gagnon, Pete

    2015-01-01

    Protease activity from host cell lines may cause product loss or affect the quality of recombinant proteins. In this study, we showed that excipients like glycine and sorbitol reduce the proteolysis of an immunoglobulin M (IgM) in the presence of added proteases like α-chymotrypsin, papain, and pepsin. The activity of the proteases in the IgM-protective environments was conserved or even enhanced as tested using low molecular weight substrates. Thus, a higher resistance against proteolytic degradation appears to be caused by the conformational stabilization of the IgM due to preferential exclusion of sorbitol and glycine. PMID:26839826

  20. Splenectomy Associated Changes in IgM Memory B Cells in an Adult Spleen Registry Cohort

    PubMed Central

    Cameron, Paul U.; Jones, Penelope; Gorniak, Malgorzata; Dunster, Kate; Paul, Eldho; Lewin, Sharon; Woolley, Ian; Spelman, Denis

    2011-01-01

    Asplenic patients have a lifelong risk of overwhelming post-splenectomy infection and have been reported to have low numbers of peripheral blood IgM memory B cells. The clinical value of quantitation of memory B cells as an indicator of splenic abnormality or risk of infection has been unclear. To assess changes in B cell sub-populations after splenectomy we studied patients recruited to a spleen registry (n = 591). A subset of 209 adult asplenic or hyposplenic subjects, and normal controls (n = 140) were tested for IgM memory B cells. We also determined a) changes in IgM memory B cells with time after splenectomy using the cross-sectional data from patients on the registry and b) the kinetics of changes in haematological markers associated with splenectomy(n = 45). Total B cells in splenectomy patients did not differ from controls, but memory B cells, IgM memory B cells and switched B cells were significantly (p<0.001) reduced. The reduction was similar for different indications for splenectomy. Changes of asplenia in routine blood films including presence of Howell-Jolly bodies (HJB), occurred early (median 25 days) and splenectomy associated thrombocytosis and lymphocytosis peaked by 50 days. There was a more gradual decrease in IgM memory B cells reaching a stable level within 6 months after splenectomy. IgM memory B cells as proportion of B cells was the best discriminator between splenectomized patients and normal controls and at the optimal cut-off of 4.53, showed a true positive rate of 95% and false positive rate of 20%. In a survey of 152 registry patients stratified by IgM memory B cells around this cut-off there was no association with minor infections and no registry patients experienced OPSI during the study. Despite significant changes after splenectomy, conventional measures of IgM memory cells have limited clinical utility in this population. PMID:21829713

  1. Magnetic bead-based reverse colorimetric immunoassay strategy for sensing biomolecules.

    PubMed

    Gao, Zhuangqiang; Xu, Mingdi; Hou, Li; Chen, Guonan; Tang, Dianping

    2013-07-16

    A novel reverse colorimetric immunoassay (RCIA) strategy was for the first time designed and utilized for sensitive detection of low-abundance protein (prostate-specific antigen, PSA, used in this case) in biological fluids by coupling highly catalytic efficient catalase with magnetic bead-based peroxidase mimics. To construct such a RCIA system, two nanostructures including magnetic beads and gold nanoparticles were first synthesized and functionalized with anti-PSA capture antibody and catalase/anti-PSA detection antibody, respectively. Thereafter, a specific sandwich-type immunoassay format was employed for determination of PSA by using functional gold nanoparticles as enzymatic bioreactors and anti-PSA-conjugated magnetic beads as a colorimetric developer. The carried catalase, followed by the sandwiched immunocomplex, partially consumed the added hydrogen peroxide in the detection solution, which slowed down the catalytic efficiency of magnetic bead-based peroxidase mimics toward TMB/H2O2, thereby weakening the visible color and decreasing the colorimetric density. Different from conventional colorimetric immunoassay, the RCIA method determined the residual hydrogen peroxide in the substrate after consumption. Under the optimal conditions, the developed RCIA exhibited a wide dynamic range of 0.05-20 ng mL(-1) toward PSA with a detection limit of 0.03 ng mL(-1) at the 3Sblank level. Intra- and interassay coefficients of variation were below 6.1% and 9.3%, respectively. Additionally, the methodology was further validated for the analysis of 12 PSA clinical serum specimens, giving results in good accordance with those obtained by the commercially available enzyme-linked immunosorbent assay (ELISA) method. PMID:23806145

  2. Detection of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens

    PubMed Central

    Vojdani, Aristo

    2009-01-01

    Background Despite the first documented case of food allergy to cooked food in 1921 by Prausnitz and Kustner, all commercial food antigens are prepared from raw food. Furthermore, all IgE and IgG antibodies against dietary proteins offered by many clinical laboratories are measured against raw food antigens. Methods We developed an enzyme-linked immunosorbent assay for the measurement of IgE, IgG, IgA and IgM antibodies against raw and processed food antigens. Sera with low or high reactivity to modified food antigens were subjected to myelin basic protein, oxidized low density lipoprotein, and advanced glycation end products (AGE) such as AGE-human serum albumin and AGE-hemoglobin. Results Compared to raw food antigens, IgE antibodies showed a 3–8-fold increase against processed food antigens in 31% of the patients. Similarly, IgG, IgA and IgM antibodies against modified food antigens overall were found at much higher levels than antibody reactions against raw food antigens. Almost every tested serum with high levels of antibodies against modified food antigens showed very high levels of antibodies against myelin basic protein, oxidized low density lipoprotein, AGE-human serum albumin and AGE-hemoglobin. Conclusion We conclude that the determination of food allergy, intolerance and sensitivity would be improved by testing IgE, IgG, IgA and IgM antibodies against both raw and processed food antigens. Antibodies against modified food antigens, by reacting with AGEs and tissue proteins, may cause perturbation in degenerative and autoimmune diseases such as diabetes, atherosclerosis, inflammation, autoimmunity, neurodegeneration and neuroautoimmunity. PMID:19435515

  3. The mystifying nomenclature of cardiac troponin immunoassays.

    PubMed

    Lippi, Giuseppe

    2014-06-01

    The laboratory assessment of cardiospecific troponins(s) represents the cornerstone for the diagnosis of acute coronary syndrome (ACS). Although troponin immunoassays are classified according to either analytical imprecision or percentage of measurable values in a presumably healthy population, it is rather clear that the nomenclature of commercial methods according to these systems of classification carries several drawbacks. The leading problems in classification according to imprecision are represented by the arbitrarity of optimal imprecision threshold, the uncertain correspondence between analytical performance and clinical outcomes and the improper use of terms, which has also been magnified by the lack of specific focus on this topic by regulating bodies such as the US Food and Drug Administration (FDA) and the European Union. Additional issues emerging from classification according to percentage of measurable values include the characterization of healthy population, the variation of values according to age, gender and race, as well as the influence of comorbidities. Considering that what really matters from a clinical standpoint is the clinical performance of the assay rather than the claimed analytical characteristics, it seems reasonable at this point in time to introduce a paradigm shift and gradually abandon the former analytical classification in favour of a different approach, preferable based on clinical outcomes. PMID:24588414

  4. Clinical evaluation of teicoplanin fluorescence polarization immunoassay.

    PubMed Central

    Rybak, M J; Bailey, E M; Reddy, V N

    1991-01-01

    A teicoplanin fluorescence polarization immunoassay (FPIA) developed by International BioClinical (IBC) was evaluated by using serum samples from patients who had been receiving teicoplanin at Detroit Receiving Hospital (DRH) as part of a clinical investigation. Patient samples collected over a 1-year span were assayed at DRH and at IBC, and the results were compared with those of a standard microbiological assay performed at Merrell Dow Research Institute, Indianapolis, Ind. The FPIA has a rapid turnaround time (circa 20 min), utilizes small sample volumes (less than 100 microliters) and is sensitive and accurate in determining concentrations in the range of 5 to 100 micrograms/ml. The intra-assay and interassay coefficient of variation for controls (7, 35, and 75 micrograms/ml) was less than or equal to 13%. Concentrations greater than 100 micrograms/ml must be diluted prior to the assay, which may introduce additional error in determination. The FPIA compared well with the bioassay (r = 0.901) for 193 clinical samples. The results obtained utilizing the FPIA system were reproducible at two different sites, as illustrated by the high degree of correlation between the results at DRH and IBC (r = 0.92). There was less than 7% interference noted when teicoplanin was assayed in the presence of other antibiotics. Patient samples stored for up to 1 year retained their potency: the mean recovery rate in these samples was 107%. The FPIA should be useful for monitoring and adjusting teicoplanin dosage regimens in patients. PMID:1834014

  5. Multiplex lateral flow immunoassay for mycotoxin determination.

    PubMed

    Song, Suquan; Liu, Na; Zhao, Zhiyong; Njumbe Ediage, Emmanuel; Wu, Songling; Sun, Changpo; De Saeger, Sarah; Wu, Aibo

    2014-05-20

    A new lateral flow immunoassay (LFA) is proposed for qualitative and/or semiquantitative determination of aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and their analogues (AFs, ZEAs, DONs) in cereal samples. Each of the mycotoxin specific antibody was class specific and there was no cross reactivity to other groups of compounds. The visual limits of detection (vLOD) of the strip were 0.03, 1.6, and 10 μg/kg for AFB1, ZEA and DON, respectively. The calculated limits of detection (cLOD) were 0.05, 1, and 3 μg/kg, respectively. Meanwhile the cutoff values were achieved at 1, 50, and 60 μg/kg for AFB1, ZEA and DON, respectively. Recoveries ranged from 80% to 122% and RSD from 5% to 20%. Both the vLOD and cLOD for the three mycotoxins were lower than the EU maximum levels. Analysis of naturally contaminated maize samples resulted in a good agreement between the multiplex LFA and LC-MS/MS (100% for DONs and AFs, and 81% for ZEAs). Careful analysis of the results further explained the general overestimation of LFA compared to chromatographic methods for quantification of mycotoxins. PMID:24745689

  6. Enzyme Kinetics.

    ERIC Educational Resources Information Center

    Moe, Owen; Cornelius, Richard

    1988-01-01

    Conveys an appreciation of enzyme kinetic analysis by using a practical and intuitive approach. Discusses enzyme assays, kinetic models and rate laws, the kinetic constants (V, velocity, and Km, Michaels constant), evaluation of V and Km from experimental data, and enzyme inhibition. (CW)

  7. Vertical flow immunoassay (VFA) biosensor for a rapid one-step immunoassay.

    PubMed

    Oh, Young Kyoung; Joung, Hyou-Arm; Kim, Sanghyo; Kim, Min-Gon

    2013-03-01

    A highly rapid, one-step immunoassay of high sensitivity C-reactive protein (hsCRP) using a biosensor with a vertical flow immunoassay (VFA) was developed. The VFA biosensor was primarily composed of a sample pad, conjugate pad, FTH film and nitrocellulose (NC) membrane, which were all vertically stacked upon one another. Anti-hsCRP and secondary antibodies were consecutively immobilized on the NC membrane at the position below the holes. Gold nanoparticles (AuNPs) conjugated with another anti-hsCRP antibody were encapsulated in the conjugation pad. Various assay conditions, including the size of the hole and the sample volume, were optimized. Under optimized conditions, hsCRP concentrations from 0.01 to 10 μg mL(-1) were detected within 2 min. In comparison with a lateral flow assay (LFA) system, the VFA sensor showed a gradual increase of signal in a concentration-dependent manner without a hook effect in the tested range. PMID:23303290

  8. HCV infection in a patient with hyper IgM syndrome.

    PubMed

    Quinti, I; Giovannetti, A; Paganelli, R; Pucillo, L P; Varani, A R; Ricci, G; Scala, E; Pandolfi, F; Casato, M; Aiuti, F

    1996-11-01

    The association between an acquired form of hyper-IgM syndrome and a chronic hepatitis C virus (HCV) infection in a 71-year-old female patient is described. Both diseases were diagnosed at the age of 58 years. She was started on intramuscular and then intravenous immunoglobulin replacement therapy. HCV RNA was detected in 1992. The patient remained in well-balanced clinical condition until 1994, when total and specific anti-HCV IgM levels increased and the patient developed an IgM kappa monoclonal gammopathy. Adherent cells and B cells were HCV RNA positive, while T cells were HCV RNA negative. Anti-IgM reactivity was specifically directed to the core antigen of the HCV. The patient we describe showed a picture of a late-onset form of hypogammaglobulinemia with a progressive increase in IgM antibodies, possibly due to the concomitant HCV infection. It is possible that the immunodeficiency might also result from the HCV infection, with formation of specific antibodies belonging to the IgM class, and that the worsening of the clinical condition may be directly related to the persistent viral infection. PMID:8946276

  9. Complete amino acid sequence of the Mu heavy chain of a human IgM immunoglobulin.

    PubMed

    Putnam, F W; Florent, G; Paul, C; Shinoda, T; Shimizu, A

    1973-10-19

    The amino acid sequence of the micro, chain of a human IgM immunoglobulin, including the location of all disulfide bridges and oligosaccharides, has been determined. The homology of the constant regions of immunoglobulin micro, gamma, alpha, and epsilon heavy chains reveals evolutionary relationships and suggests that two genes code for each heavy chain. PMID:4742735

  10. Genetic parameters of IgM and IgG antibodies binding autoantigens in healthy chickens.

    PubMed

    Bao, M; Bovenhuis, H; Nieuwland, M G B; Parmentier, H K; van der Poel, J J

    2016-02-01

    Levels of natural antibodies (NAb) binding keyhole limpet hemocyanin (KLH) in layers were shown to be heritable and to be potential indicative parameters for survival. A proportion of NAb are directed to self-molecules, or slightly changed self-molecules (neo-epitopes), labeled as natural autoantibodies (NAAb). It is unknown whether the levels of NAAb are heritable and genetically correlated. In this paper, we estimated genetic parameters in plasma of healthy layers for IgM and IgG antibodies binding ovalbumin (OVA), myosin (MYO), cardiolipin (CAR), lysozyme (LYS), and the model antigen keyhole limpet hemocyanin (KLH). A linear animal model was used to estimate (co)variance components, heritabilities, and correlations. The estimates of heritabilities ranged from 0.10 to 0.17 for IgM, and 0.02 to 0.11 for IgG, respectively. For both IgM and IgG, high genetic correlations were observed between levels of NAAb binding autoantigens and NAb binding KLH, except for IgG binding KLH and LYS, for which a low genetic correlation was found. Low to moderate phenotypic correlations were found between NAAb and NAb. In addition, significant maternal environmental effects of 0.03, 0.07, and 0.04 were observed for IgM binding OVA, LYS, and KLH, respectively. Results from this study indicated that NAAb or NAb levels in plasma were heritable and could provide tools to identify the health status of birds. PMID:26706361

  11. Baryonic Content in the Warm-Hot IGM at Low Redshift

    NASA Technical Reports Server (NTRS)

    Sonneborn, George; Shull, M.; Danforth, C.; Moos, W.

    2007-01-01

    Baryons are 4.5% of the universe's mass/energy density; only 10% of these are in stars, galaxies, and clusters. At low-redshift 90% of baryons are in the IGM, 30% in Ly-alpha forest, but most are in hot gas (10(exp 5-7) K) produced by shocks during structure formation. O VI 1032-38 A are the best tracers of this gas. The distribution of O VI absorbers observed by FUSE rises as N(sup -2+/-0.2, down to 10(exp 13)/sq cm. Integrated to logN=13, 7% of baryons reside in the O VI-bearing IGM at 10% solar metallicity, T approx. 10(exp 5.5) K. At redshift z<0.1 metals have been transported less than 800/h kpc from L* galaxies and 200/h kpc from 0.1 L* galaxies. The steepness of dN/dz means that low-N absorbers contribute an equal mass of hot IGM as higher N gas. The total mass of O VI-bearing gas in the IGM depends on determining the turnover in dN/dz at low N(O VI). Future observations by FUSE are needed to reach lower N and to reduce the uncertainty in the dN/dz power law.

  12. Photoelectrochemical detection of enzymatically generated CdS nanoparticles: Application to development of immunoassay.

    PubMed

    Barroso, Javier; Saa, Laura; Grinyte, Ruta; Pavlov, Valeri

    2016-03-15

    We report an innovative photoelectrochemical process (PEC) based on graphite electrode modified with electroactive polyvinylpyridine bearing osmium complex (Os-PVP). The system relies on the in situ enzymatic generation of CdS quantum dots (QDs). Alkaline phosphatase (ALP) catalyzes the hydrolisis of sodium thiophosphate (TP) to hydrogen sulfide (H2S) which in the presence Cd(2+) ions yields CdS semiconductor nanoparticles (SNPs). Irradiation of SNPs with the standard laboratory UV-illuminator (wavelength of 365 nm) results in photooxidation of 1-thioglycerol (TG) mediated by Os-PVP complex on the surface of graphite electrode at applied potential of 0.31 V vs. Ag/AgCl. A novel immunoassay based on specific enzyme linked immunosorbent assay (ELISA) combined with the PEC methodology was developed. Having selected the affinity interaction between bovine serum albumine (BSA) with anti-BSA antibody (AB) as a model system, we built the PEC immunoassay for AB. The new assay displays a linear range up to 20 ngmL(-1) and a detection limit (DL) of 2 ngmL(-1) (S/N=3) which is lower 5 times that of the traditional chromogenic ELISA test employing p-nitro-phenyl phosphate (pNPP). PMID:26432195

  13. Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations.

    PubMed

    Stave, James W

    2002-01-01

    Immunoassay methods are available for detection and quantitation of proteins expressed by most biotechnology-derived crops in commercial production. The 2 most common test formats are enzyme-linked immunosorbent assay (ELISA) and immunochromatographic (lateral flow) strip tests. Two ELISA methods, one for Roundup Ready soybeans and one for MON810 CrylAb corn, were the subject of large international collaborative studies and were demonstrated to quantitatively determine the concentrations of biotech crops in samples of ground grain. Quantitative ELISA methods are also useful for analysis of processed fractions of agricultural commodities such as soybean toasted meal or corn flour. Both strip tests and ELISAs for biotech crops are currently being used on a large scale in the United States to manage the sale and distribution of grain. In these applications, tests are used to determine if the concentration of biotech grain is above or below specified threshold limits. Using existing U.S. Department of Agriculture sampling techniques, the reliability of the threshold determination is expressed in terms of statistical confidence rather than analytical precision. Combining the use of protein immunoassays with Identity Preservation systems provides an effective means of characterizing the raw and processed agricultural inputs to the food production system in a way that allows food producers to comply with labeling laws. PMID:12083275

  14. Dirofilaria immitis: performance and standardization of specific antibody immunoassays for filariasis.

    PubMed

    Hamilton, R G; Scott, A L; D'Antonio, R; Levy, D A; Adkinson, N F

    1983-12-01

    The factors associated with the development, optimization, and validation of immunoassays for the detection of parasite-specific antibody in filariasis infections were investigated using the dog heartworm, Dirofilaria immitis as a model. We examined two assays, the Protein A solid-phase radioimmunoassay (SPRIA) and enzyme-linked immunosorbent assay (ELISA), for quantitation of specific antibody to the parasite in canine serum. Precision, reproducibility, and parallelism were examined using response-error relationships and precision profile analyses. A staphylococcal Protein A saturation analysis was applied to the standardization of IgG anti-parasite antibody reference sera in weight per volume units (microgram/ml). Using the mean minimal detectable dose + 3 SD and an intraassay precision profile less than 10% coefficient of variation (CV) as criteria for assay sensitivity, the SPRIA and ELISA displayed comparable positive thresholds of 1 microgram/ml IgG anti-parasite antibody/ml of serum. Both assays demonstrated good reproducibility (less than 15% interassay CV) and parallelism (less than 20% interdilutional CV) over their working ranges (SPRIA: 1-40 micrograms/ml; ELISA: 1-5 micrograms/ml). Specificity of each assay was enhanced by preadsorption of cross-reacting antibodies in canine serum (i.e., specific for Toxocara canis antigens) with solid-phase antigen prior to assay. Methods for comparing different immunoassay designs are considered in relation to the variables that influence the assays' performance characteristics. PMID:6357832

  15. Ultrasensitive On-Chip Immunoassays with a Nanoparticle-Assembled Photonic Crystal

    PubMed Central

    Han, Jin-Hee; Sudheendra, L.; Kim, Hee-Joo; Gee, Shirley J.; Hammock, Bruce D.; Kennedy, Ian M.

    2012-01-01

    Electrophoretic particle entrapment system (EPES) is employed to generate 2D array of nanoparticles coated with biological molecules (i.e. antibodies). Phase matching of the excitation and the emission in the 2D arrays with particles produces a highly enhanced fluorescence signal that was shown to improve the limit of detection in immunoassays. The phase matching is achieved when the particle are in the sub-100 nm range. A comparison between different size particles shows that the sensitivity of an immunoassay is extended to a range that is difficult to achieve with standard technology (e.g. Enzyme-linked immunosorbent assay-ELISA). The effectiveness of this novel configuration of particle-in-a well was demonstrated with an assay for human epidermal growth factor receptor 2 (HER2; breast cancer biomarker), with a detection limit as low as 10 aM in less than 10 μl of serum-based sample. The limit of detection of HER2 indicated far superior assay performance compared to the corresponding standard 96-well plate-based ELISA. The particle-based photonic platform reduces the reagent volume and the time for performing an assay in comparison to competing methods. The simplicity of operation and the level of sensitivity demonstrated here can be used for rapid and early-stage detection of biomarkers. PMID:22957818

  16. B-1 B cell IgM antibody initiates T cell elicitation of contact sensitivity.

    PubMed

    Askenase, P W; Tsuji, R F

    2000-01-01

    Although B-1 B cells have received considerable attention, their actual role in the normal functioning of the immune system is unclear. The hypothesized role of B-1 cell IgM in natural protective immunity is just being established. We have uncovered a separate and novel role for B-1 cell IgM in initiating the elicitation of acquired T cell-dependent contact sensitivity (CS), the prototype of in vivo T cell immunity, early after immunization (within 4 days). The recent recognition of a similarly unanticipated role of B cells in a variety of T cell responses, may indicate that B-1 cell IgM has a broader role in immunity than thought previously. We showed that 24 hr CS responses, and rises in local IFN-gamma levels at 24 hrs later after antigen (Ag) challenge the ears, were absent in pan B cell and antibody deficient mice. The mechanism of B cell involvement in CS-initiation is via local C5a generation early (1-2 hrs) after antigen (Ag) challenge of the ears, in 4 day contact sensitized mice. C5a activates local mast cells to release serotonin (5-HT) and TNF alpha to induce endothelial ICAM-1 and VCAM-1, leading to T cell recruitment. We hypothesized that C5a was generated via complement activation due to antibodies forming local AgAb complexes, and that B-1 cell IgM was involved because isotype switching of B-2 cells to produce C-activating IgG isotypes, could not occur as early as day 4. Indeed, B-1 cell deficient CBA/N-xid mice lacked C5a in 2 hr ear extracts, and had impaired CS ear swelling and elaboration of IFN-gamma at 24 hrs. Importantly, adoptive transfer of purified normal peritoneal B-1 cells, or just i.v. injection of Ag-specific IgM monoclonal antibodies in sensitized xid, restored deficient early C5a and late 24 hr ear swelling. These results suggest that early after Ag challenge, specific B-1 cell IgM, produced at distant sites by prior sensitization, forms AgAb complexes that trigger elaboration of C5a, to activate mast cell release of vasoactive TNF

  17. AquaLite, a bioluminescent label for immunoassay and nucleic acid detection: quantitative analyses at the attomol level

    NASA Astrophysics Data System (ADS)

    Smith, David F.; Stults, Nancy L.

    1996-04-01

    AquaLiteR is a direct, bioluminescent label capable of detecting attomol levels of analyte in clinical immunoassays and assays for the quantitative measurement of nucleic acids. Bioluminescent immunoassays (BIAs) require no radioisotopes and avoid complex fluorescent measurements and many of the variables of indirect enzyme immunoassays (EIAs). AquaLite, a recombinant form of the photoprotein aequorin from a bioluminescent jellyfish, is coupled directly to antibodies to prepare bioluminescent conjugates for assay development. When the AquaLite-antibody complex is exposed to a solution containing calcium ions, a flash of blue light ((lambda) max equals 469 nm) is generated. The light signal is measured in commercially available luminometers that simultaneously inject a calcium solution and detect subattomol photoprotein levies in either test tubes or microtiter plates. Immunometric or 'sandwich' type assays are available for the quantitative measurement of human endocrine hormones and nucleic acids. The AquaLite TSH assay can detect 1 attomol of thyroid stimulating hormone (TSH) in 0.2 mL of human serum and is a useful clinical tool for diagnosing hyperthyroid patients. AquaLite-based nucleic acid detection permits quantifying attomol levels of specific nucleic acid markers and represents possible solution to the difficult problem of quantifying the targets of nucleic acid amplification methods.

  18. Bioconjugation of Antibodies and Enzyme Labels onto Magnetic Beads.

    PubMed

    Otieno, B A; Krause, C E; Rusling, J F

    2016-01-01

    Immunoassays employ antibodies and labels to capture and detect target macromolecular analytes, often from complex sample matrices such as serum, plasma, or saliva. The high affinity and specificity of antibody-antigen interactions makes immunoassays critically important analytical techniques for clinical diagnostics as well as other research applications in the areas of pharmaceutical and environmental analysis. Integration of magnetic beads (MBs) into immunoassays and other bioanalytical methodologies is a valuable approach to allow efficient target capture, enrichment, and convenient separation. In addition, large signal amplification can be achieved by preconcentration of the target and by attaching many thousands of enzyme labels to the MBs. These features have enabled MB-based biosensors to achieve ultra-low detection limits needed for advanced clinical diagnostics that are challenging or impossible using traditional immunoassays. MBs are employed either as mobile substrates for target analyte capture, as detection labels (or label carriers), or simultaneously as substrates and labels. For optimal assay performance, it is crucial to apply an easy, efficient, and robust bead-probe conjugation protocol, and to thoroughly characterize the bioconjugated products. Herein, we describe methods used in our laboratory to functionalize MBs with antibodies and enzyme labels for ultrasensitive detection of protein analytes. We also present detailed strategies for characterizing the MB bioconjugates. PMID:27112398

  19. Placebo-controlled trial of rituximab in IgM anti-myelin–associated glycoprotein neuropathy

    PubMed Central

    Viala, Karine; Nicolas, Guillaume; Créange, Alain; Vallat, Jean-Michel; Pouget, Jean; Clavelou, Pierre; Vial, Christophe; Steck, Andreas; Musset, Lucile; Marin, Benoit

    2013-01-01

    Objective: To determine whether rituximab 375 mg/m2 was efficacious in patients with immunoglobulin M (IgM) anti-myelin–associated glycoprotein antibody demyelinating neuropathy (IgM anti-MAG demyelinating neuropathy). Methods: Fifty-four patients with IgM anti-MAG demyelinating neuropathy were enrolled in this randomized, double-blind, placebo-controlled trial. The inclusion criteria were inflammatory neuropathy cause and treatment (INCAT) sensory score (ISS) ≥4 and visual analog pain scale >4 or ataxia score ≥2. The primary outcome was mean change in ISS at 12 months. Results: Twenty-six patients were randomized to a group receiving 4 weekly infusions of 375 mg/m2 rituximab, and 28 patients to placebo. Intention-to-treat analysis, with imputation of missing ISS values by the last observation carried forward method, showed a lack of mean change in ISS at 12 months, 1.0 ± 2.7 in the rituximab group, and 1.0 ± 2.8 in the placebo group. However, changes were observed, in per protocol analysis at 12 months, for the number of patients with an improvement of at least 2 points in the INCAT disability scale (p = 0.027), the self-evaluation scale (p = 0.016), and 2 subscores of the Short Form–36 questionnaire. Conclusions: Although primary outcome measures provide no evidence to support the use of rituximab in IgM anti-MAG demyelinating neuropathy, there were improvements in several secondary outcomes in per protocol analysis. Level of evidence: This study provides Class I evidence that rituximab is ineffective in improving ISS in patients with IgM anti-MAG demyelinating neuropathy. PMID:23667063

  20. IgM Repertoire Biodiversity is Reduced in HIV-1 Infection and Systemic Lupus Erythematosus

    PubMed Central

    Yin, Li; Hou, Wei; Liu, Li; Cai, Yunpeng; Wallet, Mark Andrew; Gardner, Brent Paul; Chang, Kaifen; Lowe, Amanda Catherine; Rodriguez, Carina Adriana; Sriaroon, Panida; Farmerie, William George; Sleasman, John William; Goodenow, Maureen Michels

    2013-01-01

    Background: HIV-1 infection or systemic lupus erythematosus (SLE) disrupt B cell homeostasis, reduce memory B cells, and impair function of IgG and IgM antibodies. Objective: To determine how disturbances in B cell populations producing polyclonal antibodies relate to the IgM repertoire, the IgM transcriptome in health and disease was explored at the complementarity determining region 3 (CDRH3) sequence level. Methods: 454-deep pyrosequencing in combination with a novel analysis pipeline was applied to define populations of IGHM CDRH3 sequences based on absence or presence of somatic hypermutations (SHM) in peripheral blood B cells. Results: HIV or SLE subjects have reduced biodiversity within their IGHM transcriptome compared to healthy subjects, mainly due to a significant decrease in the number of unique combinations of alleles, although recombination machinery was intact. While major differences between sequences without or with SHM occurred among all groups, IGHD and IGHJ allele use, CDRH3 length distribution, or generation of SHM were similar among study cohorts. Antiretroviral therapy failed to normalize IGHM biodiversity in HIV-infected individuals. All subjects had a low frequency of allelic combinations within the IGHM repertoire similar to known broadly neutralizing HIV-1 antibodies. Conclusion: Polyclonal expansion would decrease overall IgM biodiversity independent of other mechanisms for development of the B cell repertoire. Applying deep sequencing as a strategy to follow development of the IgM repertoire in health and disease provides a novel molecular assessment of multiple points along the B cell differentiation pathway that is highly sensitive for detecting perturbations within the repertoire at the population level. PMID:24298273

  1. Capillary Isoelectric Focusing Immunoassay for Fat Cell Differentiation Proteomics

    PubMed Central

    Johlfs, Mary G.; Gorjala, Priyatham; Urasaki, Yasuyo; Le, Thuc T.; Fiscus, Ronald R.

    2015-01-01

    Profiling cellular proteome is critical to understanding signal integration during cell fate determination. In this study, the capability of capillary isoelectric focusing (cIEF) immunoassays to detect post-translational modifications (PTM) of protein isoforms is demonstrated. cIEF immunoassays exhibit protein detection sensitivity at up to 5 orders of magnitude higher than traditional methods. This detection ultra-sensitivity permits proteomic profiling of several nanograms of tissue samples. cIEF immunoassays are employed to simultaneously profile three protein kinases during fat cell differentiation: cGMP-dependent protein kinase type I (PKG-I) of the nitric oxide (NO) signaling pathway, protein kinase B (Akt) of the insulin signaling pathway, and extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway. Interestingly, a switch in the expression level of PKG- isoforms is observed during fat cell differentiation. While both PKG-Iα and PKG-Iβ isoforms are present in preadipocytes, only PKG-Iβ isoform is expressed in adipocytes. On the other hand, the phosphorylation level increases for Akt while decreases for ERK1 and ERK2 following the maturation of preadipocytes into adipocytes. Taken together, cIEF immunoassay provides a highly sensitive means to study fat cell differentiation proteomics. cIEF immunoassay should be a powerful proteomics tool to study complex protein signal integration in biological systems. PMID:26132171

  2. Immunoassay Analysis of Kanamycin in Serum Using the Tobramycin Kit.

    PubMed

    Dijkstra, J A; Voerman, A J; Greijdanus, B; Touw, D J; Alffenaar, J W C

    2016-08-01

    Kanamycin is one of the aminoglycosides used in the treatment of multidrug-resistant tuberculosis. Blood concentrations of kanamycin are predictive for the treatment efficacy and the occurrence of side effects, and dose adjustments can be needed to optimize therapy. However, an immunoassay method for the quantification of kanamycin is not commercially available. We modified the existing tobramycin immunoassay to analyze kanamycin. This modified method was tested in a concentration range of 0.3 to 80.0 mg/liter for inaccuracy and imprecision. In addition, the analytical results of the immunoassay method were compared to those obtained by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) analytical method using Passing and Bablok regression. Within-day imprecision varied from 2.3 to 13.3%, and between-day imprecision ranged from 0.0 to 11.3%. The inaccuracy ranged from -5.2 to 7.6%. No significant cross-reactivity with other antimicrobials and antiviral agents was observed. The results of the modified immunoassay method were comparable with the LC-MS/MS analytical outcome. This new immunoassay method enables laboratories to perform therapeutic drug monitoring of kanamycin without the need for complex and expensive LC-MS/MS equipment. PMID:27185806

  3. High-Throughput Optical Sensing Immunoassays on Smartphone.

    PubMed

    Wang, Li-Ju; Sun, Rongrong; Vasile, Tina; Chang, Yu-Chung; Li, Lei

    2016-08-16

    We present an optical sensing platform on a smartphone for high-throughput screening immunoassays. For the first time, a designed microprism array is utilized to achieve a one-time screening of 64 samples. To demonstrate the capability and the reliability of this optical sensing platform on smartphone, human interleukin 6 (IL-6) protein and six types of plant viruses are immunoassayed. The ability of quantification is shown by a sigmoidal dose-response curve fitting to analyze IL-6 protein. The accuracy in measuring the concentrations of IL-6 protein achieves 99.1%. On the other hand, to validate on-field immunoassays by our device, a total of 1030 samples are assayed using three immunoassay methods to detect six types of plant viruses. The accuracy is up to 96.2-99.9%; in addition, there is a high degree of agreement with lab instruments. The total cost for this high-throughput optical screening platform is ∼$50 USD. The reading time is only 2 s for 64 samples. The size is just as big as a portable hard drive. Our optical sensing platform on the smartphone offers a route toward in situ high-throughput screening immunoassays for viruses, pathogens, biomarkers, and toxins by decentralizing laboratory tests. With this mobile point-of-care optical platform, the spread of disease can be timely stopped within a very short turnaround time. PMID:27434250

  4. Enzyme Informatics

    PubMed Central

    Alderson, Rosanna G.; Ferrari, Luna De; Mavridis, Lazaros; McDonagh, James L.; Mitchell, John B. O.; Nath, Neetika

    2012-01-01

    Over the last 50 years, sequencing, structural biology and bioinformatics have completely revolutionised biomolecular science, with millions of sequences and tens of thousands of three dimensional structures becoming available. The bioinformatics of enzymes is well served by, mostly free, online databases. BRENDA describes the chemistry, substrate specificity, kinetics, preparation and biological sources of enzymes, while KEGG is valuable for understanding enzymes and metabolic pathways. EzCatDB, SFLD and MACiE are key repositories for data on the chemical mechanisms by which enzymes operate. At the current rate of genome sequencing and manual annotation, human curation will never finish the functional annotation of the ever-expanding list of known enzymes. Hence there is an increasing need for automated annotation, though it is not yet widespread for enzyme data. In contrast, functional ontologies such as the Gene Ontology already profit from automation. Despite our growing understanding of enzyme structure and dynamics, we are only beginning to be able to design novel enzymes. One can now begin to trace the functional evolution of enzymes using phylogenetics. The ability of enzymes to perform secondary functions, albeit relatively inefficiently, gives clues as to how enzyme function evolves. Substrate promiscuity in enzymes is one example of imperfect specificity in protein-ligand interactions. Similarly, most drugs bind to more than one protein target. This may sometimes result in helpful polypharmacology as a drug modulates plural targets, but also often leads to adverse side-effects. Many cheminformatics approaches can be used to model the interactions between druglike molecules and proteins in silico. We can even use quantum chemical techniques like DFT and QM/MM to compute the structural and energetic course of enzyme catalysed chemical reaction mechanisms, including a full description of bond making and breaking. PMID:23116471

  5. PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Immunoassays are extensively utilized in disease diagnostics with monoclonal antibodies serving as critical tools within the assay. Detection of scrapie in sheep and goats relies heavily on immunoassays including immunohistochemistry, western blotting, and ELISA. In the United States, regulatory tes...

  6. SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION (SITE) REPORT FOR THE WESTINGHOUSE BIOANALYTICAL SYSTEMS PENTACHLOROPHENOL (PCP) IMMUNOASSAYS

    EPA Science Inventory

    The results of the demonstration of two Westinghouse Bio-Analytic Systems (WBAS) immunoassay technologies are described in this report. The immunoassays measure parts per billion concentrations of pentachlorophenol in environmental water samples. The study was conducted under the...

  7. SUPERFUND INNOVATIVE TECHNOLOGY EVALUATION PROGRAM DEMONSTRATION PLAN FOR WESTINGHOUSE BIO-ANALYTIC SYSTEMS PENTACHLOROPHENOL IMMUNOASSAYS

    EPA Science Inventory

    This plan provides a detailed design and description of the demonstration and evaluation program for the Westinghouse Bio-Analytic Systems immunoassay technologies specific for the analysis of pentachlorophenol. he immunoassays measure parts per billion concentrations of pentachl...

  8. The Effects of Sample Matrices on Immunoassays to Detect Microcystin-LR in Water

    EPA Science Inventory

    Abstract: Immunoassays are widely used biochemical techniques to detect microcystins in environmental samples. The use of immunoassays for the detection of microcystins is vulnerable to matrix components and other interferents. This study is an evaluation of the effects of interf...

  9. Evaluation of a new immunoassay for determination of phenytoin and phenobarbital: results of a European collaborative control study.

    PubMed

    Schmidt, D; Goldberg, V; Guelen, P J; Johannessen, S; Kleijn, E; Meijer, J W; Meinardi, H; Richens, A; Schneider, H; Stein-Lavie, Y; Symann-Louette, N

    1977-09-01

    The performance of a new enzyme multiplied immunoassay technique (EMIT) was compared with other current methods, namely gas-liquid chromatography and thin-layer chromatography, for the determination of phenytoin and phenobarbital in serum. Forty-three serum samples were sent as unknowns to the participating laboratories for determination. The precision of repeated determinations was very similar for EMIT and chromatography. There was good agreement among gas chromatography, thin-layer chromatography, and EMIT results. Interlaboratory variability was lower for EMIT determinations of both antiepileptic drugs. The rapid analysis of small samples, made possible by the EMIT system, could have beneficial effects on the treatment of epilepsies. PMID:891490

  10. Potential applications of immunoassays in studies of flatfish recruitment

    NASA Astrophysics Data System (ADS)

    Feller, Robert J.

    The fisheries recruitment-stock problem, a lack of correlation between measures of reproductive output of the parent stock and recruitment to the fishery, has several potential biotic and abiotic causes. Immunoassays may be useful in examining several aspects of this and several other problems in flatfish ecology: stock identification, parasitism and disease, and trophic interactions. Given stage-specific antisera capable of recognozing antigenic moieties of, for instance, eggs, larvae, or newly-settled juveniles, it is possible to screen stomach contents of many putative predators ( e.g., shrimp or crabs) rapidly for the presence and amounts of platfish prey. This trophic application of immunological methods has great promise for measuring loss of potential recruits to predation. All immunoassays are limited by the quality of antisera used and the researcher's ability to interpret quantitative data in an ecologically meaningful way. Key references for applications of immunoassays in fish-related questions are provided with recommendations for their utilization.

  11. Pre-cut Filter Paper for Detecting Anti-Japanese Encephalitis Virus IgM from Dried Cerebrospinal Fluid Spots

    PubMed Central

    Bharucha, Tehmina; Chanthongthip, Anisone; Phuangpanom, Soumphou; Phonemixay, Ooyanong; Sengvilaipaseuth, Onanong; Vongsouvath, Manivanh; Lee, Sue; Newton, Paul N.; Dubot-Pérès, Audrey

    2016-01-01

    Background The use of filter paper as a simple, inexpensive tool for storage and transportation of blood, ‘Dried Blood Spots’ or Guthrie cards, for diagnostic assays is well-established. In contrast, there are a paucity of diagnostic evaluations of dried cerebrospinal fluid (CSF) spots. These have potential applications in low-resource settings, such as Laos, where laboratory facilities for central nervous system (CNS) diagnostics are only available in Vientiane. In Laos, a major cause of CNS infection is Japanese encephalitis virus (JEV). We aimed to develop a dried CSF spot protocol and to evaluate its diagnostic performance using the World Health Organisation recommended anti-JEV IgM antibody capture enzyme-linked immunosorbent assay (JEV MAC-ELISA). Methodology and Principal Findings Sample volumes, spotting techniques and filter paper type were evaluated using a CSF-substitute of anti-JEV IgM positive serum diluted in Phosphate Buffer Solution (PBS) to end-limits of detection by JEV MAC-ELISA. A conventional protocol, involving eluting one paper punch in 200μl PBS, did not detect the end-dilution, nor did multiple punches utilising diverse spotting techniques. However, pre-cut filter paper enabled saturation with five times the volume of CSF-substitute, sufficiently improving sensitivity to detect the end-dilution. The diagnostic accuracy of this optimised protocol was compared with routine, neat CSF in a pilot, retrospective study of JEV MAC-ELISA on consecutive CSF samples, collected 2009–15, from three Lao hospitals. In comparison to neat CSF, 132 CSF samples stored as dried CSF spots for one month at 25–30°C showed 81.6% (65.7–92.3 95%CI) positive agreement, 96.8% (91.0–99.3 95%CI) negative agreement, with a kappa coefficient of 0.81 (0.70–0.92 95%CI). Conclusions/Significance The novel design of pre-cut filter paper saturated with CSF could provide a useful tool for JEV diagnostics in settings with limited laboratory access. It has the

  12. A comparison of salivary testosterone measurement using immunoassays and tandem mass spectrometry.

    PubMed

    Welker, Keith M; Lassetter, Bethany; Brandes, Cassandra M; Prasad, Smrithi; Koop, Dennis R; Mehta, Pranjal H

    2016-09-01

    Enzyme immunoassays (EIAs) are widely used to measure salivary testosterone. However, little is known about how accurately different EIAs assess testosterone, partially because estimates across various EIAs differ considerably. We compared testosterone concentrations across EIAs of three commonly used manufacturers (DRG International, Salimetrics, and IBL International) to liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative to EIAs from Salimetrics and IBL International, EIAs supplied by DRG International provided the closest approximation to LC-MS/MS testosterone concentrations, followed closely by EIAs from Salimetrics, and then IBL. Additionally, EIAs tended to inflate estimates of lower testosterone concentrations in women. Examining our results and comparing them to existing data revealed that testosterone EIAs had decreased linear correspondence with LC-MS/MS in comparison to cortisol EIAs. Overall, this paper provides researchers with information to better measure testosterone in their research and more accurately compare testosterone measurements across different methods. PMID:27295182

  13. Development of a highly sensitive and specific immunoassay for enrofloxacin based on heterologous coating haptens.

    PubMed

    Wang, Zhanhui; Zhang, Huiyan; Ni, Hengjia; Zhang, Suxia; Shen, Jianzhong

    2014-04-11

    In the paper, an enzyme-linked immunosorbent immunoassay (ELISA) for detection of enrofloxacin was described using one new derivative of enrofloxacin as coating hapten, resulting in surprisingly high sensitivity and specificity. Incorporation of aminobutyric acid (AA) in the new derivative of enrofloxacin had decreased the IC50 of the ELISA for enrofloxacin from 1.3 μg L(-1) to as low as 0.07 μg L(-1). The assay showed neglect cross-reactivity for other fluoroquinolones but ofloxacin (8.23%), marbofloxacin (8.97%) and pefloxacin (7.29%). Analysis of enrofloxacin fortified chicken muscle showed average recoveries from 81 to 115%. The high sensitivity and specificity of the assay makes it a suitable screening method for the determination of low levels of enrofloxacin in chicken muscle without clean-up step. PMID:24745749

  14. Measuring nerve growth factor in saliva by immunoassay: A cautionary note.

    PubMed

    Matin, Marla J; Li, Daming; Peterson, Jon; Taylor, Marcus K; Laurent, Heidemarie K; Lucas, Todd; Granger, Steve J; Granger, Douglas A; Granger, Steve W

    2016-01-01

    Nerve growth factor (NGF), a neurotrophin, modulates a diverse set of physiologic processes in the nervous, immune, and endocrine systems. Studies suggest that NGF can be measured in saliva (sNGF). Historically, the method for measuring sNGF involves the off-label use of an enzyme immunoassay designed for use with cell-culture supernatants/tissue extracts (Nam et al., 2007; Ruhl et al., 2004). In a series of experiments we reveal this measurement strategy is subject to non-specific interference by constituents present in oral fluids. We conclude that the measurement of sNGF by this assay is not optimal for use with oral fluid specimens. PMID:26519777

  15. Survivability of immunoassay reagents exposed to the space radiation environment on board the ESA BIOPAN-6 platform as a prelude to performing immunoassays on Mars.

    PubMed

    Derveni, Mariliza; Allen, Marjorie; Sawakuchi, Gabriel O; Yukihara, Eduardo G; Richter, Lutz; Sims, Mark R; Cullen, David C

    2013-01-01

    The Life Marker Chip (LMC) instrument is an immunoassay-based sensor that will attempt to detect signatures of life in the subsurface of Mars. The molecular reagents at the core of the LMC have no heritage of interplanetary mission use; therefore, the design of such an instrument must take into account a number of risk factors, including the radiation environment that will be encountered during a mission to Mars. To study the effects of space radiation on immunoassay reagents, primarily antibodies, a space study was performed on the European Space Agency's 2007 BIOPAN-6 low-Earth orbit (LEO) space exposure platform to complement a set of ground-based radiation studies. Two antibodies were used in the study, which were lyophilized and packaged in the intended LMC format and loaded into a custom-made sample holder unit that was mounted on the BIOPAN-6 platform. The BIOPAN mission went into LEO for 12 days, after which all samples were recovered and the antibody binding performance was measured via enzyme-linked immunosorbent assays (ELISA). The factors expected to affect antibody performance were the physical conditions of a space mission and the exposure to space conditions, primarily the radiation environment in LEO. Both antibodies survived inactivation by these factors, as concluded from the comparison between the flight samples and a number of shipping and storage controls. This work, in combination with the ground-based radiation tests on representative LMC antibodies, has helped to reduce the risk of using antibodies in a planetary exploration mission context. PMID:23286207

  16. Performance evaluation of two immunoassays for 25-hydroxyvitamin D

    PubMed Central

    Li, Lusha; Zeng, Qin; Yuan, Jingjing; Xie, Zhongjian

    2016-01-01

    Although immunoassays in measuring 25-hydroxyvitamin D [25(OH)D] have been improved recently, relatively large differences are still seen between results of 25(OH)D measured by immunoassays and by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the present studies, we compared two immunoassays with LC-MS/MS for measuring 25(OH)D concentrations. Concentrations of 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] in serum samples from 59 healthy subjects were measured by two immunoassays including Siemens ADVIA Centaur Vitamin D Total (Centaur) and Roche Elecsys Vitamin D Total (Elecsys) and LC-MS/MS. To determine the cross reactivity of Elecsys and Centaur toward 25(OH)D2, a dosage of 200,000 IU vitamin D2 was given after first sampling. Serum samples were obtained 30 days later and concentrations of 25(OH)D2 and 25(OH)D3 were measured again. The results showed poor agreement between the immunoassays and LC-MS/MS in 25(OH)D2 and 25(OH)D3 measurements. The percentage of 25(OH)D2 cross-reactivity was 45.3% for Centaur and 41.2% for Elecsys and there was no significant difference between Centaur and Elecsys. In conclusion, Centaur and Elecsys perform unsatisfactorily in measuring 25(OH)D levels, especially for 25(OH)D2 cross-reactivity. Therefore, clinicians need to be aware of the underestimation of vitamin D status when using these immunoassays for measuring individuals supplemented with vitamin D2. PMID:27257343

  17. Fenton reaction-based colorimetric immunoassay for sensitive detection of brevetoxin B.

    PubMed

    Lai, Wenqiang; Wei, Qiaohua; Zhuang, Junyang; Lu, Minghua; Tang, Dianping

    2016-06-15

    We designed a new colorimetric immunoassay for sensitive monitoring of brevetoxin B (BTB) using enzyme-controlled Fenton reaction with a high-resolution 3,3',5,5'-tetramethylbenzidine (TMB)-based visual colored system. Upon addition of hydrogen peroxide (H2O2), the equivalent iron(II) could be first converted into iron(III) and free hydroxyl radical (•OH) via the classical Fenton reaction. Then the as-produced iron(III) and •OH could cause a perceptible change from colorless to blue with the increasing H2O2 concentration in the presence of TMB. Based on Fenton reaction-triggered visual colored system, a novel competitive-type colorimetric enzyme immunoassay was developed for the quantitative screening of target BTB on the bovine serum albumin-BTB-modified magnetic bead using glucose oxidase/anti-BTB antibody-labeled gold nanoparticle as the signal-transduction tag. Upon target BTB introduction, the analyte competed with the conjugated BTB on the magnetic bead for anti-BTB antibody on gold nanoparticle. The carried glucose oxidase with the gold nanoparticle could implement the oxidation of glucose to produce H2O2, and the generated H2O2 promoted the above-mentioned Fenton reaction for color development. Under the optimal conditions, the absorbance decreased with the increasing target BTB in the range from 0.1 to 150 ng kg(-1) with a low detection limit (LOD) of 0.076 ng kg(-1). The LOD was 500-fold lower than that of commercialized Abraxis BTB ELISA kit. Non-specific adsorption was not observed. The precision, reproducibility and specificity were acceptable. Finally, the method accuracy was also validated for monitoring spiked seafood samples, giving results well matched with the referenced brevetoxin ELISA kit. PMID:26851583

  18. Robust detection of peak signals for lateral flow immunoassays

    NASA Astrophysics Data System (ADS)

    Kim, Jongwon; Kim, Jong Dae; Nahm, Kie Bong; Choi, Eui Yul; Lee, Geumyoung

    2011-02-01

    Template matching method is presented to identify the peaks from the scanned signals of lateral flow immunoassay strips. The template is composed of two pulses separated by the distance of the control and the target ligand line in the assay, and is convolved with the scanned signal to deliver the maximum at the center of the two peaks. The peak regions were identified with the predefined distances from the center. Glycosylated haemoglobin immunoassay strips and fluorescent strip readers from Boditechmed Inc. were tested to estimate the lot and reader variations of the concentration measurands. The results showed the robustness of the propose method.

  19. Architecture of Human IgM in Complex with P. falciparum Erythrocyte Membrane Protein 1.

    PubMed

    Akhouri, Reetesh Raj; Goel, Suchi; Furusho, Hirotoshi; Skoglund, Ulf; Wahlgren, Mats

    2016-02-01

    Plasmodium falciparum virulence is associated with sequestration of infected erythrocytes. Microvascular binding mediated by PfEMP1 in complex with non-immune immunoglobulin M (IgM) is common among parasites that cause both severe childhood malaria and pregnancy-associated malaria. Here, we present cryo-molecular electron tomography structures of human IgM, PfEMP1 and their complex. Three-dimensional reconstructions of IgM reveal that it has a dome-like core, randomly oriented Fab2s units, and the overall shape of a turtle. PfEMP1 is a C- shaped molecule with a flexible N terminus followed by an arc-shaped backbone and a bulky C terminus that interacts with IgM. Our data demonstrate that the PfEMP1 binding pockets on IgM overlap with those of C1q, and the bulkiness of PfEMP1 limits the capacity of IgM to interact with PfEMP1. We suggest that P. falciparum exploits IgM to cluster PfEMP1 into an organized matrix to augment its affinity to host cell receptors. PMID:26776517

  20. Metabolite-specific (IgG) and drug-specific antibodies (IgG, IgM) in two cases of trimethoprim-sulfamethoxazole-induced immune thrombocytopenia.

    PubMed

    Kiefel, V; Santoso, S; Schmidt, S; Salama, A; Mueller-Eckhardt, C

    1987-01-01

    Two cases of trimethoprim-sulfamethoxazole (TMP-SMX)-induced immune thrombocytopenia are reported in which unusual drug-dependent platelet antibodies were demonstrated by immunofluorescence and enzyme-linked immunosorbent assay. Whereas two distinct sulfamethoxazole-dependent antibodies of the IgG and IgM class were detectable in the serum of one patient, the serum of the other patient contained a platelet antibody exclusively reactive with N-4-acetyl-sulfamethoxazole, a metabolite of sulfamethoxazole. Urine from a healthy volunteer collected after administration of therapeutic doses of TMP-SMX proved to be an appropriate source of ex vivo metabolites for antibody testing. The results of this study stress the role of metabolite-specific antibodies in drug-dependent immune thrombocytopenia and underscore the necessity of including metabolite preparations of drugs in serologic analyses. PMID:3296342

  1. A selective defect in IgM antigen receptor synthesis and transport causes loss of cell surface IgM expression on tolerant B lymphocytes.

    PubMed Central

    Bell, S E; Goodnow, C C

    1994-01-01

    To explore the biochemical basis for maintaining immunological tolerance by functional inactivation of self-reactive B lymphocytes, transgenic mice carrying rearranged anti-lysozyme immunoglobulin transgenes and a lysozyme transgene were used as a source of large numbers of tolerant self-reactive B cells. Antigen receptors of the IgD isotype were expressed at normal levels on tolerant B cells, contained the heterodimeric MB1/B29 signalling component of the receptor complex and were structurally indistinguishable from IgD on nontolerant B cells. In contrast, cell surface expression of IgM receptor complexes on tolerant B cells was greatly reduced, despite normal expression of mRNA encoding the receptor components. Three-fold fewer immunoreactive mu heavy chains were detectable after a short period of biosynthetic labelling and the immunoreactive mu chains produced were paired with kappa light chains and assembled normally into intact receptor complexes containing the MB1/B29 heterodimer. Nascent IgM receptor complexes nevertheless failed to be processed into an endoglycosidase H-resistant form in the tolerant B cells and thus appeared to be selectively blocked in their transport from the endoplasmic reticulum to the medial Golgi. These findings demonstrate that intracellular trafficking of antigen receptor complexes is regulated by exposure to receptor stimuli at the cell surface causing a long-lasting decrease in surface receptor expression on tolerant B cells. Images PMID:8112296

  2. Enzymes, Industrial

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enzymes serve key roles in numerous biotechnology processes and products that are commonly encountered in the forms of food and beverages, cleaning supplies, clothing, paper products, transportation fuels, pharmaceuticals, and monitoring devices. Enzymes can display regio- and stereo-specificity, p...

  3. Understanding Enzymes.

    ERIC Educational Resources Information Center

    Sinnott, M. L.

    1979-01-01

    Describes the way enzymes operate through reaction energetics, and explains that most of the catalytic power of enzymes lies in the strong noncovalent forces responsible for initial binding of substrate, which are only manifested at the transition state of the reaction. (Author/GA)

  4. Soil Enzymes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The functionality and resilience of natural and managed ecosystems mainly rely on the metabolic abilities of microbial communities, the main source of enzymes in soils. Enzyme mediated reactions are critical in the decomposition of organic matter, cycling of nutrients, and in the breakdown of herbic...

  5. Solid-phase enzyme immunoassay or radioimmunoassay for the detection of immune complexes based on their recognition by conglutinin: conglutinin-binding test. A comparative study with 125I-labelled C1q binding and Raji-cell RIA tests

    PubMed Central

    Casali, P.; Bossus, A.; Carpentier, Nicole A.; Lambert, P.-H.

    1977-01-01

    Bovine conglutinin was used in a solid-phase assay for the detection of immune complexes. In a first step, the tested serum sample is incubated in polypropylene tubes coated with conglutinin to allow C3-coated immune complexes to bind to solid-phase conglutinin. In a second step, the conglutinin-bound complexes are detected using an enzyme-conjugated or radiolabelled anti-immunoglobulin antibody. The conglutinin-binding (KgB) test does not suffer from the interference of DNA, heparin or endotoxins. Its limit of sensitivity for aggregated IgG is 3 μg/ml undiluted human serum. Immune complexes prepared in vitro using tetanus toxoid, or DNA, and corresponding antibodies in human sera could be detected at various antigen/antibody ratios and at antibody concentrations lower than 8 μg/ml. The KgB test allowed for the detection of immune complexes in sera from patients with systemic lupus erythematosus, rheumatoid arthritis, idiopathic vasculitis, leprosy and leukemia. These sera were also tested using the 125I-labelled Clq-binding activity (BA) test and the KgB test simultaneously, and a significant rank order correlation was observed. In patients with leukemia, a significant correlation was observed using three tests, KgB, 125I-labelled Clq BA and Raji-cell radioimmunoassay (RIA). Therefore, the KgB test appears as a simple and reproducible method, utilizing a very stable reagent, with a sensitivity and specificity comparable to the other tests studied and allowing for clinical application. PMID:332422

  6. Respiratory allergy to Aspergillus-derived enzymes in bakers' asthma.

    PubMed

    Quirce, S; Cuevas, M; Díez-Gómez, M; Fernández-Rivas, M; Hinojosa, M; González, R; Losada, E

    1992-12-01

    Baking and food industry workers are exposed to several powdered Aspergillus-derived enzymes with carbohydrate-cleaving activity that are commonly used to enhance baked products. We describe a retrospective study of sensitization to fungal alpha-amylase and cellulase on bakers. Five bakers in whom respiratory allergy symptoms developed when they were exposed to bread "improvers" that contained fungal alpha-amylase and cellulase were investigated by in vivo and in vitro tests. Type I hypersensitivity to these enzymes was demonstrated in the five patients by means of skin testing, histamine release test, positive reverse enzyme-immunoassay for specific IgE antibodies, and bronchial provocation test response to alpha-amylase or cellulase or both. Isolated immediate and dual responses to the bronchial challenge tests with these enzymes were observed. Immunoblot analysis with use of a pooled serum identified IgE-binding components in both enzymes. In the reverse-enzyme immunoassay-inhibition assays cross-reactivity between alpha-amylase and cellulase was not found, but some degree of cross-reactivity between alpha-amylase and A. oryzae, and between cellulase and A. niger was demonstrated. Four of the patients were also sensitized to cereal flour. Aspergillus-derived enzymes used as flour additives can elicit IgE-mediated respiratory allergy, and this fact has to be considered in the diagnosis and clinical management of bakers' asthma. PMID:1281180

  7. Prevalence, specificity and functionality of anti-ganglioside antibodies in neuropathy associated with IgM monoclonal gammopathy.

    PubMed

    Stork, Abraham C J; Jacobs, Bart C; Tio-Gillen, Anne P; Eurelings, Marijke; Jansen, Marc D; van den Berg, Leonard H; Notermans, Nicolette C; van der Pol, W-Ludo

    2014-03-15

    IgM antibodies against gangliosides and their complexes were studied in sera from 54 patients with polyneuropathy and IgM monoclonal gammopathy (IgM-PNP) without anti-MAG antibodies. Anti-ganglioside antibodies were found in 19 (35%) patients. Five (9%) patients had antibodies against ganglioside complexes. IgM antibodies against gangliosides activated complement in vitro. Light chain usage was restricted to kappa or lambda in most, but not all patients. In conclusion, anti-ganglioside antibodies in IgM-PNP are common, display pathogenic properties and do not always arise from a monoclonal B cell proliferation. PMID:24529728

  8. Toxicological Effects of Nickel Chloride on IgA+ B Cells and sIgA, IgA, IgG, IgM in the Intestinal Mucosal Immunity in Broilers

    PubMed Central

    Wu, Bangyuan; Cui, Hengmin; Peng, Xi; Fang, Jing; Zuo, Zhicai; Deng, Junliang; Huang, Jianying

    2014-01-01

    The objective of this study was to investigate the toxicological effects of dietary NiCl2 on IgA+ B cells and the immunoglobulins including sIgA, IgA, IgG and IgM in the small intestine and cecal tonsil of broilers by the methods of immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Two hundred and forty one-day-old avian broilers were randomly divided into four groups and fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg NiCl2 for 42 days. Compared with the control group, the IgA+ B cell number and the sIgA, IgA, IgG, and IgM contents in the NiCl2-treated groups were significantly decreased (p < 0.05 or p < 0.01). It was concluded that dietary NiCl2 in the excess of 300 mg/kg had negative effects on the IgA+ B cell number and the abovementioned immunoglobulin contents in the small intestine and the cecal tonsil. NiCl2-reduced sIgA, IgA, IgG and IgM contents is due to decrease in the population and/or the activation of B cell. The results suggest that NiCl2 at high levels has intestinal mucosal humoral immunotoxicity in animals. PMID:25116637

  9. Peroxidase-like activity of mesoporous silica encapsulated Pt nanoparticle and its application in colorimetric immunoassay.

    PubMed

    Wang, Zhifei; Yang, Xia; Yang, Jingjing; Jiang, Yanyun; He, Nongyue

    2015-03-01

    Nanomaterial-based artificial enzymes have received great attention in recent year due to their potential application in immunoassay techniques. However, such potential is usually limited by poor dispersion stability or low catalytic activity induced by the capping agent essentially required in the synthesis. In an attempt to address these challenges, here, we studied the novel Pt nanoparticles (NPs) based peroxidase-like mimic by encapsulating Pt NP in mesoporous silica (Pt@mSiO2 NPs). Compared with other nanomaterial-based artificial enzymes, the obtained Pt@mSiO2 NPs not only exhibit high peroxidase-like activity but also have good dispersion stability in buffer saline solution when grafted with spacer PEG. Results show that when the thickness of silica shell is about 9 nm the resulting Pt@mSiO2 NPs exhibit the catalytic activity similar to that of Pt NPs, which is approximately 26 times higher than that of Fe3O4 NPs (in terms of Kcat for H2O2). Due to the protection of silica shell, the subsequent surface modification with antibody has little effect on their catalytic activity. The analytical performance of this system in detecting hCG shows that after 5 min incubation the limit of detection can reach 10 ng mL(-1) and dynamic linear working range is 5-200 ng mL(-1). Our findings pave the way for design and development of novel artificial enzyme labeling. PMID:25682428

  10. Further comparisons of assays for detecting MAG IgM autoantibodies.

    PubMed

    Jaskowski, Troy D; Prince, Harry E; Greer, Ryan W; Litwin, Christine M; Hill, Harry R

    2007-07-01

    Anti-MAG antibodies are commonly found in the sera of patients with demyelinating sensorimotor neuropathy and IgM paraproteinemia. Our objective here was to compare MAG results obtained by two different laboratories using similar methods (Western blot, EIA, IFA). Western blot (WB) employing MAG from monkey was less sensitive (72.5%) than myelin IFA (92.5%; monkey nerve) and EIA (97.5%; human MAG) when compared to WB using human MAG and is most likely due to methodology (not antigen source). EIA detected low titers of MAG IgM antibodies in suspected patient sera (negative by other methods) that were also SGPG IgM-positive. Patients having low titers by EIA, but negative by WB may have other autoimmune neuropathies without demyelination. PMID:17537521

  11. Human cord blood contains an IGM antibody to the 41KD flagellar antigen of Borrelia burgdorferi.

    PubMed

    Cooke, W D; Orr, A S; Wiseman, B L; Rouse, S B; Murray, W C; Ranck, S G

    1993-10-01

    Natural antibodies are the IgM products of fetal and neonatal B cells. These are germline encoded low affinity antibodies with multiple specificities to self and exogenous antigens. Lyme borreliosis is the disease resulting from infection with the spirochete, Borrelia burgdorferi. The humoral response to this organism is brisk, directed at multiple proteins, and persistent. Antibody to the 41kd flagellar antigen is found early in disease, but may also be found in non-exposed individuals. These properties suggest that the anti-41kd antibody may be a natural antibody. We report here the finding of an IgM anti-41kd reactivity in 29% of cord blood samples from patients in an area non-endemic for Lyme disease. The results are consistent with the hypothesis that antibody to flagellin may be a germline encoded natural antibody, and could be important in the immunopathogenesis of Lyme arthritis and other arthritides. PMID:8211003

  12. Blood-nerve barrier in IgM paraproteinemic neuropathy: a clinicopathologic assessment.

    PubMed

    Kanda, T; Usui, S; Beppu, H; Miyamoto, K; Yamawaki, M; Oda, M

    1998-02-01

    We report the pathologic findings in a patient with sensorimotor neuropathy associated with Waldenström's macroglobulinemia, particularly in relation to blood-nerve barrier defects. The monoclonal IgM was of kappa type and possessed anti-HNK-1 activity. A sural nerve biopsy specimen revealed severe loss of myelinated and unmyelinated nerve fibers and gaps between adjacent endothelial cells of small endoneurial vessels. Postmortem findings 3 years later included severe loss of myelinated nerve fibers and diffuse infiltration by lymphoplasmacytic B cells throughout the peripheral nervous system, sparing the central nervous system. Findings in this case suggest an immune attack against endoneurial endothelial cells with permeation of IgM into peripheral nerve tissue. PMID:9498055

  13. Protection against gram-negative bacteremia and endotoxemia with human monoclonal IgM antibodies.

    PubMed Central

    Teng, N N; Kaplan, H S; Hebert, J M; Moore, C; Douglas, H; Wunderlich, A; Braude, A I

    1985-01-01

    Hybridomas producing human monoclonal IgM antibodies (mAbs) against bacterial lipopolysaccharide (LPS) were generated by fusion of B lymphocytes from sensitized human spleen with heteromyeloma cells. The splenocytes were from patients undergoing splenectomy during staging for Hodgkin disease after vaccination with the J5 mutant of Escherichia coli, which is deficient in O antigenic side chains. This deficiency exposes the core oligosaccharide, common to LPS of all Gram-negative bacteria. The mAbs cross-reacted strongly with endotoxins from a wide range of unrelated species of Gram-negative bacteria. The mAbs also gave strong protection against LPS in the dermal Shwartzman reaction and against lethal Gram-negative bacteremia in mice. These findings indicate that monoclonal IgM against LPS endotoxin can neutralize its toxicity in vivo and might be valuable for treatment of patients with Gram-negative bacteremia. Analysis of one of the hybridoma clones, A6(H4C5), showed that the IgM mAb is directed against the covalently bound lipid A, which represents the most conservative and least variable structural element of LPS. Images PMID:3856860

  14. Diagnosis of autoimmune neutropenia by neutrophil-bound IgG and IgM antibodies.

    PubMed

    Ito, Taichi; Taniuchi, Shoichiro; Tsuji, Shoji; Iharada, Anna; Hasui, Masafumi; Kaneko, Kazunari

    2011-10-01

    Autoimmune neutropenia (AIN) in infancy is caused by antineutrophil (granulocyte-specific) autoantibodies. These antibodies are rarely found in circulation because their serum levels are extremely low. We hypothesized that a direct granulocyte immunofluore