[Standardized indirect immunofluorescence. Differentiation of mitochondrial, microsomal and ribosomal antibodies].

PubMed

Storch, W

1977-02-15

By an extensive standardisation of the indirect immunofluorescence for the demonstration espeially of mitochondrial antibodies we succeeded in recognizing atypical fluorescence patterns and in describing their exact localisation. On the basis of absorption studies with mitochondrias, microsomas and ribosomas by comparative observation of sections of liver, stomach and kidneys of rats the preferred sort of reaction and the intensity of fluorescence of antibodies against mitochondria, microsomas and ribosomas were empirically established. Antimitochondrial antibodies react above all with the parietal cells of the stomach and the distal epithelia of the tubulus of the kidney. Antibodies against microsomas of liver and kidney are characterized by a brilliant diffuse cytoplasmatic fluorescence of the hepatocytes and by a comparatively weaker fluorescence of exclusively proximal tubuli of the kidneys of rats. Antibodies against ribosomas lead to a fluorescence especially of the main cells of the stomach. The differentiation of several cytoplasmatic antibodies is among others of interest for the diagnosis of certain autoimmune diseases. Although there are numerous still unclear findings and "overlap" phenomena the existence of high titre antibodies against mitochondrias speaks for a primarily biliary cirrhosis or a pseudo-LE-syndrome, the existence of antibodies against microsomas of kidney and liver of rats for a special form of a chronically active hepatitis and the existence of the very rare antibodies against ribosomas for an active lupus erythematodes disseminatus.

Development and decline of antiplasmodial indirect fluorescent antibodies in mice infected with Plasmodium berghei (NK65) and treated with drugs

PubMed Central

Waki, Seiji; Suzuki, Mamoru

1974-01-01

Malaria parasites in mice present a simplified rodent model for the immunological study of malaria. Experiments have been performed to determine the pattern and persistence of malaria antibody as detected by the indirect fluorescent antibody (IFA) test utilizing specific antimouse IgM and IgG conjugates. The antibody levels in mice inoculated with Plasmodium berghei and treated with antimalarial drugs were traced after complete elimination of the parasites from the host. Within 1-2 weeks after inoculation, both specific IgM and IgG reached peak levels, which thereafter declined rapidly. The results suggest that a high IFA titre may be taken as an indication of recent parasitaemia when the parasites are absent from the host. The protective role of the specific immunoglobulin was not found in the cured animals at the time when the animals showed a high IFA titer. It seems that the detected IFA may not reflect protective immunity against reinfection with malaria parasites. PMID:4617640

Comparison of PCR and culture to the indirect fluorescent-antibody test for diagnosis of Potomac horse fever.

PubMed

Mott, J; Rikihisa, Y; Zhang, Y; Reed, S M; Yu, C Y

1997-09-01

Potomac horse fever is an acute systemic equine disease caused by Ehrlichia risticii. Currently, serologic methods are widely used to diagnose this disease. However, serologic methods cannot determine whether the horse is presently infected or has been exposed to ehrlichial antigens in the past. The purpose of the present study was to compare the sensitivities of the nested PCR and cell culture with that of the indirect fluorescent-antibody (IFA) test for the diagnosis of Potomac horse fever. Blood and fecal specimens serially collected from a pony experimentally infected with E. risticii Maryland, blood specimens serially collected from mice inoculated with E. risticii Ohio 380, and blood and/or fecal specimens collected from 27 horses which had clinical signs compatible with Potomac horse fever were examined. These horses resided in Kentucky, Indiana, Pennsylvania, and Vermont. The IFA test titer became positive after 6 days postinoculation (p.i.) for the pony. A culture of the blood of the pony was positive for E. risticii starting on day 1 and was positive through day 28 p.i. By the nested PCR, E. risticii was detectable in the blood and feces of the pony starting on day 1 and was detectable through day 32 p.i. E. risticii was detected in the blood of subclinically infected mice by the nested PCR. Twenty-two clinical specimens were seropositive for E. risticii by the IFA test, with titers ranging from 1:20 to 1:1,280. E. risticii was cultured from 95% (20 of 21) of seropositive clinical blood specimens. E. risticii was detected in the blood by PCR in 81% (17 of 20) of the culture-positive clinical specimens. The study indicated that the nested PCR is as sensitive as culture for detecting infection with E. risticii.

Indirect ELISA (iELISA) for routine detection of antibodies against Minute Virus of Mice (MVM) in mice colonies.

PubMed

Laborde, Juan M; Sguazza, Guillermo H; Fuentealba, Nadia A; Corva, Santiago G; Carbone, Cecilia; Galosi, Cecilia M

In this study we developed an indirect ELISA to detect antibodies against Minute Virus of Mice (MVM) using an antigen produced from BHK-21 cells infected with a prototype strain of the virus. The optimal antigen concentration and serum dilutions were established. In order to analyze variability in the laboratory, reproducibility and repeatability within and between plates were determined. Then, a panel of 460 sera from conventional facilities and previously classified as positive or negative by the indirect fluorescent antibody assay was analyzed. The cutoff value was determined by a receiver operating characteristic (ROC) curve. The results of the indirect ELISA were compared with those of the indirect fluorescent antibody assay. The ELISA assay showed 100% sensitivity and 99% specificity. ELISA is a useful tool to be developed in standard virology laboratories and can be used for screening animals faster than the traditional indirect fluorescent antibody assay. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Evaluation and comparison of an indirect fluorescent antibody test for detection of antibodies to Sarcocystis neurona, using serum and cerebrospinal fluid of naturally and experimentally infected, and vaccinated horses.

PubMed

Duarte, Paulo C; Daft, Barbara M; Conrad, Patricia A; Packham, Andrea E; Saville, William J; MacKay, Robert J; Barr, Bradd C; Wilson, W David; Ng, Terry; Reed, Stephen M; Gardner, Ian A

2004-04-01

The objectives of this study were to evaluate the accuracy of the indirect fluorescent antibody test (IFAT) using serum and cerebrospinal fluid (CSF) of horses naturally and experimentally infected with Sarcocystis neurona, to assess the correlation between serum and CSF titers, and to determine the effect of S. neurona vaccination on the diagnosis of infection. Using receiver-operating characteristic analysis, the areas under the curve for the IFAT were 0.97 (serum) and 0.99 (CSF). Sensitivity and specificity were 83.3 and 96.9% (serum, cutoff 80) and 100 and 99% (CSF, cutoff 5), respectively. Titer-specific likelihood ratios (LRs) ranged from 0.03 to 187.8 for titers between <10 and 640. Median time to conversion was 22-26 days postinfection (DPI) (serum) and 30 DPI (CSF). The correlation between serum and CSF titers was moderately strong (r = 0.6) at 30 DPI. Percentage of vaccinated antibody-positive horses ranged from 0 to 95% between 0 and 112 days after the second vaccination. Thus, the IFAT was reliable and accurate using serum and CSF. Use of LRs potentially improves clinical decision making. Correlation between serum and CSF titers affects the joint accuracy of the IFAT; therefore, the ratio of serum to CSF titers has potential diagnostic value. The S. neurona vaccine could possibly interfere with equine protozoal myeloencephalitis diagnosis.

Tracking Silent Hypersensitivity Reactions to Asparaginase during Leukemia Therapy Using Single-Chip Indirect Plasmonic and Fluorescence Immunosensing.

PubMed

Charbonneau, David M; Breault-Turcot, Julien; Sinnett, Daniel; Krajinovic, Maja; Leclerc, Jean-Marie; Masson, Jean-François; Pelletier, Joelle N

2017-12-22

Microbial asparaginase is an essential component of chemotherapy for the treatment of childhood acute lymphoblastic leukemia (cALL). Silent hypersensitivity reactions to this microbial enzyme need to be monitored accurately during treatment to avoid adverse effects of the drug and its silent inactivation. Here, we present a dual-response anti-asparaginase sensor that combines indirect SPR and fluorescence on a single chip to perform ELISA-type immunosensing, and correlate measurements with classical ELISA. Analysis of serum samples from children undergoing cALL therapy revealed a clear correlation between single-chip indirect SPR/fluorescence immunosensing and ELISA used in clinical settings (R 2 > 0.9). We also report that the portable SPR/fluorescence system had a better sensitivity than classical ELISA to detect antibodies in clinical samples with low antigenicity. This work demonstrates the reliability of dual sensing for monitoring clinically relevant antibody titers in clinical serum samples.

SEROLOGICAL TYPING OF STAPHYLOCOCCI BY MEANS OF FLUORESCENT ANTIBODIES I.

PubMed Central

Cohen, Jay O.; Oeding, Per

1962-01-01

Cohen, Jay O. (Communicable Disease Center, Atlanta, Ga.) and Per Oeding. Serological typing of staphylococci by means of fluorescent antibodies. I. Development of specific reagents for seven serological factors. J. Bacteriol. 84:735–741. 1962—Fluorescent antibody reagents for identifying seven antigenic factors of staphylococci have been prepared. The fluorescent staining reactions of these reagents were compared to the agglutination reactions with diagnostic cultures of coagulase-positive staphylococci. Correlation between the two serological tests was almost complete with factors a, b, i, and k. The c fluorescent antibody reagent had a somewhat broader spectrum of activity than the corresponding agglutination serum, whereas the m fluorescent antibody reagent stained fewer strains than were agglutinated in m serum. The fluorescent antibody reagent for h factor stained strains possessing h1 factor but not strains possessing only h2 factor. Fluorescent antibody reagents for specific staphylococcal factors did not stain strains of group A streptococci. PMID:14022057

STUDIES ON FLUORESCENT ANTIBODY STAINING

PubMed Central

Goldstein, Gerald; Slizys, Irene S.; Chase, Merrill W.

1961-01-01

1. A study has been made of the non-specific fluorescent staining of splenic imprints treated with fluorescent sheep antibody globulins. 2. In tissue imprints made with the spleens of antigen-stimulated animals, no morphological distinction was evident between areas showing non-specific fluorescence and specific fluorescence. 3. Elimination of non-specific fluorescence was not achieved by any one, or any combination of the following: (a) conjugating only gamma globulins with fluorescein isothiocyanate; (b) removal of dialyzable fluorescent products on sephadex, followed by concentration through the use of pressure dialysis; (c) use of crystalline preparations of fluorescein isothiocyanate. 4. Individual preparations of fluorescent antibodies were separated by gradient elution chromatography on diethylaminoethyl (DEAE) cellulose into fractions possessing different numbers of fluorescein radicals per molecule of globulin. 5. The coupling ratio of 50 mg fluorescein isothiocyanate (FITC) per gm of protein, as commonly advocated, can not be recommended for the precise localization of antibody globulin in tissues owing to the capacity of the coupled products to give non-specific fluorescent staining. When crystalline preparations of FITC are used instead of the amorphous product at 50 mg/gm protein, far too high non-specific fluorescence results. 6. A fraction with bright specific fluorescence and no or negligible nonspecific fluorescence was obtained from each fluorescent antibody that was prepared by using 6 to 8 mg of crystalline fluorescein isothiocyanate per gm of globulin and was then subjected to DEAE-cellulose chromatography and gradient elution to eliminate the most highly coupled molecules. PMID:13706641

Cross-sectional study of serum antibodies against Sarcocystis neurona in cats tested for antibodies against Toxoplasma gondii.

PubMed

Rossano, Mary G; Murphy, Alice J; Vrable, Ruth A; Vanzo, Nicole E; Lewis, Stacy K; Sheline, Katherine D; Kaneene, John B; Mansfield, Linda S

2002-08-15

To determine apparent seroprevalence of antibodies against Sarcocystis neurona in a population of domestic cats previously tested for antibodies against Toxoplasma gondii. Cross-sectional study. Serum from 196 domestic cats. Banked serum samples submitted to the Michigan State University Animal Health Diagnostic Laboratory for T. gondii diagnostic testing were tested for antibodies against S. neurona by use of an indirect fluorescent antibody (IFA) test and a western blot test. Submission records were analyzed to determine descriptive statistics and test for associations between positive results of a test for S. neurona and other variables in the data set. 10 of 196 (5%) samples yielded positive results for antibodies against S. neurona by use of western blot analysis, whereas 27 samples yielded positive results by use of the IFA. No association was found between S. neurona western blot test results and T. gondii test results, age, sex, or the reason for T. gondii testing. The S. neurona IFA titer was positively and significantly associated with positive results of western blot analysis. Domestic cats are not likely to play a substantial role as intermediate hosts in the natural life cycle of S. neurona. Results indicate that natural infection of domestic cats may occur, and small animal practitioners should be aware of this fact when evaluating cats with neurologic disease. The S. neurona IFA test had lower specificity than western blot analysis.

Comparison of visual microscopic and computer-automated fluorescence detection of rabies virus neutralizing antibodies.

PubMed

Péharpré, D; Cliquet, F; Sagné, E; Renders, C; Costy, F; Aubert, M

1999-07-01

The rapid fluorescent focus inhibition test (RFFIT) and the fluorescent antibody virus neutralization test (FAVNT) are both diagnostic tests for determining levels of rabies neutralizing antibodies. An automated method for determining fluorescence has been implemented to reduce the work time required for fluorescent visual microscopic observations. The automated method offers several advantages over conventional visual observation, such as the ability to rapidly test many samples. The antibody titers obtained with automated techniques were similar to those obtained with both the RFFIT (n = 165, r = 0.93, P < 0.001) and the FAVNT (n = 52, r = 0.99, P < 0.001).

[Pediatric pneumonia, pleural effusion, and pericarditis following cat scratch disease and serological cross-reactions among Bartonella henselae and Rickettsia japonica determined by indirect fluorescence antibodies].

PubMed

Takeda, Nobue; Ishiwada, Naruhiko; Fukasawa, Chie; Furuya, Yumiko; Tsuneoka, Hidehiro; Tsukahara, Masato; Kohno, Yoichi

2007-03-01

Cat scratch disease is associated with a variety of systemic manifestations. We report a pediatric case associated with pneumonia, pleural effusion, and pericarditis. A 3-year-old boy developed prolonged fever unresponsive to antibiotic treatment, including azithromycin and minocycline. Although the fever resolved with corticosteroid treatment, Bartonella henselae IgG titer was positive in indirect fluorescence antibodies, as was Rickettsia japonica IgG titer. Both titers were significantly reduced by serum absorption with B. henselae antigens, and we observed a serological cross-reaction between B. henselae and R. japonica.

Qualitative evaluation of selective tests for detection of Neospora hughesi antibodies in serum and cerebrospinal fluid of experimentally infected horses.

PubMed

Packham, Andrea E; Conrad, Patricia A; Wilson, W David; Jeanes, Lisa V; Sverlow, Karen W; Gardner, Ian A; Daft, Barbara M; Marsh, Antoinette E; Blagburn, Byron L; Ferraro, Gregory L; Barr, Bradd C

2002-12-01

Neospora hughesi is a newly recognized protozoan pathogen in horses that causes a myeloencephalitis similar to Sarcocystis neurona. There are no validated serologic tests using the gold standard sera that are currently available to detect specific N. hughesi antibodies and, thus, no tests available to detect antemortem exposure or estimate seroprevalence in the horse. The objectives of the present study were to establish a bank of gold standard equine sera through experimental infections with N. hughesi and to assess several serologic tests for the detection of related protozoan antibodies. Seven horses were inoculated with N. hughesi tachyzoites, and 7 horses received uninfected cell culture material. The horses were monitored, and blood and cerebrospinal fluid were collected repeatedly over a 4-mo period. With the sera, 4 different serologic techniques were evaluated. including a whole-parasite lysate enzyme-linked immunosorbent assay (ELISA), a recombinant protein ELISA, a modified direct agglutination test, and an indirect fluorescent antibody test. Qualitative and quantitative evaluation of the results showed that the N. hughesi indirect fluorescent antibody test (IFAT) consistently discriminated between experimentally infected and noninfected horses, using a cutoff of 1:640. Sera from 3 naturally infected horses had titers >1:640. Cerebrospinal fluid in all but I infected horse had very low N. hughesi IFAT titers (<1:160), starting at postinoculation day 30.

Evaluation of Two rK39 Dipstick Tests, Direct Agglutination Test, and Indirect Fluorescent Antibody Test for Diagnosis of Visceral Leishmaniasis in a New Epidemic Site in Highland Ethiopia

PubMed Central

Cañavate, Carmen; Herrero, Merce; Nieto, Javier; Cruz, Israel; Chicharro, Carmen; Aparicio, Pilar; Mulugeta, Abate; Argaw, Daniel; Blackstock, Anna J.; Alvar, Jorge; Bern, Caryn

2011-01-01

We assessed the performance characteristics of two rK39 immunochromatographic tests, a direct agglutination test (DAT), and an indirect immunofluorescent antibody test (IFAT) in the site of a new epidemic of visceral leishmaniasis (VL) in northwestern Ethiopia. The study population was composed of 179 patients with suspected VL and 67 controls. The sensitivities of Kalazar Detect®, DiaMed-IT Leish®, DAT, and IFAT in 35 polymerase chain reaction–confirmed VL cases were 94.3%, 91.4%, 91.4%, and 100%, respectively, and the specificities were 98.5%, 94%, 98.5%, and 98.5%, respectively. In a Bayesian latent class analysis of all 246 specimens, the estimated sensitivities were 90.5%, 89%, 88.8%, and 96% for Kalazar Detect®, DiaMed-IT Leish®, DAT, and IFAT, respectively; DAT showed the highest estimated specificity (97.4%). Both rK39 immunochromatographic tests perform as well as DAT, and are suitable for VL diagnosis in first-level health centers in this area of Ethiopia. PMID:21212210

Cell-free measurements of brightness of fluorescently labeled antibodies

PubMed Central

Zhou, Haiying; Tourkakis, George; Shi, Dennis; Kim, David M.; Zhang, Hairong; Du, Tommy; Eades, William C.; Berezin, Mikhail Y.

2017-01-01

Validation of imaging contrast agents, such as fluorescently labeled imaging antibodies, has been recognized as a critical challenge in clinical and preclinical studies. As the number of applications for imaging antibodies grows, these materials are increasingly being subjected to careful scrutiny. Antibody fluorescent brightness is one of the key parameters that is of critical importance. Direct measurements of the brightness with common spectroscopy methods are challenging, because the fluorescent properties of the imaging antibodies are highly sensitive to the methods of conjugation, degree of labeling, and contamination with free dyes. Traditional methods rely on cell-based assays that lack reproducibility and accuracy. In this manuscript, we present a novel and general approach for measuring the brightness using antibody-avid polystyrene beads and flow cytometry. As compared to a cell-based method, the described technique is rapid, quantitative, and highly reproducible. The proposed method requires less than ten microgram of sample and is applicable for optimizing synthetic conjugation procedures, testing commercial imaging antibodies, and performing high-throughput validation of conjugation procedures. PMID:28150730

Rabies direct fluorescent antibody test does not inactivate rabies or eastern equine encephalitis viruses.

PubMed

Jarvis, Jodie A; Franke, Mary A; Davis, April D

2016-08-01

An examination using the routine rabies direct fluorescent antibody test was performed on rabies or Eastern equine encephalitis positive mammalian brain tissue to assess inactivation of the virus. Neither virus was inactivated with acetone fixation nor the routine test, thus laboratory employees should treat all samples as rabies and when appropriate Eastern equine encephalitis positive throughout the whole procedure. Copyright © 2016 Elsevier B.V. All rights reserved.

Assessing antibody microarrays for space missions: effect of long-term storage, gamma radiation, and temperature shifts on printed and fluorescently labeled antibodies.

PubMed

de Diego-Castilla, Graciela; Cruz-Gil, Patricia; Mateo-Martí, Eva; Fernández-Calvo, Patricia; Rivas, Luis A; Parro, Víctor

2011-10-01

Antibody microarrays are becoming frequently used tools for analytical purposes. A key factor for optimal performance is the stability of the immobilized (capturing) antibodies as well as those that have been fluorescently labeled to achieve the immunological test (tracers). This is especially critical for long-distance transport, field testing, or planetary exploration. A number of different environmental stresses may affect the antibody integrity, such as dryness, sudden temperature shift cycles, or, as in the case of space science, exposure to large quantities of the highly penetrating gamma radiation. Here, we report on the effect of certain stabilizing solutions for long-term storage of printed antibody microarrays under different conditions. We tested the effect of gamma radiation on printed and freeze- or vacuum-dried fluorescent antibodies at working concentrations (tracer antibodies), as well as the effect of multiple cycles of sudden and prolonged temperature shifts on the stability of fluorescently labeled tracer antibody cocktails. Our results show that (i) antibody microarrays are stable at room temperature when printed on stabilizing spotting solutions for at least 6 months, (ii) lyophilized and vacuum-dried fluorescently labeled tracer antibodies are stable for more than 9 months of sudden temperature shift cycles (-20°C to 25°C and 50°C), and (iii) both printed and freeze- or vacuum-dried fluorescent tracer antibodies are stable after several-fold excess of the dose of gamma radiation expected during a mission to Mars. Although different antibodies may exhibit different susceptibilities, we conclude that, in general, antibodies are suitable for use in planetary exploration purposes if they are properly treated and stored with the use of stabilizing substances.

Immunochemical Parameters of Some Commercial Conjugates for the Fluorescent Treponemal Antibody-Absorption Test

PubMed Central

Hunter, E. F.; Smith, J. F.; Lewis, J. S.; McGrew, B. E.; Schmale, J. D.

1972-01-01

Fluorescein-labeled anti-human globulins were examined to determine the need for standardization of conjugates used in the fluorescent treponemal antibody-absorption (FTA-ABS) test. Twenty-one of 33 conjugates submitted by commercial manufacturers to the Reagents Control Activity, Venereal Disease Research Laboratory, for evaluation in the FTA-ABS test were available for study. Conjugates, after evaluation in FTA-ABS performance tests, were examined by immunoelectrophoresis, by titration against immunoglobulins G and M (IgG, IgM) with FTA-ABS techniques, and by the biuret protein and fluorescein diacetate methods for determining fluorescein to protein (F/P) ratios. The conjugates were predominately anti-IgG globulin with anti-light-chain activity. Differences were noted in the ability of some conjugates to detect IgM antibody. The F/P ratios of those conjugates that could be determined varied from 2.6 to 17.8 μg of fluorescein per mg of protein. The need to identify and standardize both the immunologic capabilities and the optimum F/P ratio for FTA-ABS test conjugates is presented. PMID:4564403

Development of a sensitive and specific indirect enzyme-linked immunosorbent assay based on a baculovirus recombinant antigen for detection of specific antibodies against Ehrlichia canis.

PubMed

López, Lissett; Venteo, Angel; Aguirre, Enara; García, Marga; Rodríguez, Majosé; Amusátegui, Inmaculada; Tesouro, Miguel A; Vela, Carmen; Sainz, Angel; Rueda, Paloma

2007-11-01

An indirect enzyme-linked immunosorbent assay (ELISA) based on baculovirus recombinant P30 protein of Ehrlichia canis and the 1BH4 anticanine IgG monoclonal antibody was developed and evaluated by examining a panel of 98 positive and 157 negative sera using the indirect fluorescent antibody (IFA) test as the reference technique. The P30-based ELISA appeared to be sensitive and specific (77.55% and 95.54%, respectively) when qualitative results (positive/negative) were compared with those of the IFA test; the coefficient of correlation (R) between the 2 tests was 0.833. Furthermore, it was possible to establish a mathematical formula for use in comparing the results of both techniques. These results indicate that recombinant P30 antigen-based ELISA is a suitable alternative of the IFA test for simple, consistent, and rapid serodiagnosis of canine ehrlichiosis. Moreover, the use of this recombinant protein as antigen offers a great advantage for antigen preparation in comparison with other techniques in which the whole E. canis organism is used as antigen.

Indirect-blocking ELISA for detecting antibodies against glycoprotein B (gB) of porcine cytomegalovirus (PCMV).

PubMed

Liu, Xiao; Zhu, Ling; Shi, Xiaohong; Xu, Zhiwen; Mei, Miao; Xu, Weiwei; Zhou, Yuancheng; Guo, Wanzhu; Wang, Xiaoyu

2012-12-01

The major epitope region of the glycoprotein B (gB) gene of the porcine cytomegalovirus (PCMV), with a length of 270 bp, was cloned and expressed in Escherichia coli Rosetta (DE3). The major gB epitope was detected using an agar gel precipitation and Western blot analysis with the polyclonal antibodies specific for the major epitope. An indirect-blocking enzyme-linked immunosorbent assay (ELISA) was developed using the expressed major gB epitope as a coating antigen for the detection of PCMV antibodies. The results of the tests show that the indirect-blocking ELISA has 98% specificity and 97.8% sensitivity. No cross-reactions were observed between the major gB epitope and the antibodies against other virus, which indicates that the gB epitope is specific for PCMV antibodies. The indirect-blocking ELISA is a highly specific, sensitive method for detecting anti-PCMV gB antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

One-day fluorescent-antibody procedure for detecting salmonellae in frozen and dried foods.

PubMed

Goepfert, J M; Mann, M E; Hicks, R

1970-12-01

The indirect fluorescent-antibody technique was used to examine 422 food samples for the presence of salmonellae. A cultural phase involving a 16-hr preenrichment in buffered nutrient broth-milk medium followed by a 4- to 5-hr subculture into fresh medium of the same composition was evaluated. This procedure yielded a sufficient population of salmonellae so that no false-negative results were obtained. Of the 31 false-positives obtained, 12 samples yielded positive cultural results upon extensive subculture of the original enrichment broths. Yeast cells and both vegetative and spore forms of bacilli were observed to fluoresce when stained with anti-Salmonella serum. Efforts to ascertain the cause of these cross-reactions and several alternate explanations are discussed.

Evaluation of fluorescent-antibody tests as a means of confirming infant botulism.

PubMed Central

Glasby, C; Hatheway, C L

1984-01-01

Fluorescent-antibody techniques were evaluated for confirming infant botulism. Seventy-seven stool specimens from suspected cases were examined. All 34 specimens containing viable Clostridium botulinum at time of study gave positive results (29 on direct smears and 34 on enrichments). Two false-positive reactions were observed. Images PMID:6394626

Comparison of two commercial rapid in-clinic serological tests for detection of antibodies against Leishmania spp. in dogs.

PubMed

Athanasiou, Labrini V; Petanides, Theodoros A; Chatzis, Manolis K; Kasabalis, Dimitrios; Apostolidis, Kosmas N; Saridomichelakis, Manolis N

2014-03-01

Antibodies against Leishmania spp. are detected in most dogs with clinical signs of leishmaniasis due to Leishmania infantum. Accurate, rapid in-clinic serological tests may permit immediate confirmation of the diagnosis and implementation of therapeutic measures. The aim of the current study was to evaluate the diagnostic accuracy of 2 commercial, rapid in-clinic serological tests for the detection of anti-Leishmania antibodies in sera of dogs, the Snap Canine Leishmania Antibody Test kit (IDEXX Laboratories Inc., Westbrook, Maine) and the ImmunoRun Antibody Detection kit (Biogal Galed Labs, Kibbutz Galed, Israel), using indirect fluorescent antibody test (IFAT) as the reference method. A total of 109 sera collected from 65 seropositive and 44 seronegative dogs were used. The sensitivities of the Snap and ImmunoRun kits were 89.23% (95% confidence interval: 79.05-95.54%) and 86.15% (95% confidence interval: 75.33-93.45%), respectively, and the specificity of both tests was 100%. A good agreement between each of the rapid in-clinic serological tests and IFAT and between the 2 rapid in-clinic serological tests was witnessed. Both rapid in-clinic serological tests showed an adequate diagnostic accuracy and can be used for the fast detection of antibodies against L. infantum in dogs.

Modification of the fluorescent antibody virus neutralisation test--elimination of the cytotoxic effect for the detection of rabies virus neutralising antibodies.

PubMed

Bedeković, Tomislav; Lemo, Nina; Lojkić, Ivana; Mihaljević, Zeljko; Jungić, Andreja; Cvetnić, Zeljko; Cač, Zeljko; Hostnik, Peter

2013-04-01

The virus neutralisation test is used for the quantitation of specific antibodies in serum samples. However, the success of the test depends on the quality of samples. In the case of poor quality samples, a cytotoxic effect can be observed and the results of the test can be compromised. Additionally, the cytotoxic effect limits the use of different substances, such as muscle extract or liquid from thoracic cavity (thoracic liquid), as a sample for the detection of rabies virus neutralising antibodies in the follow-up of fox oral vaccination campaigns. To eliminate the cytotoxic effect, a modified fluorescent antibody virus neutralisation (mFAVN) test was developed and evaluated. In the mFAVN test, inocula were removed after a 1h and the cytotoxic effect was prevented. According to the results obtained, the specificity of the mFAVN test compared to the FAVN test was 88.8% and the sensitivity was 94.4%. The diagnostic validity of the test was 0.99 (CI=0.98-1.00). To evaluate the possibility of using muscle extract and thoracic liquid as samples for the virus neutralisation test, 102 sera, muscle extract and thoracic liquid samples of dog origin were tested with the mFAVN test. The correlation between sera and muscle extracts was 87.9% (r=0.88, p<0.001). The correlation between sera and thoracic liquid was 94.2% (r=0.94, p<0.001). These findings indicated that both muscle extract and thoracic liquid could be used as samples for detection of rabies virus neutralising antibodies in the follow-up of oral vaccination campaigns. To evaluate the level of elimination of the cytotoxic effect, the 102 samples of sera, muscle extracts and thoracic liquid of dog origin were also tested in parallel using the mFAVN and FAVN tests. In the mFAVN test, no instance of cytotoxic effect was observed in the cells. In the FAVN test, two sera (1.9%), 35 muscle extracts (34.3%) and 56 thoracic liquid samples (54.9%) showed cytotoxic effect. The results of this study strongly suggest that

An ultra-sensitive monoclonal antibody-based fluorescent microsphere immunochromatographic test strip assay for detecting aflatoxin M1 in milk

USDA-ARS?s Scientific Manuscript database

A rapid lateral flow fluorescent microspheres immunochromatography test strip (FMs-ICTS) has been developed for the detection of aflatoxin M1 (AFM1) residues in milk. For this purpose, an ultra-sensitive anti-AFM1 monoclonal antibody (MAb) 1D3 was prepared and identified. The IC50 value of the MA...

Comparison of a direct and indirect ELISA for quantitating antisperm antibody in semen.

PubMed

Lynch, D M; Howe, S E

1987-01-01

A direct and an indirect quantitative ELISA for antisperm antibody were compared using the spermatozoa and cell-free seminal fluid of 66 infertile males. The normal concentration of sperm binding immunoglobulin was less than or equal to 1.5 fg Ig per spermatozoon for the indirect seminal plasma assay and less than or equal to 1.5 fg Ig per spermatozoon by the direct assay. Of the 66 infertile males, 21% (14/66) had elevated levels of antisperm antibody in their seminal plasma and 26% (17/66) had elevated levels bound directly to their spermatozoa. The direct correlation between the results of these assays was 94%. A simple linear regression analysis between the indirect and direct measurements of antisperm antibody resulted in a correlation coefficient of r = 0.907. There was no statistically significant difference between results from the direct and indirect methods of the patients as a group. However, there was evidence of autospecificity in a small percentage of males who had elevated levels of antisperm antibody by the direct assay that was not detected by the indirect assay using pooled donor spermatozoa.

Antibody-mediated fluorescence enhancement based on shifting the intramolecular dimer<-->monomer equilibrium of fluorescent dyes.

PubMed

Wei, A P; Blumenthal, D K; Herron, J N

1994-05-01

A novel concept is described for directly coupling fluorescence emission to protein-ligand binding. It is based on shifting the intramolecular monomer<-->dimer equilibrium of two fluorescent dyes linked by a short spacer. A 13-residue peptide, recognized by a monoclonal antibody against human chorionic gonadotrophin (hCG), was labeled with fluorescein (F) and tetramethylrhodamine (T) at its N- and C-terminus, respectively. Spectral evidence suggests that when the conjugate is free in solution, F and T exist as an intramolecular dimer. Fluorescence quenching of fluorescein and rhodamine is approximately 98% and approximately 90%, respectively, due to dimerization. When the double-labeled peptide is bound to anti-hCG, however, the rhodamine fluorescence increases by up to 7.8-fold, depending upon the excitation wavelength. This is attributed to the dissociation of intramolecular dimers brought about by conformational changes of the conjugate upon binding. Fluorescein fluorescence, on the other hand, was still quenched because of excited-state energy transfer and residual ground-state interactions. Antibody binding also resulted in a approximately 3.4-fold increase in fluorescence anisotropy of the peptide. These changes in intensity and anisotropy allow direct measurement of antigen-antibody binding with a fluorescence plate reader or a polarization analyzer, without the need for separation steps and labeling antibodies. Because recent advances in peptide technology have allowed rapid and economical identification of antigen-mimicking peptides, the double-labeled peptide approach offers many opportunities for developing new diagnostic assays and screening new therapeutic drugs. It also has many potential applications to techniques involving recombinant antibodies, biosensors, cell sorting, and DNA probes.

Comparison of stabilities in translucency, fluorescence and opalescence of direct and indirect composite resins.

PubMed

Yu, Bin; Lee, Young-Keun

2013-01-01

To evaluate translucency, fluorescence and opalescence stabilities of direct and indirect composite resins after aging. One direct (16 shades) and two indirect composite resins (16 and 26 shades) were investigated. Resins were filled in a mold (1 mm thick) and light cured; post-curings were performed for indirect resins. Color was measured before and after 5,000 cycles of thermocycling on a reflection spectrophotometer in reflectance and transmittance modes to calculate parameters for translucency (TP), fluorescence (FL) and opalescence (OP). Differences in the changes of TP, FL and OP after aging by the type of resin were determined by t test, and those were also determined by one-way ANOVA with the factor of the brand or the shade group (P < 0.05). Changes in TP, FL and OP were -1.2 to 0.7, -0.2 to 0.4 and -0.6 to 1.3, respectively, for direct resins; and were -2.0 to 1.8, -0.9 to 0.4 and -2.9 to 3.7, respectively, for indirect resins. Changes in TP were not significantly different by the type of resin, but those in FL and OP were different (P = 0.05). Changes in optical parameters were influenced by the brand or the shade group of the resins (P < 0.05). Stabilities in optical properties of resins varied depending on type, brand or shade group. Aging significantly affected fluorescence and opalescence, but not translucency, of indirect resins compared to those of direct resins.

Immunoglobulin M indirect-fluorescent antibody test for the diagnosis of acute toxoplasmosis during pregnancy in the avidity era: A 14-year experience at the Tuscany Reference Center for Infectious Diseases in Pregnancy, Florence, Italy.

PubMed

Trotta, Michele; Borchi, Beatrice; Zammarchi, Lorenzo; Sterrantino, Gaetana; Brogi, Michela; Kiros, Seble Tekle; Lorini, Chiara; Bonaccorsi, Guglielmo; Colao, Maria Grazia; Bartoloni, Alessandro

2016-12-01

The aim of this study was to evaluate immunoglobulin M indirect-fluorescent antibody test (IgM IFAT) for the diagnosis of acute or chronic Toxoplasma infection in pregnancy. Pregnant women with suspected acute toxoplasmosis referred to the Tuscany Reference Center for Infectious Diseases in Pregnancy during the period 1998-2012 were retrospectively enrolled. All women were tested with a panel of serological tests, including enzyme-linked immunosorbent assay for IgG avidity and IgM IFAT. On the basis of anamnestic, clinical, and serological criteria, pregnant women were classified into three groups: recently infected (RI), latently infected (LI), and doubtful latently infected (DLI). Patients classified as DLI were excluded from the analysis. The association between IgM IFAT (positive or negative) and the diagnosis of infection (acute or chronic) was assessed. Positive predictive value and negative predictive value of the IgM IFAT were calculated. A total of 810 pregnant women were enrolled in the study: 302 in the RI group and 508 in the LI group. Fifty-two women classified as DLI were excluded. IgM IFAT was positive in 172 out of 302 (56.9%) pregnant women in the RI group and in 29 out of 508 (5.7%) in the LI group. The positive predictive value and negative predictive value of IgM IFAT in predicting RI was 85.6% and 78.6%, respectively. IgM IFAT has reasonable sensitivity and specificity in diagnosing recent infection and, mostly in case of borderline avidity test, could be considered as a further aid for an accurate diagnosis of acute toxoplasmosis in pregnancy. © 2016 Japan Society of Obstetrics and Gynecology.

Development, characterization, and use of monoclonal and polyclonal antibodies against the myxosporean, Ceratomyxa shasta

USGS Publications Warehouse

Bartholomew, J.L.; Rohovec, J.S.; Fryer, J.L.

1989-01-01

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.

Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.

PubMed

Nath, Nidhi; Godat, Becky; Zimprich, Chad; Dwight, Stephen J; Corona, Cesear; McDougall, Mark; Urh, Marjeta

2016-04-01

Receptor-mediated antibody internalization is a key mechanism underlying several anti-cancer antibody therapeutics. Delivering highly toxic drugs to cancer cells, as in the case of antibody drug conjugates (ADCs), efficient removal of surface receptors from cancer cells and changing the pharmacokinetics profile of the antibody drugs are some of key ways that internalization impacts the therapeutic efficacy of the antibodies. Over the years, several techniques have been used to study antibody internalization including radiolabels, fluorescent microscopy, flow cytometry and cellular toxicity assays. While these methods allow analysis of internalization, they have limitations including a multistep process and limited throughput and are generally endpoint assays. Here, we present a new homogeneous method that enables time and concentration dependent measurements of antibody internalization. The method uses a new hydrophilic and bright pH sensor dye (pHAb dye), which is not fluorescent at neutral pH but becomes highly fluorescent at acidic pH. For receptor mediated antibody internalization studies, antibodies against receptors are conjugated with the pHAb dye and incubated with the cells expressing the receptors. Upon binding to the receptor, the dyes conjugated to the antibody are not fluorescent because of the neutral pH of the media, but upon internalization and trafficking into endosomal and lysosomal vesicles the pH drops and dyes become fluorescent. The enabling attributes of the pHAb dyes are the hydrophilic nature to minimize antibody aggregation and bright fluorescence at acidic pH which allows development of simple plate based assays using a fluorescent reader. Using two different therapeutic antibodies--Trastuzumab (anti-HER2) and Cetuximab (anti-EGFR)--we show labeling with pHAb dye using amine and thiol chemistries and impact of chemistry and dye to antibody ration on internalization. We finally present two new approaches using the pHAb dye, which will be

Comparison between available serologic tests for detecting antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi in horses in Canada.

PubMed

Schvartz, Gili; Epp, Tasha; Burgess, Hilary J; Chilton, Neil B; Lohmann, Katharina L

2015-07-01

To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalence-adjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted. © 2015 The Author(s).

The relationship between fluorescent, agglutinating, and precipitating antibodies to Candida albicans and their immunoglobin classes

PubMed Central

Lehner, T.; Buckley, Helen R.; Murray, I. G.

1972-01-01

A parallel study of fluorescent, agglutinating, and precipitating antibodies to Candida albicans revealed that precipitating antibodies belong to the IgG class, whereas agglutinating antibodies reside in the IgG, IgM, and IgA classes. The three types as well as the three classes of antibodies were found in Candida endocarditis and mucocutaneous candidiasis. Immuno-absorption studies suggest that the three serological tests estimate antibodies to mannan determinants of Candida albicans. Images PMID:4555044

Performance analysis of automated evaluation of Crithidia luciliae-based indirect immunofluorescence tests in a routine setting - strengths and weaknesses.

PubMed

Hormann, Wymke; Hahn, Melanie; Gerlach, Stefan; Hochstrate, Nicola; Affeldt, Kai; Giesen, Joyce; Fechner, Kai; Damoiseaux, Jan G M C

2017-11-27

Antibodies directed against dsDNA are a highly specific diagnostic marker for the presence of systemic lupus erythematosus and of particular importance in its diagnosis. To assess anti-dsDNA antibodies, the Crithidia luciliae-based indirect immunofluorescence test (CLIFT) is one of the assays considered to be the best choice. To overcome the drawback of subjective result interpretation that inheres indirect immunofluorescence assays in general, automated systems have been introduced into the market during the last years. Among these systems is the EUROPattern Suite, an advanced automated fluorescence microscope equipped with different software packages, capable of automated pattern interpretation and result suggestion for ANA, ANCA and CLIFT analysis. We analyzed the performance of the EUROPattern Suite with its automated fluorescence interpretation for CLIFT in a routine setting, reflecting the everyday life of a diagnostic laboratory. Three hundred and twelve consecutive samples were collected, sent to the Central Diagnostic Laboratory of the Maastricht University Medical Centre with a request for anti-dsDNA analysis over a period of 7 months. Agreement between EUROPattern assay analysis and the visual read was 93.3%. Sensitivity and specificity were 94.1% and 93.2%, respectively. The EUROPattern Suite performed reliably and greatly supported result interpretation. Automated image acquisition is readily performed and automated image classification gives a reliable recommendation for assay evaluation to the operator. The EUROPattern Suite optimizes workflow and contributes to standardization between different operators or laboratories.

Utility of feline coronavirus antibody tests.

PubMed

Addie, Diane D; le Poder, Sophie; Burr, Paul; Decaro, Nicola; Graham, Elizabeth; Hofmann-Lehmann, Regina; Jarrett, Oswald; McDonald, Michael; Meli, Marina L

2015-02-01

Eight different tests for antibodies to feline coronavirus (FCoV) were evaluated for attributes that are important in situations in veterinary practice. We compared four indirect immunofluorescent antibody tests (IFAT), one enzyme-linked immunosorbent assay (ELISA) (FCoV Immunocomb; Biogal) and three rapid immunochromatographic (RIM) tests against a panel of samples designated by consensus as positive or negative. Specificity was 100% for all but the two IFATs based on transmissible gastroenteritis virus (TGEV), at 83.3% and 97.5%. The IFAT and ELISA tests were best for obtaining an antibody titre and for working in the presence of virus. The RIM tests were the best for obtaining a result quickly (10-15 mins); of these, the Speed F-Corona was the most sensitive, at 92.4%, followed by FASTest feline infectious peritonitis (FIP; 84.6%) and Anigen Rapid FCoV antibody test (64.1%). Sensitivity was 100% for the ELISA, one FCoV IFAT and one TGEV IFAT; and 98.2% for a second TGEV IFA and 96.1% for a second FCoV IFAT. All tests worked with effusions, even when only blood products were stipulated in the instruction manual. The ELISA and Anigen RIM tests were best for small quantities of sample. The most appropriate FCoV antibody test to use depends on the reason for testing: in excluding a diagnosis of FIP, sensitivity, specificity, small sample quantity, rapidity and ability to work in the presence of virus all matter. For FCoV screening, speed and sensitivity are important, and for FCoV elimination antibody titre is essential. © ISFM and AAFP 2014.

Fluorescently labeled therapeutic antibodies for detection of microscopic melanoma

PubMed Central

Day, Kristine E.; Beck, Lauren N.; Deep, Nicholas L.; Kovar, Joy; Zinn, Kurt R; Rosenthal, Eben L.

2013-01-01

Objective Detection of microscopic disease during surgical resection of melanoma remains a significant challenge. To assess real-time optical imaging for visualization of microscopic cancer, we evaluated three FDA-approved therapeutic monoclonal antibodies. Study Design Prospective, basic science Methods Melanoma cell lines (A375 and SKMEL5) were xenografted into the ears of immunodeficient mice. Bevacizumab, panitumumab, tocilizumab, or a non-specific IgG were covalently linked to a near-infrared (NIR) fluorescent probe (IRDye800CW) and systemically injected. Primary tumors were imaged and then resected under fluorescent guidance using the SPY, an NIR imaging system used in plastic and reconstructive surgeries to evaluate perfusion. Mice were also imaged with the Pearl Impulse small animal imager, an NIR imaging system designed for use with IRDye800CW. Post-resection, small tissue fragments were fluorescently imaged and presence of tumor subsequently confirmed by correlation with histology. Results All fluorescently-labeled therapeutic monoclonal antibodies could adequately delineate tumor from normal tissue based on tumor-to-background ratios (TBR) compared to IgG-IRDye800CW. On serial imaging, panitumumab achieved the highest TBRs with both SPY and Pearl (3.8 and 6.6). When used to guide resections, the antibody-dye conjugates generated TBRs in the range of 1.3-2.2 (average=1.6) using the SPY and 1.9-6.3 (average=2.7) using the Pearl. There was no significant difference amongst the antibodies with either imaging modality or cell line (one-way ANOVA). Conclusion Our data suggests that FDA approved antibodies may be suitable targeting agents for the intraoperative fluorescent detection of melanoma. Level of Evidence N/A PMID:23616260

Seroprevalences of anti-Sarcocystis neurona and anti-Neospora hughesi antibodies among healthy equids in the United States.

PubMed

James, Kaitlyn E; Smith, Woutrina A; Conrad, Patricia A; Packham, Andrea E; Guerrero, Leopoldo; Ng, Mitchell; Pusterla, Nicola

2017-06-01

OBJECTIVE To describe the general seroprevalence of anti-Sarcocystis neurona and anti-Neospora hughesi antibodies among healthy equids by use of indirect fluorescent antibody tests and determine potential risk factors for seropositivity. DESIGN Cross-sectional study. SAMPLE Whole blood samples collected from 5,250 equids (1 sample/animal) across 18 states in the United States during October 2013. PROCEDURES Information regarding potential risk factors (geographic region, breed, primary use, sex, and age) was collected along with the blood samples. For each equid, an indirect fluorescent antibody test was used to determine serum titers of antibody against each of the 2 protozoal parasites. Mixed-effects logistic regression models were created to determine ORs for seropositivity. RESULTS The overall seroprevalence of anti-S neurona and anti-N hughesi antibodies in the tested equids was 78% and 34%, respectively. Of the equids, 31% were seropositive and 18% were seronegative for antibodies against both parasites. Factors associated with equids being seropositive for anti-S neurona antibodies were residence in the South, warmblood breed, and age > 5 years. Seroprevalence of anti-N hughesi antibodies did not differ among equids in different states across the country, but warmblood breed and age > 5 years were associated with seropositivity. CONCLUSIONS AND CLINICAL RELEVANCE With regard to risk factors for S neurona and N hughesi exposure and antibody response among tested equids, older age was not unexpected; however, the influences of warmblood breed and geographic location on seropositivity for anti-S neurona antibody but not for anti-N hughesi antibody deserve further investigation.

Means and method for capillary zone electrophoresis with laser-induced indirect fluorescence detection

DOEpatents

Yeung, Edward S.; Kuhr, Werner G.

1996-02-20

A means and method for capillary zone electrphoresis with laser-induced indirect fluorescence detection. A detector is positioned on the capillary tube of a capillary zone electrophoresis system. The detector includes a laser which generates a laser beam which is imposed upon a small portion of the capillary tube. Fluorescence of the elutant electromigrating through the capillary tube is indirectly detected and recorded.

Means and method for capillary zone electrophoresis with laser-induced indirect fluorescence detection

DOEpatents

Yeung, Edwards; Kuhr, Werner G.

1991-04-09

A means and method for capillary zone electrphoresis with laser-induced indirect fluorescence detection. A detector is positioned on the capillary tube of a capillary zone electrophoresis system. The detector includes a laser which generates a laser beam which is imposed upon a small portion of the capillary tube. Fluorescence of the elutant electromigrating through the capillary tube is indirectly detected and recorded.

Novel Fitc-Labeled Igy Antibody: Fluorescence Imaging Toxoplasma Gondii In Vitro.

PubMed

Sert, Mehtap; Cakir Koc, Rabia; Budama Kilinc, Yasemin

2017-04-12

Toxoplasmosis is caused by T. gondii and can create serious health problems in humans and also worldwide economic harm. Because of the clinical and veterinary importance of toxoplasmosis, its timely and accurate diagnosis has a major impact on disease-fighting strategies. T. gondii surface antigen 1 (SAG1), an immunodominant-specific antigen, is often used as a diagnostic tool. Therefore, the aim of this study was the optimization of novel fluorescein isothiocyanate (FITC) labeling of the SAG1-specific IgY antibody to show the potential for immunofluorescence imaging of T. gondii in vitro. The specificity of IgY antibodies was controlled by an enzyme-linked immunosorbent assay (ELISA), and the concentration of the IgY antibody was detected using a spectrophotometer. The optimum incubation time and FITC concentration were determined with a fluorescence spectrometer. The obtained FITC-labeled IgY was used for marking T. gondii tachyzoites, which were cultured in vitro and viewed using light microscopy. The interaction of the fluorescence-labeled antibody and the T. gondii tachyzoites was examined with a fluorescence microscope. In this study, for the first time, a FITC-labeled anti-SAG1 IgY antibody was developed according to ELISA, fluorescence spectrometer, and fluorescence imaging of cell culture. In the future, the obtained FITC-labeled T. gondii tachyzoites' specific IgY antibodies may be used as diagnostic tools for the detection of T. gondii infections in different samples.

Clinico-laboratory aspects of anti-nuclear and anti-native DNA antibody tests.

PubMed

Webb, J

1978-01-01

Available techniques for detection of anti-nuclear antibodies are here briefly reviewed. The relatively insensitive LE cell test has been largely supplanted by the indirect immunofluorescent ANA test which should be reported in terms of titre and pattern. Specific measurement of nDNA antibodies is now a regular technique in SLE diagnosis and management.

Seroprevalence of bovine viral diarrhea virus neutralizing antibodies in finisher hogs in Ontario swine herds and targeted diagnostic testing of 2 suspect herds

PubMed Central

O’Sullivan, Terri; Friendship, Robert; Carman, Susy; Pearl, David L.; McEwen, Beverly; Dewey, Catherine

2011-01-01

A pilot study was initiated to determine the seroprevalence of bovine viral diarrhea virus (BVDV) neutralizing antibodies in finisher hogs in Ontario swine herds, including 2 swine herds with clinical syndromes suspicious of BVDV. No herds were positive for BVDV antibodies by virus neutralization. The 2 swine herds with clinical disease suggestive of pestivirus infection were also negative for antibodies to BVDV in indirect fluorescent antibody assays. Prevalence of BVDV in Ontario swine farms is negligible. PMID:22654141

Plasmodium berghei infection in pregnant rats: effects on antibody response and course of infection in offspring.

PubMed

Palmer, T T

1978-06-01

The effects of primary, patent Plasmodium berghei infection in Sprague-Dawley rats during pregnancy upon the course of infection and the humoral antibody response to malaria in their offspring were examined. Malaria specific antibody determined by an indirect fluorescent antibody test correlated well with the parasitologic profiles of each experimental group. Utilization of foster mother groups indicated passive transfer of protective antibody through milk. Evidence for in utero sensitization by soluble malaria antigens was shown by an anamnestic-like antibody response during subsequent infection of offspring from infected mothers.

Direct agglutination test for serologic diagnosis of Neospora caninum infection.

PubMed

Romand, S; Thulliez, P; Dubey, J P

1998-01-01

A direct agglutination test was evaluated for the detection and quantitation of IgG antibodies to Neospora caninum in both experimental and natural infections in various animal species. As compared with results obtained by the indirect fluorescent antibody test, the direct agglutination test appeared reliable for the serologic diagnosis of neosporosis in a variety of animal species. The direct agglutination test should provide easily available and inexpensive tools for serologic testing for antibodies to N. caninum in many host species.

The interaction of antibodies with lipid membranes unraveled by fluorescence methodologies

NASA Astrophysics Data System (ADS)

Figueira, Tiago N.; Veiga, Ana Salomé; Castanho, Miguel A. R. B.

2014-12-01

The interest and investment in antibody therapies has reached an overwhelming scale in the last decade. Yet, little concern has been noticed among the scientific community to unravel important interactions of antibodies with biological structures other than their respective epitopes. Lipid membranes are particularly relevant in this regard as they set the stage for protein-protein recognition, a concept potentially inclusive of antibody-antigen recognition. Fluorescence techniques allow experimental monitoring of protein partition between aqueous and lipid phases, deciphering events of adsorption, insertion and diffusion. This review focuses on the available fluorescence spectroscopy methodologies directed to the study of antibody-membrane interactions.

[Evaluation of a Computer-Aided Microscope System and Its Anti-Nuclear Antibody Test Kit for Indirect Immunofluorescence Assay].

PubMed

Hayashi, Nobuhide; Saegusa, Jun; Uto, Kenichi; Oyabu, Chinami; Saito, Toshiharu; Sato, Itsuko; Kawano, Seiji; Kumagai, Shunichi

2016-02-01

Antinuclear antibody (ANA) testing is indispensable for diagnosing and understanding clinical conditions of autoimmune diseases. The indirect immunofluorescence assay (IFA) is the gold standard for ANA screening, and it can detect more than 100 different antibodies, such as anti-PCNA as well as anti-cytoplasmic antibodies. However, complicated procedures of conventional IFA and visual interpretation require highly skilled laboratory staff. This study evaluates the capability, characteristics, and applicability of the recently developed ANA detection system (EUROPattern Cosmic IFA System, EPA) using HEp20-10 cells and the automated pattern recognition microscope. Findings using EPA and conventional methods were compared in 282 sera obtained from connective tissue disease patients and 250 sera from healthy individuals. The concordance of the positivity rate, antibody titer (within +/- 1 tube difference), and the accurate recognition rate of ANA patterns between the automated EPA method and the microscopic judgement of the EPA image by eye was 98.9, 97.4, and 55.3%, respectively. The EPA method showed concordance of the positivity rate as high as 93.3% and concordance of the antibody titer as high as 94.0% (within +/- 1 titer) compared with the conventional method. Regarding the four typical patterns of ANA (homogeneous, speckled, nucleolar, and centromere), large differences between the EPA and conventional methods were not observed, and the rate of concordance between the final EPA result and the conventional method was from 94.1 to 100%. The positivity rate of ANA using the EPA and conventional methods showed marked agreement among the six connective tissue diseases (SLE, MCTD, SSc, PM/DM, and SS) and healthy individuals. Although the EPA system is not considered a complete system and laboratory staff should verify the results, it is a useful system for routine ANA analysis because it contributes to ANA standardization and an efficient workflow.

Production, characterization and application of monoclonal antibody to spherulocytes: A subpopulation of coelomocytes of Apostichopus japonicus

USDA-ARS?s Scientific Manuscript database

One monoclonal antibody (mAb 3F6) against coelomocytes of sea cucumber Apostichopus japonicus was developed by immunization of Balb/C mice. Analyzed by indirect immunofluorescence assay test (IIFAT), immunocytochemical assay (ICA),Western blotting and fluorescence-activated cell sorter (FACS), mAb 3...

Comparison of desmoglein ELISA and indirect immunofluorescence using two substrates (monkey oesophagus and normal human skin) in the diagnosis of pemphigus.

PubMed

Ng, Patricia P L; Thng, Steven T G; Mohamed, Khatija; Tan, Suat Hoon

2005-11-01

A prospective study was performed to assess the usefulness of desmoglein enzyme-linked immunosorbent assay testing compared with indirect immunofluorescence in the diagnosis of new cases of pemphigus, as well as to compare the relative sensitivities of monkey oesophagus and normal human skin as substrates for indirect immunofluorescence. These tests were performed on the sera of 29 consecutive new cases of pemphigus diagnosed over a 2-year period based on clinical, histological and direct immunofluorescence findings. Desmoglein enzyme-linked immunosorbent assay was positive in all patients whereas indirect immunofluorescence was positive in only 25 of 29 patients. All four patients with negative indirect immunofluorescence had positive antinuclear antibodies or cytoplasmic fluorescence that could have masked the anti-intercellular antibodies. Desmoglein enzyme-linked immunosorbent assay appeared to reflect the disease activity better than indirect immunofluorescence in a few patients who had active disease of recent onset. Monkey oesophagus was found to be superior or equal to human skin as a substrate for indirect immunofluorescence in both pemphigus vulgaris and foliaceus.

Indirect Competitive Enzyme-Linked Immunosorbent Assay (ELISA).

PubMed

Kohl, Thomas O; Ascoli, Carl A

2017-07-05

The indirect competitive ELISA (indirect cELISA) pits plate-immobilized antigen against antigens in solution for binding to antigen-specific antibody. The antigens in solution are in the test sample and are first incubated with antigen-specific antibody. These antibody-antigen complexes are then added to microtiter plates whose wells have been coated with purified antigen. The wells are washed to remove unbound antigen-antibody complexes and free antigen. A reporter-labeled secondary antibody is then added followed by the addition of substrate. Substrate hydrolysis yields a signal that is inversely proportional to antigen concentration within the sample. This is because when antigen concentration is high in the test sample, most of the antibody is bound before adding the solution to the plate. Most of the antibody remains in solution (as complexes) and is thus washed away before the addition of the reporter-labeled secondary antibody and substrate. Thus, the higher the antigen concentration in the test sample, the weaker the resultant signal in the detection step. The indirect cELISA is often used for competitive detection and quantification of antibodies against viral diseases in biological samples. © 2017 Cold Spring Harbor Laboratory Press.

Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA.

PubMed

Giménez-Lirola, Luis G; Mur, Lina; Rivera, Belen; Mogler, Mark; Sun, Yaxuan; Lizano, Sergio; Goodell, Christa; Harris, D L Hank; Rowland, Raymond R R; Gallardo, Carmina; Sánchez-Vizcaíno, José Manuel; Zimmerman, Jeff

2016-01-01

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

Limited value of testing for intrinsic factor antibodies with negative gastric parietal cell antibodies in pernicious anaemia.

PubMed

Khan, S; Del-Duca, C; Fenton, E; Holding, S; Hirst, J; Doré, P C; Sewell, W A C

2009-05-01

The appropriate testing strategy for diagnosing pernicious anaemia using gastric parietal cell (GPC) and/or intrinsic factor antibodies (IFA) is controversial. Intrinsic factor antibodies are found in only about 70% of cases. Indirect immunofluorescence screening for gastric parietal cell antibodies is more sensitive, labour intensive, and less specific. The frequency of antibody positivity (IFA and/or GPC) was retrospectively examined in patients tested for both autoantibodies over a three-year period. It was investigated whether B12 levels were related to antibody status. These findings were validated in a prospective study of IFA in 91 GPC negative patients with low B12 levels. Of 847 samples identified in the retrospective study, 4 (0.47%) were positive for only intrinsic factor antibodies, 731 (86.3%) positive for GPC alone, and 112 (13.2%) for both. Student t test on log-transformed data showed B12 levels had no bearing on autoantibody status. 91 consecutive patients with low B12 levels were tested for both autoantibodies; all were negative for gastric parietal cell antibodies. Only one sample was positive for intrinsic factor antibody using the porcine intrinsic factor assay, but was negative by a human recombinant intrinsic factor-based ELISA. This study provides evidence that testing for gastric parietal cell antibodies is an appropriate screening test for pernicious anaemia, with intrinsic factor antibodies reserved for confirmatory testing or in patients with other autoantibodies that mask the GPC pattern; B12 levels are not related to autoantibody status.

Development of a homogeneous assay format for p53 antibodies using fluorescence correlation spectroscopy

NASA Astrophysics Data System (ADS)

Neuweiler, Hannes; Scheffler, Silvia; Sauer, Markus

2005-08-01

The development of reliable methods for the detection of minute amounts of antibodies directly in homogeneous solution represents one of the major tasks in the current research field of molecular diagnostics. We demonstrate the potential of fluorescence correlation spectroscopy (FCS) in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing two parameters simultaneously: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Our data demonstrate that FCS in combination with fluorescence-quenched peptide epitopes opens new possibilities for the reliable detection of antibody binding events in homogeneous solution.

Prevalence of antibodies against Neospora spp. and Sarcocystis neurona in donkeys from northeastern Brazil.

PubMed

Gennari, Solange Maria; Pena, Hilda Fátima de Jesus; Lindsay, David Scott; Lopes, Marcos Gomes; Soares, Herbert Sousa; Cabral, Aline Diniz; Vitaliano, Sérgio Netto; Amaku, Marcos

2016-01-01

Sarcocystis neurona and Neospora hughesi are coccidian protozoa that can cause neurological illness in horses in America. In this study we report seroprevalence of Neospora spp. andS. neurona in sera of 333 donkeys from the northeastern region of Brazil. Antibodies to Neospora spp. were detected in 2% (7 donkeys) of 333 sera tested by the indirect fluorescent antibody test (IFAT) with a cut-off dilution of 1:40. Antibodies to S. neurona were found in 3% (10 donkeys) of the samples tested by IFAT (cut-off ≥50) and 21% (69 donkeys) by the direct agglutination test (SAT ≥50). The SAT and IFAT results for S. neurona showed a poor concordance (value of Kappa=0.051). This is the first report of Neospora spp. antibodies in Brazilian donkeys and the first detection of antibodies against S. neurona in this animal species.

Evaluation of an In-house indirect ELISA for detection of antibody against haemorrhagic septicemia in Asian elephants.

PubMed

Tankaew, Pallop; Singh-La, Thawatchai; Titaram, Chatchote; Punyapornwittaya, Veerasak; Vongchan, Preeyanat; Sawada, Takuo; Sthitmatee, Nattawooti

2017-03-01

Pasteurella multocida causes haemorrhagic septicemia in livestock and wild animals, including elephants. The disease has been reported in Asian elephants in India and Sri Lanka, but to date there have been no reported cases in Thailand. ELISA or indirect hemagglutination assays (IHA) have been demonstrated to be able to detect the antibody against the disease in cattle, but no data are available for elephants. The present study reports a novel in-house indirect ELISA for antibody detection of haemorrhagic septicemia in Asian elephants, and evaluates the sensitivity and specificity of the method using a Bayesian approach. The characteristics of ELISA and IHA were analyzed using a one population Bayesian model assuming conditional dependence between these two diagnostic tests. The IHA was performed as recommended by the World Organization for Animal Health (OIE) manual for haemorrhagic septicemia. An in-house indirect ELISA was developed with a heat extract antigen of P. multocida strain M-1404 (serovar B:2) as a coating antigen and rabbit anti-immunoglobulin G conjugated with horseradish peroxidase (eIgG-HRP). The checkerboard titration method was done using elephant sera immunized with P. multocida bacterin and negative sera from colostrum-deprived elephant calves. The concentrations of heat extract antigen (160μg/ml), sample serum (1:100), and eIgG-HRP (1:1000) were optimal for the assay. The calculated cut-off value was 0.103. Of the elephant sera, 50.59% (43/85) were considered seropositive by ELISA. The sensitivity of the ELISA test was higher than that of the IHA test [median=86.5%, 95% posterior probability interval (PPI)=52.5-98.9%] while the specificity was lower (median=54.1%, PPI=43.6-64.7%). The median sensitivity and specificity of IHA were 80.5% (PPI=43.8-98.0%) and 78.4% (PPI=69.0-87.0%), respectively. These findings suggest that our in-house indirect ELISA can be used as a tool to detect the antibody against haemorrhagic septicemia in Asian

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

PubMed Central

Yim, Sung Sun; Bang, Hyun Bae; Kim, Young Hwan; Lee, Yong Jae; Jeong, Gu Min; Jeong, Ki Jun

2014-01-01

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. PMID:25303314

Comparative efficacy of antigen and antibody detection tests for human trichinellosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ivanoska, D.; Cuperlovic, K.; Gamble, H.R.

1989-02-01

Sera collected from patients with suspected or confirmed exposure to Trichinella spiralis were tested for circulating parasite antigens and antiparasite antibodies. Using an immunoradiometric assay, excretory--secretory antigens from muscle-stage larvae of T. spiralis were detected in the sera of 47% of 62 patients with clinical trichinellosis and 13% of 39 patients without clinical signs but suspected of exposure to infected meat. In comparison, antibodies were detected using an indirect immunofluorescent test in the circulation of 100% of the 62 patients with clinical trichinellosis and 46% of the 39 patients with suspected exposure. The presence of antibodies specific to excretory-secretory productsmore » of T. spiralis muscle larvae was confirmed in the majority of the samples tested by a monoclonal antibody-based competitive inhibition assay. These results indicate that antibody detection is a more sensitive diagnostic method for human trichinellosis, but that antigen detection might be a useful confirmatory test because it is a direct demonstration of parasite products in the circulation.« less

Paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

PubMed

Yeow, Natasha; McLiesh, Heather; Guan, Liyun; Shen, Wei; Garnier, Gil

2016-07-01

A rapid and simple paper-based elution assay for red blood cell antigen typing by the indirect antiglobulin test (IAT) was established. This allows to type blood using IgG antibodies for the important blood groups in which IgM antibodies do not exist. Red blood cells incubated with IgG anti-D were washed with saline and spotted onto the paper assay pre-treated with anti-IgG. The blood spot was eluted with an elution buffer solution in a chromatography tank. Positive samples were identified by the agglutinated and fixed red blood cells on the original spotting area, while red blood cells from negative samples completely eluted away from the spot of origin. Optimum concentrations for both anti-IgG and anti-D were identified to eliminate the washing step after the incubation phase. Based on the no-washing procedure, the critical variables were investigated to establish the optimal conditions for the paper-based assay. Two hundred ten donor blood samples were tested in optimal conditions for the paper test with anti-D and anti-Kell. Positive and negative samples were clearly distinguished. This assay opens up new applications of the IAT on paper including antibody detection and blood donor-recipient crossmatching and extends its uses into non-blood typing applications with IgG antibody-based diagnostics. Graphical abstract A rapid and simple paper-based assay for red blood cell antigen typing by the indirect antiglobulin test.

Fluorescent-Antibody Measurement Of Cancer-Cell Urokinase

NASA Technical Reports Server (NTRS)

Morrison, Dennis R.

1993-01-01

Combination of laboratory techniques provides measurements of amounts of urokinase in and between normal and cancer cells. Includes use of fluorescent antibodies specific against different forms of urokinase-type plasminogen activator, (uPA), fluorescence microscopy, quantitative analysis of images of sections of tumor tissue, and flow cytometry of different uPA's and deoxyribonucleic acid (DNA) found in suspended-tumor-cell preparations. Measurements provide statistical method for indicating or predicting metastatic potentials of some invasive tumors. Assessments of metastatic potentials based on such measurements used in determining appropriate follow-up procedures after surgical removal of tumors.

Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica.

PubMed

Hemadi, Ahmad; Ekrami, Alireza; Oormazdi, Hormozd; Meamar, Ahmad Reza; Akhlaghi, Lame; Samarbaf-Zadeh, Ali Reza; Razmjou, Elham

2015-05-01

Rapid detection of Entamoeba histolytica based on fluorescent silica nanoparticle (FSNP) indirect immunofluorescence microscopy was evaluated. Silica nanoparticles were synthesized using Stöber's method, with their surface activated to covalently bind to, and immobilize, protein A. For biolabeling, FSNP was added to conjugated E. histolytica trophozoites with monoclonal anti-E. histolytica IgG1 for microscopic observation of fluorescence. Fluorescent silica nanoparticle sensitivity was determined with axenically cultured E. histolytica serially diluted to seven concentrations. Specificity was evaluated using other intestinal protozoa. Fluorescent silica nanoparticles detected E. histolytica at the lowest tested concentration with no cross-reaction with Entamoeba dispar, Entamoeba moshkovskii, Blastocystis sp., or Giardia lamblia. Visualization of E. histolytica trophozoites with anti-E. histolytica antibody labeled with fluorescein isothiocyanate (FITC) was compared with that using anti-E. histolytica antibody bioconjugated FSNP. Although FITC and FSNP produced similar results, the amount of specific antibody required for FITC to induce fluorescence of similar intensity was fivefold that for FSNP. Fluorescent silica nanoparticles delivered a rapid, simple, cost-effective, and highly sensitive and specific method of detecting E. histolytica. Further study is needed before introducing FSNP for laboratory diagnosis of amoebiasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Detection of antibodies in human serum using trimellityl-erythrocytes: direct and indirect haemagglutination and haemolysis.

PubMed

Turner, E S; Pruzansky, J J; Patterson, R; Zeiss, C R; Roberts, M

1980-02-01

Utilizing trimellityl-erythrocytes (TM-E), antibodies were detected in sera of seven workers with trimellitic anhydride (TMA) induced airway syndromes by direct haemagglutination, indirect haemagglutination with anti-human IgG, IgA or IgM or by haemolysis. Detectable levels of antibody were obtained with all three methods. The most sensitive technique was indirect haemagglutination using anti-IgG. When added as an inhibitor, TM-human serum albumin produced a 10- to 800-fold reduction in titres. TM-ovalbumin of similar epitope density was less inhibitory and sodium trimellitate the least inhibitory on a molar basis. All of the assays using haptenized human red cells were also capable of detecting anti-TM antibodies in Rhesus monkeys whose airways had been exposed to TMA. These assays are useful for detecting anti-TM antibodies and may also be adapted to demonstrate antibodies induced against other inhaled haptens in sera of environmentally exposed individuals or in animal models of such exposure.

Fluorescent antibody detection of microorganisms in terrestrial environments

NASA Technical Reports Server (NTRS)

Schmidt, E. L.

1972-01-01

The fluorescent antibody technique and its use in direct microscopic examination of the soil is discussed. Feasibility analyses were made to determine if the method could be used to simultaneously observe and recognize microorganisms in the soil. Some data indicate this may be possible. Data are also given on two related problems involving the interaction of soil microorganisms with plant roots to form symbiotic structures. One was concerned with the developmental ecology and biology of the root nodule of alder and the second was concerned with the ectotrophic mycorrhizal structure on forest trees, especially pines. In both, the fluorescent antibody detection of the microbial symbiont both as a free living form in soil, and as a root inhabiting form in the higher plant was emphasized. A third aspect of the research involved the detection of autotrophic ammonia oxidizing microorganisms in soil.

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

PubMed

Klimushina, M V; Gumanova, N G; Metelskaya, V A

2017-05-06

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum. Copyright © 2017 Elsevier Inc. All rights reserved.

Differentiation of Cariogenic Streptococci by Fluorescent Antibody1

PubMed Central

Jablon, James M.; Zinner, Doran D.

1966-01-01

Jablon, J. M. (University of Miami, Miami, Fla.), and D. D. Zinner. Differentiation of cariogenic streptococci by fluorescent antibody. J. Bacteriol. 92:1590–1596. 1966.—Eight strains of streptococci were isolated from human carious lesions by the fluorescent-antibody (FA) technique. Seven of these strains produced experimental caries in hamsters or rats maintained on a high sucrose diet. The eighth strain was noncariogenic in animals but possessed some antigenic components in common with the cariogenic strains. On the basis of antigen-antibody reactions by microprecipitin and agar-gel diffusion patterns, the strains were divided into four groups; these groups differed with regard to their cariogenic activity in hamsters. Fluorescein-conjugated antisera, prepared against the human strains, showed some cross-reactions which interfered with the efficacy of the FA technique in differentiating between the related streptococcal groups. To eliminate these cross-reactions, a small amount of related-strain antisera was added to the fluorescein-conjugated antisera to the cariogenic strains. This technique is effective in blocking cross-reactions and should be tried wherever cross-reactions are encountered in the FA technique. Images PMID:5334765

Decline of antibody response in indirect ELISA tests during the periparturient period caused diagnostic gaps in Coxiella burnetii and BVDV serology in pluriparous cows within a Holstein dairy herd.

PubMed

Walraph, J; Zoche-Golob, V; Weber, J; Freick, M

2018-06-01

In cattle, indirect enzyme-linked immunosorbent assay (ELISA) tests are commonly used in serological routine diagnostics. In a longitudinal study design, changes in relative optical density (OD) from drying-off until week 11 after calving were analyzed in blood and milk samples from pluriparous dairy cows (n=21) using a commercial indirect anti-C. burnetii ELISA test. In a second part of this study, changes over time were evaluated in blood and milk samples of 11 of these cows in an indirect ELISA detecting anti-Bovine viral diarrhea virus (BVDV) antibodies. Regarding the cows which were still in the herd at the time of calving, blood serological qualitative changes from positive to negative or indeterminate were demonstrated in 7/20 cows (35%) for the anti-C. burnetii indirect ELISA and in 2/10 cows (20%) for the anti-BVDV indirect ELISA, respectively, during the period from 14days ante partum to calving. Relative OD in the anti-C. burnetii and the anti-BVDV indirect ELISA tests followed basically similar courses over time. In blood serum, relative OD initially increased after drying-off, before a drop around calving was observed. After the colostrum period, relative OD in blood serum showed an increase until week 11 of lactation. In colostrum samples, relative OD levels were higher than in milk samples obtained one day before drying-off. After parturition, relative OD in milk decreased until week 6 of lactation in the anti-C. burnetii indirect ELISA and until week 11 in the anti-BVDV indirect ELISA, respectively. In conclusion, blood serological investigations in periparturient dairy cows using indirect ELISA kits should be avoided. Copyright © 2018 Elsevier Ltd. All rights reserved.

Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing.

PubMed

Ricchiuti, Vincent; Adams, Joseph; Hardy, Donna J; Katayev, Alexander; Fleming, James K

2018-01-01

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6-97.5% of patterns and 71.0-93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity.

Automated Processing and Evaluation of Anti-Nuclear Antibody Indirect Immunofluorescence Testing

PubMed Central

Ricchiuti, Vincent; Adams, Joseph; Hardy, Donna J.; Katayev, Alexander; Fleming, James K.

2018-01-01

Indirect immunofluorescence (IIF) is considered by the American College of Rheumatology (ACR) and the international consensus on ANA patterns (ICAP) the gold standard for the screening of anti-nuclear antibodies (ANA). As conventional IIF is labor intensive, time-consuming, subjective, and poorly standardized, there have been ongoing efforts to improve the standardization of reagents and to develop automated platforms for assay incubation, microscopy, and evaluation. In this study, the workflow and performance characteristics of a fully automated ANA IIF system (Sprinter XL, EUROPattern Suite, IFA 40: HEp-20-10 cells) were compared to a manual approach using visual microscopy with a filter device for single-well titration and to technologist reading. The Sprinter/EUROPattern system enabled the processing of large daily workload cohorts in less than 8 h and the reduction of labor hands-on time by more than 4 h. Regarding the discrimination of positive from negative samples, the overall agreement of the EUROPattern software with technologist reading was higher (95.6%) than when compared to the current method (89.4%). Moreover, the software was consistent with technologist reading in 80.6–97.5% of patterns and 71.0–93.8% of titers. In conclusion, the Sprinter/EUROPattern system provides substantial labor savings and good concordance with technologist ANA IIF microscopy, thus increasing standardization, laboratory efficiency, and removing subjectivity. PMID:29780386

Enumeration of Vibrio cholerae O1 in Bangladesh waters by fluorescent-antibody direct viable count.

PubMed Central

Brayton, P R; Tamplin, M L; Huq, A; Colwell, R R

1987-01-01

A field trial to enumerate Vibrio cholerae O1 in aquatic environments in Bangladesh was conducted, comparing fluorescent-antibody direct viable count with culture detection by the most-probable-number index. Specificity of a monoclonal antibody prepared against the O1 antigen was assessed and incorporated into the fluorescence staining method. All pond and water samples yielded higher counts of viable V. cholerae O1 by fluorescent-antibody direct viable count than by the most-probable-number index. Fluorescence microscopy is a more sensitive detection system than culture methods because it allows the enumeration of both culturable and nonculturable cells and therefore provides more precise monitoring of microbiological water quality. PMID:3324967

Neospora caninum and Toxoplasma gondii antibodies in red foxes (Vulpes vulpes) in the Czech Republic.

PubMed

Bártová, Eva; Slezáková, Radka; Nágl, Ivan; Sedlák, Kamil

2016-01-01

Neospora caninum and Toxoplasma gondii are worldwide spread parasites, causing serious illnesses in sensitive animals; toxoplasmosis is also important zoonosis. Although neosporosis is not considered as a zoonosis, it leads to aborted births in cattle, as well as paresis and paralysis in dogs. The aim of this study was to discover the prevalence of N. caninum and T. gondii antibodies in red foxes (Vulpes vulpes) in the Czech Republic. Sera of 80 foxes from 8 regions of the Czech Republic were tested for antibodies to N. caninum and T. gondii by competitive enzyme linked immunosorbent assay (cELISA) and indirect ELISA. All samples were simultaneously tested by indirect fluorescent antibody test (IFAT) to detect both N. caninum and T. gondii antibodies. Antibodies to N. caninum were found by IFAT in 3 (3.8%) red foxes with titre 50 and in 2 (2.5%) red foxes with inhibition 42.7% and 30.2 %. Antibodies to T. gondii were found in all tested animals in both IFAT (titres 50-6400) and in ELISA (S/P ranging from 34%-133%). This is the first prevalence study of N. caninum and T. gondii antibodies in red foxes in the Czech Republic. The results obtained show that red foxes are exposed at different levels to both protozoan infections, and thus could play an important role in the transmission cycle of N. caninum and T. gondii in sylvatic cycle.

A Comparative Analysis of Polymerase Chain Reaction and Direct Fluorescent Antibody Test for Diagnosis of Genital Herpes.

PubMed

Patwardhan, Vrushali; Bhalla, Preena; Rawat, Deepti; Garg, Vijay Kumar; Sardana, Kabir; Sethi, Sumit

2017-01-01

To compare laboratory tests that can simultaneously detect and type herpes simplex virus (HSV) directly from the genital ulcer specimens in clinically suspected cases of genital herpes. A study was conducted over 10 months and 44 adult male and female patients clinically suspected with genital herpes were recruited. Genital ulcer swab specimens were subjected to glycoprotein-G gene-based conventional polymerase chain reaction (PCR) and commercially available direct fluorescent antibody (DFA) test and the results were compared. PCR for HSV was positive in 82% (36/44) cases. DFA was positive in 68.2% (30/44) cases. There was 100% agreement between HSV types detected by DFA and PCR. The strength of agreement between the results was better in primary genital herpes than recurrent cases. PCR was found to be better in the detection of HSV in recurrent genital herpes patients. It is a better modality, especially when genital herpes clinically presents with ulcerative or crusted lesions, and is also a cheaper alternative as compared to DFA.

Anti-epidermal growth factor receptor (anti-EGFR) antibody conjugated fluorescent nanoparticles probe for breast cancer imaging

NASA Astrophysics Data System (ADS)

Hun, Xu; Zhang, Zhujun

2009-10-01

Fluorescent nanoparticles (FNs) with unique optical properties may be useful as biosensors in living cancer cell imaging and cancer targeting. In this study, anti-EGFR antibody conjugated fluorescent nanoparticles (FNs) (anti-EGFR antibody conjugated FNs) probe was used to detect breast cancer cells. FNs with excellent character such as non-toxicity and photostability were first synthesized with a simple, cost-effective and environmentally friendly modified Stőber synthesis method, and then successfully modified with anti-EGFR antibody. This kind of fluorescence probe based on the anti-EGFR antibody conjugated FNs has been used to detect breast cancer cells with fluorescence microscopy imaging technology. The experimental results demonstrate that the anti-EGFR antibody conjugated FNs can effectively recognize breast cancer cells and exhibited good sensitivity and exceptional photostability, which would provide a novel way for the diagnosis and curative effect observation of breast cancer cells and offer a new method in detecting EGFR.

Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida.

PubMed

Jacobson, Elliott R; Johnson, April J; Hernandez, Jorge A; Tucker, Sylvia J; Dupuis, Alan P; Stevens, Robert; Carbonneau, Dwayne; Stark, Lillian

2005-01-01

In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.

Platelet indirect radioactive Coombs test. Its utilization for PLA1 grouping.

PubMed

Soulier, J P; Patereau, C; Drouet, J

1975-01-01

A platelet indirect radioactive Coombs test (PIRC) has been described. The technique for purification and labelling the antiglobulin has been precised. This test allowed the typing of platelets in the PLA system by using an absorbed serum from a mother of a thrombocytopenic child. Six other families of neonatal thrombocytopenias were tested. In three of them, the mothers were found PLA1 negative (PLA2, PLA2). Among a panel of 93 platelets, two (0.022) were found PLA1, negative. This PIRC test has many advantages compared to other tests such as platelet complement fixation, assay for blocking antibodies or antiglobulin consumption: it gives objective and quantitative results and is highly reproducible; anticomplementary serum may be tested.

Seroepidemiologic Survey of Varicella-Zoster Virus in Korean Adults Using Glycoprotein Enzyme Immuno Assay and Fluorescent Antibody to Membrane Antigen Test

PubMed Central

Kim, Yun Hwa; Hwang, Ji Young; Lee, Kyung Min; Choi, Jin Hee; Lee, Tae Yoon; Choi, Jong Soo

2011-01-01

Background Herpes zoster (HZ) occurs mainly in the elderly and Korea is rapidly becoming an aging society. Therefore, it is important to know the immune status against varicella-zoster virus (VZV) in Korean adults to prevent the disease. Objective The aim of this study was to survey the immune status of Korean adults over 40 years of age against VZV. Methods Antibody titer was measured using a VaccZyme™ VZV glycoprotein enzyme immunoassay (gpEIA) (Binding Site, UK). Fluorescent antibody to membrane antigen (FAMA) test was performed to measure the seropositive rate. Results HZ incidence in the 214 adults enrolled in this study was 10.3%. The gpEIA geometric mean titer (GMT) was 490 mIU/ml and 90.2% of the subjects had a protective level of gpEIA antibody titer against varicella. The average gpEIA GMT of adults who previously had HZ was 1,122 mIU/ml, which was higher than the average gpEIA GMT of 457 mIU/ml in adults who had not had HZ. The FAMA positive rate was 98.6%. Conclusion Most (90.2%) Korean adults ≥40-years-of-age have a protective level of gpEIA antibody against varicella and 98.6% were FAMA seropositive. The GMT of gpEIA antibody was significantly increased with age, and was higher in adults with a history of HZ. PMID:21738361

Identification of the optimal therapeutic antibody for fluorescent imaging of cutaneous squamous cell carcinoma

PubMed Central

Day, Kristine E.; Beck, Lauren N.; Heath, C. Hope; Huang, Conway C.; Zinn, Kurt R.; Rosenthal, Eben L.

2013-01-01

Intraoperative, real-time fluorescence imaging may significantly improve tumor visualization and resection and postoperatively, in pathological assessment. To this end, we sought to determine the optimal FDA approved therapeutic monoclonal antibody for optical imaging of human cutaneous squamous cell carcinoma (cSCC). A near-infrared (NIR) fluorescent probe (IRDye800) was covalently linked to bevacizumab, panitumumab or tocilizumab and injected systemically into immunodeficient mice bearing either cutaneous tumor cell lines (SCC13) or cutaneous human tumor explants. Tumors were then imaged and resected under fluorescent guidance with the SPY, an FDA-approved intraoperative imaging system, and the Pearl Impulse small animal imaging system. All fluorescently labeled antibodies delineated normal tissue from tumor in SCC13 xenografts based on tumor-to-background (TBR) ratios. The conjugated antibodies produced TBRs of 1.2–2 using SPY and 1.6–3.6 using Pearl; in comparison, isotype control antibody IgG-IRDye produced TBRs of 1.0 (SPY) and 0.98 (Pearl). Comparison between antibodies revealed them to be roughly equivalent for imaging purposes with both the SPY and Pearl (p = 0.89 SPY, p = 0.99 Pearl; one way ANOVA). Human tumor explants were also imaged and tumor detection was highest with panitumumab-IRDye800 when using the SPY (TBR 3.0) and Pearl (TBR 4.0). These data suggest that FDA approved antibodies may be clinically used for intraoperative detection of cSCC. PMID:23298904

A Comparative Study of the RAPINA and the Virus-Neutralizing Test (RFFIT) for the Estimation of Antirabies-Neutralizing Antibody Levels in Dog Samples.

PubMed

Manalo, D L; Yamada, K; Watanabe, I; Miranda, M E G; Lapiz, S M D; Tapdasan, E; Petspophonsakul, W; Inoue, S; Khawplod, P; Nishizono, A

2017-08-01

The mass vaccination of dogs against rabies is a highly rational strategy for interrupting the natural transmission of urban rabies. According to the World Organization for Animal Health (OIE) and the World Health Organization (WHO), the immunization of at least 70% of the total dog population minimizes the risk of endemic rabies. Knowledge of the virus-neutralizing antibody (VNA) level against the rabies virus (RABV) is required to evaluate protective immunity and vaccine coverage of dogs in the field. The rapid focus fluorescent inhibition test (RFFIT) and the fluorescent antibody virus neutralization (FAVN) test are recommended by OIE and WHO to determine the VNA levels in serum. However, these tests are cell culture based and require the use of live viruses and specialized equipment. The rapid neutralizing antibody test (RAPINA) is a novel, immunochromatographic test that uses inactivated virus to estimate the VNA level qualitatively. It is a simple, rapid and inexpensive, although indirect, assay for the detection of VNA levels. The RAPINA has shown good positive and negative predictive values and a high concordance with the RFFIT results. In this study, we compared the performance of the two tests for evaluating the vaccination status of dogs in the Philippines, Thailand and Japan. A total of 1135 dog sera were analysed by the RAPINA and compared to the VNA levels determined by the RFFIT. The overall positive and negative predictive values of the RAPINA were 96.2-99.3% and 84.5-94.8%, respectively, with a concordance (kappa) of 0.946-0.97 among the three countries. The RAPINA results were highly homologous and reproducible among different laboratories. These results suggest that this test is appropriate to survey vaccination coverage in countries with limited resources. © 2016 The Authors. Zoonoses and Public Health published by Blackwell Verlag GmbH.

A fluorescent immunochromatographic strip test using a quantum dot-antibody probe for rapid and quantitative detection of 1-aminohydantoin in edible animal tissues.

PubMed

Le, Tao; Zhang, Zhihao; Wu, Juan; Shi, Haixing; Cao, Xudong

2018-01-01

A rapid, simple, and sensitive fluorescent immunochromatographic strip test (ICST) based on quantum dots (QDs) has been developed to detect 1-aminohydantoin (AHD), a major metabolite of nitrofurantoin in animal tissues. To achieve this, QD-labeled antibody conjugates, which consist of CdSe/ZnS QDs and monoclonal antibodies, were prepared by an activated ester method. Under optimal conditions, with the nitrophenyl derivative of AHD as the target, the ICST had a linear range from 0.1 to 100 ng/mL, with a correlation coefficient of 0.9656 and a 50% inhibitory concentration of 4.51 ng/mL. The limit of detection was 0.14 ng/g, which was below the minimum required performance limit of 1 μg/kg for AHD established by the European Commission. The recoveries for AHD ranged from 81.5% to 108.2%, with coefficients of variation below 13%, based on intraday and interday analysis. Furthermore, for AHD in real samples, the ICST showed high reliability and high correlation with liquid chromatography-tandem mass spectrometry (correlation coefficient of 0.9916). To the best of our knowledge, this is the first report of a novel and sensitive method based on a fluorescent ICST to detect AHD below the minimum required performance limit. The ICST demonstrated high reliability, and could be ideally suited for rapid, simple, and on-site screening of AHD contamination in animal tissues. Graphical abstract A rapid, simple, and sensitive fluorescent immunochromatographic strip test that is based on quantum dots was developed to detect 1-aminohydantoin (AHD), a major metabolite of nitrofurantoin in animal tissues. 2-NBA 2-nitrobenzaldehyde, NP nitrophenyl.

[Value of using polyethylene glycol in the indirect antiglobulin test with erythrocytes].

PubMed

Michalewska, B; Walewska, I; Seyfriedowa, H; Kuśnierz-Alejska, G

1991-01-01

A series of comparative investigations was carried out for establishing the value of the indirect antiglobulin test (IAT) in its first phase. The results showed that PEG, in relation to NaCl solution of low ionic strength, increased the reactivity of various antibodies of different specificity present in 72 studied sera. A significant superiority of IAT-PEG to IAT-BISS was noted in 45.8% of these sera. In search for antibodies in sera of 1160 patients IAT-PEG detected 7 antibodies not reacting in the IAT-LISS, and 5 of them had Rh system specificity, one was anti-K and one anti-Jka. Although the IAT-PEG was more sensitive than IAT-LISS in the detection of anti-Rh antibodies, it was less sensitive in this range than the enzymatic LEN test with which not only 5 of these antibodies were demonstrated but which detected also 8 antibodies not demonstrated in IAT-PEG or IAT-LISS. All 5 antibodies not detected by the IAT-PEG but demonstrated in the IAT-LISS were specific for the Lewis system. The per cent of reactions regarded as non-specific in the IAT-PEG was 1.3%. The IAT-PEG modification may be recommended as a sensitive, simple and specific method extending the possibilities of immunohematological diagnosis, especially in cases of post-transfusion reactions and in difficulties of interpretation connected with the occurrence of weakly positive reactions in the IAT-LISS.

Microdistribution of fluorescently-labeled monoclonal antibody in a peritoneal dissemination model of ovarian cancer

NASA Astrophysics Data System (ADS)

Kosaka, Nobuyuki; Ogawa, Mikako; Paik, David S.; Paik, Chang H.; Choyke, Peter L.; Kobayashi, Hisataka

2010-02-01

The microdistribution of therapeutic monoclonal antibodies within a tumor is important for determining clinical response. Nonuniform microdistribution predicts therapy failure. Herein, we developed a semiquantitative method for measuring microdistribution of an antibody within a tumor using in situ fluorescence microscopy and sought to modulate the microdistribution by altering the route and timing of antibody dosing. The microdistribution of a fluorescently-labeled antibody, trastuzumab (50-μg and 150-μg intraperitoneal injection (i.p.), and 100-μg intravenous injection (i.v.)) was evaluated in a peritoneal dissemination mouse model of ovarian cancer. In addition, we evaluated the microdistribution of concurrently-injected (30-μg i.p. and 100-μg i.v.) or serial (two doses of 30-μg i.p.) trastuzumab using in situ multicolor fluorescence microscopy. After the administration of 50-μg i.p. and 100-μg i.v. trastuzumab fluorescence imaging showed no significant difference in the central to peripheral signal ratio (C/P ratio) and demonstrated a peripheral-dominant accumulation, whereas administration of 150-μg i.p. trastuzumab showed relatively uniform, central dominant accumulation. With concurrent-i.p.-i.v. injections trastuzumab showed slightly higher C/P ratio than concurrently-injected i.p. trastuzumab. Moreover, in the serial injection study, the second injection of trastuzumab distributed more centrally than the first injection, while no difference was observed in the control group. Our results suggest that injection routes do not affect the microdistribution pattern of antibody in small peritoneal disseminations. However, increasing the dose results in a more uniform antibody distribution within peritoneal nodules. Furthermore, the serial i.p. injection of antibody can modify the microdistribution within tumor nodules. This work has implications for the optimal delivery of antibody based cancer therapies.

Prevalence of anti- Neospora caninum and anti- Toxoplasma gondii antibodies in dogs from two different indigenous communities in the Brazilian Amazon Region.

PubMed

Minervino, Antonio H H; Cassinelli, Ana Beatriz M; de Lima, Julia T R; Soares, Herbert S; Malheiros, Antonio F; Marcili, Arlei; Gennari, Solange M

2012-12-01

Prevalence of Toxoplasma gondii and Neospora caninum antibodies in sera of 325 dogs in 11 villages inhabited by the Tapirapé and Karajá ethnic groups in the south of the Brazilian Amazon was determined by the use of an indirect fluorescence antibody test. Antibodies (cutoff 1:16) to T. gondii were found in 169 (52%) and to N. caninum (cut-off 1:50) in 32 (9.8%) of 325 dogs. Seropositivity for both parasitic infections was widely prevalent in dogs from these villages and was higher in older dogs, indicating post-natal transmission.

Breaking the color barrier - a multi-selective antibody reporter offers innovative strategies of fluorescence detection.

PubMed

Gallo, Eugenio; Jarvik, Jonathan W

2017-08-01

A novel bi-partite fluorescence platform exploits the high affinity and selectivity of antibody scaffolds to capture and activate small-molecule fluorogens. In this report, we investigated the property of multi-selectivity activation by a single antibody against diverse cyanine family fluorogens. Our fluorescence screen identified three cell-impermeant fluorogens, each with unique emission spectra (blue, green and red) and nanomolar affinities. Most importantly, as a protein fusion tag to G-protein-coupled receptors, the antibody biosensor retained full activity - displaying bright fluorogen signals with minimal background on live cells. Because fluorogen-activating antibodies interact with their target ligands via non-covalent interactions, we were able to perform advanced multi-color detection strategies on live cells, previously difficult or impossible with conventional reporters. We found that by fine-tuning the concentrations of the different color fluorogen molecules in solution, a user may interchange the fluorescence signal (onset versus offset), execute real-time signal exchange via fluorogen competition, measure multi-channel fluorescence via co-labeling, and assess real-time cell surface receptor traffic via pulse-chase experiments. Thus, here we inform of an innovative reporter technology based on tri-color signal that allows user-defined fluorescence tuning in live-cell applications. © 2017. Published by The Company of Biologists Ltd.

Recombinant blood group proteins for use in antibody screening and identification tests.

PubMed

Seltsam, Axel; Blasczyk, Rainer

2009-11-01

The present review elucidates the potentials of recombinant blood group proteins (BGPs) for red blood cell (RBC) antibody detection and identification in pretransfusion testing and the achievements in this field so far. Many BGPs have been eukaryotically and prokaryotically expressed in sufficient quantity and quality for RBC antibody testing. Recombinant BGPs can be incorporated in soluble protein reagents or solid-phase assays such as ELISA, color-coded microsphere and protein microarray chip-based techniques. Because novel recombinant protein-based assays use single antigens, a positive reaction of a serum with the recombinant protein directly indicates the presence and specificity of the target antibody. Inversely, conventional RBC-based assays use panels of human RBCs carrying a huge number of blood group antigens at the same time and require negative reactions of samples with antigen-negative cells for indirect determination of antibody specificity. Because of their capacity for single-step, direct RBC antibody determination, recombinant protein-based assays may greatly facilitate and accelerate the identification of common and rare RBC antibodies.

HAI-178 antibody-conjugated fluorescent magnetic nanoparticles for targeted imaging and simultaneous therapy of gastric cancer

NASA Astrophysics Data System (ADS)

Wang, Can; Bao, Chenchen; Liang, Shujing; Zhang, Lingxia; Fu, Hualin; Wang, Yutian; Wang, Kan; Li, Chao; Deng, Min; Liao, Qiande; Ni, Jian; Cui, Daxiang

2014-05-01

The successful development of safe and highly effective nanoprobes for targeted imaging and simultaneous therapy of in vivo gastric cancer is a great challenge. Herein we reported for the first time that anti-α-subunit of ATP synthase antibody, HAI-178 monoclonal antibody-conjugated fluorescent magnetic nanoparticles, was successfully used for targeted imaging and simultaneous therapy of in vivo gastric cancer. A total of 172 specimens of gastric cancer tissues were collected, and the expression of α-subunit of ATP synthase in gastric cancer tissues was investigated by immunohistochemistry method. Fluorescent magnetic nanoparticles were prepared and conjugated with HAI-178 monoclonal antibody, and the resultant HAI-178 antibody-conjugated fluorescent magnetic nanoparticles (HAI-178-FMNPs) were co-incubated with gastric cancer MGC803 cells and gastric mucous GES-1 cells. Gastric cancer-bearing nude mice models were established, were injected with prepared HAI-178-FMNPs via tail vein, and were imaged by magnetic resonance imaging and small animal fluorescent imaging system. The results showed that the α-subunit of ATP synthase exhibited high expression in 94.7% of the gastric cancer tissues. The prepared HAI-178-FMNPs could target actively MGC803 cells, realized fluorescent imaging and magnetic resonance imaging of in vivo gastric cancer, and actively inhibited growth of gastric cancer cells. In conclusion, HAI-178 antibody-conjugated fluorescent magnetic nanoparticles have a great potential in applications such as targeted imaging and simultaneous therapy of in vivo early gastric cancer cells in the near future.

Fluorescence quenching as an indirect detection method for nitrated explosives.

PubMed

Goodpaster, J V; McGuffin, V L

2001-05-01

A novel approach based on fluorescence quenching is presented for the analysis of nitrated explosives. Seventeen common explosives and their degradation products are shown to be potent quenchers of pyrene, having Stern-Volmer constants that generally increase with the degree of nitration. Aromatic explosives such as 2,4,6-trinitrotoluene (2,4,6-TNT) are more effective quenchers than aliphatic or nitramine explosives. In addition, nitroaromatic explosives are found to have unique interactions with pyrene that lead to a wavelength dependence of their Stern-Volmer constants. This phenomenon allows for their differentiation from other nitrated explosives. The fluorescence quenching method is then applied to the determination of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazine(HMX), 2,4,6-TNT, nitromethane, and ammonium nitrate in various commercial explosive samples. The samples are separated by capillary liquid chromatography with post-column addition of the pyrene solution and detection by laser-induced fluorescence. The indirect fluorescence quenching method shows increased sensitivity and selectivity over traditional UV-visible absorbance as well as the ability to detect a wider range of organic and inorganic nitrated compounds.

A Quantitative Method for Comparing the Brightness of Antibody-dye Reagents and Estimating Antibodies Bound per Cell.

PubMed

Kantor, Aaron B; Moore, Wayne A; Meehan, Stephen; Parks, David R

2016-07-01

We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Antibody response to Ehrlichia risticii and antibody reactivity to the component antigens in horses with induced Potomac horse fever.

PubMed

Dutta, S K; Mattingly, B L; Shankarappa, B

1989-10-01

The antibody response and the antibody reactivity to component antigens of Ehrlichia risticii were studied in horses with induced Potomac horse fever. These horses had no detectable antibodies to E. risticii in their preinoculation (PrI) sera by indirect fluorescent-antibody assay and enzyme-linked immunosorbent assay (ELISA). All the horses exhibited typical disease features following experimental infection and responded with specific antibodies, as measured by ELISA and indirect fluorescent-antibody assay. A primary antibody response was detected in 70% of the horses, while a secondary-type antibody response was detected in 30% of the horses by ELISA. In the primary antibody response, a distinct titer was observed at 2 weeks postinoculation (PI), when the immunoglobulin M (IgM)/IgG ratio was 2 to 5, and the overall antibody titer peaked at 6 to 8 weeks PI. The secondary-type antibody response exhibited a characteristic titer at 1 week PI, the IgM and IgG titers were about equal at 2 weeks PI, and the overall antibody titer peaked at 6 weeks PI. A transient depression in the IgG response at 4 weeks PI was observed in both response types. The antibody was maintained at a high titer for over a year in all horses. Western immunoblot reactivity showed that the antisera collected from these infected horses at 4 to 5 weeks PI recognized some or all of the six major E. risticii component antigens (70, 55, 51, 44, 33, and 28 kilodaltons), all of which were apparent surface components. The 6- to 8-week PI antisera recognized up to 16 component antigens, including 9 major antigens (110, 86, 70, 55, 51, 49, 44, 33, and 28 kilodaltons). However, the PrI sera of these horses showed reactivity at various intensities with one to seven of the component antigens. There was no apparent correlation between this reactivity pattern and the subsequent antibody response types.

Serological tests for detecting Rift Valley fever viral antibodies in sheep from the Nile Delta.

PubMed Central

Scott, R M; Feinsod, F M; Allam, I H; Ksiazek, T G; Peters, C J; Botros, B A; Darwish, M A

1986-01-01

To determine the accuracy of serological methods in detecting Rift Valley fever (RVF) viral antibodies, we examined serum samples obtained from 418 sheep in the Nile Delta by using five tests. The plaque reduction neutralization test (PRNT) was considered the standard serological method against which the four other tests were compared. Twenty-four serum samples had RVF viral antibodies detected by PRNT. Hemagglutination inhibition and enzyme-linked immunosorbent assay antibodies to RVF virus were also present in the same 24 serum samples. Indirect immunofluorescence was less sensitive in comparison with PRNT, and complement fixation was the least sensitive. These results extend observations made with laboratory animals to a large field-collected group of Egyptian sheep. PMID:3533977

Development of Fluorescent Polymerization-based Signal Amplification for Sensitive and Non-enzymatic Biodetection in Antibody Microarrays

PubMed Central

Avens, Heather J.; Bowman, Christopher N.

2009-01-01

Antibody microarrays are a critical tool for proteomics, requiring broad, highly sensitive detection of numerous low abundance biomarkers. Fluorescent polymerization-based amplification (FPBA) is presented as a novel, non-enzymatic signal amplification method that takes advantage of the chain-reaction nature of radical polymerization to achieve a highly amplified fluorescent response. A streptavidin-eosin conjugate localizes eosin photoinitiators for polymerization on the chip where biotinylated target protein is bound. The chip is contacted with acrylamide as a monomer, N-methyldiethanolamine as a coinitiator and yellow/green fluorescent nanoparticles (NPs) which, upon initiation, combine to form a macroscopically visible and highly fluorescent film. The rapid polymerization kinetics and the presence of cross-linker favor entrapment of the fluorescent NPs in the polymer, enabling highly sensitive fluorescent biodetection. This method is demonstrated as being appropriate for antibody microarrays and is compared to detection approaches which utilize streptavidin-FITC (SA-FITC) and streptavidin-labeled yellow/green NPs (SA-NPs). It is found that FPBA is able to detect 0.16 (+/− 0.01) biotin-antibody/µm2 (or 40 zeptomole surface-bound target molecules), while SA-FITC has a limit of detection of 31 (+/− 1) biotin-antibody/µm2 and SA-NPs fail to achieve any significant signal under the conditions evaluated here. Further, FPBA in conjunction with fluorescent stereomicroscopy yields equal or better sensitivity compared to fluorescent detection of SA-eosin using a much more costly microarray scanner. By facilitating highly sensitive detection, FPBA is expected to enable detection of low abundance antigens and also make possible a transition towards less expensive fluorescence detection instrumentation. PMID:19508906

Antibody incubation at 37°C improves fluorescent immunolabeling in free-floating thick tissue sections.

PubMed

Xiao, Xia; Feng, Ya-Ping; Du, Bin; Sun, Han-Ru; Ding, You-Quan; Qi, Jian-Guo

2017-03-01

Fluorescent immunolabeling and imaging in free-floating thick (50-60 μm) tissue sections is relatively simple in practice and enables design-based non-biased stereology, or 3-D reconstruction and analysis. This method is widely used for 3-D in situ quantitative biology in many areas of biological research. However, the labeling quality and efficiency of standard protocols for fluorescent immunolabeling of these tissue sections are not always satisfactory. Here, we systematically evaluate the effects of raising the conventional antibody incubation temperatures (4°C or 21°C) to mammalian body temperature (37°C) in these protocols. Our modification significantly enhances the quality (labeling sensitivity, specificity, and homogeneity) and efficiency (antibody concentration and antibody incubation duration) of fluorescent immunolabeling of free-floating thick tissue sections.

Prevalence of Neospora caninum and Toxoplasma gondii antibodies in sera from camels (Camelus dromedarius) in Riyadh Province, Saudi Arabia.

PubMed

Al-Anazi, Abdullah D

2011-08-01

From April to December 2010, blood samples were collected from 412 healthy camels in Riyadh province, Saudi Arabia and used to evaluate serological screening for Neospora caninum and Toxoplasma gondii infection by an indirect fluorescent antibody test (IFAT). Of the 412 camels, antibodies to N. caninum were found in sixteen in titers of 1:20 and in seven in titers of 1:40 using whole N. caninum tachyzoites as IFAT slide (VMRD Inc., Pullman, WA 99163, USA). Antibodies to T. gondii were found in nineteen camels in titers 1:20 and in eight camels in titers 1:40 using whole T. gondii tachyzoites as IFAT slide.

Fluorescent detection of C-reactive protein using polyamide beads

NASA Astrophysics Data System (ADS)

Jagadeesh, Shreesha; Chen, Lu; Aitchison, Stewart

2016-03-01

Bacterial infection causes Sepsis which is one of the leading cause of mortality in hospitals. This infection can be quantified from blood plasma using C - reactive protein (CRP). A quick diagnosis at the patient's location through Point-of- Care (POC) testing could give doctors the confidence to prescribe antibiotics. In this paper, the development and testing of a bead-based procedure for CRP quantification is described. The size of the beads enable them to be trapped in wells without the need for magnetic methods of immobilization. Large (1.5 mm diameter) Polyamide nylon beads were used as the substrate for capturing CRP from pure analyte samples. The beads captured CRP either directly through adsorption or indirectly by having specific capture antibodies on their surface. Both methods used fluorescent imaging techniques to quantify the protein. The amount of CRP needed to give a sufficient fluorescent signal through direct capture method was found suitable for identifying bacterial causes of infection. Similarly, viral infections could be quantified by the more sensitive indirect capture method. This bead-based assay can be potentially integrated as a disposable cartridge in a POC device due to its passive nature and the small quantities needed.

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum.

PubMed

Muleme, Michael; Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M; Nguyen, Chelsea; Stevenson, Mark A; Wilks, Colin R; Firestone, Simon M

2016-06-01

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Bayesian Validation of the Indirect Immunofluorescence Assay and Its Superiority to the Enzyme-Linked Immunosorbent Assay and the Complement Fixation Test for Detecting Antibodies against Coxiella burnetii in Goat Serum

PubMed Central

Stenos, John; Vincent, Gemma; Campbell, Angus; Graves, Stephen; Warner, Simone; Devlin, Joanne M.; Nguyen, Chelsea; Stevenson, Mark A.; Wilks, Colin R.; Firestone, Simon M.

2016-01-01

Although many studies have reported the indirect immunofluorescence assay (IFA) to be more sensitive in detection of antibodies to Coxiella burnetii than the complement fixation test (CFT), the diagnostic sensitivity (DSe) and diagnostic specificity (DSp) of the assay have not been previously established for use in ruminants. This study aimed to validate the IFA by describing the optimization, selection of cutoff titers, repeatability, and reliability as well as the DSe and DSp of the assay. Bayesian latent class analysis was used to estimate diagnostic specifications in comparison with the CFT and the enzyme-linked immunosorbent assay (ELISA). The optimal cutoff dilution for screening for IgG and IgM antibodies in goat serum using the IFA was estimated to be 1:160. The IFA had good repeatability (>96.9% for IgG, >78.0% for IgM), and there was almost perfect agreement (Cohen's kappa > 0.80 for IgG) between the readings reported by two technicians for samples tested for IgG antibodies. The IFA had a higher DSe (94.8%; 95% confidence interval [CI], 80.3, 99.6) for the detection of IgG antibodies against C. burnetii than the ELISA (70.1%; 95% CI, 52.7, 91.0) and the CFT (29.8%; 95% CI, 17.0, 44.8). All three tests were highly specific for goat IgG antibodies. The IFA also had a higher DSe (88.8%; 95% CI, 58.2, 99.5) for detection of IgM antibodies than the ELISA (71.7%; 95% CI, 46.3, 92.8). These results underscore the better suitability of the IFA than of the CFT and ELISA for detection of IgG and IgM antibodies in goat serum and possibly in serum from other ruminants. PMID:27122484

A rapid immunochromatographic test strip for detecting rabies virus antibody.

PubMed

Wang, Hualei; Feng, Na; Yang, Songtao; Wang, Chengyu; Wang, Tiecheng; Gao, Yuwei; Su, Jianqing; Zheng, Xuexing; Hou, Xiaoqiang; Huang, Hainan; Yang, Ruimei; Zou, Xiaohuan; Huang, Geng; Xia, Xianzhu

2010-12-01

An immunochromatographic test strip (ICTS) for detecting antibodies to rabies virus was developed, using colloidal gold particles labeled with rabies virus glycoprotein as the tracer. The assay was evaluated using sera from dogs immunized with various commercial rabies vaccines, or from dogs in the clinics and sera from dogs immunized with vaccines against pathogens other than rabies virus, and negative sera from a wide variety of animal sources, including dogs, mice, and cats which had never been vaccinated. The ICTS was found to be highly specific for antibodies against rabies virus, with a detection limit of 0.5IU/ml as measured by the fluorescent antibody virus neutralization (FAVN) test. Compared with the FAVN test, the specificity and sensitivity of ICTS were 98.2% and 90.4%, respectively. There was an excellent agreement between results obtained by the ICTS and FAVN tests (kappa=0.888). Strips stored at 4°C in a plastic bag with a desiccant retained their specificity and sensitivity for at least 15 months, and strips stored at ambient temperature remained stable for 12 months. The immunochromatographic test strip may therefore be useful for clinical laboratories lacking specialized equipment and for diagnosis in the field for rapid detection of rabies virus-specific antibodies. Copyright © 2010 Elsevier B.V. All rights reserved.

Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris.

PubMed

Klei, T R; Chapman, M R; Torbert, B J; McClure, J R

1983-05-01

An indirect fluorescent antibody assay (IFA) was developed using Strongylus vulgaris third stage larvae (L3) as antigens. Observations using the IFA indicate that a species-specific antibody response to S. vulgaris L3 develops in S. vulgaris-infected ponies and that some surface L3 antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-naive and in immune ponies following initial and challenge infections using the IFA and an indirect hemagglutination assay (IHA). Antibody levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase following challenge infections of immune ponies when measured with the IFA, but not with the IHA. Significant differences in antibody titers were not seen between ponies which developed colic following challenge infections and those that did not develop colic. Antibodies were not detectable in ponies unexposed to larval migrations, but which received surgical implantation of S. vulgaris adults into the cecum.

Evaluation of monoclonal antibody-based direct, rapid immunohistochemical test for rabies diagnosis.

PubMed

Feng, Ye; Wang, Muyang; Liu, Tingfang; Zhang, Yan; Tu, Zhongzhong; Guo, Huancheng; Zhang, Cuijuan; Zhu, Renying; Ren, Wenlin; Sun, Le; Xu, Weidi; Wang, Yuyang; Li, Maohua; Tu, Changchun

2018-06-01

Rabies is a major public health problem in developing countries in Asia and Africa. Although a number of laboratory diagnoses can be used for rabies control, the WHO and OIE recommended gold standard for rabies diagnosis is the direct fluorescent antibody test (FAT). However, FAT is not widely used in developing countries because of deficient financial sources to procure fluorescent microscope. Recently the direct rapid immunohistochemical test (dRIT) has been developed and has a worldwide promising application, particularly in developing countries, since its result can be read by inexpensive light microscopy, in addition to be consistent with that of FAT. However, no commercial conjugated antibody is available to meet the laboratory demand. We describe here the production of a monoclonal antibody (MAb) against rabies virus (RABV) N protein and its use as a biotinylated conjugate in a dRIT. Tested against a batch of 107 brain specimens representing a wide phylogenetic diversity of RABV collected from different animal species with multiple geographical origins in China, results showed that the dRIT had 100% specificity (95% CI 0.93-1.00) and 96.49% sensitivity (95% CI 0.88-1.00) as compared with the gold standard FAT. It therefore provides a simple, economical alternative to FAT, particularly for use in rabies diagnosis in developing countries. Copyright © 2018 Elsevier B.V. All rights reserved.

Rapid Identification of Dengue Virus Serotypes Using Monoclonal Antibodies in an Indirect Immunofluorescence Test.

DTIC Science & Technology

1982-06-18

areas !i). Presently, the only certain method of identification is through the use of rigidly standardized reference antiserum in a virus plaque...low passaged or unpassaged dengue virus from humans or insects using an indirect immunofluorescence 71 test. MATERIALS AND METHODS :, j Cell cultures...streptomycin. Maintanance medium for infected cell cultures consisted of the appropriate growth medium containing 0.4% bovine plasma albumin instead of FBS

Detection of serum antibodies against Ehrlichia risticii in Potomac horse fever by enzyme-linked immunosorbent assay.

PubMed

Dutta, S K; Rice, R M; Hughes, T D; Savage, P K; Myrup, A C

1987-01-01

An indirect enzyme-linked immunosorbent assay (ELISA) was developed which was specific and sensitive in detecting antibodies to Ehrlichia risticii in Potomac horse fever (PHF). The ELISA antibody titers were correlated with the indirect fluorescent antibody (IFA) titers. E. risticii propagated in human histiocyte culture was purified on renografin gradient and the band of the organisms at a density of 1.182 g/ml was used as antigen. ELISA antibody titers were determined through computer assisted analysis, the observed antibody titers were derived by serial serum dilutions and using a resultant standard curve the predicted antibody titers were obtained from a single serum dilution. The standard curve had a correlation coefficient of 0.8975. The observed and predicted antibody titers were in good agreement, as the respective titers fell within a two-fold range. There was a good correlation between ELISA and IFA test results, but the ELISA titers were several times higher. In experimental infections of horses produced with the infected equine whole blood and the Ehrlichia infected macrophage culture, the antibodies were first detected in two weeks and one week postinoculation (PI), respectively. In both cases the titers reached a peak in about 4 weeks PI with a mean titer of 1:16558 and 1:4030, respectively. The antibody titers of the convalescent sera of field cases of PHF were comparatively lower than the experimentally infected horses.

Diagnosis of equine monocytic ehrlichiosis (Potomac horse fever) by indirect immunofluorescence.

PubMed

Ristic, M; Holland, C J; Dawson, J E; Sessions, J; Palmer, J

1986-07-01

The recent establishment of a system for the continuous in vitro propagation of Ehrlichia risticii, the causative agent of equine monocytic ehrlichiosis (EME; synonym, Potomac horse fever), has facilitated the development of an indirect fluorescent antibody test for the diagnosis of this disease under laboratory and field conditions. The field diagnostic application of the test has aided in the recognition of the disease in 16 states of the United States and in 1 province of Canada. A limited epidemiologic study conducted between January and September 1985, in an area where the disease is known to be enzootic, revealed that conversion from seronegative to seropositive status is not always accompanied by clinical manifestations of the disease. Confirmatory findings in experimentally inoculated horses suggest the existence of clinically undetectable infections.

High degree of correlation between Ebola virus BSL-4 neutralization assays and pseudotyped VSV BSL-2 fluorescence reduction neutralization test.

PubMed

Konduru, Krishnamurthy; Shurtleff, Amy C; Bavari, Sina; Kaplan, Gerardo

2018-04-01

Ebola virus (EBOV), classified as a category A agent by the CDC and NIH, requires BSL-4 containment and induces high morbidity and mortality in humans. The 2013-2015 epidemic in West Africa underscored the urgent need to develop vaccines and therapeutics to prevent and treat EBOV disease. Neutralization assays are needed to evaluate the efficacy of EBOV vaccines and antibody therapies. Pseudotyped viruses based on nonpathogenic or attenuated vectors reduce the risks involved in the evaluation of neutralizing antibodies against highly pathogenic viruses. Selectable markers, fluorescent proteins, and luciferase have been introduced into pseudotyped viruses for detection and quantitation purposes. The current study describes the development of a BSL-2 fluorescence reduction neutralization test (FRNT) using a recombinant vesicular stomatitis virus (VSV) in which the VSV-G envelope gene was replaced with the EBOV glycoprotein (GP) and green fluorescent protein (GFP) genes (rVSV-EBOVgp-GFP). Cells infected with rVSV-EBOVgp-GFP express GFP. Anti-GP neutralizing monoclonal and polyclonal antibodies blocked rVSV-EBOVgp-GFP infection preventing or reducing GFP fluorescence. The high degree of correlation between the EBOV BSL-2 FRNT and the BSL-4 plaque reduction neutralization test (PRNT), the accepted standard of EBOV neutralization tests, supports the use of the EBOV BSL-2 FRNT to evaluate neutralizing antibodies in clinical trials. Published by Elsevier B.V.

Schmallenberg virus antibodies detected in Poland.

PubMed

Kaba, J; Czopowicz, M; Witkowski, L

2013-02-01

Between 24 and 30 July 2012 230 adult goats from three western provinces of Poland bordering on Germany (Western Pomerania, Lubuskie and Lower Silesia) were blood-sampled and tested for antibodies to Schmallenberg virus (SBV) using indirect immunoenzymatic test (ID Screen® Schmallenberg virus indirect, IDvet Innovative Diagnostics). The ELISA test identified 21 seropositive goats - 15 in Western Pomerania (16% of all goats tested in this province), five in Lubuskie (6%) and one in Lower Silesia (2%). Our study demonstrates for the first time the presence of antibodies to SBV in Poland. © 2012 Blackwell Verlag GmbH.

Evaluation of a serological test (indirect ELISA) for the diagnosis of sarcoptic mange in red foxes (Vulpes vulpes).

PubMed

Bornstein, Set; Frössling, Jenny; Näslund, Katarina; Zakrisson, Göran; Mörner, Torsten

2006-12-01

Sarcoptic mange occurs in many parts of the world and is common in populations of domestic and wild canids, including red foxes (Vulpes vulpes). In recent years, an indirect antibody enzyme-linked immunosorbent assay (ELISA), with higher sensitivity and specificity than traditional diagnostic methods, has been successfully applied in the diagnosis of sarcoptic mange in dogs. The same ELISA has also demonstrated specific antibodies to Sarcoptes scabiei in experimentally infected red foxes. The aim of this study was to evaluate the indirect ELISA when used to detect antibodies to S. scabiei in field sera from Swedish red foxes. One cohort of both infected and non-infected red foxes (cohort 1; n = 88), and one cohort of apparently non-infected foxes (cohort 2; n = 67) were examined for skin lesions and presence of S. scabiei by thorough visual examination at autopsy and skin scrapings. Samples of blood-tinted body liquid from the abdomen or thorax cavity were collected and analysed by the indirect ELISA. The relative sensitivity and specificity of the ELISA at different cut-offs (OD values) were estimated by comparing the test results to the infection status as determined by examination and skin scrapings. The highest combination of relative sensitivity and specificity, calculated based on cohort 1, was 95.4 and 100.0%, respectively. These estimates were constant for cut-offs 0.150-0.225, which included the cut-off based on the mean plus three standard deviations of test results from cohort 2 (0.165). It is concluded that this test can be useful in diagnosis and epidemiological studies of S. scabiei infection in red foxes.

First production of fluorescent anti-ribonucleoproteins conjugate for diagnostic of rabies in Brazil.

PubMed

Medeiros Caporale, Graciane Maria; Rodrigues da Silva, Andréa de Cássia; Peixoto, Zélia Maria Pinheiro; Chaves, Luciana Botelho; Carrieri, Maria Luiza; Vassão, Ruth Camargo

2009-01-01

The laboratory tests recommended by the World Health Organization for detection of rabies virus and evaluation of specific antibodies are performed with fluorescent antibodies against the virus, the ribonucleoproteins (RNPs), or by monoclonal antibodies. In this study, we purified the rabies virus RNPs for the production of a conjugate presenting sensibility and specificity compatible with commercial reagents. The method employed for the purification of RNPs was ultracentrifugation in cesium chloride gradient, the obtained product being used for immunizing rabbits, from which the hyperimmune sera were collected. The serum used for conjugate production was the one presenting the highest titer (1/2,560) when tested by indirect immunofluorescence. The antibodies were purified by anion exchange chromatography (QAE-Sephadex A-50),conjugated to fluorescein isothiocyanate and separated by gel filtration (Sephadex G-50). The resulting conjugate presented titers of 1/400 and 1/500 when assayed by direct immunofluorescence (DIF) and simplified fluorescence inhibition microtest, respectively. Sensibility and specificity tests were performed by DIF in 100 central nervous system samples of different animal species, presenting 100% matches when compared with the commercial reagent used as standard, independent of the conservation state of the samples. The quality reached by our conjugate will enable the standardization of this reagent for use by the laboratories performing diagnosis of rabies in Brazil, contributing to the intensification of the epidemiological vigilance and research on this disease. Copyright 2009 Wiley-Liss, Inc.

Anti-insulin antibody test

MedlinePlus

Insulin antibodies - serum; Insulin Ab test; Insulin resistance - insulin antibodies; Diabetes - insulin antibodies ... You appear to have an allergic response to insulin Insulin no longer seems to control your diabetes

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle.

PubMed

Campero, Lucía M; Moreno-Gonzalo, Javier; Venturini, María C; Moré, Gastón; Dellarupe, Andrea; Rambeaud, Magdalena; Echaide, Ignacio E; Valentini, Beatriz; Campero, Carlos M; Moore, Dadín P; Cano, Dora B; Fort, Marcelo; Mota, Rinaldo A; Serrano-Martínez, Marcos E; Cruz-Vázquez, Carlos; Ortega-Mora, Luis M; Álvarez-García, Gema

2018-01-01

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1-7) and three enzyme-linked immunosorbent assays (ELISAs 1-3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the "Pre-test information," which uses previous epidemiological and serological data; (2) the "Majority of tests," which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.

Scanning force microscopy and fluorescence microscopy of microcontact printed antibodies and antibody fragments.

PubMed

LaGraff, John R; Chu-LaGraff, Quynh

2006-05-09

Unlabeled primary immunoglobulin G (IgG) antibodies and its F(ab')2 and Fc fragments were attached to oxygen-plasma-cleaned glass substrates using either microcontact printing (MCP) or physical adsorption during bath application from dilute solutions. Fluorescently labeled secondary IgGs were then bound to surface-immobilized IgG, and the relative surface coverage was determined by measuring the fluorescence intensity. Results indicated that the surface coverage of IgG increased with increasing protein solution concentration for both MCP and bath-applied IgG and that a greater concentration of IgG was transferred to a glass substrate using MCP than during physisorption during bath applications. Scanning force microscopy (SFM) showed that patterned MCP IgG monolayers were 5 nm in height, indicating that IgG molecules lie flat on the substrate. After incubation with a secondary IgG, the overall line thickness increased to around 15 nm, indicating that the secondary IgG was in a more vertical orientation with respect to the substrate. The surface roughness of these MCP patterned IgG bilayers as measured by SFM was observed to increase with increasing surface coverage. Physisorption of IgG to both unmodified patterned polydimethylsiloxane (PDMS) stamps and plasma-cleaned glass substrates was modeled by Langmuir adsorption kinetics yielding IgG binding constants of K(MCP) = 1.7(2) x 10(7) M(-1) and K(bath) = 7.8(7) x 10(5) M(-1), respectively. MCP experiments involving primary F(ab')2 and Fc fragments incubated in fluorescently labeled fragment-specific secondary IgGs were carried out to test for the function and orientation of IgG. Finally, possible origins of MCP stamping defects such as pits, pull outs, droplets, and reverse protein transfer are discussed.

The application of anti-ESAT-6 monoclonal antibody fluorescent probe in ex vivo near-infrared fluorescence imaging in mice with pulmonary tuberculosis.

PubMed

Feng, Feng; Zhang, Haoling; Zhu, Zhaoqin; Li, Cong; Shi, Yuxin; Zhang, Zhiyong

2014-09-01

Here, we aimed to assess the feasibility of anti-ESAT-6 monoclonal antibody (mAb) coupling with IR783 and rhodamine fluorescent probe in the detection of ESAT-6 expression in tuberculosis tissue of mice using near-infrared fluorescence imaging. IR783 and rhodamine were conjugated to the anti-ESAT-6 mAb or IgG. Mice in the experimental group were injected with fluorescence-labeled mAb probe, and mice in the control group were injected with fluorescence-labeled non-specific IgG antibody. Twenty-four hours later, the lung tissue of mice was examined using ex vivo near-infrared fluorescence imaging. In addition, the contrast-to-noise ratio (CNR) was calculated by measuring the signal intensities of the pulmonary lesions, normal lung tissue and background noise. The frozen lung tissue section was examined under fluorescence microscopy and compared with hemoxylin and eosin (HE) staining. The ex vivo near-infrared fluorescence imaging showed that the fluorescence signal in the lung tuberculosis lesions in the experimental group was significantly enhanced, whereas there was only a weak fluorescence signal or even no fluorescence signal in the control group. CNR values were 64.40 ± 7.02 (n = 6) and 8.75 ± 3.87 (n = 6), respectively (t = 17.01, p < 0.001). The fluorescence accumulation distribution detected under fluorescence microscopy was consistent with HE staining of the tuberculosis region. In conclusion, anti-ESAT-6 mAb fluorescent probe could target and be applied in specific ex vivo imaging of mice tuberculosis, and may be of further use in tuberculosis in living mice. Copyright © 2013 John Wiley & Sons, Ltd.

Direct and Indirect Electron Emission from the Green Fluorescent Protein Chromophore

NASA Astrophysics Data System (ADS)

Toker, Y.; Rahbek, D. B.; Klærke, B.; Bochenkova, A. V.; Andersen, L. H.

2012-09-01

Photoelectron spectra of the deprotonated green fluorescent protein chromophore have been measured in the gas phase at several wavelengths within and beyond the S0-S1 photoabsorption band of the molecule. The vertical detachment energy (VDE) was determined to be 2.68±0.1eV. The data show that the first electronically excited state is bound in the Franck-Condon region, and that electron emission proceeds through an indirect (resonant) electron-emission channel within the corresponding absorption band.

A fluorescent-photochrome method for the quantitative characterization of solid phase antibody orientation.

PubMed

Ahluwalia, Arti; De Rossi, Danilo; Giusto, Giuseppe; Chen, Oren; Papper, Vladislav; Likhtenshtein, Gertz I

2002-06-15

A fluorescent-photochrome method of quantifying the orientation and surface density of solid phase antibodies is described. The method is based on measurements of quenching and rates of cis-trans photoisomerization and photodestruction of a stilbene-labeled hapten by a quencher in solution. These experimental parameters enable a quantitative description of the order of binding sites of antibodies immobilized on a surface and can be used to characterize the microviscosity and steric hindrance in the vicinity of the binding site. Furthermore, a theoretical method for the determination of the depth of immersion of the fluorescent label in a two-phase system was developed. The model exploits the concept of dynamic interactions and is based on the empirical dependence of parameters of static exchange interactions on distances between exchangeable centers. In the present work, anti-dinitrophenyl (DNP) antibodies and stilbene-labeled DNP were used to investigate three different protein immobilization methods: physical adsorption, covalent binding, and the Langmuir-Blodgett technique. Copyright 2002 Elsevier Science (USA).

Role of enzyme-treated cells in RBC antibody screening using the gel test: a study of anti-RH1, -RH2, and -RH3 antibodies.

PubMed

Conne, Jocelyne; Schneider, Philippe; Tissot, Jean-Daniel

2007-01-01

The role of enzyme-treated cells (ETCs) in red blood cell (RBC) antibody screening has been the subject of controversy, and its place in the clinical routine remains to be determined. In this work, plasma samples containing anti-RH1 (anti-D; N = 10), anti-RH2 (anti-C; N = 10), or anti-RH3 (anti-E; N = 10) antibodies were studied. The samples were diluted in nonbuffered or buffered normal saline, as well as in a pool of AB plasma samples. Titers and scores were determined by means of the gel test, using the indirect antiglobulin test (IAT) as well as ETCs, with R(0)r, r'r, or r''r test cells. Our results showed that compared to the IAT, ETCs allowed a clearer detection of anti-RH2 and anti-RH3, but not of anti-RH1 antibodies. Based on our study, it is not clear whether the ETC phase of the gel test should be maintained for RBC antibody screening. 2007 Wiley-Liss, Inc.

Application of Tryptophan Fluorescence Bandwidth-Maximum Plot in Analysis of Monoclonal Antibody Structure.

PubMed

Huang, Cheng-Yen; Hsieh, Ming-Ching; Zhou, Qinwei

2017-04-01

Monoclonal antibodies have become the fastest growing protein therapeutics in recent years. The stability and heterogeneity pertaining to its physical and chemical structures remain a big challenge. Tryptophan fluorescence has been proven to be a versatile tool to monitor protein tertiary structure. By modeling the tryptophan fluorescence emission envelope with log-normal distribution curves, the quantitative measure can be exercised for the routine characterization of monoclonal antibody overall tertiary structure. Furthermore, the log-normal deconvolution results can be presented as a two-dimensional plot with tryptophan emission bandwidth vs. emission maximum to enhance the resolution when comparing samples or as a function of applied perturbations. We demonstrate this by studying four different monoclonal antibodies, which show the distinction on emission bandwidth-maximum plot despite their similarity in overall amino acid sequences and tertiary structures. This strategy is also used to demonstrate the tertiary structure comparability between different lots manufactured for one of the monoclonal antibodies (mAb2). In addition, in the unfolding transition studies of mAb2 as a function of guanidine hydrochloride concentration, the evolution of the tertiary structure can be clearly traced in the emission bandwidth-maximum plot.

Fluorescence Adherence Inhibition Assay: A Novel Functional Assessment of Blocking Virus Attachment by Vaccine-Induced Antibodies

PubMed Central

Asati, Atul; Kachurina, Olga; Karol, Alex; Dhir, Vipra; Nguyen, Michael; Parkhill, Robert; Kouiavskaia, Diana; Chumakov, Konstantin; Warren, William; Kachurin, Anatoly

2016-01-01

Neutralizing antibodies induced by vaccination or natural infection play a critically important role in protection against the viral diseases. In general, neutralization of the viral infection occurs via two major pathways: pre- and post-attachment modes, the first being the most important for such infections as influenza and polio, the latter being significant for filoviruses. Neutralizing capacity of antibodies is typically evaluated by virus neutralization assays that assess reduction of viral infectivity to the target cells in the presence of functional antibodies. Plaque reduction neutralization test, microneutralization and immunofluorescent assays are often used as gold standard virus neutralization assays. However, these methods are associated with several important prerequisites such as use of live virus requiring safety precautions, tedious evaluation procedure and long assessment time. Hence, there is a need for a robust, inexpensive high throughput functional assay that can be performed rapidly using inactivated virus, without extensive safety precautions. Herein, we report a novel high throughput Fluorescence Adherence Inhibition assay (fADI) using inactivated virus labeled with fluorescent secondary antibodies virus and Vero cells or erythrocytes as targets. It requires only few hours to assess pre-attachment neutralizing capacity of donor sera. fADI assay was tested successfully on donors immunized with polio, yellow fever and influenza vaccines. To further simplify and improve the throughput of the assay, we have developed a mathematical approach for calculating the 50% titers from a single sample dilution, without the need to analyze multi-point titration curves. Assessment of pre- and post-vaccination human sera from subjects immunized with IPOL®, YF-VAX® and 2013–2014 Fluzone® vaccines demonstrated high efficiency of the assay. The results correlated very well with microneutralization assay performed independently by the FDA Center of

Development and Evaluation of a Fluorescent Antibody-Drug Conjugate for Molecular Imaging and Targeted Therapy of Pancreatic Cancer

PubMed Central

Knutson, Steve; Raja, Erum; Bomgarden, Ryan; Nlend, Marie; Chen, Aoshuang; Kalyanasundaram, Ramaswamy; Desai, Surbhi

2016-01-01

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and

ANA (Antinuclear Antibody) Test: MedlinePlus Lab Test Information

MedlinePlus

... medlineplus.gov/labtests/anaantinuclearantibodytest.html ANA (Antinuclear Antibody) Test To use the sharing features on this page, ... enable JavaScript. What is an ANA (Antinuclear Antibody) Test? An ANA test looks for antinuclear antibodies in ...

Inadequacy of IgM antibody tests for diagnosis of Rocky Mountain Spotted Fever.

PubMed

McQuiston, Jennifer H; Wiedeman, Caleb; Singleton, Joseph; Carpenter, L Rand; McElroy, Kristina; Mosites, Emily; Chung, Ida; Kato, Cecilia; Morris, Kevin; Moncayo, Abelardo C; Porter, Susan; Dunn, John

2014-10-01

Among 13 suspected Rocky Mountain spotted fever (RMSF) cases identified through an enhanced surveillance program in Tennessee, antibodies to Rickettsia rickettsii were detected in 10 (77%) patients using a standard indirect immunofluorescent antibody (IFA) assay. Immunoglobulin M (IgM) antibodies were observed for 6 of 13 patients (46%) without a corresponding development of IgG, and for 3 of 10 patients (30%) at least 1 year post-onset. However, recent infection with a spotted fever group rickettsiae could not be confirmed for any patient, based on a lack of rising antibody titers in properly timed acute and convalescent serologic specimens, and negative findings by polymerase chain reaction testing. Case definitions used in national surveillance programs lack specificity and may capture cases that do not represent current rickettsial infections. Use of IgM antibodies should be reconsidered as a basis for diagnosis and public health reporting of RMSF and other spotted fever group rickettsiae in the United States. © The American Society of Tropical Medicine and Hygiene.

Inadequacy of IgM Antibody Tests for Diagnosis of Rocky Mountain Spotted Fever

PubMed Central

McQuiston, Jennifer H.; Wiedeman, Caleb; Singleton, Joseph; Carpenter, L. Rand; McElroy, Kristina; Mosites, Emily; Chung, Ida; Kato, Cecilia; Morris, Kevin; Moncayo, Abelardo C.; Porter, Susan; Dunn, John

2014-01-01

Among 13 suspected Rocky Mountain spotted fever (RMSF) cases identified through an enhanced surveillance program in Tennessee, antibodies to Rickettsia rickettsii were detected in 10 (77%) patients using a standard indirect immunofluorescent antibody (IFA) assay. Immunoglobulin M (IgM) antibodies were observed for 6 of 13 patients (46%) without a corresponding development of IgG, and for 3 of 10 patients (30%) at least 1 year post-onset. However, recent infection with a spotted fever group rickettsiae could not be confirmed for any patient, based on a lack of rising antibody titers in properly timed acute and convalescent serologic specimens, and negative findings by polymerase chain reaction testing. Case definitions used in national surveillance programs lack specificity and may capture cases that do not represent current rickettsial infections. Use of IgM antibodies should be reconsidered as a basis for diagnosis and public health reporting of RMSF and other spotted fever group rickettsiae in the United States. PMID:25092818

PERFORMANCE EVALUATION OF A PROTOTYPE ARCHITECT ANTIBODY ASSAY FOR BABESIA MICROTI.

PubMed

Cheng, Kevin; Coller, Kelly E; Marohnic, Christopher C; Pfeiffer, Zachary A; Fino, James R; Elsing, Randee R; Bergsma, Janet; Marcinkus, Marilee A; Kar, Alak K; Gumbs, Orlando H; Otis, Kathy S; Fishpaugh, Jeffrey; Schultz, Phillip W; Pope, Mark R; Narvaez, Alfredo R; Wong, Susan J; Madison-Antenucci, Susan; Leary, Thomas P; Dawson, George J

2018-05-09

The tick-borne protozoan Babesia microti is responsible for more than 200 cases of transfusion-transmitted babesiosis (TTB) infection in the United States over the last 30 years. Measures to mitigate the risk of TTB include nucleic acid testing (NAT) and B. microti antibody testing. A fully automated prototype B. microti antibody test was developed on the ARCHITECT instrument. The specificity was determined to be 99.98% in volunteer blood donors (n=28,740) from areas considered as low endemic for B. microti The sensitivity of the prototype test was studied in experimentally-infected macaques; a total of 128 samples were detected compared to 125 with the indirect fluorescent antibody test (IFA), additionally, 83 (89.2%) of the PCR positive samples were detected compared to 81 (87.1%) using the IFA test. All PCR positive samples that tested negative in the prototype antibody test were pre-seroconversion period samples. Following seroconversion, periods of intermittent parasitemia occurred; 17 PCR negative samples drawn in-between PCR positive bleed dates, tested positive both by the prototype test (robust reactivity) and IFA (marginal reactivity) prior to the administration of therapeutic drugs, indicating that the PCR test failed to detect samples from persistently infected macaques. The prototype assay detected 56 of 58 (96.6%) human subjects diagnosed with clinical babesiosis by both PCR and IFA testing. Overall, the prototype anti-babesia assay provides a highly sensitive and specific test for the diagnosis of B. microti infection. While PCR is preferred for detection of window period parasitemia, antibody tests detect infected subjects during periods of low level parasitemia. Copyright © 2018 Cheng et al.

"Quenchbodies": quench-based antibody probes that show antigen-dependent fluorescence.

PubMed

Abe, Ryoji; Ohashi, Hiroyuki; Iijima, Issei; Ihara, Masaki; Takagi, Hiroaki; Hohsaka, Takahiro; Ueda, Hiroshi

2011-11-02

Here, we describe a novel reagentless fluorescent biosensor strategy based on the antigen-dependent removal of a quenching effect on a fluorophore attached to antibody domains. Using a cell-free translation-mediated position-specific protein labeling system, we found that an antibody single chain variable region (scFv) that had been fluorolabeled at the N-terminal region showed a significant antigen-dependent fluorescence enhancement. Investigation of the enhancement mechanism by mutagenesis of the carboxytetramethylrhodamine (TAMRA)-labeled anti-osteocalcin scFv showed that antigen-dependency was dependent on semiconserved tryptophan residues near the V(H)/V(L) interface. This suggested that the binding of the antigen led to the interruption of a quenching effect caused by the proximity of tryptophan residues to the linker-tagged fluorophore. Using TAMRA-scFv, many targets including peptides, proteins, and haptens including morphine-related drugs could be quantified. Similar or higher sensitivities to those observed in competitive ELISA were obtained, even in human plasma. Because of its versatility, this "quenchbody" is expected to have a range of applications, from in vitro diagnostics, to imaging of various targets in situ.

Application of the indirect enzyme-labeled antibody microtest to the detection and surveillance of animal diseases. [Brucellosis, cholera, and trichinosis in cattle and swine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saunders, G.C. Clinard, E.H.; Bartlett, M.L.; Sanders, W.M.

1976-01-01

The rapid, indirect enzyme-labeled antibody (ELA) microplate test has been developed as a diagnostic and surveillance tool to aid in the control of animal disease. Data are presented, which illustrate the application of the test to viral (hog cholera), parasitic (trichinosis), and bacterial (brucellosis) diseases of animals. A greater than 95 percent correlation was observed between the hog cholera ELA test and the hog cholera serum neutralization test performed on over 2000 mixed hog cholera positive and negative field samples obtained during the 1976 New Jersey epizootic. Of 56 swine naturally infected with Trichinella spiralis at a level considered dangerousmore » to man, all were ELA positive, while only one of 360 T. spiralis negative packing house sera was ELA positive. Preliminary experiments with bovine brucellosis (Brucella abortus) indicate that the ELA test is more sensitive than other test methods currently in use. ELA procedures should soon become tests of choice for the detection of antibodies to animal disease agents.« less

Development of a qualitative indirect ELISA for the measurement of rabies virus-specific antibodies from vaccinated dogs and cats.

PubMed

Cliquet, F; McElhinney, L M; Servat, A; Boucher, J M; Lowings, J P; Goddard, T; Mansfield, K L; Fooks, A R

2004-04-01

A protocol suitable for the detection of rabies virus-specific antibodies in serum samples from companion animals using an enzyme linked immunosorbent assay (ELISA) is described. This method has been used successfully for the qualitative assessment of rabies virus-specific antibodies in serum samples from a cohort of vaccinated dogs and cats. In two initial field studies, a variable population of field samples from the Veterinary Laboratories Agency (VLA), United Kingdom were tested. In the first study (n = 1000), the number of false-positive and false-negative results was 11 samples (1.1%) and 67 samples (6.7%), respectively. In the second study (n = 920), the number of false-positive and false-negative results was 7 samples (0.8%) and 52 samples (5.7%). In a third study, undertaken at l'Agence Française de Sécurité Sanitaire des Aliments (AFSSA), Nancy, France (n = 440), 1 false-positive sample (0.23%) and 91 (20.7%) false-negative samples were identified. Data generated using this prototype ELISA indicate a strong correlation for specificity when compared to the gold standard fluorescent antibody virus neutralisation (FAVN) test. Although the ELISA has a lower sensitivity than the FAVN test, it is a useful tool for rapidly screening serum samples from vaccinated companion animals. Using a cut-off value of 0.6 EU/ml, the sensitivity (R = % from VLA and 79% from AFSSA) and specificity (R = 97.3%) indices between the ELISA compared favourably with data generated using the FAVN test. The major advantages of the ELISA test are that it is a qualitative tool that can be completed in four hours, does not require the use of live virus and can be performed without the need for specialised laboratory containment. This contrasts with 4 days using conventional rabies antibody virus neutralisation assays. Using the current format, the ELISA assay described would be a valuable screening tool for the detection of rabies antibodies from vaccinated domestic animals in

Improved decision making for prioritizing tumor targeting antibodies in human xenografts: Utility of fluorescence imaging to verify tumor target expression, antibody binding and optimization of dosage and application schedule.

PubMed

Dobosz, Michael; Haupt, Ute; Scheuer, Werner

2017-01-01

Preclinical efficacy studies of antibodies targeting a tumor-associated antigen are only justified when the expression of the relevant antigen has been demonstrated. Conventionally, antigen expression level is examined by immunohistochemistry of formalin-fixed paraffin-embedded tumor tissue section. This method represents the diagnostic "gold standard" for tumor target evaluation, but is affected by a number of factors, such as epitope masking and insufficient antigen retrieval. As a consequence, variances and discrepancies in histological staining results can occur, which may influence decision-making and therapeutic outcome. To overcome these problems, we have used different fluorescence-labeled therapeutic antibodies targeting human epidermal growth factor receptor (HER) family members and insulin-like growth factor-1 receptor (IGF1R) in combination with fluorescence imaging modalities to determine tumor antigen expression, drug-target interaction, and biodistribution and tumor saturation kinetics in non-small cell lung cancer xenografts. For this, whole-body fluorescence intensities of labeled antibodies, applied as a single compound or antibody mixture, were measured in Calu-1 and Calu-3 tumor-bearing mice, then ex vivo multispectral tumor tissue analysis at microscopic resolution was performed. With the aid of this simple and fast imaging method, we were able to analyze the tumor cell receptor status of HER1-3 and IGF1R, monitor the antibody-target interaction and evaluate the receptor binding sites of anti-HER2-targeting antibodies. Based on this, the most suitable tumor model, best therapeutic antibody, and optimal treatment dosage and application schedule was selected. Predictions drawn from obtained imaging data were in excellent concordance with outcome of conducted preclinical efficacy studies. Our results clearly demonstrate the great potential of combined in vivo and ex vivo fluorescence imaging for the preclinical development and characterization of

The use of serology by titering of fluorescent antibodies to evaluate levels of transmission of schistosomiasis in Rhodesia.

PubMed

Shiff, C J; Yiannakis, C

1976-05-01

The prevalence of schistosomiasis has been measured by parasitological examination and by fluorescent antibody titering in a number of communities in different parts of Rhodesia. The samples were taken from areas of high, medium, and low transmission of the disease. A correlation was detected between the mean titer and prevalence of infection, particularly in the younger people (4-13 years old). It is suggested that fluorescent antibody titrating may be a useful epidemiological tool.

Characterizing fluorescent imaging properties of antibodies conjugated to IRDye800CW for use in imaging of head and neck cancer

NASA Astrophysics Data System (ADS)

Foster, Robert C.; Krell, Asher M.; Chung, Thomas K.; Warram, Jason M.; Zinn, Kurt R.; Rosenthal, Eben L.

2014-03-01

Introduction: Proteins conjugated to the near infrared (NIR) moieties for detection of head and neck cancers are being translated to the clinic. However, little is known about the fluorescent properties of IRDye800CW after conjugation to antibodies. We investigated factors that may alter the real-time observed fluorescence of antibody conjugated dye and the rate of fluorescent signal loss. Methods: Signal loss was examined using three FDA approved monoclonal antibodies conjugated to IRDye800CW (LICOR) over a period of 15 days. Temperature effects on fluorescence were examined for conjugated dye in both solution and a mouse tumor model. Samples were cooled to -20°C then warmed to predetermined temperatures up to 60°C with imaging performed using the PEARL Impulse (LI-COR) and LUNA (Novadaq) systems. Results: Short term fluorescent signal loss (< 1 hour) was linear, while long term loss (15 days) was exponential with significant increases in rate observed with light exposure and increased temperatures. Cooling of tumor tissue at -20°C was shown to significantly increase tumor fluorescence on both imaging modalities when compared to room temperature (p=0.008, p=0.019). Concurrently the ratio of tumor to background fluorescent signal (TBR) increased with decreasing temperature with statistically significant increases seen at -20°C and 4°C (p=0.0015, p=0.03). Conclusions: TBR is increased with decreasing sample temperature, suggesting that the clinical exam of fluorescently labeled tissues may be improved at cooler temperatures. Our results indicate that both the rate of signal loss and the change in fluorescence with temperature observed for IRDye800CW are independent of the conjugating antibody.

Probing antibody internal dynamics with fluorescence anisotropy and molecular dynamics simulations.

PubMed

Kortkhonjia, Ekaterine; Brandman, Relly; Zhou, Joe Zhongxiang; Voelz, Vincent A; Chorny, Ilya; Kabakoff, Bruce; Patapoff, Thomas W; Dill, Ken A; Swartz, Trevor E

2013-01-01

The solution dynamics of antibodies are critical to antibody function. We explore the internal solution dynamics of antibody molecules through the combination of time-resolved fluorescence anisotropy experiments on IgG1 with more than two microseconds of all-atom molecular dynamics (MD) simulations in explicit water, an order of magnitude more than in previous simulations. We analyze the correlated motions with a mutual information entropy quantity, and examine state transition rates in a Markov-state model, to give coarse-grained descriptors of the motions. Our MD simulations show that while there are many strongly correlated motions, antibodies are highly flexible, with F(ab) and F(c) domains constantly forming and breaking contacts, both polar and non-polar. We find that salt bridges break and reform, and not always with the same partners. While the MD simulations in explicit water give the right time scales for the motions, the simulated motions are about 3-fold faster than the experiments. Overall, the picture that emerges is that antibodies do not simply fluctuate around a single state of atomic contacts. Rather, in these large molecules, different atoms come in contact during different motions.

Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus.

PubMed

Aoki, Takashi; Takano, Tomokazu; Unajak, Sasimnanas; Takagi, Madoka; Kim, Young Rim; Park, Seong Bin; Kondo, Hidehiro; Hirono, Ikuo; Saito-Taki, Tatsuo; Hikima, Jun-Ichi; Jung, Tae Sung

2011-05-01

Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysate of KHV-infected koi fin cells (KF-1 cells) but not with lysates of CHV- or CyHV-2-infected KF-1 cells in an immuno-blotting analysis. Similar results were shown in the following tests: (1) a indirect fluorescent antibody test using infected KF-1 cells and (2) an immunohistochemical investigation by fast red stain (infected liver) or FITC detection (infected spleen). These results suggested that mAb-7C6 specifically reacts with KHV ORF68 protein. Copyright © 2010 Elsevier Ltd. All rights reserved.

Antiparietal cell antibody test

MedlinePlus

... Gastric ulcer - anti-gastric parietal cell antibody; Pernicious anemia - anti-gastric parietal cell antibody; Vitamin B12 - anti- ... may use this test to help diagnose pernicious anemia. Pernicious anemia is a decrease in red blood ...

IgG western blot for confirmatory diagnosis of equivocal cases of toxoplasmosis by EIA-IgG and fluorescent antibody test.

PubMed

Khammari, Imen; Saghrouni, Fatma; Yaacoub, Alia; Gaied Meksi, Sondoss; Ach, Hinda; Garma, Lamia; Fathallah, Akila; Ben Saïd, Moncef

2013-08-01

The performance values of available techniques used in serodiagnosis of toxoplasmosis are satisfactory but they raise problems of equivocal and discordant results for very low IgG titers. Recently marketed, LDBio-Toxo II IgG Western blot (IB) showed an excellent correlation with the dye test. We estimated the proportion of equivocal and discordant results between the enzyme immunoassay Platelia Toxo IgG (EIA-IgG) and fluorescent antibody test (FAT) and assessed the usefulness of the IB as a confirmatory test. Out of 2,136 sera collected from pregnant women, 1,644 (77.0%) tested unequivocally positive and 407 (19.0%) were negative in both EIA-IgG and FAT. The remaining 85 (4%) sera showed equivocal or discordant results. Among them, 73 (85.9%) were positive and 12 (14.1%) were negative in IB. Forty-one (89.1%) equivocal sera in EIA-IgG and 46 (86.8%) equivocal sera in FAT were positive in IB. Reducing the cut-off values of both screening techniques improved significantly their sensitivity in detecting very low IgG titers at the expense of their specificity. In conclusion, equivocal results in routine-used techniques and their discordance in determination of the immune status in pregnancy women were not uncommon. IB test appeard to be highly useful in these situations as a confirmatory technique.

RSV antibody test

MedlinePlus

Respiratory syncytial virus antibody test; RSV serology; Bronchiolitis - RSV test ... Crowe JE. Respiratory syncytial virus. In: Kliegman RM, Stanton BF, St. Geme JW, Schor NF, eds. Nelson Textbook of Pediatrics . 20th ...

Seroprevalences of antibodies to Neospora caninum and Toxoplasma gondii in zoo animals.

PubMed

Sedlák, K; Bártová, E

2006-03-31

Neospora caninum is an apicomplexan parasite that causes neuromuscular disease in dogs and abortions in cattle. Little is known about the prevalence of antibodies to this parasite in zoo animals. Sera from 556 animals, from 13 Czech and Slovak zoos were tested for antibodies to N. caninum and Toxoplasma gondii by indirect fluorescent antibody test. Antibodies to N. caninum were found in 31 of 556 zoo animals (5.6%), representing 18 of 114 species tested: Eurasian wolf (Canis lupus lupus), Maned wolf (Chrysocyon brachyurus), fennec (Vulpes zerda), cheetah (Acinonyx jubatus), jaguarundi (Herpailurus yaguarondi), Eurasian lynx (Lynx lynx), Indian lion (Panthera leo goojratensis), fisher (Martes pennanti), blackbuck (Antilope cervicapra), European bison (Bison bonasus), lechwe (Kobus leche), African buffalo (Syncerus caffer caffer), eland (Taurotragus oryx), sitatunga (Tragelaphus spekei gratus), Thorold's deer (Cervus albirostris), Eastern elk (C. elaphus canadensis), Vietnam sika deer (C. nippon pseudaxis) and Père David's deer (Elaphurus davidianus). Titres ranged from 1:40 to 1:2560. The highest prevalence 50% was found in family mustelidae of the order carnivora. Antibodies to T. gondii were detected in 193 of 556 zoo animals (34.7%) representing 72 of 114 species tested, with titres ranging from 1:40 to 1:40960. The highest prevalence 100% was found in families: hyaenidae, mustelidae, ursidae and viveridae of the order carnivora. The results of this study indicate that zoo animals have more exposure to T. gondii than to N. caninum. It is the first report of seroprevalence of antibodies to N. caninum in European zoo animals.

Fluorescent immunolabeling of cancer cells by quantum dots and antibody scFv fragment.

PubMed

Zdobnova, Tatiana A; Dorofeev, Sergey G; Tananaev, Piter N; Vasiliev, Roman B; Balandin, Taras G; Edelweiss, Eveline F; Stremovskiy, Oleg A; Balalaeva, Irina V; Turchin, Ilya V; Lebedenko, Ekaterina N; Zlomanov, Vladimir P; Deyev, Sergey M

2009-01-01

Semiconductor quantum dots (QDs) coupled with cancer-specific targeting ligands are new promising agents for fluorescent visualization of cancer cells. Human epidermal growth factor receptor 2/neu (HER2/neu), overexpressed on the surface of many cancer cells, is an important target for cancer diagnostics. Antibody scFv fragments as a targeting agent for direct delivery of fluorophores offer significant advantages over full-size antibodies due to their small size, lower cross-reactivity, and immunogenicity. We have used quantum dots linked to anti-HER2/neu 4D5 scFv antibody to label HER2/neu-overexpressing live cells. Labeling of target cells was shown to have high brightness, photostability, and specificity. The results indicate that construction based on quantum dots and scFv antibody can be successfully used for cancer cell visualization.

[Indirect ELISA for simultaneous detection of antibodies against duck hepatitis A type 1 and 3 viruses].

PubMed

Zhang, Yuyao; Ma, Xiuli; Huang, Bing; Li, Yufeng; Yu, Kexiang; Li, Jianliang; Liu, Cunxia; Han, Hongyu; Cui, Yanshun

2015-04-04

To simultaneously detect antibodies against Duck hepatitis A type 1 (DHAV-1) and type 3 (DHAV-3) viruses, we developed an indirect enzyme-linked immunosobent assay (ELISA) with bacterially expressed recombinant viral protein as antigen in Escherichia coli. We amplified the full-length VP3 gene of DHAV-1 and the full-length VP1 gene of DHAV-3 through reverse transcription-polymerase chain reaction (RT-PCR) and then cloned them into pET-32a expression vector, designated as pET-1VP3-3VP1. The fusion protein DHAV-1VP3-3VP1 expressed correctly and was subsequently used to develop an indirect ELISA assay. DHAV-1VP3-3VP1 fusion protein expressed in BL21 (DE3) cells following induction by Isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The expressed protein was very antigenic and reactive to virus-specific antibodies in western blot assay. The optimal working concentration for coating antigen was 1.0 microg per well and the working concentration of serum samples was 1:200 dilution and the cut-off value that distinguished the positive from negative serum samples was OD650 > OR = 0.38. The ELISA method based on the prokaryotic expression of VP3 (DHAV-1) and VP1 proteins (DHAV-3) can be used effectively for the clinical detection antibodies against DHAV-1 and DHAV-3.

The determination of antibody to Streptococcus mutans serotypes in saliva for children ages 3 to 7 years.

PubMed

Everhart, D L; Rothenberg, K; Carter, W H; Klapper, B

1978-04-01

The saliva of 29 children ages 3 to 7 years was followed by indirect immunoflourescence to determine the antibody reacting with the 5 different serotypes of S mutans. Fluorescent antisera specific for alpha chain and gamma chain were used. Statistical analysis of the data demonstrated a significant negative correlation between antibody of immunoglobin class (IgA) to S mutans type b and the decayed, extracted and filled surfaces of deciduous teeth.

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W

PubMed Central

Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

2018-01-01

Objective: Using compound W (a 3,3′-diiodothyronine sulfate [T2S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Method: Photosensitive particles (donor beads) coated with compound W or T2S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. Results: The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. Conclusions: The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero. PMID:29449777

A Homogeneous Time-Resolved Fluorescence Immunoassay Method for the Measurement of Compound W.

PubMed

Huang, Biao; Yu, Huixin; Bao, Jiandong; Zhang, Manda; Green, William L; Wu, Sing-Yung

2018-01-01

Using compound W (a 3,3'-diiodothyronine sulfate [T 2 S] immuno-crossreactive material)-specific polyclonal antibodies and homogeneous time-resolved fluorescence immunoassay assay techniques (AlphaLISA) to establish an indirect competitive compound W (ICW) quantitative detection method. Photosensitive particles (donor beads) coated with compound W or T 2 S and rabbit anti-W antibody were incubated with biotinylated goat anti-rabbit antibody. This constitutes a detection system with streptavidin-coated acceptor particle. We have optimized the test conditions and evaluated the detection performance. The sensitivity of the method was 5 pg/mL, and the detection range was 5 to 10 000 pg/mL. The intra-assay coefficient of variation averages <10% with stable reproducibility. The ICW-AlphaLISA shows good stability and high sensitivity and can measure a wide range of compound W levels in extracts of maternal serum samples. This may have clinical application to screen congenital hypothyroidism in utero.

ANAlyte: A modular image analysis tool for ANA testing with indirect immunofluorescence.

PubMed

Di Cataldo, Santa; Tonti, Simone; Bottino, Andrea; Ficarra, Elisa

2016-05-01

The automated analysis of indirect immunofluorescence images for Anti-Nuclear Autoantibody (ANA) testing is a fairly recent field that is receiving ever-growing interest from the research community. ANA testing leverages on the categorization of intensity level and fluorescent pattern of IIF images of HEp-2 cells to perform a differential diagnosis of important autoimmune diseases. Nevertheless, it suffers from tremendous lack of repeatability due to subjectivity in the visual interpretation of the images. The automatization of the analysis is seen as the only valid solution to this problem. Several works in literature address individual steps of the work-flow, nonetheless integrating such steps and assessing their effectiveness as a whole is still an open challenge. We present a modular tool, ANAlyte, able to characterize a IIF image in terms of fluorescent intensity level and fluorescent pattern without any user-interactions. For this purpose, ANAlyte integrates the following: (i) Intensity Classifier module, that categorizes the intensity level of the input slide based on multi-scale contrast assessment; (ii) Cell Segmenter module, that splits the input slide into individual HEp-2 cells; (iii) Pattern Classifier module, that determines the fluorescent pattern of the slide based on the pattern of the individual cells. To demonstrate the accuracy and robustness of our tool, we experimentally validated ANAlyte on two different public benchmarks of IIF HEp-2 images with rigorous leave-one-out cross-validation strategy. We obtained overall accuracy of fluorescent intensity and pattern classification respectively around 85% and above 90%. We assessed all results by comparisons with some of the most representative state of the art works. Unlike most of the other works in the recent literature, ANAlyte aims at the automatization of all the major steps of ANA image analysis. Results on public benchmarks demonstrate that the tool can characterize HEp-2 slides in terms of

A novel indirect ELISA based on glycoprotein Gn for the detection of IgG antibodies against Rift Valley fever virus in small ruminants.

PubMed

Jäckel, S; Eiden, M; Balkema-Buschmann, A; Ziller, M; van Vuren, P Jansen; Paweska, J T; Groschup, M H

2013-10-01

Rift Valley fever virus (RVFV) is an emerging zoonotic pathogen that causes high morbidity and mortality in humans and livestock. In this paper, we describe the cloning, expression and purification of RVFV glycoprotein Gn and its application as a diagnostic antigen in an indirect ELISA for the specific detection of RVF IgG antibodies in sheep and goats. The performance of this Gn based ELISA is validated using a panel of almost 2000 field samples from sheep and goats from Mozambique, Senegal, Uganda and Yemen. All serum samples were also tested by virus neutralization test (VNT), the gold standard method for RVFV serological testing. Compared to the VNT results the Gn based ELISA proved to have an excellent sensitivity (94.56%) and specificity (95.57%). Apart from establishing this new diagnostic assay, these results also demonstrate a close correlation between the presence of RVFV Gn and neutralizing antibodies. Copyright © 2013 Elsevier Ltd. All rights reserved.

A novel method for detecting IgA endomysial antibodies by using human umbilical vein endothelial cells.

PubMed

Castellino, F; Scaglione, N; Grosso, S B; Sategna-Guidetti, C

2000-01-01

Although tissue transglutaminase was recently identified as the main autoantigen recognized by endomysial antibodies in coeliac patients, anti-endomysium antibody detection still persists as the gold standard for coeliac disease screening and diagnosis. (1) To evaluate human umbilical vein cells (HUVEC) as an alternative source of endomysial antigen and to assess their suitability in the diagnosis of coeliac disease. (2) To verify whether tissue transglutaminase is one target antigen eliciting the endomysial antibody fraction of coeliac serum IgA. University teaching hospital. Sera from 123 untreated adults with biopsy-proven coeliac disease and 84 controls (40 healthy and 44 diseased) were assessed by indirect immunofluorescence, using HUVEC on glass slides prepared by cytocentrifugation and permeabilized by using Triton X (0.5%). Indirect immunofluorescence was performed: (1) using coeliac disease serum samples on HUVEC with or without prior incubation with tissue transglutaminase; and (2) incubating both HUVEC and monkey oesophagus with goat anti-guinea pig tissue transglutaminase antibody. All the coeliac patients, who were also positive on monkey oesophagus, showed the typical fluorescent homogeneous cytoplasmic stain on HUVEC. All control sera were negative both on HUVEC and on monkey oesophagus. IgA antibodies did not react with non-permeabilized cells, with intact membrane. Preincubation of coeliac sera with tissue transglutaminase abolished the typical fluorescent pattern. The incubation of anti-tissue transglutaminase antibody with monkey oesophagus and HUVEC resulted in an immunofluorescence staining pattern identical to that obtained with positive coeliac sera. (1) As a substrate for anti-endomysial antibody, HUVEC may provide the same diagnostic accuracy as monkey oesophagus, thus bypassing economical and ethical problems. The HUVEC antigen reacting with IgA from coeliac disease sera is an intracellular rather than a cell-surface antigen, as Ig

21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

Code of Federal Regulations, 2010 CFR

2010-04-01

... heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs..., specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. The...) Specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. [50...

Coexistence of antibodies to tick-borne pathogens of babesiosis, ehrlichiosis, and Lyme borreliosis in human sera.

PubMed

Magnarelli, L A; Dumler, J S; Anderson, J F; Johnson, R C; Fikrig, E

1995-11-01

Serum specimens from persons with or without Lyme borreliosis were analyzed by indirect fluorescent antibody staining methods for total immunoglobulins to Babesia microti, Ehrlichia chaffeensis (Arkansas strain), and Ehrlichia equi (MRK strain). There was serologic evidence of human exposure to multiple tick-borne agents in 15 (6.6%) of 227 serum samples obtained in Connecticut and Minnesota. Of these, 10 serum samples were from Connecticut patients who had erythema migrans and antibodies to Borrelia burgdorferi (range, 1:160 to 1:40, 960). A maximal antibody titer of 1:640 was noted for a B. microti infection, whereas titration end points of 1:640 and 1:1,280 were recorded for E. chaffeensis and E. equi seropositives, respectively. In specificity tests, there was no cross-reactivity among the antisera and antigens tested for the four tick-borne pathogens. On the basis of serologic testing, a small group of persons who had Lyme borreliosis had been exposed to one or more other tick-borne agents, but there was no clinical diagnosis of babesiosis or ehrlichiosis. Therefore, if the clinical picture is unclear or multiple tick-associated illnesses are suspected, more extensive laboratory testing is suggested.

Fluorescently labeled chimeric anti-CEA antibody improves detection and resection of human colon cancer in a patient-derived orthotopic xenograft (PDOX) nude mouse model.

PubMed

Metildi, Cristina A; Kaushal, Sharmeela; Luiken, George A; Talamini, Mark A; Hoffman, Robert M; Bouvet, Michael

2014-04-01

The aim of this study was to evaluate a new fluorescently labeled chimeric anti-CEA antibody for improved detection and resection of colon cancer. Frozen tumor and normal human tissue samples were stained with chimeric and mouse antibody-fluorophore conjugates for comparison. Mice with patient-derived orthotopic xenografts (PDOX) of colon cancer underwent fluorescence-guided surgery (FGS) or bright-light surgery (BLS) 24 hr after tail vein injection of fluorophore-conjugated chimeric anti-CEA antibody. Resection completeness was assessed using postoperative images. Mice were followed for 6 months for recurrence. The fluorophore conjugation efficiency (dye/mole ratio) improved from 3-4 to >5.5 with the chimeric CEA antibody compared to mouse anti-CEA antibody. CEA-expressing tumors labeled with chimeric CEA antibody provided a brighter fluorescence signal on frozen human tumor tissues (P = 0.046) and demonstrated consistently lower fluorescence signals in normal human tissues compared to mouse antibody. Chimeric CEA antibody accurately labeled PDOX colon cancer in nude mice, enabling improved detection of tumor margins for more effective FGS. The R0 resection rate increased from 86% to 96% with FGS compared to BLS. Improved conjugating efficiency and labeling with chimeric fluorophore-conjugated antibody resulted in better detection and resection of human colon cancer in an orthotopic mouse model. © 2013 Wiley Periodicals, Inc.

Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

PubMed

Halling, V W; Jones, M F; Bestrom, J E; Wold, A D; Rosenblatt, J E; Smith, T F; Cockerill, F R

1999-10-01

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

Clinical Comparison of the Treponema pallidum CAPTIA Syphilis-G Enzyme Immunoassay with the Fluorescent Treponemal Antibody Absorption Immunoglobulin G Assay for Syphilis Testing

PubMed Central

Halling, V. W.; Jones, M. F.; Bestrom, J. E.; Wold, A. D.; Rosenblatt, J. E.; Smith, T. F.; Cockerill, F. R.

1999-01-01

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of ≥0.650 and ≤0.900) were retested; if the second analysis produced an index of >0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a

Frequency of antibodies against Sarcocystis neurona and Neospora caninum in domestic cats in the state of Bahia, Brazil.

PubMed

Meneses, Iris Daniela Santos de; Andrade, Müller Ribeiro; Uzêda, Rosângela Soares; Bittencourt, Marta Vasconcelos; Lindsay, David Scott; Gondim, Luís Fernando Pita

2014-01-01

Sarcocystis neurona is the major agent of equine protozoal myeloencephalitis. It infects several mammalian species in the Americas, where the definitive hosts, marsupials of the genus Didelphis (D. virginiana and D. albiventris) are found. Domestic cats are one of the confirmed intermediate hosts of the parasite; however, antibodies against S. neurona had never before been demonstrated in Brazilian cats. The aim of this study was to determine whether cats in Bahia, Brazil, are exposed to the parasite. A total of 272 feline serum samples (134 from feral and 138 from house cats) were subjected to an indirect fluorescent antibody test using cultured merozoites of S. neurona as antigen. Positivity was detected in 4.0% (11/272) of the tested samples, with titers ranging from 25 to 800. The feline sera were also tested for antibodies against the protozoan Neospora caninum, with an observed antibody frequency of 2.9%. To the author's knowledge, this is the first study to report antibodies against S. neurona in Brazilian cats. We conclude that cats are exposed to the parasite in the region of this study. Further investigations are needed to confirm the role of cats in the transmission cycle of S. neurona in Brazil.

Cold Regions Test of Indirect Fire Weapons Ammunition

DTIC Science & Technology

1983-03-08

COLD REGIONS TEST OF INDIRECT FIRE WEAPONS AMMUNITION Paragraph 1 . SCOPE. 1 2. FACILITIES AND INSTRUMENTATION .......... 3. PREPARATION FOR TEST...A- 1 B. Data Collection Sheets ..... .............. B- 1 C. References ..... .................... ... C- 1 D. Cold-Dry...Uniform .D...... .. ... .. ... 0- 1 1 . SCOPE. The procedures outlined in this TOP are designed to determine the c-h-arac-teristics of indirect artillery

High-sensitivity immunochromatographic assay for fumonisin B1 based on indirect antibody labeling.

PubMed

Urusov, Alexandr E; Petrakova, Alina V; Gubaydullina, Milyausha K; Zherdev, Anatoly V; Eremin, Sergei A; Kong, Dezhao; Liu, Liqiang; Xu, Chuanlai; Dzantiev, Boris B

2017-05-01

To develop a high-sensitivity immunochromatographic test for fumonisin B1 in plant extracts. Unlike conventional immunochromatographic tests, this assay is performed in two stages: competitive reaction with free specific antibodies and identifying immune complexes by their interaction with the anti-species antibody-conjugated gold nanoparticles. The use of a new geometry for the test strip membranes and a novel reagent application method ensures the proper order of these stages without additional manipulations. The contact of the ready-to-use test strip with the liquid sample suffices in initiating all stages of the assay and obtaining test results. The developed test was used on corn extracts; its instrumental limit of fumonisin B1 detection was 0.6 ng ml -1 at 15 min of assay duration. The proposed approach is flexible and can be used for a wide range of low molecular compounds. The use of anti-species antibody-conjugated gold nanoparticles in immunochromatography significantly facilitates the development of test systems by eliminating the need to synthesize and characterize the conjugates with specific antibodies for each new compound to be detected.

Indirect calculation of monoclonal antibodies in nanoparticles using the radiolabeling process with technetium 99 metastable as primary factor: Alternative methodology for the entrapment efficiency.

PubMed

Helal-Neto, Edward; Cabezas, Santiago Sánchez; Sancenón, Félix; Martínez-Máñez, Ramón; Santos-Oliveira, Ralph

2018-05-10

The use of monoclonal antibodies (Mab) in the current medicine is increasing. Antibody-drug conjugates (ADCs) represents an increasingly and important modality for treating several types of cancer. In this area, the use of Mab associated with nanoparticles is a valuable strategy. However, the methodology used to calculate the Mab entrapment, efficiency and content is extremely expensive. In this study we developed and tested a novel very simple one-step methodology to calculate monoclonal antibody entrapment in mesoporous silica (with magnetic core) nanoparticles using the radiolabeling process as primary methodology. The magnetic core mesoporous silica were successfully developed and characterised. The PXRD analysis at high angles confirmed the presence of magnetic cores in the structures and transmission electron microscopy allowed to determine structures size (58.9 ± 8.1 nm). From the isotherm curve, a specific surface area of 872 m 2 /g was estimated along with a pore volume of 0.85 cm 3 /g and an average pore diameter of 3.15 nm. The radiolabeling process to proceed the indirect determination were well-done. Trastuzumab were successfully labeled (>97%) with Tc-99m generating a clear suspension. Besides, almost all the Tc-99m used (labeling the trastuzumab) remained trapped in the surface of the mesoporous silica for a period as long as 8 h. The indirect methodology demonstrated a high entrapment in magnetic core mesoporous silica surface of Tc-99m-traztuzumab. The results confirmed the potential use from the indirect entrapment efficiency methodology using the radiolabeling process, as a one-step, easy and cheap methodology. Copyright © 2018 Elsevier B.V. All rights reserved.

A correlative study of Papanicolaou smear, fluorescent antibody, and culture for the diagnosis of Chlamydia trachomatis.

PubMed

Spence, M R; Barbacci, M; Kappus, E; Quinn, T

1986-11-01

A prospective study of 300 patients undergoing therapeutic termination of pregnancy was conducted. A Papanicolaou smear was obtained and a clinical evaluation of the cervix was made. Specimens from the cervix were examined by both direct fluorescent antibody and culture techniques for the presence of Chlamydia trachomatis. The presence of inflammation on Papanicolaou smear could be correlated with C trachomatis isolation. Papanicolaou smear findings consistent with C trachomatis lacked both sensitivity and specificity when compared with direct fluorescent antibody and/or culture techniques. A correlation was found between the clinical diagnosis of cervicitis and C trachomatis. This interrelationship was absent when the component findings of cervicitis (ectopy, friability, and purulent mucus) were examined independently.

An ultrasensitive quantum dots fluorescent polarization immunoassay based on the antibody modified Au nanoparticles amplifying for the detection of adenosine triphosphate.

PubMed

He, Yanlong; Tian, Jianniao; Hu, Kun; Zhang, Juanni; Chen, Sheng; Jiang, Yixuan; Zhao, Yanchun; Zhao, Shulin

2013-11-13

In this work, an ultrasensitive fluorescent polarization immunoassay (FPIA) method based on the quantum dot/aptamer/antibody/gold nanoparticles ensemble has been developed for the detection of adenosine triphosphate (ATP). DNA hybridization is formed when ATP is present in the PBS solution containing the DNA-conjugated quantum dots (QDs) and antibody-AuNPs. The substantial sensitivity improvement of the antibody-AuNPs-enhanced method is mainly attributed to the slower rotation of fluorescent unit when QDs-labeled oligonucleotides hybridize with antibody modified the gold nanoparticle. As a result, the fluorescent polarization (FP) values of the system increase significantly. Under the optimal conditions, a linear response with ATP concentration is ranged from 8×10(-12) M to 2.40×10(-4) M. The detection limit reached as low as 1.8 pM. The developed work provides a sensitive and selective immunoassay protocol for ATP detection, which could be applied in more bioanalytical systems. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment.

PubMed

Jemima, Ebenezer Angel; Manoharan, Seeralan; Kumanan, Kathaperumal

2014-08-01

The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/concordance was found to be 97.39 %. A rapid field test-a latex agglutination test (LAT)-was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.

Occurrence of antibodies against Toxoplasma gondii and its isolation and genotyping in donkeys, mules, and horses in Brazil.

PubMed

Gennari, Solange M; Esmerini, Patrícia de O; Lopes, Marcos G; Soares, Herbert S; Vitaliano, Sérgio N; Cabral, Aline D; Pena, Hilda F J; Horta, Maurício C; Cavalcante, Paulo H; Fortes, Kleber P; Villalobos, Eliana M C

2015-04-15

The occurrence of antibodies against Toxoplasma gondii was determined in donkeys, mules, and horses from different regions of Brazil. Serum samples from 304 donkeys (67.11%), 118 horses (26.05%), and 31 mules (6.84%) were analyzed by means of the indirect fluorescent antibody test (cutoff=64). Antibodies against T. gondii were detected in 129 equids (28.47%) (82 donkeys, 32 horses, and 15 mules). Tissue samples from 19 seropositive and 50 seronegative animals were obtained in order to isolate the parasite by means of mouse bioassay, and T. gondii was isolated from a donkey. Through genotypic characterization of the isolate, by means of polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) using 11 genotypic markers, the genotype #163 (TgCkBr220), which has already been described in chickens in Brazil, was identified. Copyright © 2015 Elsevier B.V. All rights reserved.

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

PubMed

Bazl, M Rajabi; Rasaee, M J; Foruzandeh, M; Rahimpour, A; Kiani, J; Rahbarizadeh, F; Alirezapour, B; Mohammadi, M

2007-02-01

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

Development and preliminary application of an indirect ELISA for detecting antibodies against Avian Influenza Virus.

PubMed

Wang, G; Ding, J; Hu, S; Yang, X

2012-10-01

Fragment of 759 bp DNA spanning the Matrix 1 (M1) gene of Avian Influenza Virus (AIV) was inserted into an expression vector pET28c to construct a recombinant plasmid pET28c-M1. The pET28c-M1 plasmid was transformed into the Escherichia coli BL21 (DE3) competent cell to produce a recombinant strain E. coli 21 (DE3). After being induced by Isopropyl-b-D-galactopyranoside (IPTG), E. coli 21 (DE3) expressed a 28-kDa fusion protein at a high level. This protein can bind anti-AIV (H5N1) positive serum by Western-blot analysis. After being denatured, renatured, and purified by Ni(2+)-column, the fusion protein was used as an antigen to develop Matrix 1 Enzyme-Linked Immunosorbent Assay (M1-ELISA) for detecting antibodies against AIV from chicken serum. We found that this indirect M1-ELISA was sensitive for differentiating antisera against AIV and antisera against other six kinds of avian viruses apart from AIV and this method is more sensitive than Hemagglutination Inhibition (HI) test. When compared with HI test and ELISA (IDEXX) in evaluating 581 serum samples from field vaccinated chickens, this assay showed 93.3% agreement ratio with the HI test, as well as 96.0% agreement ratio with ELISA (IDEXX). In a preliminary application, the assay successfully detected 19 AIVs from 51 nonvaccinated chicken lungs. It concludes that an indirect ELISA was successfully developed for detecting AIV. The assay is specific and sensitive. The application will greatly contribute to the long-term prevention and control of avian influenza in China. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis

PubMed Central

Jaramillo Ortiz, José Manuel; Montenegro, Valeria Noely; de la Fournière, Sofía Ana María; Sarmiento, Néstor Fabián; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-01-01

The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known—positive and 300 known—negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen’s kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68–80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present. PMID:29360801

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

PubMed

Jaramillo Ortiz, José Manuel; Montenegro, Valeria Noely; de la Fournière, Sofía Ana María; Sarmiento, Néstor Fabián; Farber, Marisa Diana; Wilkowsky, Silvina Elizabeth

2018-01-23

The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known-positive and 300 known-negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen's kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68-80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.

Development of an indirect ELISA for detection of antibody to wobbly possum disease virus in archival sera of Australian brushtail possums (Trichosurus vulpecula) in New Zealand.

PubMed

Giles, J C; Johnson, W; Jones, G; Heuer, C; Dunowska, M

2018-07-01

To develop an indirect ELISA based on recombinant nucleocapsid (rN) protein of wobbly possum disease (WPD) virus for investigation of the presence of WPD virus in Australian brushtail possums (Trichosurus vulpecula) in New Zealand. Pre- and post-infection sera (n=15 and 16, respectively) obtained from a previous experimental challenge study were used for ELISA development. Sera were characterised as positive or negative for antibody to WPD virus based on western-blot using WPD virus rN protein as antigen. An additional 215 archival serum samples, collected between 2000-2016 from five different regions of New Zealand, were also tested using the ELISA. Bayesian modelling of corrected optical density at 450 nm (OD 450 ) results from the ELISA was used to obtain estimates of receiver operating characteristic (ROC) curves to establish cut-off values for the ELISA, and to estimate the prevalence of antibody to WPD virus. Western blot analysis showed 5/14 (36%) pre-infection sera and 11/11 (100%) post-infection sera from experimentally infected possums were positive for antibodies to WPD virus. Bayesian estimates of the ROC curves established cut-off values of OD 450 ≥0.41 for samples positive, and OD 450 <0.28 for samples negative for antibody to WPD virus, for sera diluted 1:100 for the ELISA. Based on the model, the estimated proportion of samples with antibodies to WPD virus was 0.30 (95% probability interval=0.196-0.418). Of the 230 archival serum samples tested using the ELISA, 48 (20.9%) were positive for antibody to WPD virus, 155 (67.4%) were negative and 27 (11.7%) equivocal, using the established cut-off values. The proportion of samples positive for WPD virus antibody differed between geographical regions (p<0.001). The results suggested that WPD virus or a related virus has circulated among possums in New Zealand with differences in the proportion of antibody-positive samples from different geographical regions. Antibodies to WPD virus did not seem to

A fluorescence microplate screen assay for the detection of neurite outgrowth and neurotoxicity using an antibody against βIII-tubulin.

PubMed

Popova, Dina; Jacobsson, Stig O P

2014-04-01

The majority of environmental and commercial chemicals have not been evaluated for their potential to cause neurotoxicity. We have investigated if neuron specific anti-βIII-tubulin antibodies are useful in a microplate assay of neurite outgrowth of retinoic acid-induced neurons from mouse P19 embryonal carcinoma cells. By incubating the P19-derived neurons with the primary anti-βIII-tubulin antibody and a secondary Alexa Fluor 488-conjugated antibody, followed by measuring the fluorescence in a microplate reader, a time-dependent increase in anti-βIII-tubulin immunofluorescence was observed. The relative fluorescence units increased by 4.3-fold from 2 to 10 days in culture. The results corresponded well with those obtained by semi-automatic tracing of neurites in fluorescence microscopy images of βIII-tubulin-labeled neurons. The sensitivity of the neurite outgrowth assay using a microplate reader to detect neurotoxicity produced by nocodazole, methyl mercury chloride and okadaic acid was significantly higher than for a cell viability assay measuring intracellular fluorescence of calcein-AM. The microplate-based method to measure toxicity targeting neurites using anti-βIII-tubulin antibodies is however less sensitive than the extracellular lactate dehydrogenase activity assay to detect general cytotoxicity produced by high concentrations of clomipramine, or glutamate-induced excitotoxicity. In conclusion, the fluorescence microplate assay for the detection of neurite outgrowth by measuring changes in βIII-tubulin immunoreactivity is a rapid and sensitive method to assess chemical- or toxin-induced neurite toxicity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Red Blood Cell Antibody Screen

MedlinePlus

... Internet]. Ann Arbor (MI): The Regents of the University of Michigan; c1995-2017. Coombs Antibody Test (Indirect ... gov/health/health-topics/topics/bdt/with NorthShore University Health System [Internet]. NorthShore University Health System; c2017. ...

Development and evaluation of a truncated recombinant NS3 antigen-based indirect ELISA for detection of pestivirus antibodies in sheep and goats.

PubMed

Kalaiyarasu, Semmannan; Mishra, Niranjan; Rajukumar, Katherukamem; Nema, Ram Kumar; Behera, Sthita Pragnya

2015-01-01

The aim of this study was to develop an indirect ELISA using the helicase domain of bovine viral diarrhoea virus (BVDV) NS3 protein instead of full-length NS3 protein for detection of BVDV and BDV antibodies in sheep and goats and its validation by comparing its sensitivity and specificity with virus neutralization test (VNT) as the reference test. The purified 50 kDa recombinant NS3 protein was used as the coating antigen in the ELISA. The optimal concentration of antigen was 320 ng/well at a serum dilution of 1:20 and the optimal positive cut-off optical density value was 0.40 based on test results of 418 VNT negative sheep and goat sera samples. When 569 serum samples from sheep (463) and goats (106) were tested, the ELISA showed a sensitivity of 91.71% and specificity of 94.59% with BVDV VNT. A good correlation (93.67%) was observed between the two tests. It showed a sensitivity of 85% and specificity of 86.6% with VNT in detecting BDV antibody positive or negative samples. This study demonstrates the efficacy of truncated recombinant NS3 antigen based ELISA for seroepidemiological study of pestivirus infection in sheep and goats.

INCIDENCE AND DETECTION OF PLEUROPNEUMONIA-LIKE ORGANISMS IN CELL CULTURES BY FLUORESCENT ANTIBODY AND CULTURAL PROCEDURES1

PubMed Central

Barile, Michael F.; Malizia, Walter F.; Riggs, Donald B.

1962-01-01

Barile, Michael F. (National Institutes of Health, Bethesda, Md.), Walter F. Malizia, and Donald B. Riggs. Incidence and detection of pleuropneumonia-like organisms in cell cultures by fluorescent antibody and cultural procedures. J. Bacteriol. 84:130–136. 1962—A total of 102 tissue-cell cultures from 17 separate laboratories was examined for pleuropneumonia-like organisms (PPLO) by the fluorescent antibody and cultural procedures. PPLO were isolated from 48 of the 49 tissue-cell cultures found positive for PPLO by the fluorescent antibody procedure, and results of the two procedures agreed in 101 of the 102 (99%) cases. PPLO were isolated from none of 10 primary-cell cultures prepared from six animal species and from 48 of 92 (52%) continuous-cell cultures prepared from eight animal species. Cells grown in media containing antibiotics were more frequently contaminated with PPLO (72%) than cells grown in antibiotic-free media (7%). Cultures (91%) from tissue-culture-producing laboratories and cultures (76%) used for propagation of microorganisms were contaminated with PPLO, although none used for tissue-culture metabolic studies was contaminated. In addition, our findings support the view that PPLO contamination of cell cultures is probably owing to bacterial contaminants which revert to L forms in the presence of antibiotics. Images PMID:13865001

Antibody immobilization on to polystyrene substrate--on-chip immunoassay for horse IgG based on fluorescence.

PubMed

Darain, Farzana; Gan, Kai Ling; Tjin, Swee Chuan

2009-06-01

A simple microfluidic immunoassay card was developed based on polystyrene (PS) substrate for the detection of horse IgG, an inexpensive model analyte using fluorescence microscope. The primary antibody was captured onto the PS based on covalent bonding via a self-assembled monolayer (SAM) of thiol to pattern the surface chemistry on a gold-coated PS. The immunosensor chip layers were fabricated from sheets by CO(2) laser ablation. The functionalized PS surfaces after each step were characterized by contact angle measurement, X-ray photoelectron spectroscopy (XPS), and atomic force microscopy (AFM). After the antibody-antigen interaction as a sandwich immunoassay with a fluorescein isothiocyanate (FITC)-conjugated secondary antibody, the intensity of fluorescence was measured on-chip to determine the concentration of the target analyte. The present immunosensor chip showed a linear response range for horse IgG between 1 microg/ml and 80 microg/ml (r = 0.971, n = 3). The detection limit was found to be 0.71 microg/ml. The developed microfluidic system can be extended for various applications including medical diagnostics, microarray detection and observing protein-protein interactions.

In vivo imaging of the inflammatory receptor CD40 after cerebral ischemia using a fluorescent antibody.

PubMed

Klohs, Jan; Gräfe, Michael; Graf, Kristof; Steinbrink, Jens; Dietrich, Thore; Stibenz, Dietger; Bahmani, Peyman; Kronenberg, Golo; Harms, Christoph; Endres, Matthias; Lindauer, Ute; Greger, Klaus; Stelzer, Ernst H K; Dirnagl, Ulrich; Wunder, Andreas

2008-10-01

Brain inflammation is a hallmark of stroke, where it has been implicated in tissue damage as well as in repair. Imaging technologies that specifically visualize these processes are highly desirable. In this study, we explored whether the inflammatory receptor CD40 can be noninvasively and specifically visualized in mice after cerebral ischemia using a fluorescent monoclonal antibody, which we labeled with the near-infrared fluorescence dye Cy5.5 (Cy5.5-CD40MAb). Wild-type and CD40-deficient mice were subjected to transient middle cerebral artery occlusion. Mice were either intravenously injected with Cy5.5-CD40MAb or control Cy5.5-IgGMAb. Noninvasive and ex vivo near-infrared fluorescence imaging was performed after injection of the compounds. Probe distribution and specificity was further assessed with single-plane illumination microscopy, immunohistochemistry, and confocal microscopy. Significantly higher fluorescence intensities over the stroke-affected hemisphere, compared to the contralateral side, were only detected noninvasively in wild-type mice that received Cy5.5-CD40MAb, but not in CD40-deficient mice injected with Cy5.5-CD40MAb or in wild-type mice that were injected with Cy5.5-IgGMAb. Ex vivo near-infrared fluorescence showed an intense fluorescence within the ischemic territory only in wild-type mice injected with Cy5.5-CD40MAb. In the brains of these mice, single-plane illumination microscopy demonstrated vascular and parenchymal distribution, and confocal microscopy revealed a partial colocalization of parenchymal fluorescence from the injected Cy5.5-CD40MAb with activated microglia and blood-derived cells in the ischemic region. The study demonstrates that a CD40-targeted fluorescent antibody enables specific noninvasive detection of the inflammatory receptor CD40 after cerebral ischemia using optical techniques.

Development and Use of Fluorescent Antibody and qPCR Protocols for the Electrostatic Spore Trap

USDA-ARS?s Scientific Manuscript database

Fluorescent antibody (FA) and qPCR protocols were evaluated for the newly developed aerobiological sampler (Ionic Spore Trap), which depends upon electrostatic deposition of particulates onto a 25 mm aluminum disk (stub). This device was originally designed for assessment of captured particulates by...

Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

PubMed

Chan, Kwok-Hung; Chan, Jasper Fuk-Woo; Tse, Herman; Chen, Honglin; Lau, Candy Choi-Yi; Cai, Jian-Piao; Tsang, Alan Ka-Lun; Xiao, Xincai; To, Kelvin Kai-Wang; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Zheng, Bo-Jiang; Wang, Ming; Yuen, Kwok-Yung

2013-08-01

A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of <1:10. No blood donor had antibody against either virus. Surprisingly, 17/28 (60.7%) of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Development of an indirect enzyme-linked immunosorbent assay test for detecting antibodies to chicken astrovirus in chicken sera.

PubMed

Skibinska, A; Lee, A; Wylie, M; Smyth, V J; Welsh, M D; Todd, D

2015-01-01

The development of an indirect enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of Group B chicken astrovirus (CAstV) infections is described. The test was based on the use of an affinity-purified capsid antigen, specific to CAstV isolate 11672, produced as a glutathione-S-transferase N-terminal fusion protein by a recombinant baculovirus. Strongly positive ELISA signals were elicited against experimentally produced antisera raised to CAstVs from Group B (subgroups i and ii) but were negative for antisera raised to a Group A CAstV. Using a panel of 240 selected serum samples, 99% agreement was observed when the results obtained by ELISA were compared to those from an indirect immunofluorescence test for CAstV 11672. The ELISA test was applied to 68 serum sets comprising 1864 samples, which were obtained from parent and grandparent flocks originating mainly in the UK. Of the 52 sets containing ELISA-positive samples, 24 sets had >75% samples positive and nine sets had <25% samples positive and were regarded as having high and low seropositivities, respectively. Of the 1864 serum samples tested 1090 (58.5%) were ELISA positive and of these, 234 sera (21.5%) produced strongly positive signals, whereas moderately positive and weakly positive signals were produced by 562 (51.5%) and 294 (27%) sera. When used for flock screening purposes, this ELISA test can be used to (i) investigate the occurrence of first-time CAstV infections of parent flocks during lay and the possible adverse effects caused by vertically transmitted CAstV infections on broiler hatchability and performance and (ii) diagnose Group B CAstV infections within specific pathogen free flocks.

Antibody Blood Tests

MedlinePlus

... out for sure? If antibody tests and/or symptoms suggest celiac disease, the physician needs to establish the diagnosis by ... who is still experiencing symptoms, to establish the diagnosis or to rule out celiac disease as a part of establishing another diagnosis. Find ...

Clinical use of serum parvovirus and distemper virus antibody titers for determining revaccination strategies in healthy dogs.

PubMed

Twark, L; Dodds, W J

2000-10-01

To assess whether serum canine parvovirus (CPV) and canine distemper virus (CDV) antibody titers can be used to determine revaccination protocols in healthy dogs. Case series. 1,441 dogs between 6 weeks and 17 years old. CPV and CDV antibody titers in serum samples submitted to a commercial diagnostic laboratory were measured by use of indirect fluorescent antibody (IFA) tests. On the basis of parallel measurements of CPV and CDV serum antibody titers in 61 paired serum samples determined by use of hemagglutination inhibition and serum neutralization methods, respectively, we considered titers > or = 1:5 (IFA test) indicative of an adequate antibody response. Age, breed, and sex were not significantly associated with adequate CPV- or CDV-specific antibody responses. Of 1,441 dogs, 1,370 (95.1%) had adequate and 71 (4.9%) had inadequate antibody responses to CPV, whereas 1,346 of 1,379 (97.6%) dogs had adequate and 33 (2.4%) had inadequate responses to CDV. Vaccination histories were available for 468 dogs (468 for CPV, 457 for CDV). Interval between last vaccination and antibody measurement was 1 to 2 years for the majority (281/468; 60.0%) of dogs and 2 to 7 years for 142 of 468 (30.3%) dogs. Interval was < 1 year in only 45 of 468 (9.6%) dogs. The high prevalence of adequate antibody responses (CPV, 95.1%; CDV, 97.6%) in this large population of dogs suggests that annual revaccination against CPV and CDV may not be necessary.

Part-per-trillion level detection of estradiol by competitive fluorescence immunoassay using DNA/dye conjugate as antibody multiple labels.

PubMed

Zhu, Shengchao; Zhang, Qin; Guo, Liang-Hong

2008-08-22

Fluorescent organic dyes are currently the standard signal-generating labels used in microarray quantification. However, new labeling strategies are needed to meet the demand for high sensitivity in the detection of low-abundance proteins and small molecules. In this report, a long-chain DNA/dye conjugate was used to attach multiple fluorescence labels on antibodies to improve signal intensity and immunoassay sensitivity. Compared with the 30 base-pair (bp) oligonucleotide used in our previous work [Q. Zhang, L.-H. Guo, Bioconjugate Chem. 18 (2007) 1668-1672], conjugation of a 219 bp DNA in solution with a fluorescent DNA binder SYBR Green I resulted in more than sixfold increase in signal intensity, consistent with the increase in bp number. In a direct immunoassay for the detection of goat anti-mouse IgG in a mouse IgG-coated 96-well plate, the long DNA conjugate label also produced higher fluorescence than the short one, accompanied by about 15-fold improvement in the detection limit. To demonstrate its advantage in real applications, the DNA/dye conjugate was employed in the competitive immunoassay of 17beta-estradiol, a clinically and environmentally important analyte. The biotin-terminated DNA was attached to biotinylated anti-estradiol antibody through the biotin/streptavidin/biotin bridge after the immuno-reaction was completed, followed by conjugation with SYBR Green I. The limit of detection for 17beta-estradiol is 1.9 pg mL(-1), which is 200-fold lower than the assay using fluorescein-labeled antibodies. The new multiple labeling strategy uses readily available reagents, and is also compatible with current biochip platform. It has great potential in the sensitive detection of protein and antibody microarrays.

Nuclear fluorescence serum reactivity on monkey oesophagus: a new antibody for the follow-up of coeliac disease?

PubMed Central

Picarelli, A; Sabbatella, L; Di Tola, M; Silano, M; Nicolussi, A; D'Inzeo, S; Coppa, A

2010-01-01

We have identified previously a nuclear fluorescence reactivity (NFR) pattern on monkey oesophagus sections exposed to coeliac disease (CD) patients' sera positive for anti-endomysium antibodies (EMA). The aim of the present work was to characterize the NFR, study the time–course of NFR-positive results in relation to gluten withdrawal and evaluate the potential role of NFR in the follow-up of CD. Twenty untreated, 87 treated CD patients and 15 healthy controls were recruited and followed for 12 months. Their sera were incubated on monkey oesophagus sections to evaluate the presence of NFR by indirect immunofluorescence analysis. Duodenal mucosa samples from treated CD patients were challenged with gliadin peptides, and thus the occurrence of NFR in culture supernatants was assessed. The NFR immunoglobulins (Igs) reactivity with the nuclear extract of a human intestinal cell line was investigated. Serum NFR was present in all untreated CD patients, persisted up to 151 ± 37 days from gluten withdrawal and reappeared in treated CD patients under dietary transgressions. Serum NFR was also detected in two healthy controls. In culture supernatants of coeliac intestinal mucosa challenged with gliadin peptides, NFR appeared before EMA. The Igs responsible for NFR were identified as belonging to the IgA2 subclass. The NFR resulted differently from EMA and anti-nuclear antibodies, but reacted with two nuclear antigens of 65 and 49 kDa. A new autoantibody, named NFR related to CD, was described. Furthermore, NFR detection might become a valuable tool in monitoring adherence to a gluten-free diet and identifying slight dietary transgressions. PMID:20529089

Behavioral and Psychological Responses to HIV Antibody Testing.

ERIC Educational Resources Information Center

Jacobsen, Paul B.; And Others

1990-01-01

Considers effects of informing individuals of their antibody status as determined by human immunodeficiency virus (HIV) antibody testing. Reviews research examining changes in psychological distress and in behaviors associated with HIV infections among individuals who have undergone antibody testing. Identifies methodological issues in studying…

Heparin-Induced Thrombocytopenia Antibody Test

MedlinePlus

... HIT II is clinically suspected. There is a pre-test scoring system that is typically used to determine ... The HIT antibody test is performed when this pre-scoring test shows that a person has a moderate to ...

Immunity to Escherichia coli in pigs: Serum Gamma Globulin Levels, Indirect Hemagglutinating Antibody Titres and Bactericidal Activity Against E. coli in pigs up to five Weeks of Age

PubMed Central

Wilson, M. R.; Svendsen, J.

1972-01-01

Serum gamma globulin levels, indirect hemagglutinating antibody titres and bactericidal activity against the 0149:K91;K88ac:H10 Serotype of Escherichia coli were determined in pigs up to five weeks of age from vaccinated and non-vaccinated sows. Gamma globulin levels at two days of age were approximately twice adult levels, by three weeks of age they were one quarter of adult levels and remained so until five weeks of age. Indirect hemagglutinating antibody activity was highest at two days of age, fell until three weeks of age and then rose. Little or no indirect hemagglutinating antibody activity was detected in sera taken at two days of age from pigs from non-vaccinated sows. Only three of 26 two day old pigs had demonstrable bactericidal activity; by three weeks of age 16 of 26 had bactericidal activity. Serum from piglets of vaccinated sows had no more bactericidal activity than did sera from non-vaccinated sows. PMID:4110608

21 CFR 866.5110 - Antiparietal antibody immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5110 Antiparietal antibody immunological test system. (a) Identification. An antiparietal antibody... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antiparietal antibody immunological test system...

21 CFR 866.5100 - Antinuclear antibody immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5100 Antinuclear antibody immunological test system. (a) Identification. An antinuclear antibody... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antinuclear antibody immunological test system...

Nucleoprotein-based indirect enzyme-linked immunosorbent assay (indirect ELISA) for detecting antibodies specific to Ebola virus and Marbug virus.

PubMed

Huang, Yi; Zhu, Youjie; Yang, Mengshi; Zhang, Zhenqing; Song, Donglin; Yuan, Zhiming

2014-12-01

Full-length nucleoproteins from Ebola and Marburg viruses were expressed as His-tagged recombinant proteins in Escherichia coli and nucleoprotein-based enzyme-linked immunosorbent assays (ELISAs) were established for the detection of antibodies specific to Ebola and Marburg viruses. The ELISAs were evaluated by testing antisera collected from rabbit immunized with Ebola and Marburg virus nucleoproteins. Although little cross-reactivity of antibodies was observed in anti-Ebola virus nucleoprotein rabbit antisera, the highest reactions to immunoglobulin G (IgG) were uniformly detected against the nucleoprotein antigens of homologous viruses. We further evaluated the ELISA's ability to detect antibodies to Ebola and Marburg viruses using human sera samples collected from individuals passing through the Guangdong port of entry. With a threshold set at the mean plus three standard deviations of average optical densities of sera tested, the ELISA systems using these two recombinant nucleoproteins have good sensitivity and specificity. These results demonstrate the usefulness of ELISA for diagnostics as well as ecological and serosurvey studies of Ebola and Marburg virus infection.

Indirect scaling methods for testing quantitative emotion theories.

PubMed

Junge, Martin; Reisenzein, Rainer

2013-01-01

Two studies investigated the utility of indirect scaling methods, based on graded pair comparisons, for the testing of quantitative emotion theories. In Study 1, we measured the intensity of relief and disappointment caused by lottery outcomes, and in Study 2, the intensity of disgust evoked by pictures, using both direct intensity ratings and graded pair comparisons. The stimuli were systematically constructed to reflect variables expected to influence the intensity of the emotions according to theoretical models of relief/disappointment and disgust, respectively. Two probabilistic scaling methods were used to estimate scale values from the pair comparison judgements: Additive functional measurement (AFM) and maximum likelihood difference scaling (MLDS). The emotion models were fitted to the direct and indirect intensity measurements using nonlinear regression (Study 1) and analysis of variance (Study 2). Both studies found substantially improved fits of the emotion models for the indirectly determined emotion intensities, with their advantage being evident particularly at the level of individual participants. The results suggest that indirect scaling methods yield more precise measurements of emotion intensity than rating scales and thereby provide stronger tests of emotion theories in general and quantitative emotion theories in particular.

Hatchery workers' IgG antibody profiles to airborne bacteria.

PubMed

Brauner, Paul; Gromöller, Silvana; Pfeifer, Yvonne; Wilharm, Gottfried; Jäckel, Udo

2017-04-01

Occupational exposure to high concentrations of airborne bacteria in poultry production is related to an increased risk of respiratory disorders. However, etiology and in particular microorganisms' potential role in pathogenesis still needs to be elucidated. Thus, detection of specific antibodies against occupational microbial antigens may lead to identification of potentially harmful species. For the purpose of IgG titer determination, indirect immunofluorescence on various bacterial isolates from duck hatchery air was combined with image-based quantification of fluorescence intensity. Moreover, in addition to established assays with pure bacterial cultures, a new approach utilized complex bioaerosol samples for detection of anti-microbial antibodies in human sera by determination of percentages of antibody-bound cells in different serum dilutions. Mean titers in sera from hatchery workers and a non-exposed control group did not display significant differences for most tested isolates and application of comprehensive cluster analysis to entire titer data revealed no structure reflecting workers and controls group. Furthermore, determination of immunoreactivity to the complete microbial community in workplace air displayed similar proportions of antibody-bound cells in both groups. Although no general differences in immunoreaction patterns were observed, mean titers to a Proteus mirabilis isolate and to 3 of 4 distinct Acinetobacter baumannii isolates were higher in the group of hatchery workers than in the reference group indicating a potential applicability as exposure markers. We conclude, despite long term bioaerosol exposure, hatchery workers' IgG antibody profiles to tested antigens did not differ substantially from those of the control group. However, increased workers' titers to A. baumannii and clinical relevance of this species should lead to further investigations regarding potential involvement in pathogenesis of occupational respiratory disorders

Improved method for fluorescence cytometric immunohematology testing.

PubMed

Roback, John D; Barclay, Sheilagh; Hillyer, Christopher D

2004-02-01

A method for accurate immunohematology testing by fluorescence cytometry (FC) was previously described. Nevertheless, the use of vacuum filtration to wash RBCs and a standard-flow cytometer for data acquisition hindered efforts to incorporate this method into an automated platform. A modified procedure was developed that used low-speed centrifugation of 96-well filter plates for RBC staining. Small-footprint benchtop capillary cytometers (PCA and PCA-96, Guava Technologies, Inc.) were used for data acquisition. Authentic clinical samples from hospitalized patients were tested for ABO group and the presence of D antigen (n = 749) as well as for the presence of RBC alloantibodies (n = 428). Challenging samples with mixed-field reactions and weak antibodies were included. Results were compared to those obtained by column agglutination technology (CAT), and discrepancies were resolved by standard tube methods. Detailed investigations of FC sensitivity and reproducibility were also performed. The modified FC method with the PCA determined the correct ABO group and D type for 98.7 percent of 520 samples, compared to 98.8 percent for CAT (p > 0.05). No-type-determined (NTD) rates were 1.2 percent for both methods. In testing for unexpected alloantibodies, FC determined the correct result for 98.6 percent of 215 samples, compared to 96.3 percent for CAT (p > 0.05). When samples were automatically acquired in the 96-well plate format with the PCA-96, 98.7 percent of 229 samples had correct ABO group and D type determined by FC, compared to 97.4 percent for CAT (p > 0.05). NTD rates were 0.9 and 2.6 percent, respectively. Antibody screens were accurate for 99.1 percent of 213 samples with the PCA-96, compared to 99.5 percent for CAT (p > 0.05). Further investigations demonstrated that FC with the PCA-96 was better than CAT at detecting weak anti-A (p < 0.0001) and alloantibodies. An improved method for FC immunohematology testing has been described. This assay was comparable

Theoretical and testing performance of an innovative indirect evaporative chiller

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jiang, Yi; Xie, Xiaoyun

2010-12-15

An indirect evaporative chiller is a device used to produce chilled water at a temperature between the wet bulb temperature and dew point of the outdoor air, which can be used in building HVAC systems. This article presents a theoretical analysis and practical performance of an innovative indirect evaporative chiller. First, the process of the indirect evaporative chiller is introduced; then, the matching characteristics of the process are presented and analyzed. It can be shown that the process that produces cold water by using dry air is a nearly-reversible process, so the ideal produced chilled water temperature of the indirectmore » evaporative chiller can be set close to the dew point temperature of the chiller's inlet air. After the indirect evaporative chiller was designed, simulations were done to analyze the output water temperature, the cooling efficiency relative to the inlet dew point temperature, and the COP that the chiller can performance. The first installation of the indirect evaporative chiller of this kind has been run for 5 years in a building in the city of Shihezi. The tested output water temperature of the chiller is around 14-20 C, which is just in between of the outdoor wet bulb temperature and dew point. The tested COP{sub r,s} of the developed indirect evaporative chiller reaches 9.1. Compared with ordinary air conditioning systems, the indirect evaporative chiller can save more than 40% in energy consumption due to the fact that the only energy consumed is from pumps and fans. An added bonus is that the indirect evaporative chiller uses no CFCs that pollute to the aerosphere. The tested internal parameters, such as the water-air flow rate ratio and heat transfer area for each heat transfer process inside the chiller, were analyzed and compared with designed values. The tested indoor air conditions, with a room temperature of 23-27 C and relative humidity of 50-70%, proved that the developed practical indirect evaporative chiller

Conjugation of Specifically Developed Antibodies for High- and Low-Molecular-Weight Glutenins with Fluorescent Quantum Dots as a Tool for Their Detection in Wheat Flour Dough.

PubMed

Bonilla, Jose C; Ryan, Valerie; Yazar, Gamze; Kokini, Jozef L; Bhunia, Arun K

2018-04-25

The importance of gluten proteins, gliadins and glutenins, is well-known in the quality of wheat products. To gain more specific information about the role of glutenins in wheat dough, the two major subunits of glutenin, high- and low-molecular-weight (HMW and LMW) glutenins, were extracted, isolated, and identified by mass spectrometry. Antibodies for HMW and LMW glutenins were developed using the proteomic information on the characterized glutenin subunits. The antibodies were found to be specific to each subunit by western immunoblots and were then conjugated to quantum dots (QDs) using site-click conjugation, a new method to keep antibody integrity. A fluorescence-link immunosorbent assay tested the successful QD conjugation. The QD-conjugated antibodies were applied to dough samples, where they recognized glutenin subunits and were visualized using a confocal laser scanning microscope.

Comparison of Biotinylated Monoclonal and Polyclonal Antibodies in an Evaluation of a Direct Rapid Immunohistochemical Test for the Routine Diagnosis of Rabies in Southern Africa

PubMed Central

Coetzer, Andre; Sabeta, Claude T.; Markotter, Wanda; Rupprecht, Charles E.; Nel, Louis H.

2014-01-01

The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus. PMID:25254652

Comparison of biotinylated monoclonal and polyclonal antibodies in an evaluation of a direct rapid immunohistochemical test for the routine diagnosis of rabies in southern Africa.

PubMed

Coetzer, Andre; Sabeta, Claude T; Markotter, Wanda; Rupprecht, Charles E; Nel, Louis H

2014-09-01

The major etiological agent of rabies, rabies virus (RABV), accounts for tens of thousands of human deaths per annum. The majority of these deaths are associated with rabies cycles in dogs in resource-limited countries of Africa and Asia. Although routine rabies diagnosis plays an integral role in disease surveillance and management, the application of the currently recommended direct fluorescent antibody (DFA) test in countries on the African and Asian continents remains quite limited. A novel diagnostic assay, the direct rapid immunohistochemical test (dRIT), has been reported to have a diagnostic sensitivity and specificity equal to that of the DFA test while offering advantages in cost, time and interpretation. Prior studies used the dRIT utilized monoclonal antibody (MAb) cocktails. The objective of this study was to test the hypothesis that a biotinylated polyclonal antibody (PAb) preparation, applied in the dRIT protocol, would yield equal or improved results compared to the use of dRIT with MAbs. We also wanted to compare the PAb dRIT with the DFA test, utilizing the same PAb preparation with a fluorescent label. The PAb dRIT had a diagnostic sensitivity and specificity of 100%, which was shown to be marginally higher than the diagnostic efficacy observed for the PAb DFA test. The classical dRIT, relying on two-biotinylated MAbs, was applied to the same panel of samples and a reduced diagnostic sensitivity (83.50% and 90.78% respectively) was observed. Antigenic typing of the false negative samples indicated all of these to be mongoose RABV variants. Our results provided evidence that a dRIT with alternative antibody preparations, conjugated to a biotin moiety, has a diagnostic efficacy equal to that of a DFA relying on the same antibody and that the antibody preparation should be optimized for virus variants specific to the geographical area of focus.

Indirect Immunofluorescence Assay for the Simultaneous Detection of Antibodies against Clinically Important Old and New World Hantaviruses

PubMed Central

Lederer, Sabine; Lattwein, Erik; Hanke, Merle; Sonnenberg, Karen; Stoecker, Winfried; Lundkvist, Åke; Vaheri, Antti; Vapalahti, Olli; Chan, Paul K. S.; Feldmann, Heinz; Dick, Daryl; Schmidt-Chanasit, Jonas; Padula, Paula; Vial, Pablo A.; Panculescu-Gatej, Raluca; Ceianu, Cornelia; Heyman, Paul; Avšič-Županc, Tatjana; Niedrig, Matthias

2013-01-01

In order to detect serum antibodies against clinically important Old and New World hantaviruses simultaneously, multiparametric indirect immunofluorescence assays (IFAs) based on biochip mosaics were developed. Each of the mosaic substrates consisted of cells infected with one of the virus types Hantaan (HTNV), Puumala (PUUV), Seoul (SEOV), Saaremaa (SAAV), Dobrava (DOBV), Sin Nombre (SNV) or Andes (ANDV). For assay evaluation, serum IgG and IgM antibodies were analyzed using 184 laboratory-confirmed hantavirus-positive sera collected at six diagnostic centers from patients actively or previously infected with the following hantavirus serotypes: PUUV (Finland, n = 97); SEOV (China, n = 5); DOBV (Romania, n = 7); SNV (Canada, n = 23); ANDV (Argentina and Chile, n = 52). The control panel comprised 89 sera from healthy blood donors. According to the reference tests, all 184 patient samples were seropositive for hantavirus-specific IgG (n = 177; 96%) and/or IgM (n = 131; 72%), while all control samples were tested negative. In the multiparametric IFA applied in this study, 183 (99%) of the patient sera were IgG and 131 (71%) IgM positive (accordance with the reference tests: IgG, 96%; IgM, 93%). Overall IFA sensitivity for combined IgG and IgM analysis amounted to 100% for all serotypes, except for SNV (96%). Of the 89 control sera, 2 (2%) showed IgG reactivity against the HTNV substrate, but not against any other hantavirus. Due to the high cross-reactivity of hantaviral nucleocapsid proteins, endpoint titrations were conducted, allowing serotype determination in >90% of PUUV- and ANDV-infected patients. Thus, multiparametric IFA enables highly sensitive and specific serological diagnosis of hantavirus infections and can be used to differentiate PUUV and ANDV infection from infections with Murinae-borne hantaviruses (e.g. DOBV and SEOV). PMID:23593524

Direct fluorescent antibody technique for the detection of bacterial kidney disease in paraffin-embedded tissues

USGS Publications Warehouse

Ochiai, T.; Yasutake, W.T.; Gould, R.W.

1985-01-01

The direct fluorescent antibody technique (FAT) was successfully used to detect the causative agent of bacterial kidney disease (BKD), Renibacterium salmoninarum, in Bouin's solution flexed and paraffinembedded egg and tissue sections. This method is superior to gram stain and may be particularly useful in detecting the BKD organism in fish with low-grade infection.

Molecular imaging needles: dual-modality optical coherence tomography and fluorescence imaging of labeled antibodies deep in tissue

PubMed Central

Scolaro, Loretta; Lorenser, Dirk; Madore, Wendy-Julie; Kirk, Rodney W.; Kramer, Anne S.; Yeoh, George C.; Godbout, Nicolas; Sampson, David D.; Boudoux, Caroline; McLaughlin, Robert A.

2015-01-01

Molecular imaging using optical techniques provides insight into disease at the cellular level. In this paper, we report on a novel dual-modality probe capable of performing molecular imaging by combining simultaneous three-dimensional optical coherence tomography (OCT) and two-dimensional fluorescence imaging in a hypodermic needle. The probe, referred to as a molecular imaging (MI) needle, may be inserted tens of millimeters into tissue. The MI needle utilizes double-clad fiber to carry both imaging modalities, and is interfaced to a 1310-nm OCT system and a fluorescence imaging subsystem using an asymmetrical double-clad fiber coupler customized to achieve high fluorescence collection efficiency. We present, to the best of our knowledge, the first dual-modality OCT and fluorescence needle probe with sufficient sensitivity to image fluorescently labeled antibodies. Such probes enable high-resolution molecular imaging deep within tissue. PMID:26137379

Prevalence of Toxoplasma gondii antibodies among veterinary college staff and students, Iowa State University.

PubMed Central

1976-01-01

Serum samples obtained from two groups within the College of Veterinary Medicine, Iowa State University, were examined for Toxoplasma gondii antibodies. Serum samples from 65 (26.0%) of the 250 persons examined yielded a titer of 1:16 or greater by the indirect fluorescent antibody test. A prevalence of 33.3% was obtained for 108 Veterinary Medical Research Institute (VMRI) personnel. Fof 142 veterinary students, the reactive rate was 20.4%. There was a marked difference of results within the student group--the reactive rate of 31.0% of 58 members of the class of 1975 was significantly higher (p less than 0.05) than the 13.1% for 84 members of the class of 1976. Studies on multiple serum samples were carried out on 55 VMRI personnel; 17 showed a change in serologic reactivity but only 5 had titers of 1:64 or greater. No definitive relationships could be determined between the epidemiologic characteristics examined and the presence of T. gondii antibodies. Characteristics examined included age, sex, primary residency, food habits (eating of rare beef and smoked pork products), and extent of animal contact. Images p526-a PMID:825918

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

PubMed Central

Lagerkvist, Ann Catrin; Földes-Papp, Zeno; Persson, Mats A.A.; Rigler, Rudolf

2001-01-01

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. PMID:11468349

Graft Immunocomplex Capture Fluorescence Analysis to Detect Donor-Specific Antibodies and HLA Antigen Complexes in the Allograft.

PubMed

Nakamura, Tsukasa; Ushigome, Hidetaka; Watabe, Kiyoko; Imanishi, Yui; Masuda, Koji; Matsuyama, Takehisa; Harada, Shumpei; Koshino, Katsuhiro; Iida, Taku; Nobori, Shuji; Yoshimura, Norio

2017-04-01

Immunocomplex capture fluorescence analysis (ICFA) is an attractive method to detect donor-specific anti-HLA antibodies (DSA) and HLA antigen complexes. Currently, antibody-mediated rejection (AMR) due to DSA is usually diagnosed by C4d deposition and serological DSA detection. Conversely, there is a discrepancy between these findings frequently. Thereupon, our graft ICFA technique may contribute to establish the diagnosis of AMR. Graft samples were obtained by a percutaneous needle biopsy. Then, the specimen was dissolved in PBS by the lysis buffer. Subsequently, HLA antigens were captured by anti-HLA beads. Then, DSA-HLA complexes were detected by PE-conjugated anti-human IgG antibodies, where DSA had already reacted with the allograft in vivo, analyzed by a Luminex system. A ratio (sample MFI/blank beads MFI) was calculated: ≥ 1.0 was determined as positive. We found that DSA-HLA complexes in the graft were successfully detected from only slight positive 1.03 to 79.27 in a chronic active AMR patient by graft ICFA. Next, positive graft ICFA had predicted the early phase of AMR (MFI ratio: 1.38) even in patients with no serum DSA. Finally, appropriate therapies for AMR deleted DSA deposition (MFI ratio from 0.3 to 0.7) from allografts. This novel application would detect early phase or incomplete pathological cases of AMR, which could lead to a correct diagnosis and initiation of appropriate therapies. Moreover, graft ICFA might address a variety of long-standing questions in terms of DSA. AMR: Antibody-mediated rejection; DSA: Donor-specific antibodies; ICFA: Immunocomplex capture fluorescence analysis.

Nuclear fluorescence serum reactivity on monkey oesophagus: a new antibody for the follow-up of coeliac disease?

PubMed

Picarelli, A; Sabbatella, L; Di Tola, M; Silano, M; Nicolussi, A; D'Inzeo, S; Coppa, A

2010-09-01

We have identified previously a nuclear fluorescence reactivity (NFR) pattern on monkey oesophagus sections exposed to coeliac disease (CD) patients' sera positive for anti-endomysium antibodies (EMA). The aim of the present work was to characterize the NFR, study the time-course of NFR-positive results in relation to gluten withdrawal and evaluate the potential role of NFR in the follow-up of CD. Twenty untreated, 87 treated CD patients and 15 healthy controls were recruited and followed for 12 months. Their sera were incubated on monkey oesophagus sections to evaluate the presence of NFR by indirect immunofluorescence analysis. Duodenal mucosa samples from treated CD patients were challenged with gliadin peptides, and thus the occurrence of NFR in culture supernatants was assessed. The NFR immunoglobulins (Igs) reactivity with the nuclear extract of a human intestinal cell line was investigated. Serum NFR was present in all untreated CD patients, persisted up to 151 ± 37 days from gluten withdrawal and reappeared in treated CD patients under dietary transgressions. Serum NFR was also detected in two healthy controls. In culture supernatants of coeliac intestinal mucosa challenged with gliadin peptides, NFR appeared before EMA. The Igs responsible for NFR were identified as belonging to the IgA2 subclass. The NFR resulted differently from EMA and anti-nuclear antibodies, but reacted with two nuclear antigens of 65 and 49 kDa. A new autoantibody, named NFR related to CD, was described. Furthermore, NFR detection might become a valuable tool in monitoring adherence to a gluten-free diet and identifying slight dietary transgressions. © 2010 British Society for Immunology.

Technical note: Protozoa-specific antibodies raised in sheep plasma bind to their target protozoa in the rumen.

PubMed

Williams, Y J; Rea, S M; Popovski, S; Skillman, L C; Wright, A-D G

2014-12-01

Binding of IgG antibodies to Entodinium spp. in the rumen of sheep (Ovis aries) was investigated by adding IgG, purified from plasma, directly into the rumen. Plasma IgG was sourced from sheep that had or had not been immunized with a vaccine containing whole fixed Entodinium spp. cells. Ruminal fluid was sampled approximately 2 h after each antibody dosing. Binding of protozoa by a specific antibody was detected using an indirect fluorescent antibody test. An antibody titer in the ruminal fluid was determined by ELISA, and the concentration of ruminal fluid ammonia-N and ruminal pH were also determined. Entodinium spp. and total protozoa from IgG-infused sheep were enumerated by microscopic counts. Two-hourly additions of IgG maintained a low antibody titer in the rumen for 12 h and the binding of the antibody to the rumen protozoa was demonstrated. Increased ammonia-N concentrations and altered ruminal fluid pH patterns indicated that additional fermentation of protein was occurring in the rumen after addition of IgG. No reduction in numbers of Entodinium spp. was observed (P>0.05). Although binding of antibodies to protozoa has been demonstrated in the rumen, it is unclear how much cell death occurred. On the balance of probability, it would appear that the antibody was degraded or partially degraded, and the impact of this on protozoal populations and the measurement of a specific titer is also unclear.

21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Systems § 866.5090 Antimitochondrial antibody immunological test system. (a) Identification. An antimitochondrial antibody immunological test system is a device that consists of the reagents used to measure by... of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's own...

Cross-reactivity of stimulants found in sports drug testing by two fluorescence polarization immunoassays.

PubMed

de la Torre, R; Badia, R; Gonzàlez, G; García, M; Pretel, M J; Farré, M; Segura, J

1996-01-01

We investigated the usefulness of immunological methods for presumptive detection of stimulants found in sports drug testing. The ingestion of substances that show no cross-reactivity in tests commercially available for the detection of amphetamines can produce positive results in the urine. Human metabolism contributes to the positive results of some urine samples when the parent compound does not cross-react with the antibodies of the assay. Urine samples from healthy volunteers given stimulants were tested by chromatographic methods and by two different fluorescence polarization immunoassays (FPIA) from Abbott Laboratories for the analysis of amphetamines. According to the results obtained, we classified stimulants into four groups: detectable stimulants that gave rise to amphetamine by human metabolism (group 1); detectable ephedrines and related compounds, appearing in the urine either as parent compounds or originated by metabolism (group 2); detectable stimulants that displayed actual cross-reactivity with amphetamine tests (group 3); and stimulants not detected by FPIA (group 4). Most of the true doping cases due to the ingestion of stimulants may be detected by FPIA. The specificity of the results may be increased by combining immunological assays with different antibodies.

Evaluation of Gamma Interferon and Antibody Tuberculosis Tests in Alpacas

PubMed Central

Holder, Tom; Clifford, Derek; Dexter, Ian; Brewer, Jacky; Smith, Noel; Waring, Laura; Crawshaw, Tim; Gillgan, Steve; Lyashchenko, Konstantin; Lawrence, John; Clarke, John; de la Rua-Domenech, Ricardo; Vordermeier, Martin

2012-01-01

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a “test package,” although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations. PMID:22914362

Evaluation of gamma interferon and antibody tuberculosis tests in alpacas.

PubMed

Rhodes, Shelley; Holder, Tom; Clifford, Derek; Dexter, Ian; Brewer, Jacky; Smith, Noel; Waring, Laura; Crawshaw, Tim; Gillgan, Steve; Lyashchenko, Konstantin; Lawrence, John; Clarke, John; de la Rua-Domenech, Ricardo; Vordermeier, Martin

2012-10-01

We describe the performance of cell-based and antibody blood tests for the antemortem diagnosis of tuberculosis (TB) in South American camelids (SAC). The sensitivity and specificity of the gamma interferon (IFN-γ) release assay, two lateral flow rapid antibody tests (Stat-Pak and Dual Path Platform [DPP]), and two enzyme-linked immunosorbent assay (ELISA)-based antibody tests (Idexx and Enferplex) were determined using diseased alpacas from Mycobacterium bovis culture-confirmed breakdown herds and TB-free alpacas from geographical areas with no history of bovine TB, respectively. Our results show that while the sensitivities of the IFN-γ and antibody tests were similar (range of 57.7% to 66.7%), the specificity of the IFN-γ test (89.1%) was lower than those of any of the antibody tests (range of 96.4% to 97.4%). This lower specificity of the IFN-γ test was at least in part due to undisclosed Mycobacterium microti infection in the TB-free cohort, which stimulates a positive purified protein derivative (PPD) response. The sensitivity of infection detection could be increased by combining two antibody tests, but even the use of all four antibody tests failed to detect all diseased alpacas. These antibody-negative alpacas were IFN-γ positive. We found that the maximum sensitivity could be achieved only by the combination of the IFN-γ test with two antibody tests in a "test package," although this resulted in decreased specificity. The data from this evaluation of tests with defined sensitivity and specificity provide potential options for antemortem screening of SAC for TB in herd breakdown situations and could also find application in movement testing and tracing investigations.

Indirect competitive ELISA based on monoclonal antibody for the detection of 5-hydroxymethyl-2-furfural in milk, compared with HPLC.

PubMed

Guan, Yongguang; Wu, Xinlan; Meng, Hecheng

2013-08-01

In this study, a method for rapid detection of 5-hydroxymethyl-2-furfural (HMF) was investigated. Monoclonal antibody (anti-HMF) was prepared and evaluated by an indirect competitive ELISA (ic-ELISA) format. The optimized standard curve was y=-0.2097x+1.0432 [where x is the logarithm (base 10) of the values of the HMF concentration and y is the absorbance of ic-ELISA results tested at 490 nm] and the linear detection range was 0.008 to 32.768 mg/L. The percentage of cross-reactivity of HMF with 5 major furfural derivatives was less than 2.92%. Finally, the established ic-ELISA format was used to test HMF in milk, and compared with the result obtained by HPLC, which produced an error of about 0.3%. Based on the data in this experiment, we concluded that the established ic-ELISA format was reliable with a high specificity. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Antibody Responses to MAP 1B and Other Cowdria ruminantium Antigens Are Down Regulated in Cattle Challenged with Tick-Transmitted Heartwater

PubMed Central

Semu, S. M.; Peter, T. F.; Mukwedeya, D.; Barbet, A. F.; Jongejan, F.; Mahan, S. M.

2001-01-01

Serological diagnosis of heartwater or Cowdria ruminantium infection has been hampered by severe cross-reactions with antibody responses to related ehrlichial agents. A MAP 1B indirect enzyme-linked immunosorbent assay that has an improved specificity and sensitivity for detection of immunoglobulin G (IgG) antibodies has been developed to overcome this constraint (A. H. M. van Vliet, B. A. M. Van der Zeijst, E. Camus, S. M. Mahan, D. Martinez, and F. Jongejan, J. Clin. Microbiol. 33:2405–2410, 1995). When sera were tested from cattle in areas of endemic heartwater infection in Zimbabwe, only 33% of the samples tested positive in this assay despite a high infection pressure (S. M. Mahan, S. M. Samu, T. F. Peter, and F. Jongejan, Ann. N.Y. Acad. Sci 849:85–87, 1998). To determine underlying causes for this observation, the kinetics of MAP 1B-specific IgG antibodies in cattle after tick-transmitted C. ruminantium infection and following recovery were investigated. Sera collected weekly over a period of 52 weeks from 37 cattle, which were naturally or experimentally infected with C. ruminantium via Amblyomma hebraeum ticks, were analyzed. MAP 1B-specific IgG antibody responses developed with similar kinetics in both field- and laboratory-infected cattle. IgG levels peaked at 4 to 9 weeks after tick infestation and declined to baseline levels between 14 and 33 weeks, despite repeated exposure to infected ticks and the establishment of a carrier state as demonstrated by PCR and xenodiagnosis. Some of the serum samples from laboratory, and field-infected cattle were also analyzed by immunoblotting and an indirect fluorescent-antibody test (IFAT) to determine whether this observed seroreversion was specific to the MAP 1B antigen. Reciprocal IFAT and immunoblot MAP 1-specific antibody titres peaked at 5 to 9 weeks after tick infestation but also declined between 30 and 45 weeks. This suggests that MAP 1B-specific IgG antibody responses and antibody responses to other C

Submicron polymer particles containing fluorescent semiconductor nanocrystals CdSe/ZnS for bioassays.

PubMed

Generalova, Alla N; Sizova, Svetlana V; Zdobnova, Tatiana A; Zarifullina, Margarita M; Artemyev, Michail V; Baranov, Alexander V; Oleinikov, Vladimir A; Zubov, Vitaly P; Deyev, Sergey M

2011-02-01

This study aimed to design a panel of uniform particulate biochemical reagents and to test them in specific bioassays. These reagents are polymer particles of different sizes doped with semiconductor nanocrystals and conjugated with either full-size antibodies or recombinant mini-antibodies (4D5 scFv fragment) designed by genetic engineering approaches. A panel of highly fluorescent polymer particles (150-800 nm) were formed by embedding CdSe/ZnS nanocrystals (quantum dots) into preformed polyacrolein and poly(acrolein-co-styrene) particles. Morphology, content and fluorescence characteristics of the prepared materials were studied by laser correlation spectroscopy, spectrophotometry, optical and fluorescent microscopy and fluorimetry. The obtained fluorescent particles sensitized by anti-Yersinia pestis antibodies were used for rapid agglutination glass test suitable for screening analysis of Y. pestis antigen and for microtiter particle agglutination, which, owing to its speed and simplicity, is very beneficial for diagnostic detection of Y. pestis antigen. Recombinant 4D5 scFv antibodies designed and conjugated with polymer particles containing quantum dots provide multipoint highly specific binding with cancer marker HER2/neu on the surface of SKOV-3 cell.

Comparative analysis of monoclonal antibody N-glycosylation using stable isotope labelling and UPLC-fluorescence-MS.

PubMed

Millán Martín, Silvia; Delporte, Cédric; Farrell, Amy; Navas Iglesias, Natalia; McLoughlin, Niaobh; Bones, Jonathan

2015-03-07

A twoplex method using (12)C6 and (13)C6 stable isotope analogues (Δmass = 6 Da) of 2-aminobenzoic acid (2-AA) is described for quantitative analysis of N-glycans present on monoclonal antibodies and other glycoproteins using ultra performance liquid chromatography with sequential fluorescence and accurate mass tandem quadrupole time of flight (QToF) mass spectrometric detection.

Development and validation of a fluorescent microsphere immunoassay for soluble CD30 testing.

PubMed

Pavlov, Igor; Martins, Thomas B; Delgado, Julio C

2009-09-01

Testing for soluble CD30 (sCD30), an indicator of Th2 immune response, is a useful prognostic marker in solid organ transplantation, lymphoproliferative disorders, autoimmunity, and various parasitic diseases. In this study we report the development and validation of a fluorescent microsphere immunoassay for the detection of sCD30 in serum, plasma, and culture supernatants. The dynamic range of this assay is 1 to 400 ng/ml, and the rate of recovery of various concentrations of recombinant sCD30 ranges from 97 to 116% (average recovery, 105%). The test showed a high degree of precision in both intra-assay and interassay studies (coefficients of variation, as high as 7% and 8%, respectively), with a sensitivity of 1 ng/ml. The normal reference range calculated for a cohort of 151 healthy individuals was 1 to 29 ng/ml. The clinical usefulness of the sCD30 fluorescent microsphere immunoassay was demonstrated by showing that levels of sCD30 have a positive correlation with specimens containing high titers of anti-double-stranded DNA antibodies and high titers of immunoglobulin G against Leishmania species. Given the multiplexing potential of the sCD30 fluorescent microsphere immunoassay reported in this study, it is expected that testing of sCD30 concentrations along with those of other cytokines will become an important diagnostic tool for selected immunological and inflammatory diseases where Th2-type cytokine responses have been reported.

Seroprevalence of Mycoplasma gallisepticum antibody by ELISA and serum plate agglutination test of laying chicken

PubMed Central

Ali, Md. Zulfekar; Rahman, Md. Mostafizer; Sultana, Shirin

2015-01-01

Aim: Mycoplasma gallisepticum (MG) is important avian pathogens responsible for chronic respiratory diseases of chicken and turkeys, which result in large economic loss for the poultry industry. The objectives of this study were determination of seroprevalence of MG antibody of commercial layer chicken at laying period in selected areas of Bangladesh. Materials and Methods: A total of 563 blood samples were collected randomly from selected commercial layer chickens at laying period during the period from July to December, 2013. Indirect enzyme linked immunosorbent assay (iELISA) and serum plate agglutination (SPA) test were performed to detect the presence of antibodies against MG. Results: Of 563 samples, 64.47% and 56.13% showed an overall prevalence of MG antibodies in iELISA and SPA test respectively. Prevalence of MG was recorded the highest (69.63%) at 50-55 weeks of age compared with lowest (53.26%) at 56-61 weeks of age (p<0.05). Significant (p<0.05) effect of breed were observed in the seroprevalence of MG infection in layer birds in the present study. The overall, 68.77%, 63.74% and 59.37% prevalence were found respectively in sonali, ISA Brown and White leg horn. The prevalence of MG antibodies was the highest (70.13%) in December followed by November (68%), October (65.67%), August (63.46%), September (58.54%) and July (51.78%) month. The seroprevalence of MG antibodies was higher (69.63%) in most of the large flocks and lower (56.82%) in small flocks. Conclusion: Therefore, might be suggested that the commercial layer farms should be routinely checked to monitor MG infection and the reactor birds should be culled since MG organism has the potential to transmit vertically. The correlation between MG antibody in month and flock size was not significant (p=0.359 and p=0.868, respectively). PMID:27046987

Feasibility of Using Convalescent Plasma Immunotherapy for MERS-CoV Infection, Saudi Arabia.

PubMed

Arabi, Yaseen M; Hajeer, Ali H; Luke, Thomas; Raviprakash, Kanakatte; Balkhy, Hanan; Johani, Sameera; Al-Dawood, Abdulaziz; Al-Qahtani, Saad; Al-Omari, Awad; Al-Hameed, Fahad; Hayden, Frederick G; Fowler, Robert; Bouchama, Abderrezak; Shindo, Nahoko; Al-Khairy, Khalid; Carson, Gail; Taha, Yusri; Sadat, Musharaf; Alahmadi, Mashail

2016-09-01

We explored the feasibility of collecting convalescent plasma for passive immunotherapy of Middle East respiratory syndrome coronavirus (MERS-CoV) infection by using ELISA to screen serum samples from 443 potential plasma donors: 196 patients with suspected or laboratory-confirmed MERS-CoV infection, 230 healthcare workers, and 17 household contacts exposed to MERS-CoV. ELISA-reactive samples were further tested by indirect fluorescent antibody and microneutralization assays. Of the 443 tested samples, 12 (2.7%) had a reactive ELISA result, and 9 of the 12 had reactive indirect fluorescent antibody and microneutralization assay titers. Undertaking clinical trials of convalescent plasma for passive immunotherapy of MERS-CoV infection may be feasible, but such trials would be challenging because of the small pool of potential donors with sufficiently high antibody titers. Alternative strategies to identify convalescent plasma donors with adequate antibody titers should be explored, including the sampling of serum from patients with more severe disease and sampling at earlier points during illness.

Development of a monoclonal antibody-based indirect competitive immunosorbent assay for 4(5)-Methylimidazole detection in caramels.

PubMed

Wu, Xin-Lan; Yu, Shu-Juan; Kang, Ke-Ren

2015-03-01

In this study, an indirect competitive enzyme-linked immunoassay (ic-ELISA) based on monoclonal antibody for 4(5)-Methylimidazole (4-MI) detection was described. The artificial antigens were prepared by conjugating bovine serum albumin (BSA) or ovalbumin (OVA) with the hapten of 4-MI. And monoclonal antibody, evaluated by ic-ELISA, was obtained by immunizing BABL/c mice. After optimizing, a standard curve for ic-ELISA detection on 4-MI was obtained with the linear detection range of 0.64-20.48 mg/L. The cross-reactivity (CR) of all the structural analogues of 4-MI was less than 5.62%. The recoveries of 4-MI in caramels detection were ranged from 88.69% to 114.09%, with relative standard deviation (n=3) below 8.07%. The results suggested that the established ic-ELISA is promising for 4-MI commercial detection in caramels. Copyright © 2014 Elsevier Ltd. All rights reserved.

A step towards standardization: A method for end-point titer determination by fluorescence index of an automated microscope. End-point titer determination by fluorescence index.

PubMed

Carbone, Teresa; Gilio, Michele; Padula, Maria Carmela; Tramontano, Giuseppina; D'Angelo, Salvatore; Pafundi, Vito

2018-05-01

Indirect Immunofluorescence (IIF) is widely considered the Gold Standard for Antinuclear Antibody (ANA) screening. However, the high inter-reader variability remains the major disadvantage associated with ANA testing and the main reason for the increasing demand of the computer-aided immunofluorescence microscope. Previous studies proposed the quantification of the fluorescence intensity as an alternative for the classical end-point titer evaluation. However, the different distribution of bright/dark light linked to the nature of the self-antigen and its location in the cells result in different mean fluorescence intensities. The aim of the present study was to correlate Fluorescence Index (F.I.) with end-point titers for each well-defined ANA pattern. Routine serum samples were screened for ANA testing on HEp-2000 cells using Immuno Concepts Image Navigator System, and positive samples were serially diluted to assign the end-point titer. A comparison between F.I. and end-point titers related to 10 different staining patterns was made. According to our analysis, good technical performance of F.I. (97% sensitivity and 94% specificity) was found. A significant correlation between quantitative reading of F.I. and end-point titer groups was observed using Spearman's test and regression analysis. A conversion scale of F.I. in end-point titers for each recognized ANA-pattern was obtained. The Image Navigator offers the opportunity to improve worldwide harmonization of ANA test results. In particular, digital F.I. allows quantifying ANA titers by using just one sample dilution. It could represent a valuable support for the routine laboratory and an effective tool to reduce inter- and intra-laboratory variability. Copyright © 2018. Published by Elsevier B.V.

21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5090 Antimitochondrial antibody immunological test system. (a) Identification. An... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antimitochondrial antibody immunological test...

Comparison of Xpert Flu rapid nucleic acid testing with rapid antigen testing for the diagnosis of influenza A and B.

PubMed

DiMaio, Michael A; Sahoo, Malaya K; Waggoner, Jesse; Pinsky, Benjamin A

2012-12-01

Influenza infections are associated with thousands of hospital admissions and deaths each year. Rapid detection of influenza is important for prompt initiation of antiviral therapy and appropriate patient triage. In this study the Cepheid Xpert Flu assay was compared with two rapid antigen tests, BinaxNOW Influenza A & B and BD Directigen EZ Flu A+B, as well as direct fluorescent antibody testing for the rapid detection of influenza A and B. Using real-time, hydrolysis probe-based, reverse transcriptase PCR as the reference method, influenza A sensitivity was 97.3% for Xpert Flu, 95.9% for direct fluorescent antibody testing, 62.2% for BinaxNOW, and 71.6% for BD Directigen. Influenza B sensitivity was 100% for Xpert Flu and direct fluorescent antibody testing, 54.5% for BinaxNOW, and 48.5% for BD Directigen. Specificity for influenza A was 100% for Xpert Flu, BinaxNOW, and BD Directigen, and 99.2% for direct fluorescent antibody testing. All methods demonstrated 100% specificity for influenza B. These findings support the use of the Xpert Flu assay in settings requiring urgent diagnosis of influenza A and B. Copyright © 2012 Elsevier B.V. All rights reserved.

Chikungunya virus RNA and antibody testing at a National Reference Laboratory since the emergence of Chikungunya virus in the Americas.

PubMed

Prince, Harry E; Seaton, Brent L; Matud, Jose L; Batterman, Hollis J

2015-03-01

Since first reported in the Americas in December 2013, chikungunya virus (CHIKV) infections have been documented in travelers returning from the Caribbean, with many cases identified by CHIKV antibody and/or RNA testing at our laboratory. We used our large data set to characterize the relationship between antibody titers and RNA detection and to estimate IgM persistence. CHIKV RNA was measured by nucleic acid amplification and CHIKV IgG/IgM by indirect immunofluorescence. Of the 1,306 samples submitted for RNA testing in January through September 2014, 393 (30%) were positive; for 166 RNA-positive samples, CHIKV antibody testing was also ordered, and 84% were antibody negative. Of the 6,971 sera submitted for antibody testing in January through September 2014, 1,811 (26%) were IgM positive; 1,461 IgM positives (81%) were also IgG positive. The relationship between the CHIKV antibody titers and RNA detection was evaluated using 376 IgM-positive samples (138 with RNA testing ordered and 238 deidentified and tested for RNA). RNA detection showed no significant association with the IgM titer but was inversely related to the IgG titer; 63% of the IgG negative sera were RNA positive, compared to 36% of sera with low IgG titers (1:10 to 1:80) and 16% with IgG titers of ≥1:160. Using second-sample results from 62 seroconverters, we estimated that CHIKV IgM persists for 110 days (95% confidence interval, 78 to 150 days) after the initial antibody-negative sample. These findings indicate that (i) RNA detection is more sensitive than antibody detection early in CHIKV infection, (ii) in the absence of RNA results, the IgG titer of the IgM-positive samples may be a useful surrogate for viremia, and (iii) CHIKV IgM persists for approximately 4 months after symptom onset. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Utility of HLA Antibody Testing in Kidney Transplantation

PubMed Central

Konvalinka, Ana

2015-01-01

HLA antigens are polymorphic proteins expressed on donor kidney allograft endothelium and are critical targets for recipient immune recognition. HLA antibodies are risk factors for acute and chronic rejection and allograft loss. Solid-phase immunoassays for HLA antibody detection represent a major advance in sensitivity and specificity over cell-based methods and are widely used in organ allocation and pretransplant risk assessment. Post-transplant, development of de novo donor–specific HLA antibodies and/or increase in donor-specific antibodies from pretransplant levels are associated with adverse outcomes. Although single antigen bead assays have allowed sensitive detection of recipient HLA antibodies and their specificities, a number of interpretive considerations must be appreciated to understand test results in clinical and research contexts. This review, which is especially relevant for clinicians caring for transplant patients, discusses the technical aspects of single antigen bead assays, emphasizes their quantitative limitations, and explores the utility of HLA antibody testing in identifying and managing important pre- and post-transplant clinical outcomes. PMID:25804279

Cross-Sectional Serosurvey and Risk Factors Associated with the Presence of Toxoplasma gondii Antibodies in Pigs in Greece.

PubMed

Papatsiros, Vasileios G; Athanasiou, Labrini V; Stougiou, Despina; Papadopoulos, Elias; Maragkakis, Giorgios G; Katsoulos, Panagiotis D; Lefkaditis, Menelaos; Kantas, Dimitrios; Tzika, Eleni D; Tassis, Panagiotis D; Boutsini, Sofia

2016-01-01

Toxoplasmosis is a worldwide reported zoonotic infection caused by the protozoon Toxoplasma gondii. Pigs may become infected by ingesting feed or water contaminated with cat faeces, by cannibalism, and/or by eating infected rodents. T. gondii infected edible tissues of pigs are a source of infection for humans. This study was undertaken to detect seropositivity of pigs against T. gondii and identify possible risk factors as well as to compare two serological methods. A total of 609 blood samples were collected from 10% of the sows from 65 pig farms located in mainland Greece. Information about the geographical location, size, and biosecurity level of the herd was recorded. Samples were tested for the presence of antibodies against T. gondii employing the indirect fluorescent antibody test and the enzyme-linked immunosorbent assay. Twenty-six positive samples were detected (4.3%) in 17 out of the 65 farms tested (26.2%). The average seroprevalence in affected herds was 26.8% (95% confidence interval 21.0-32.6%). Among the categorical data evaluated, the biosecurity level (odds ratio 0.17, p < 0.01) and the geographical characteristics (odds ratio 13.55, p < 0.05) significantly affected the presence of toxoplasmosis in the herd. Toxoplasmosis was recorded in significantly higher percentages in mountainous farms as compared to lowlands (p < 0.05) and in those with poor biosecurity levels than in those with very good (p < 0.05). A very good agreement (κ = 0.958) was observed between the two serological methods. The presence of antibodies in pigs is indirect information on the risk of the infection and an indication of the necessity of biosecurity measures to be taken in order to control infection at least in the areas at highest risk.

Adaptive virus detection using filament-coupled antibodies.

PubMed

Stone, Gregory P; Lin, Kelvin S; Haselton, Frederick R

2006-01-01

We recently reported the development of a filament-antibody recognition assay (FARA), in which the presence of virions in solution initiates the formation of enzyme-linked immunosorbent assay (ELISA)-like antibody complexes. The unique features of this assay are that processing is achieved by motion of a filament and that, in the presence of a virus, antibody-virus complexes are coupled to the filament at known locations. In this work, we combine the unique features of this assay with a 638-nm laser-based optical detector to enable adaptive control of virus detection. Integration of on-line fluorescence detection yields approximately a five-fold increase in signal-to-noise ratio (SNR) compared to the fluorescence detection method reported previously. A one-minute incubation with an M13K07 test virus is required to detect 10(10) virionsml, and 40 min was required to detect 10(8) virionsml. In tests of the components of an adaptive strategy, a 30-min virus (3.3 x 10(10) virionsml) incubation time, followed by repositioning the filament-captured virus either within the detecting antibody chamber, (20 microg ml) or within the virus chamber, found an increase in signal roughly proportional to the cumulative residence times in these chambers. Furthermore, cumulative fluorescence signals observed for a filament-captured virus after repeated positioning of the filament within the virus chamber are similar to those observed for a single long incubation time. The unique features of the FARA-like design combined with online optical detection to direct subsequent bioprocessing steps provides new flexibility for developing adaptive molecular recognition assays.

An intradermal skin test for determination of immunity to varicella

PubMed Central

Somekh, E; Bujanover, Y; Tal, G; Dalal, I; Tanay, A; Lehman, D

2001-01-01

AIMS—To evaluate the usefulness of a diluted, inactivated solution of attenuated varicella vaccine in predicting susceptibility to varicella and its correlation with specific antibody titre to varicella.
METHODS—In a prospective blinded study, 63 healthy subjects (aged 2-43 years) were studied. Skin test solution was prepared from vials of OKA strain virus which was inactivated by exposure of the vials to room temperature for 10 days; solution was diluted at 1/50 with normal saline and kept at 4°C until used for skin testing. The material was injected intradermally. Serum samples were drawn prior to skin testing and kept at −70°C until analysis for antibody assay by the indirect fluorescent antibody (IFA) method.
RESULTS—Forty three patients were IFA antibody positive; 41 of them reacted to the skin test. One of the 20 IFA negative patients reacted to the skin test. Sixteen patients had two serological tests performed, one month apart. Four out of these 16 patients tested negative with the skin test. All four had negative serology on both samples. Six of the 12 IFA positive patients showed a boost in the antibody titre one month after application of the skin test. The specificity and sensitivity of the skin test compared to the IFA assay were both 95%, and the positive and negative predictive values were 97% and 90% respectively.
CONCLUSIONS—Results suggest that a varicella skin test prepared using this simple and relatively cheap method is a safe, sensitive, and specific tool by which to assess immunity to varicella.

 PMID:11719333

Immunofluorescent detection in the ovary of host antibodies against a secretory ferritin injected into female Haemaphysalis longicornis ticks.

PubMed

Galay, Remil Linggatong; Matsuo, Tomohide; Hernandez, Emmanuel Pacia; Talactac, Melbourne Rio; Kusakisako, Kodai; Umemiya-Shirafuji, Rika; Mochizuki, Masami; Fujisaki, Kozo; Tanaka, Tetsuya

2018-04-01

Due to the continuous threat of ticks and tick-borne diseases to human and animal health worldwide, and the drawbacks of chemical acaricide application, many researchers are exploring vaccination as an alternative tick control method. Earlier studies have shown that host antibodies can circulate in the ticks, but it has not been confirmed whether these antibodies can be passed on to the eggs. We previously reported that ticks infesting rabbits immunized with a recombinant secretory ferritin of Haemaphysalis longicornis (HlFER2) had reduced egg production and hatching. Here we attempted to detect the presence of antibodies against HlFER2 in the ovary and eggs of female ticks through immunofluorescent visualization. Purified anti-HlFER2 antibodies or rabbit IgG for control was directly injected to engorged female H. longicornis. Ovaries and eggs after oviposition were collected and prepared for an indirect immunofluorescent antibody test. Positive fluorescence was detected in ovaries one day post-injection of anti-HlFER2 antibodies. Through silencing of Hlfer2 gene, we also determined whether the injected antibodies can specifically bind to native HlFER2. Immunofluorescence was observed in the oocytes of dsLuciferase control ticks injected with anti-HlFER2 antibodies, but not in the oocytes of Hlfer2-silenced ticks also injected with anti-HlFER2 antibodies. Our current findings suggest that host antibodies can be passed on to the oocytes, which is significant in formulating a vaccine that can disrupt tick reproduction. Copyright © 2017 Elsevier B.V. All rights reserved.

Membrane filtration – Fluorescent antibody staining procedure for detecting and quantifying Renibacterium salmoninarum in coelomic fluid of Chinook Salmon (Oncorhynchus tshawytscha)

USGS Publications Warehouse

Elliott, D.G.; Barila, T.Y.

1987-01-01

We developed a rapid method for detecting and quantifying the pathogen Renibacterium salmoninarum in coelomic fluid of spring chinook salmon (Oncorhynchus tshawytscha) by concentrating the bacteria on 0.2-μm polycarbonate filters and staining them with specific fluorescein-labeled antibody. Centrifugation of samples and resuspension of the sedimented material in phosphate-buffered saline containing Triton X-100 increased the ease of filtration. Background fluorescence was reduced by counterstaining filters with Eriochrome black T. Postfiltration staining, rinsing, and counterstaining were done in the syringe-mounted filter holders, reducing handling of the filters and possible loss of bacteria. The number of bacteria detected by the filtration – fluorescent antibody technique in a broth culture of R. salmoninarum ranged from 6.7 × 107to7.6 × 107/mL and was slightly higher than that determined by plate count (9.6 × 106/mL). Increasing the sample dilution or decreasing the number of microscope fields examined generally increased the variability of filter counts of R. salmoninarum. Using the filtration – fluorescent antibody technique, we detected the bacterium in the coelomic fluid of 85% of spawning female spring chinook salmon sampled from a hatchery population.

Development of a fluorescent immnunochromatographic assay for the procalcitonin detection of clinical patients in China.

PubMed

Wang, Haiying; Wang, Hong; Chen, Shaopei; Dzakah, Emmanuel E; Kang, Keren; Wang, Jihua; Wang, Jufang

2015-04-15

Procalcitonin (PCT) has been recognized as a biomarker in severe inflammation, infection and sepsis. PCT detection in serum requires sensitive and specific antibodies. In this study, we generated monoclonal antibodies (mAbs) and developed fluorescent immunochromatographic assay for PCT detection. Human recombinant PCT was used as immunogen. mAbs against PCT were developed and applied to fluorescent immunochromatographic assay for PCT detection in clinical samples. Out of 35 hybridoma cell lines secreting antibodies against the recombinant PCT, five sensitive and specific cell lines were selected and designated as F6, G2, C2, D2 and E5. All these antibodies have no cross reaction with calcitonin or calcitonin gene-related peptides (CGRP). After screening for pairing, mAb F6 was labeled with fluorescent microspheres and C2 was coated on a nitrocellulose membrane for immunochromatographic test. All 35 clinical samples were detected by the mAb F6-C2 test strips and the bioMérieux PCT assay. The test strips showed high specificity and sensitivity for PCT. Good correlation was observed between our immunochromatographic test strips and the bioMérieux PCT assay (R(2):0.986). These newly developed anti-PCT mAbs and fluorescent immunochromatographic assay can serve as important diagnostic tools for a fast, reliable and point-of-care testing for easy determination of PCT in serum and diagnosis of bacterial infection, inflammation or sepsis. Copyright © 2015 Elsevier B.V. All rights reserved.

Variations in the detection of anti-PEDV antibodies in serum samples using three diagnostic tests - short communication.

PubMed

Plut, Jan; Toplak, Ivan; Štukelj, Marina

2018-06-01

Over the last few years several porcine epidemic diarrhoea (PED) outbreaks have been discovered in Europe including the first PED case in Slovenia in January 2015. The aim of this study was to determine when PED virus (PEDV) infection started in Slovenia. Serum samples collected between 2012 and 2016 were tested. Three hundred and seventy-five serum samples were collected from 132 Slovenian small, one-site pig farms. Samples were tested for PEDV antibodies utilising three different serological methods: commercially-available indirect ELISA, in-house blocking ELISA test and Immunoperoxidase Monolayer Assay (IPMA) test. One hundred and seventy (45.33%) tested samples were found positive by the commercially-available ELISA test kit, and 10 (5.68%) of these 170 samples found positive were positive by the in-house blocking ELISA. Only these 10 samples were collected from a farm where clinical signs of PED infection had been observed and PEDV was confirmed by RT-PCR methodology; the other 160 samples were collected randomly. Thirty-two samples with the highest S/P value obtained with the commercial ELISA were all negative with IPMA. Reasons for the high variance in the results obtained remain unclear; more research is required to ensure higher sensitivity and specificity in terms of PEDV antibody tests and other PED diagnostic methods.

Evaluation of a newly developed quantitative heart-type fatty acid binding protein assay based on fluorescence immunochromatography using specific monoclonal antibodies.

PubMed

Kang, Keren; Wu, Peidian; Li, Wenmei; Tang, Shixing; Wang, Jihua; Luo, Xiaochun; Xie, Mingquan

2015-01-01

To develop a rapid, sensitive and specific assay for quantification of serum heart-type fatty acid binding protein (H-FABP) based on immunofluorescence of specific monoclonal antibodies. We generated novel H-FABP-directed monoclonal antibodies by cloning of spleen cells of mice immunized with H-FABP. Epitopes were mapped and antigen affinity was assessed by surface plasmon resonance (SPR). The H-FABP specific monoclonal antibodies were coupled to fluorescent beads and sprayed onto a nitrocellulose membrane facilitating quantification of H-FABP by immunofluorescence. Reagent cross-reactivity, interference resistance, accuracy and sensitivity were examined. A total of 103 clinical samples were used to compare the sensitivity and specificity of the new assay to a commercially available Randox kit. This new assay could be finished within 15 min, with sensitivity reaching 1 ng/ml. In a trial of 103 clinical serum samples, the new testing kit results were highly correlated with those from the Randox kit (R(2) = 0.9707). Using the Randox kit as the reference kit, the sensitivity of the new assay was 98.25%, and specificity was 100%. An immunofluorescence-based H-FABP assay employing novel monoclonal antibodies could rapidly, specifically and sensitively detect H-FABP in serum samples, providing an effective method for rapid clinical assessment of H-FABP index in the clinic.

Visual Detection of Human Antibodies Using Sugar Chain-Immobilized Fluorescent Nanoparticles: Application as a Point of Care Diagnostic Tool for Guillain-Barré Syndrome.

PubMed

Shinchi, Hiroyuki; Yuki, Nobuhiro; Ishida, Hideharu; Hirata, Koichi; Wakao, Masahiro; Suda, Yasuo

2015-01-01

Sugar chain binding antibodies have gained substantial attention as biomarkers due to their crucial roles in various disorders. In this study, we developed simple and quick detection method of anti-sugar chain antibodies in sera using our previously developed sugar chain-immobilized fluorescent nanoparticles (SFNPs) for the point-of-care diagnostics. Sugar chain structure on SFNPs was modified with the sugar moieties of the GM1 ganglioside via our original linker molecule to detect anti-GM1 antibodies. The structures and densities of the sugar moieties immobilized on the nanoparticles were evaluated in detail using lectins and sera containing anti-GM1 antibodies from patients with Guillain-Barré syndrome, a neurological disorder, as an example of disease involving anti-sugar chain antibodies. When optimized SFNPs were added to sera from patients with Guillain-Barré syndrome, fluorescent aggregates were able to visually detect under UV light in three hours. The sensitivity of the detection method was equivalent to that of the current ELISA method used for the diagnosis of Guillain-Barré syndrome. These results suggest that our method using SFNPs is suitable for the point-of-care diagnostics of diseases involving anti-sugar chain antibodies.

The variability of the indirect tensile stripping test.

DOT National Transportation Integrated Search

1990-01-01

The purpose of this investigation was to determine the variability of the Virginia Department of Transportation's (VDOT) indirect tensile stripping test. Five contractor labs and eight VDOT labs participated in the study. Each lab performed three rep...

Comparison of the membrane-filtration fluorescent antibody test, the enzyme-linked immunosorbent assay, and the polymerase chain reaction to detect Renibacterium salmoninarum in salmon ovarian fluid

USGS Publications Warehouse

Pascho, Ronald J.; Chase, Dorothy M.; McKibben, Constance L.

1998-01-01

Ovarian fluid samples from naturally infected chinook salmon (Oncorhynchus tshawytscha) were examined for the presence of Renibacterium salmoninarum by the membrane-filtration fluorescent antibody test (MF-FAT), an antigen capture enzyme-linked immunosorbent assay (ELISA), and a nested polymerase chain reaction (PCR). On the basis of the MF-FAT, 64% (66/103) samples contained detectable levels of R. salmoninarum cells. Among the positive fish, the R. salmoninarum concentrations ranged from 25 cells/ml to 4.3 × 109cells/ml. A soluble antigenic fraction of R. salmoninarum was detected in 39% of the fish (40/103) by the ELISA. The ELISA is considered one of the most sensitive detection methods for bacterial kidney disease in tissues, yet it did not detect R. salmoninarum antigen consistently at bacterial cell concentrations below about 1.3 × 104cells/ml according to the MF-FAT counts. When total DNA was extracted and tested in a nested PCR designed to amplify a 320-base-pair region of the gene encoding a soluble 57-kD protein of R. salmoninarum, 100% of the 100 samples tested were positive. The results provided strong evidence that R. salmoninarum may be present in ovarian fluids thought to be free of the bacterium on the basis of standard diagnostic methods.

Fluorescence correlation spectroscopy to study antibody binding and stoichiometry of complexes

NASA Astrophysics Data System (ADS)

Swift, Kerry M.; Matayoshi, Edmund D.

2008-02-01

FCS (fluorescence correlation spectroscopy) was used to study the association at the single molecule level of tumor necrosis factor alpha (TNF-α) and two of its protein antagonists Humira (TM) (adalimumab), a fully humanized monoclonal antibody, and Enbrel (TM) (etanercept), a soluble form of the TNF receptor. Single molecule approaches potentially have the advantage not only of enhanced sensitivity, but also of observing at equilibrium the details that would otherwise be lost in classical ensemble experiments where heterogeneity is averaged. We prepared fluorescent conjugates of the protein drugs and their biological target, the trimeric soluble form of TNF-α. The bivalency of adalimumab and the trimeric nature of TNF-α potentially allow several forms of associative complexes that may differ in stoichiometry. Detailed knowledge of this reaction may be relevant to understanding adalimumab's pharmacological properties. Our FCS data showed that a single trimeric TNF-α can bind up to three adalimumab molecules. Under some conditions even larger complexes are formed, apparently the result of cross-linking of TNF-α trimers by adalimumab. In addition, distinct differences between Humira and Enbrel were observed in their association with TNF-α.

Development and evaluation of an enzyme-labeled antibody test for the rapid detection of hog cholera antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saunders, G.C.

1977-01-01

A rapid enzyme-labeled antibody (ELA) microtechnique for the screening of swine for hog cholera antibodies was developed and evaluated with a blind study, using a 640-sample hog cholera serum bank. The total time to run a group of 22 samples was approximately 1 hour. The ELA test results correlated >99% with hog cholera serum-neutralization test results on the same serums. Test results also indicated that the ELA test shares with the hog cholera serum-neutralization test the problem of cross reactions between the antibodies of hog cholera and bovine viral diarrhea.

Antibody testing against canine coronavirus by immunoperoxidase plaque staining.

PubMed

Soma, T; Hara, M; Ishii, H; Yamamoto, S

2001-05-01

The application of the immunoperoxidase (IP) plaque staining procedure (IP test) to the diagnosis of canine coronavirus (CCV) infection was investigated. The IP test did not react with sera from either 15 specific pathogen-free (SPF) dogs or 7 SPF dogs immunized with a multivalent vaccine, including canine parvovirus type 2, canine distemper virus, canine adenovirus type 2, and canine parainfluenza virus. To compare the IP test with the neutralizing test (NT), sera from 240 healthy dogs and from 3 experimentally CCV-infected dogs were examined. All 60 sera positive for NT antibody were positive for IP antibody, and all 180 sera negative for NT antibody were negative for IP antibody in the healthy dogs. The IP titres showed similar changes with time after CCV inoculation to those of the NT titres in the experimentally infected dogs. These findings indicate that the IP test specifically detected anti-CCV antibodies. When the IP test and NT were compared in dogs with diarrhoeic signs. 2.1% of 48 sera and 20.3% of 74 sera, which were all negative for NT antibody, were positive for IP antibody in the dogs of under one year of age and at least one year of age, respectively. The difference between the IP and NT titres (log10 [reciprocal of IP titre] log10 [reciprocal of NT titre]) for the diarrhoeic dogs of under one year of age (2.350 +/- 0.931) was significantly larger than that for the healthy dogs (0.982 +/- 0.447) (p<0.0001), the NT titre being negative or very low, despite a high IP titre in many diarrhoeic dogs. Hence, the IP test is more able to detect anti-CCV antibodies, especially in dogs showing clinical signs. The IP-positivity rate was significantly higher in the diarrhoeic dogs of under one year of age (48.7%) than in the healthy dogs (25.0%) (chi2 = 19.844, p<0.0001), suggesting that CCV may contribute to diarrhoea in many juvenile dogs.

Two-photon fluorescence anisotropy imaging

NASA Astrophysics Data System (ADS)

Li, Wei; Wang, Yi; Shao, Hanrong; He, Yonghong; Ma, Hui

2006-09-01

We have developed a novel method for imaging the fluorescence intensity and anisotropy by two-photon fluorescence microscopy and tested its capability in biological application. This method is applied to model sample including FITC and FITC-CD44 antibody solution and also FITC-CD44 stained cells. The fluorescence anisotropy (FA) of FITC-CD44ab solution is higher than the FITC solution with the same concentration. The fluorescence in cell sample has even higher FA than in solution because the rotation diffusion is restrained in membrane. The method is employed to study the effect of berberine a kind of Chinese medicine, on tumor metastasis. The results indicated that tumor cell membrane fluidity is decreasing with increasing the concentration of berberine in culture medium.

Multiplex Staining by Sequential Immunostaining and Antibody Removal on Routine Tissue Sections.

PubMed

Bolognesi, Maddalena Maria; Manzoni, Marco; Scalia, Carla Rossana; Zannella, Stefano; Bosisio, Francesca Maria; Faretta, Mario; Cattoretti, Giorgio

2017-08-01

Multiplexing, labeling for multiple immunostains in the very same cell or tissue section in situ, has raised considerable interest. The methods proposed include the use of labeled primary antibodies, spectral separation of fluorochromes, bleaching of the fluorophores or chromogens, blocking of previous antibody layers, all in various combinations. The major obstacles to the diffusion of this technique are high costs in custom antibodies and instruments, low throughput, and scarcity of specialized skills or facilities. We have validated a method based on common primary and secondary antibodies and diffusely available fluorescent image scanners. It entails rounds of four-color indirect immunofluorescence, image acquisition, and removal (stripping) of the antibodies, before another stain is applied. The images are digitally registered and the autofluorescence is subtracted. Removal of antibodies is accomplished by disulfide cleavage and a detergent or by a chaotropic salt treatment, this latter followed by antigen refolding. More than 30 different antibody stains can be applied to one single section from routinely fixed and embedded tissue. This method requires a modest investment in hardware and materials and uses freeware image analysis software. Multiplexing on routine tissue sections is a high throughput tool for in situ characterization of neoplastic, reactive, inflammatory, and normal cells.

Anti-ENA antibody profiles in patients with hepatitis C virus infection.

PubMed

Litwin, Christine M; Rourk, Angela R

2018-03-01

The presence of antinuclear antibodies (ANA) has been described following hepatitis C virus (HCV). Very few studies have investigated the presence of anti-extractable nuclear antigens (ENA) in HCV infection. The aim of this study was to assess the prevalence of ENA antibodies in 100 patients with HCV infection compared to the prevalence of ENA antibodies in 100 healthy control patients. Sera from patients were tested for ENA using a multiplex microbead immunoassay. Sera positive for ENA were subsequently tested for ANA using an indirect immunofluorescence assay and titered if positive. Fourteen (14%) of the 100 patients with HCV infection had anti-ENA antibodies: four each showed anti-SSA antibodies and anti-dsDNA antibodies, three each had RNP antibodies and Scl-70 antibodies, and one each had anti-SSB, centromere B, Sm, and Sm/RNP antibodies. Ten of the 14 patients positive for anti-ENA were positive by indirect immunofluorescence staining (IFA) with titers ranging from 1:40 to 1:160. Five had antinuclear patterns, one had combined antinuclear and cytoplasmic patterns, and four only had a cytoplasmic pattern. Three of the 100 healthy control patients had ANA positive titers (1:80 and 1:320) and anti-ENA antibodies: one anti-Scl-70 and two anti-RNP. The prevalence of anti-ENA antibodies was significantly higher in the patients with HCV infections than in the healthy controls. Other studies of anti-ENA profiles in patients with HCV infection have identified similar patterns of positivity for anti-SSA, anti-SSB, anti-dsDNA, anti-RNP, anti- Sm/RNP, Scl-70, centromere B, and anti-Sm. © 2017 Wiley Periodicals, Inc.

The impact of milk handling procedures on Ostertagia ostertagi antibody ELISA test results.

PubMed

Vanderstichel, Raphaël; Dohoo, Ian; Stryhn, Henrik

2010-04-19

The impact of various milk handling stressors were analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) test measuring Ostertagia ostertagi antibodies in milk from dairy cattle (Svanovir). An indirect ELISA has the ability to determine the amount of milk production losses related to intestinal parasitism. The ELISA test recommends fresh defatted milk, however, milk collected from Dairy Herd Improvement (DHI) programs in North America undergo many stressors, including, heating, freezing and are not defatted. Normalized optical density ratios (ODRs) were compared between fresh defatted milk and milk subjected to one or more stressors with a linear mixed model accounting for differences in variation between the fresh and the frozen samples. Concordance correlation coefficients were also analyzed for comparisons to other similar studies. After accounting for random cow and container effects, the treatment factors interacted with each other (p<0.001). Biologically interesting contrasts were created to explain the interaction. The estimated difference in ODR between the milk samples handled according to recommendations of the manufacturers of Svanovir and the whole milk samples that were subjected to the most extreme treatment (heated, frozen, thawed, and re-frozen for 4 weeks) was 0.062 (p<0.001). This difference represented less than 5% of the range, and was thus considered biologically negligible. Frozen whole milk processed by DHI programs, the most likely method of collecting on-farm samples in North America, will likely yield reliable results for the indirect ELISA tests, particularly, Svanovir.

Radionuclide and Fluorescence Imaging of Clear Cell Renal Cell Carcinoma Using Dual Labeled Anti-Carbonic Anhydrase IX Antibody G250.

PubMed

Muselaers, Constantijn H J; Rijpkema, Mark; Bos, Desirée L; Langenhuijsen, Johan F; Oyen, Wim J G; Mulders, Peter F A; Oosterwijk, Egbert; Boerman, Otto C

2015-08-01

Tumor targeted optical imaging using antibodies labeled with near infrared fluorophores is a sensitive imaging modality that might be used during surgery to assure complete removal of malignant tissue. We evaluated the feasibility of dual modality imaging and image guided surgery with the dual labeled anti-carbonic anhydrase IX antibody preparation (111)In-DTPA-G250-IRDye800CW in mice with intraperitoneal clear cell renal cell carcinoma. BALB/c nu/nu mice with intraperitoneal SK-RC-52 lesions received 10 μg DTPA-G250-IRDye800CW labeled with 15 MBq (111)In or 10 μg of the dual labeled irrelevant control antibody NUH-82 (20 mice each). To evaluate when tumors could be detected, 4 mice per group were imaged weekly during 5 weeks with single photon emission computerized tomography/computerized tomography and the fluorescence imaging followed by ex vivo biodistribution studies. As early as 1 week after tumor cell inoculation single photon emission computerized tomography and fluorescence images showed clear delineation of intraperitoneal clear cell renal cell carcinoma with good concordance between single photon emission computerized tomography/computerized tomography and fluorescence images. The high and specific accumulation of the dual labeled antibody conjugate in tumors was confirmed in the biodistribution studies. Maximum tumor uptake was observed 1 week after inoculation (mean ± SD 58.5% ± 18.7% vs 5.6% ± 2.3% injected dose per gm for DTPA-G250-IRDye800CW vs NUH-82, respectively). High tumor uptake was also observed at other time points. This study demonstrates the feasibility of dual modality imaging with dual labeled antibody (111)In-DTPA-G250-IRDye800CW in a clear cell renal cell carcinoma model. Results indicate that preoperative and intraoperative detection of carbonic anhydrase IX expressing tumors, positive resection margins and metastasis might be feasible with this approach. Copyright © 2015 American Urological Association Education and Research

Production, characterization and application of monoclonal antibody against immunoglobulin D heavy chain of flounder (Paralichthys olivaceus).

PubMed

Tang, Xiaoqian; Liu, Fuguo; Sheng, Xiuzhen; Xing, Jing; Zhan, Wenbin

2017-05-01

Immunoglobulin D (IgD) is considered to be an enigmatic Ig molecule because of the lack understanding of its immunological functions. In the present study, a partial δ region of the flounder IgD was recombinantly expressed, purified and used as an immunogen to produce monoclonal antibodies (MAbs) against the H chain of flounder IgD. After fusion, a total of 97 hybridomas were generated and observed under an inverted microscope One of the hybridomas, designated 5G7, gave strong positive results in an indirect enzyme-linked immunosorbent assay (ELISA) and was cloned and subcloned by limiting dilution. Western blot analysis showed that MAb 5G7 could specifically recognize a 118 kDa protein from peripheral blood lymphocytes (PBLs), which was identified to be the H chain of flounder IgD by mass spectrometric analysis. Indirect immunofluorescence assay tests (IIFAT) showed that specific fluorescence signals were observed on the membranes of the PBLs, which suggests that MAb 5G7 could recognize the membrane-bound IgD molecule. Moreover, only the subset of IgD+/IgM + B cells were observed in the PBLs of healthy flounder when tested by flow cytometry analysis. Consistent with the results of flow cytometry, a double immunofluorescence assay test (DIFAT) showed that the positive lymphocytes were stained with both green and red fluorescence signals, which represent the IgM+/IgD + lymphocytes subset. These results demonstrate that the produced MAb 5G7 could specifically recognize the flounder IgD, which provides a useful tool to study the functions of flounder IgD. Copyright © 2017 Elsevier Ltd. All rights reserved.

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus.

PubMed

Canakoglu, Nurettin; Berber, Engin; Ertek, Mustafa; Yoruk, Mustafa D; Tonbak, Sukru; Bolat, Yusuf; Aktas, Munir; Kalkan, Ahmet; Ozdarendeli, Aykut

2013-01-03

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV. Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies. Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed. The pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the measurement of CCHF neutralizing antibodies

Pseudo-plaque reduction neutralization test (PPRNT) for the measurement of neutralizing antibodies to Crimean-Congo hemorrhagic fever virus

PubMed Central

2013-01-01

Background Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus of the genus Nairovirus family Bunyaviridae, which are enveloped viruses containing tripartite, negative polarity, single-stranded RNA. CCHF is characterized by high case mortality, occurring in Asia, Africa, the Middle East and Europe. Currently, there are no specific treatments or licensed vaccines available for CCHFV. Recently, two research groups have found adult mice with defective interferon responses allowed to lethal CCHFV infection. These mouse models could provide invaluable information for further studies. Efforts to develop a vaccine against CCHFV are being made. To determine the efficacy of vaccine candidates it is important to conduct serological studies that can accurately measure levels of protective antibodies. In the present study, a pseudo-plaque reduction neutralization test (PPRNT) based on enzyme-catalyzed color development of infected cells probed with anti-CCHFV antibodies was used to measure neutralization antibody of CCHFV. Methods Sixty-nine human serum samples (20 acute and 49 convalescent) were tested. The presence of CCHFV antibodies was determined and confirmed by a commercial ELISA kit. CCHFV RNA was determined by RT-PCR. All the samples were analyzed by PPRNT and fluorescent focus reduction neutralization test (FFRNT) to measure of CCHFV-neutralizing antibodies. Results Pseudo-plaque reduction neutralization test showed a high sensitivity (98%), specificity (100%) and agreement (96,6%) in qualitative comparison with those of the FFRNT. There was a high correlation between the titers obtained in PPRNT and FFRNT (R2 = 0.92). The inter- and intra-assay variation of PPRNT revealed good reproducibility and positive cut-off of PPRNT was defined as 1:4 by the geometric mean titers for the individual samples distributed. Conclusion The pseudo-plaque reduction neutralization test described in this study is a fast, reproducible and sensitive method for the

Specific IgA and IgG antibodies in paired serum and breast milk samples in human strongyloidiasis.

PubMed

Mota-Ferreira, Daniela M L; Gonçalves-Pires, Maria do Rosário F; Júnior, Alvaro Ferreira; Sopelete, Mônica C; Abdallah, Vânia O S; Costa-Cruz, Julia M

2009-02-01

Strongyloidiasis, caused by the nematode Strongyloides stercoralis, is one of the major worldwide parasitic infections in humans. Breastfeeding may offer a potential protection against this infection. Feces, serum and milk samples were obtained from 90 lactating women from Clinical Hospital of Universidade Federal de Uberlândia, Brazil. The fecal samples were collected for parasitological diagnosis and the serum and milk samples were examined for specific S. stercoralis IgA and IgG antibodies using the indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA). Fecal examination showed that the rate of prevalence of S. stercoralis infection in the lactating women was 4.4%. IFAT manifested a 16.7% positivity rate for specific IgA antibody in serum and a 28.9% rate in milk samples; specific IgG was 41.1% in serum and 25.5% in milk samples. According to ELISA the positivity rate for specific IgA antibody was 21.1% in serum and 42.2% in milk samples; specific IgG was 40% in serum and 18.9% in milk samples. In serum samples, these immunological tests showed a concurrence of 91.1% and 94.4%, respectively, in detecting specific IgA and IgG antibodies. In milk samples, they showed a concurrence of 70% and 78.9%, respectively, in detecting specific IgA and IgG antibodies. There was a statistically significant difference between concordant and discordant results of immunological tests (P<0.0001). IFAT and ELISA highly concurred in their detection of specific S. stercoralis IgA and IgG antibodies in serum and in milk samples reconfirming prior studies that the serological method is a complement to the direct diagnosis of the parasite, and suggesting that immunological methods using milk samples can also be helpful. Furthermore, in endemic areas, infants may acquire antibodies to S. stercoralis from breast milk, possibly, contributing to the enhancement of specific mucosal immunity against this parasite.

A fluorescence quenching test for the detection of flavonoid transformation.

PubMed

Schoefer, L; Braune, A; Blaut, M

2001-11-13

A novel fluorescence quenching test for the detection of flavonoid degradation by microorganisms was developed. The test is based on the ability of the flavonoids to quench the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH). Several members of the anthocyanidins, flavones, isoflavones, flavonols, flavanones, dihydroflavanones, chalcones, dihydrochalcones and catechins were tested with regard to their quenching properties. The anthocyanidins were the most potent quenchers of DPH fluorescence, while the flavanones, dihydroflavanones and dihydrochalcones, quenched the fluorescence only weakly. The catechins had no visible impact on DPH fluorescence. The developed test allows a quick and easy differentiation between flavonoid-degrading and flavonoid-non-degrading bacteria. The investigation of individual reactions of flavonoid transformation with the developed test system is also possible.

21 CFR 864.9175 - Automated blood grouping and antibody test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... Manufacture Blood and Blood Products § 864.9175 Automated blood grouping and antibody test system. (a) Identification. An automated blood grouping and antibody test system is a device used to group erythrocytes (red... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Automated blood grouping and antibody test system...

21 CFR 864.9175 - Automated blood grouping and antibody test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... Manufacture Blood and Blood Products § 864.9175 Automated blood grouping and antibody test system. (a) Identification. An automated blood grouping and antibody test system is a device used to group erythrocytes (red... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Automated blood grouping and antibody test system...

21 CFR 864.9175 - Automated blood grouping and antibody test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Manufacture Blood and Blood Products § 864.9175 Automated blood grouping and antibody test system. (a) Identification. An automated blood grouping and antibody test system is a device used to group erythrocytes (red... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Automated blood grouping and antibody test system...

21 CFR 864.9175 - Automated blood grouping and antibody test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... Manufacture Blood and Blood Products § 864.9175 Automated blood grouping and antibody test system. (a) Identification. An automated blood grouping and antibody test system is a device used to group erythrocytes (red... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Automated blood grouping and antibody test system...

21 CFR 864.9175 - Automated blood grouping and antibody test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... Manufacture Blood and Blood Products § 864.9175 Automated blood grouping and antibody test system. (a) Identification. An automated blood grouping and antibody test system is a device used to group erythrocytes (red... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Automated blood grouping and antibody test system...

Detection of koi herpesvirus (KHV) using a monoclonal antibody against Cyprinus carpio IgM.

PubMed

Li, Yingying; Zheng, Shucheng; Wang, Qing; Bergmann, Sven M; Zeng, Weiwei; Wang, Yingying; Liu, Chun; Shi, Cunbin

2017-08-01

Koi herpesvirus disease (KHVD) is associated with high mortality in both common carp and koi carp (Cyprinus carpio L.) worldwide. The indirect detection of fish viruses based on the identification of antibodies has emerged as a practical and reliable means of diagnosis. Thus, it is important to create monoclonal antibodies (MAbs) against carp IgM. By using hybridoma-monoclonal antibody technology, one hybridoma cell line secreting MAbs against IgM from carp was established. In western blot analysis, the secreted MAb from cell line A5-E10 recognized the heavy chain of IgM from common carp or koi but did not react with immunoglobulins from three different fish species: grass carp (Ctenopharyngodon idella), tilapia (Oreochromis mossambicus) and Mandarin fish (Siniperca chuatsi). These results demonstrated that this MAb is highly specific for the IgM of carp and suggested that it can be used for monitoring the immunity level of carp, for example for indirect KHV diagnosis by antibody ELISA. We therefore established an indirect ELISA, which was tested using 200 serum samples from koi from three farms. The final results showed that 147 (73.5%) samples were confirmed to be KHV antibody negative and 53 (26.5%) were definitely positive, containing antibodies against KHV.

21 CFR 866.5120 - Antismooth muscle antibody immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5120 Antismooth muscle antibody immunological test system. (a) Identification. An antismooth... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Antismooth muscle antibody immunological test...

Clinical Utility of Acetylcholine Receptor Antibody Testing in Ocular Myasthenia Gravis.

PubMed

Peeler, Crandall E; De Lott, Lindsey B; Nagia, Lina; Lemos, Joao; Eggenberger, Eric R; Cornblath, Wayne T

2015-10-01

The sensitivity of acetylcholine receptor (AChR) antibody testing is thought to be lower in ocular myasthenia gravis (OMG) compared with generalized disease, although estimates in small-scale studies vary. There is little information in the literature about the implications of AChR antibody levels and progression from OMG to generalized myasthenia gravis. To test the hypothesis that serum AChR antibody testing is more sensitive in OMG than previously reported and to examine the association between AChR antibody levels and progression from OMG to generalized myasthenia gravis. A retrospective, observational cohort study was conducted of 223 patients (mean [SD] age, 59.2 [16.4] years; 139 [62.3%] male) diagnosed with OMG between July 1, 1986, and May 31, 2013, at 2 large, academic medical centers. Baseline characteristics, OMG symptoms, results of AChR antibody testing, and progression time to generalized myasthenia gravis (if this occurred) were recorded for each patient. Multiple logistic regression was used to measure the association between all clinical variables and antibody result. Kaplan-Meier survival analysis was performed to examine time to generalization. Among the 223 participants, AChR antibody testing results were positive in 158 participants (70.9%). In an adjusted model, increased age at diagnosis (odds ratio [OR], 1.03; 95% CI, 1.01-1.04; P = .007) and progression to generalized myasthenia gravis (OR, 2.92; 95% CI, 1.18-7.26; P = .02) were significantly associated with positive antibody test results. Women were less likely to have a positive antibody test result (OR, 0.36; 95% CI, 0.19-0.68; P = .002). Patients who developed symptoms of generalized myasthenia gravis had a significantly higher mean (SD) antibody level than those who did not develop symptoms of generalized myasthenia gravis (12.7 [16.5] nmol/L vs 4.2 [7.9] nmol/L; P = .002). We demonstrate a higher sensitivity of AChR antibody testing than previously reported in the

Mapping the distribution of specific antibody interaction forces on individual red blood cells

NASA Astrophysics Data System (ADS)

Yeow, Natasha; Tabor, Rico F.; Garnier, Gil

2017-02-01

Current blood typing methods rely on the agglutination of red blood cells (RBCs) to macroscopically indicate a positive result. An indirect agglutination mechanism is required when blood typing with IgG forms of antibodies. To date, the interaction forces between anti-IgG and IgG antibodies have been poorly quantified, and blood group related antigens have never been quantified with the atomic force microscope (AFM). Instead, the total intensity resulting from fluorescent-tagged antibodies adsorbed on RBC has been measured to calculate an average antigen density on a series of RBCs. In this study we mapped specific antibody interaction forces on the RBC surface. AFM cantilever tips functionalized with anti-IgG were used to probe RBCs incubated with specific IgG antibodies. This work provides unique insight into antibody-antigen interactions in their native cell-bound location, and crucially, on a per-cell basis rather than an ensemble average set of properties. Force profiles obtained from the AFM directly provide not only the anti-IgG - IgG antibody interaction force, but also the spatial distribution and density of antigens over a single cell. This new understanding might be translated into the development of very selective and quantitative interactions that underpin the action of drugs in the treatment of frontier illnesses.

Antismooth muscle and antiactin antibodies are indirect markers of histological and biochemical activity of autoimmune hepatitis.

PubMed

Couto, Claudia A; Bittencourt, Paulo L; Porta, Gilda; Abrantes-Lemos, Clarice P; Carrilho, Flair J; Guardia, Bianca D; Cançado, Eduardo L R

2014-02-01

Reactivity and titers of autoantibodies vary during the course of autoimmune hepatitis (AIH), and some autoantibodies have been associated with disease activity and adverse outcomes after treatment. The aim of this study was to assess the autoantibody behavior in AIH and its significance as predictors of biochemical and histological remission. A total of 117 patients with AIH (mean age 18.6 [4-69] years) were evaluated and tested for autoantibodies at disease onset and successively (mean 3.2 [2-6] times) after a mean follow-up evaluation of 70 [20-185] months. Antismooth muscle (ASMA), antiliver kidney microsome type 1 (anti-LKM1), antiliver cytosol type 1 (anti-LC1), antimitochondrial, antinuclear (ANA), and antiactin antibodies (AAA) were determined at disease onset and 379 other times during the follow-up evaluation through indirect immunofluorescence in rodent tissues, HEp-2 cells, and human fibroblasts. Anti-SLA/LP were assessed 45 times in the follow-up evaluation of 19 patients using enzyme-linked immunosorbent assay (ELISA). Upon admission, AIH types 1 and 2 were observed in 95 and 17 patients, respectively. Five subjects had AIH with anti-SLA/LP as the sole markers. Patients initially negative for AAA did not develop these antibodies thereafter. ANA were detected de novo in six and three subjects with AIH types 1 and 2, respectively. After treatment, only ASMA (>1:80) and AAA (>1:40) were significantly associated with biochemical (76.9% and 79.8%) and histological features (100% and 100%) of disease activity (P < 0.001). With the exception of ANA, the autoantibody profile does not markedly vary in the course of AIH. The persistence of high titers of ASMA and/or AAA in patients with AIH is associated with disease activity. © 2013 by the American Association for the Study of Liver Diseases.

In vitro antibody-enzyme conjugates with specific bactericidal activity.

PubMed

Knowles, D M; Sulivan, T J; Parker, C W; Williams, R C

1973-06-01

IgG with antibacterial antibody opsonic activity was isolated from rabbit antisera produced by intravenous hyperimmunization with several test strains of pneumococci, Group A beta-hemolytic streptococci, Staphylococcus aureus, Proteus mirabilis, Pseudomonas aeruginosa, and Escherichia coli. Antibody-enzyme conjugates were prepared, using diethylmalonimidate to couple glucose oxidase to IgG antibacterial antibody preparations. Opsonic human IgG obtained from serum of patients with subacute bacterial endocarditis was also conjugated to glucose oxidase. Antibody-enzyme conjugates retained combining specificity for test bacteria as demonstrated by indirect immunofluorescence. In vitro test for bactericidal activity of antibody-enzyme conjugates utilized potassium iodide, lactoperoxidase, and glucose as cofactors. Under these conditions glucose oxidase conjugated to antibody generates hydrogen peroxide, and lactoperoxidase enzyme catalyzes the reduction of hydrogen peroxide with simultaneous oxidation of I(-) and halogenation and killing of test bacteria. Potent in vitro bactericidal activity of this system was repeatedly demonstrated for antibody-enzyme conjugates against pneumococci, streptococci, S. aureus, P. mirabilis, and E. coli. However, no bactericidal effect was demonstrable with antibody-enzyme conjugates and two test strains of P. aeruginosa. Bactericidal activity of antibody-enzyme conjugates appeared to parallel original opsonic potency of unconjugated IgG preparations. Antibody-enzyme conjugates at concentrations as low as 0.01 mg/ml were capable of intense bactericidal activity producing substantial drops in surviving bacterial counts within 30-60 min after initiation of assay. These in vitro bactericidal systems indicate that the concept of antibacterial antibody-enzyme conjugates may possibly be adaptable as a mechanism for treatment of patients with leukocyte dysfunction or fulminant bacteremia.

Neospora caninum and Toxoplasma gondii antibodies in dogs from Durango City, Mexico.

PubMed

Dubey, J P; Alvarado-Esquivel, C; Liesenfeld, O; Herrera-Flores, R G; Ramírez-Sánchez, B E; González-Herrera, A; Martínez-García, S A; Bandini, L A; Kwok, O C H

2007-10-01

Toxoplasma gondii and Neospora caninum are structurally similar parasites, with many hosts in common. The prevalence of antibodies to T. gondii and N. caninum was determined in sera from dogs from Durango City, Mexico. Using a modified agglutination test, antibodies to T. gondii were found in 52 (51.5%) of the 101 dogs with titers of 1:25 in 27, 1:50 in 11, 1:100 in 5, 1:200 in 4, 1:400 in 2, 1:800 in 2, and 1:3,200 or higher in 1. Antibodies to N. caninum were determined by the indirect immunofluorescent antibody test (IFAT) and the Neospora sp. agglutination test (NAT). Two of the 101 dogs had N. caninum antibodies; these dogs did not have T. gondii antibodies, supporting the specificity of the tests used. The N. caninum antibody titers of the 2 dogs were: 1:400 by IFAT and 1:200 by NAT in 1, and 1:25 by NAT and IFAT in the other. Results indicate that these 2 structurally similar protozoans are antigenically different.

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT

PubMed Central

Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun

2017-01-01

Background Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. Methodology/principal findings The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). Conclusions/significance The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV. PMID:29267277

Development and evaluation of an anti-rabies virus phosphoprotein-specific monoclonal antibody for detection of rabies neutralizing antibodies using RFFIT.

PubMed

Um, Jihye; Chun, Byung Chul; Lee, Yeong Seon; Hwang, Kyu Jam; Yang, Dong-Kun; Park, Jun-Sun; Kim, Su Yeon

2017-12-01

Rabies is a major public health problem with a fatality rate close to 100%; however, complete prevention can be achieved through pre- or post-exposure prophylaxis. The rapid fluorescent focus inhibition test (RFFIT) is one of the recommended testing methods to determine the production of neutralizing antibodies after vaccination. Here, we report the development of a new monoclonal antibody (mAb) designed to react specifically with Rabies virus (RABV) phosphoprotein (P protein), and the evaluation of its applicability to the RFFIT and its effectiveness as a diagnostic reagent for human rabies. The mAb KGH P 16B8 was produced to target the P protein of the Korean KGH RABV strain. An indirect immunofluorescence assay (IFA) was conducted to detect various strains of RABV in various cell lines. Alexa-conjugated KGH P 16B8 (16B8-Alexa) was developed for the RFFIT. The IFA test could detect RABV up to a 1:2,500 dilution, with a detection limit comparable to that of a commercial diagnostic reagent. The sensitivity, specificity, positive predictive value, and negative predictive value of the RFFIT using 16B8-Alexa in 414 clinical specimens were 98.67%, 99.47%, 99.55%, and 98.42%, respectively. The results of the RFFIT with 16B8-Alexa were strongly correlated with those obtained using an existing commercial diagnostic reagent (r = 0.995, p<0.001). The mAb developed in this study shows high sensitivity and specificity, confirming its clinical utility with the RFFIT to measure the rabies neutralizing antibody titer and establish a diagnosis in human. Thus, 16B8-Alexa is expected to serve as an alternative diagnostic reagent that is widely accessible, with potentially broad applications beyond those of the RFFIT in Korea. Further studies with 16B8-Alexa should provide insight into the immunological mechanism of the P protein of Korean RABV.

Leptospirosis in Sheep in Western Canada

PubMed Central

Kingscote, B.

1985-01-01

A survey of 930 ovine sera and kidneys from 33 sheep was conducted to assess the rate of leptospiral infection in sheep slaughtered in Alberta. Sera were tested for the presence of agglutinins to indigenous serovars of Leptospira interrogans. Kidneys with gross lesions were examined for the presence of leptospires by means of an indirect fluorescent antibody test (FAT) and by culture. Antibodies to serovars pomona and hardjo were present at rates of 1.0% and 0.4%, respectively, in sheep from Saskatchewan, Alberta and British Columbia. Sera from 120 feedlot lambs shipped from Oregon reacted to serovars pomona, hardjo and grippotyphosa at rates of 1.7%, 61.7% and 59.1%, respectively. Fluorescent antibody test detected serovars (presumptively) hardjo in 52% of Oregon feedlot lambs and grippotyphosa in 32% of the same group, a finding supported by the isolation of both these serovars from a pool of two fluorescent antibody test-positive kidneys. The grippotyphosa strain was highly virulent for hamsters, producing intense icterus and death. Leptospires, presumptively serovar grippotyphosa were demonstrated by fluorescent antibody test in one Alberta lamb kidney. The possibility of spreading leptospirosis by movement of breeding stock through public facilities and by assembling lambs in feedlots is discussed. PMID:17422531

Can indirect tests detect a known recombination event in human mtDNA?

PubMed

White, Daniel James; Gemmell, Neil John

2009-07-01

Whether human mitochondrial DNA (mtDNA) recombines sufficiently to influence its evolution, evolutionary analysis, and disease etiology, remains equivocal. Overall, evidence from indirect studies of population genetic data suggests that recombination is not occurring at detectable levels. This may be explained by no, or low, recombination or, alternatively, current indirect tests may be incapable of detecting recombination in human mtDNA. To investigate the latter, we have tested whether six well-established indirect tests of recombination could detect recombination in a human mtDNA data set, in which its occurrence had been empirically confirmed. Three showed statistical evidence for recombination (r(2) vs. distance, the Homoplasy test, Neighborhood Similarity Score), and three did not (D' vs. distance, Max Chi Squared, Pairwise Homoplasy Index). Possible reasons for detection failure are discussed. Further, evidence from earlier studies suggesting a lack of recombination in mtDNA in humans is reconsidered, taking into account the appropriateness of the tests used, based on our new findings.

Development and validation of an anti-N3 indirect immunofluorescent antibody test to be used as a companion diagnostic test in the framework of a "DIVA" vaccination strategy for avian influenza infections in poultry.

PubMed

Cattoli, Giovanni; Milani, Adelaide; Bettini, Francesca; Serena Beato, Maria; Mancin, Marzia; Terregino, Calogero; Capua, Ilaria

2006-04-01

Avian influenza (AI) infections have become of growing importance both for animal and human health. Vaccination has become a recommended tool to support eradication efforts and limit the economic losses caused by this disease. The "DIVA" system, using a vaccine containing a heterologous neuraminidase to the field virus, has been shown to be an effective tool in increasing the resistance of birds to field challenge, preventing clinical signs and reducing viral shedding in the environment. The companion diagnostic test to the vaccine, however, has been only partially validated in the field against one subtype of neuraminidase (N1). The present paper presents the results of a full laboratory and field validation of the diagnostic test developed to detect antibodies to the N3 subtype of AI in vaccinated and unvaccinated chickens and turkeys. Antibody kinetic studies conducted in the laboratory have shown that antibodies to the N protein may be detected earlier than antibodies to the haemagglutinin. The data derived from this extensive validation trial indicate the excellent capability of this assay in detecting the presence of active AI infection at an early stage in both unvaccinated and vaccinated birds and the lack of interference with vaccine-induced antibodies.

ISOLATION AND GENOTYPING OF Toxoplasma gondii IN SERONEGATIVE URBAN RATS AND PRESENCE OF ANTIBODIES IN COMMUNICATING DOGS IN BRAZIL

PubMed Central

RUFFOLO, Bruno Bergamo; TOLEDO, Roberta dos Santos; MARTINS, Felippe Danyel Cardoso; BUGNI, Felipe Monteiro; da COSTA, Letícia; MARANA, Elizabete Regina Marangoni; NAVARRO, Italmar Teodorico; GARCIA, João Luis; SU, Chunlei; FREIRE, Roberta Lemos

2016-01-01

The role of rodents in the epidemiology of toxoplasmosis was investigated in Londrina, Paraná State, Brazil. One hundred and eighty-one Rattus rattus and one Mus musculus were caught in 37 places. Blood and tissues were collected and submitted to the indirect fluorescence antibody test (IFAT) and the bioassay. Serum samples from 61 contacting dogs were also collected. Sixteen rats (8.8%) were positive for Toxoplasma gondii, but just two of them were positive by serology and bioassay test. Antibodies were found in nine (4.9%) rats. Tissues of nine rats bioassayed were positive and four isolates were obtained. Restriction fragment length polymorphism (RFLP) analysis was performed using 12 markers (SAG1, SAG2, SAG2-alt, C22-8, C29-2, L358, PK1, BTUB, GRA6, SAG3, Apico, CS3). Genotyping revealed that the four strains isolated from this study have been isolated before in cats and chickens from Brazil. None of the isolates was identified like clonal archetypal T-types I, II, and III. The rats presented lower serologic Toxoplasma gondii prevalence (8.8%) compared to contacting dogs (70.5%). PMID:27074322

Studies on antiplague haemagglutinating antibodies

PubMed Central

Suzuki, Sosuke; Chikasato, Yoshio; Hotta, Susumu

1974-01-01

The indirect haemagglutination (IHA) test has been widely applied in the detection of antiplague antibodies in rodent sera. In the present study, acetone treatment of the test serum was tried in order to improve the specificity of the reaction. It was shown that the IHA titres of acetone-treated sera correlated well with those of untreated sera measured by the standard method recommended by WHO. Essentially the same results were obtained with sera from experimentally immunized rodents and from captured wild rats. In addition to acetone treatment, the sera were treated with 2-mercaptoethanol (ME). The results obtained indicated that the antiplague IHA antibodies produced early after the inoculation of plague bacilli were ME-sensitive, whereas those detected in the later stages or after a second inoculation were ME-resistant. The data suggest that acetone treatment of sera could be useful for the screening of antiplague antibodies, and that treatment with ME is helpful in assessing the time of past plague infections. The present survey has also shown that the positive rates of antiplague antibodies in wild rats trapped in Kobe, one of the largest sea ports in Japan, have so far been very low. PMID:4549347

Lupus erythematosus cell preparation, antinuclear factor and antideoxyribonucleic acid antibody incongruity in systemic lupus erythematosus.

PubMed

Chee, Y C

1983-01-01

'Total antinuclear antibody' (ANF) is detected by the fluorescent antinuclear antibody technique which is a screening test, positive in 99% of systemic lupus erythematosus (SLE) sera. The LE factor (positive in 75% of SLE sera), like the anti-DNA antibody, is an antinuclear antibody but directed against DNA-histone. ANF-negative SLE is a clinical entity with absence of these antibodies. A false negative ANF, in the presence of high titre anti-DNA antibody and/or LE cells, is illustrated in two cases of SLE. Postulated mechanisms for this phenomenon are interference in ANF detection by rheumatoid factor, and the prozone effect on the immunofluorescent tests.

Indirect carotid cavernous fistula mimicking ocular myasthenia.

PubMed

Leishangthem, Lakshmi; Satti, Sudhakar Reddy

2017-10-19

71-year-old woman with progressive left-sided, monocular diplopia and ptosis. Her symptoms mimicked ocular myasthenia, but she had an indirect carotid cavernous fistula (CCF). She was diagnosed with monocular myasthenia gravis (negative acetylcholinesterase antibody) after a positive ice test and started on Mestinon and underwent a thymectomy complicated by a brachial plexus injury. Months later, she developed left-sided proptosis and ocular bruit. She was urgently referred to neuro-interventional surgery and was diagnosed with an indirect high-flow left CCF, which was treated with Onyx liquid and platinum coil embolisation. Mestinon was discontinued. Her ophthalmic symptoms resolved. However, she was left with a residual left arm and hand hemiparesis and dysmetria secondary to a brachial plexus injury. Indirect CCF usually can present with subtle and progressive symptoms leading to delayed diagnosis or misdiagnosis. It is important for ophthalmologists to consider this differential in a patient with progressive ocular symptoms. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Prevalence of antibodies to Coxiella burnetii among veterinary school dairy herds in the United States, 2003.

PubMed

McQuiston, Jennifer H; Nargund, Vrinda N; Miller, Jeffrey D; Priestley, Rachael; Shaw, Edward I; Thompson, Herbert A

2005-01-01

Prevalence of antibodies to Coxiella burnetii in 24 veterinary school-associated dairy herds in the United States was assessed through laboratory testing of bulk tank milk specimens by indirect immunofluorescent antibody assay. Twenty-two herds (92%) had evidence of antibodies to C. burnetii Phase I antibodies at a titer of > or = 1:16, and nine herds (38%) had Phase I antibody titers of > or = 1:256. These results suggest that C. burnetii infection is geographically widespread among dairy herds in the United States.

[Extramembranous glomerulonephritis of acquired syphilis in a patient recently infected by the hepatitis B virus. Demonstration of the treponemal antigen in the kidney by indirect immunofluorescence].

PubMed

Schillinger, F; Montagnac, R; Goclowski, C; Dine, G; Alessandri, E; Hopfner, C; Birembaut, P

1983-01-22

A 38 years old male homosexual with active secondary syphilis presented with pure nephrotic syndrome while HBs and HBe tests were positive without clinical hepatitis. He had circulating immune complexes, IgG--IgM cryoglobulinemia and high IgA, IgM and IgE levels; the C3 and C4 complement constituents were normal. Examination of renal biopsy sections under light, fluorescent and electronic microscopy showed stage I membranous glomerulonephritis the syphilitic origin of which was confirmed by indirect immunofluorescence and by rapid cure under penicillin treatment. This case calls for the following comments: (1) glomerular deposits are extramembranous rather than subendothelial in syphilitic nephrosis, a disease now classified among circulating immune complexes diseases; (2) in the kidney, the treponema antigen can be demonstrated by indirect immunofluorescence and the anti-treponema antibody, by elution; (3) the outcome of the nephrotic syndrome is always favourable, either spontaneously or after penicillin treatment; (4) syphilis and HBs antigens are frequently associated, particularly in homosexual patients; one should be looked for when the other is discovered.

DARPA Antibody Technology Program. Standardized Test Bed for Antibody Characterization: Characterization of an MS2 ScFv Antibody Produced by Illumina

DTIC Science & Technology

2016-08-01

platforms. 15. SUBJECT TERMS Antibody Antibody Technology Program (ATP) Quality Enzyme-linked immunosorbent assay ( ELISA ) Biosurveillance Single-chain...2.6 Thermal Stress Test............................................................................................4 2.7 ELISA ...3.5 ELISA Results .................................................................................................11 3.6 SPR Results

Antibody immobilization using pneumatic spray: comparison with the avidin-biotin bridge immobilization method.

PubMed

Figueroa, Jhon; Magaña, Sonia; Lim, Daniel V; Schlaf, Rudy

2012-12-14

The formation of a thin antibody film on a glass surface using pneumatic spray was investigated as a potential immobilization technique for capturing pathogenic targets. Goat-Escherichia coli O157:H7 IgG films were made by pneumatic spray and compared against the avidin-biotin bridge immobilized films by assaying with green fluorescent protein (GFP) transformed E. coli O157:H7 cells and fluorescent reporter antibodies. Functionality, stability, and immobilization of the films were tested. The pneumatic spray films had lower fluorescence intensity values than the avidin-biotin bridge films but resulted in similar detection for E. coli O157:H7 at 10(5)-10(7)cells/ml sample concentrations with no detection of non-E. coli O157:H7 strains. Both methods also resulted in similar percent capture efficiencies. The results demonstrated that immobilization of antibody via pneumatic spray did not render the antibody non-functional and produced stable antibody films. The amount of time necessary for immobilization of the antibody was reduced significantly from 24h for the avidin-biotin bridge to 7 min using the pneumatic spray technique, with additional benefits of greatly reduced use of materials and chemicals. The pneumatic spray technique promises to be an alternative for the immobilization of antibodies on glass slides for capturing pathogenic targets and use in biosensor type devices. Copyright © 2012. Published by Elsevier B.V.

Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

NASA Astrophysics Data System (ADS)

Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

1982-08-01

Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

[Targeted detecting HER2 expression with recombinant anti HER2 ScFv-GFP fusion antibody].

PubMed

Gao, Guohui; Chen, Chong; Yang, Yanmei; Yang, Han; Wang, Jindan; Zheng, Yi; Huang, Qidi; Hu, Xiaoqu

2012-08-01

To verify the reliability of targeted detecting HER2 positive cancer cells and clinical pathological tissue specimens with a recombinant anti HER2 single chain antibody in single chain Fv fragment (scFv) format, we have constructed the fusion variable regions of the ScFv specific for HER2/neu. labeled a green-fluorescent protein(GFP). The humanized recombinant Anti HER2 ScFv-GFP gene was inserted into pFast Bac HT A, and expressed in insect cells sf9. Then the recombinant fusion protein Anti HER2 ScFv-GFP was properly purified with Ni2+-NTA affinity chromatography from the infected sf9 cells used to test the specificity of the fusion antibody for HER2 positive cancer cells. Firstly, the purified antibody incubated with HER2 positive breast cancer cells SKBR3, BT474 and HER2 negative breast cancer cells MCF7 for 12 h/24 h/48 h at 37 degrees C, in order to confirm targeted detecting HER2 positive breast cancer cells by Laser Confocal Microscopy. Furthermore, the same clinical pathological tissue samples were assessed by immunohistochemistry (IHC) and the fusion antibody Anti HER2 ScFv-GFP in the meanwhile. The data obtained indicated that the recombinant eukaryotic expression plasmid pFast Bac HT A/Anti HER2 ScFv-GFP was constructed successfully In addition, obvious green fluorescent was observed in insect cells sf9. When the purified fusion antibody was incubated with different cancer cells, much more green fluorescent was observed on the surface of the HER2 positive cancer cells SKBR3 and BT474. In contrast, no green fluorescent on the surface of the HER2 negative cancer cells MCF7 was detected. The concentration of the purified fusion antibody was 115.5 microg/mL, of which protein relative molecular weight was 60 kDa. The analysis showed the purity was about 97% and the titer was about 1:64. The detection results of IHC and fusion antibody testing indicated the conformity. In summary, the study showed that the new fusion antibody Anti HER2 ScFv-GFP can test HER2

21 CFR 866.5120 - Antismooth muscle antibody immunological test system.

Code of Federal Regulations, 2011 CFR

2011-04-01

... Systems § 866.5120 Antismooth muscle antibody immunological test system. (a) Identification. An antismooth muscle antibody immunological test system is a device that consists of the reagents used to measure by... serum. The measurements aid in the diagnosis of chronic hepatitis (inflammation of the liver) and...

Application and Comparative Evaluation of Fluorescent Antibody, Immunohistochemistry and Reverse Transcription Polymerase Chain Reaction Tests for the Detection of Rabies Virus Antigen or Nucleic Acid in Brain Samples of Animals Suspected of Rabies in India.

PubMed

Prabhu, K Nithin; Isloor, Shrikrishna; Veeresh, B Hanchinal; Rathnamma, Doddamane; Sharada, R; Das, Lekshmi J; Satyanarayana, M L; Hegde, Nagendra R; Rahman, Sira Abdul

2018-02-28

Accurate and early diagnosis of animal rabies is critical for undertaking public health measures. Whereas the direct fluorescent antibody (DFA) technique is the recommended test, the more convenient, direct rapid immunochemistry test (dRIT), as well as the more sensitive, reverse transcription polymerase chain reaction (RT-PCR), have recently been employed for the laboratory diagnosis of rabies. We compared the three methods on brain samples from domestic (dog, cat, cattle, buffalo, horse, pig and goat) and wild (leopard, wolf and jackal) animals from various parts of India. Of the 257 samples tested, 167 were positive by all the three tests; in addition, 35 of the 36 decomposed samples were positive by RT-PCR. This is the first study in which such large number of animal samples have been subjected to the three tests simultaneously. The results confirm 100% corroboration between DFA and dRIT, buttress the applicability of dRIT in the simple and rapid diagnosis of rabies in animals, and reaffirm the suitability of RT-PCR for samples unfit for testing either by DFA or dRIT.

ANCA / MPO / PR3 Antibodies Test

MedlinePlus

... normal limits." For more information, please read the article Reference Ranges and What They Mean . What is being tested? Antineutrophil cytoplasmic antibodies (ANCA) are autoantibodies produced by a person's immune system that mistakenly target and attack proteins within the ...

Indirect suppression of photosynthesis on individual leaves by arthropod herbivory

PubMed Central

Nabity, Paul D.; Zavala, Jorge A.; DeLucia, Evan H.

2009-01-01

Background Herbivory reduces leaf area, disrupts the function of leaves, and ultimately alters yield and productivity. Herbivore damage to foliage typically is assessed in the field by measuring the amount of leaf tissue removed and disrupted. This approach assumes the remaining tissues are unaltered, and plant photosynthesis and water balance function normally. However, recent application of thermal and fluorescent imaging technologies revealed that alterations to photosynthesis and transpiration propagate into remaining undamaged leaf tissue. Scope and Conclusions This review briefly examines the indirect effects of herbivory on photosynthesis, measured by gas exchange or chlorophyll fluorescence, and identifies four mechanisms contributing to the indirect suppression of photosynthesis in remaining leaf tissues: severed vasculature, altered sink demand, defence-induced autotoxicity, and defence-induced down-regulation of photosynthesis. We review the chlorophyll fluorescence and thermal imaging techniques used to gather layers of spatial data and discuss methods for compiling these layers to achieve greater insight into mechanisms contributing to the indirect suppression of photosynthesis. We also elaborate on a few herbivore-induced gene-regulating mechanisms which modulate photosynthesis and discuss the difficult nature of measuring spatial heterogeneity when combining fluorescence imaging and gas exchange technology. Although few studies have characterized herbivore-induced indirect effects on photosynthesis at the leaf level, an emerging literature suggests that the loss of photosynthetic capacity following herbivory may be greater than direct loss of photosynthetic tissues. Depending on the damage guild, ignoring the indirect suppression of photosynthesis by arthropods and other organisms may lead to an underestimate of their physiological and ecological impacts. PMID:18660492

Development of a H2 O2 -sensitive quantum dots-based fluorescent sandwich ELISA for sensitive detection of bovine β-lactoglobulin by monoclonal antibody.

PubMed

He, Shengfa; Li, Xin; Gao, Jinyan; Tong, Ping; Chen, Hongbing

2018-01-01

Bovine β-lactoglobulin (BLG) is the major allergen in cows' milk, and the specific epitope plays a key role in food allergy. Developing a method specifically bind to the IgE epitope is necessary for testing BLG and its allergenic residues. The monoclonal antibody (1G9) specific to the IgE linear epitope for BLG was identified as high affinity and specificity. Based on 1G9, a sensitive fluorescent sandwich enzyme-linked immunosorbent assay (sELISA) was successfully developed using catalase-mediated fluorescence quenching of thiolated CdTe quantum dots in the presence of hydrogen peroxide as fluorescent signal output. The fluorescent sELISA showed high sensitivity and specificity, the limit of detection was 0.49 ng mL -1 , which was 16-fold lower than horseradish peroxidase (HRP)-based sELISA. The linear range for BLG detection were 125-4000 ng mL -1 (r = 0.9939) and 0.48-62.5 ng mL -1 (r = 0.9919). The recoveries and coefficients of variation were 94.25-109.83% and 4.38-20.29%, respectively. Allergenic residues were also detected in hydrolysed infant formulas. The results of fluorescent sELISA showed good performance as HRP-based sELISA and commercial sELISA kit. This proposed fluorescent sELISA could be employed to detect BLG and its allergenic residues in food with highly sensitivity, reliability, and recovery. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

Code of Federal Regulations, 2013 CFR

2013-04-01

... immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's own... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

Code of Federal Regulations, 2014 CFR

2014-04-01

... immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's own... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

21 CFR 866.5090 - Antimitochondrial antibody immunological test system.

Code of Federal Regulations, 2012 CFR

2012-04-01

... immunochemical techniques the antimitochondrial antibodies in human serum. The measurements aid in the diagnosis of diseases that produce a spectrum of autoantibodies (antibodies produced against the body's own... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test...

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing.

PubMed

Matsumoto, Chieko; Sagara, Yasuko; Sobata, Rieko; Inoue, Yukiko; Morita, Maiko; Uchida, Shigeharu; Kiyokawa, Hiroyuki; Satake, Masahiro; Tadokoro, Kenji

2017-08-01

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens. © 2017 Wiley Periodicals, Inc.

Separation and detection of VX and its methylphosphonic acid degradation products on a microchip using indirect laser-induced fluorescence.

PubMed

Heleg-Shabtai, Vered; Gratziany, Natzach; Liron, Zvi

2006-05-01

The application of indirect LIF (IDLIF) technique for on-chip electrophoretic separation and detection of the nerve agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothiolate (VX) and its major phosphonic degradation products, ethyl methylphosphonic acid (EMPA) and methylphosphonic acid (MPA) was demonstrated. Separation and detection of MPA degradation products of VX and the nerve agent isopropyl methylphosphonofluoridate (GB) are presented. The negatively charged dye eosin was found to be a good fluorescent marker for both the negatively charged phosphonic acids and the positively charged VX, and was chosen as the IDLIF visualization fluorescent dye. Separation and detection of VX, EMPA, and MPA in a simple-cross microchip were completed within less than a minute, and consumed only a 50 pL sample volume. A characteristic system peak that appeared in all IDLIF electropherograms served as an internal standard that increased the reliability of peak identification. The negative peak of both VX and the MPAs is in agreement with indirect detection theory and with previous reports in the literature. The LOD of VX and EMPA by IDLIF was 30 and 37 microM, respectively. Despite the fact that the detection sensitivity is relatively low, the rapid simultaneous on-chip analysis of both VX and its degradation products as well as the separation and detection of the MPA degradation products of both VX and GB, increases detection reliability and may present a choice when sensitivity is not critical compared with speed and simplicity of the assay.

Establishment of a simple and quantitative immunospot assay for detecting anti-type II collagen antibody using an infrared fluorescence imaging system (IFIS).

PubMed

Ota, Shusuke; Kanazawa, Satoshi; Kobayashi, Masaaki; Otsuka, Takanobu; Okamoto, Takashi

2005-04-01

Antibodies to type II collagen (col II) have been detected in patients with rheumatoid arthritis and in animal models of collagen induced arthritis. Here, we describe a novel method to detect anti-col II antibodies using an immunospot assay with an infrared fluorescence imaging system. This method showed very high sensitivity and specificity, and was simple, with low background levels. It also showed higher reproducibility and linearity, with a dynamic range of approximately 500-fold, than the conventional immunospot assay with enhanced chemiluminescence detection. Using this method we were able to demonstrate the antibody affinity maturation process in mice immunized with col II. In these immunized mice, although cross-reactive antibodies reacting with other collagen species were detected in earlier stages of immunization, the titers of cross-reactive antibodies rapidly diminished after the antigen boost, concomitantly with the elevation of the anti-col II antibody. The method and its possible applications are discussed.

Fluorescent humanized anti-CEA antibody specifically labels metastatic pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) mouse model

NASA Astrophysics Data System (ADS)

Lwin, Thinzar M.; Miyake, Kentaro; Murakami, Takashi; DeLong, Jonathan C.; Yazaki, Paul J.; Shivley, John E.; Clary, Bryan; Hoffman, Robert M.; Bouvet, Michael

2018-03-01

Specific tumor targeting can result in selective labeling of cancer in vivo for surgical navigation. In the present study, we show that the use of an anti-CEA antibody conjugated to the near-infrared (NIR) fluorescent dye, IRDye800CW, can selectively target and label pancreatic cancer and its metastases in a clinically relevant patient derived xenograft mouse model.

Humoral immune response kinetics in Philander opossum and Didelphis marsupialis infected and immunized by Trypanosoma cruzi employing an immunofluorescence antibody test.

PubMed

Legey, A P; Pinho, A P; Chagas Xavier, S C; Leon, L L; Jansen, A M

1999-01-01

Philander opossum and Didelphis marsupialis considered the most ancient mammals and an evolutionary success, maintain parasitism by Trypanosoma cruzi without developing any apparent disease or important tissue lesion. In order to elucidate this well-balanced interaction, we decided to compare the humoral immune response kinetics of the two didelphids naturally and experimentally infected with T. cruzi and immunized by different schedules of parasite antigens, employing an indirect fluorescence antibody test (IFAT). Both didelphids responded with high serological titers to different immunization routes, while the earliest response occurred with the intradermic route. Serological titers of naturally infected P. opossum showed a significant individual variation, while those of D. marsupialis remained stable during the entire follow-up period. The serological titers of the experimentally infected animals varied according to the inoculated strain. Our data suggest that (1) IFAT was sensitive for follow-up of P. opossum in natural and experimental T. cruzi infections; (2) both P. opossum and D. marsupialis are able to mount an efficient humoral immune response as compared to placental mammals; (3) experimentally infected P. opossum and D. marsupialis present distinct patterns of infection, depending on the subpopulation of T. cruzi, (4) the differences observed in the humoral immune responses between P. opossum and D. marsupialis, probably, reflect distinct strategies selected by these animals during their coevolution with T. cruzi.

21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR...

21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR...

21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR...

21 CFR 660.54 - Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for...

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Potency tests, specificity tests, tests for heterospecific antibodies, and additional tests for nonspecific properties. 660.54 Section 660.54 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR...

Goat anti-rabbit IgG conjugated fluorescent dye-doped silica nanoparticles for human breast carcinoma cell recognition.

PubMed

Chen, Min-Yan; Chen, Ze-Zhong; Wu, Ling-Ling; Tang, Hong-Wu; Pang, Dai-Wen

2013-11-12

We report an indirect method for cancer cell recognition using photostable fluorescent silica nanoprobes as biological labels. The dye-doped fluorescent silica nanoparticles were synthesized using the water-in-oil (W/O) reverse microemulsion method. The silica matrix was produced by the controlled hydrolysis of tetraethylorthosilicate (TEOS) in water nanodroplets with the initiation of ammonia (NH3·H2O). Fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate conjugated with dextran (RBITC-Dextran) was doped in silica nanoparticles (NPs) with a size of 60 ± 5 nm as a fluorescent signal element by covalent bonding and steric hindrance, respectively. The secondary antibody, goat anti-rabbit IgG, was conjugated on the surface of the PEG-terminated modified FITC-doped or RBITC-Dextran-doped silica nanoparticles (PFSiNPs or PBSiNPs) by covalent binding to the PEG linkers using the cyanogen bromide method. The concentrations of goat anti-rabbit IgG covering the nanoprobes were quantified via the Bradford method. In the proof-of-concept experiment, an epithelial cell adhesion molecule (EpCAM) on the human breast cancer SK-Br-3 cell surface was used as the tumor marker, and the nanoparticle functionalized with rabbit anti-EpCAM antibody was employed as the nanoprobe for cancer cell recognition. Compared with fluorescent dye labeled IgG (FITC-IgG and RBITC-IgG), the designed nanoprobes display dramatically increased stability of fluorescence as well as photostability under continuous irradiation.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

PubMed

Sippel, J; Bukhtiari, N; Awan, M B; Krieg, R; Duncan, J F; Karamat, K A; Malik, I A; Igbal, L M; Legters, L

1989-06-01

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population.

Indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assays (ELISAs) and IgM capture ELISA for detection of antibodies to lipopolysaccharide in adult typhoid fever patients in Pakistan.

PubMed Central

Sippel, J; Bukhtiari, N; Awan, M B; Krieg, R; Duncan, J F; Karamat, K A; Malik, I A; Igbal, L M; Legters, L

1989-01-01

Sera from 339 adult febrile patients in Pakistan were tested for antibodies to Salmonella typhi lipopolysaccharide by indirect immunoglobulin G (IgG) and IgM enzyme-linked immunosorbent assay (ELISA) and IgM capture ELISA. A total of 55 patients had S. typhi cultured from their blood, 20 had S. typhi cultured from their stool, 24 were blood or stool culture positive for S. paratyphi A, 41 were culture negative but clinically diagnosed as having enteric fever, 41 had gastrointestinal or urinary tract infections, 41 were clinically diagnosed as having malaria, 20 were smear-positive patients with malaria, 58 had respiratory infections, and the remaining 39 individuals were placed in a miscellaneous group who did not have Salmonella infection. The sensitivities of the indirect IgG ELISA, indirect IgM ELISA, and IgM capture ELISA determined with specimens obtained from the blood culture-positive patients with typhoid fever (positive controls) were 80, 64, and 62%, respectively. The specificities of the assays determined with sera from the patients with respiratory infections (negative controls) were 95, 95, and 97%, respectively. The percentage of smear-positive patients with malaria who were positive by these assays was lower than that in the negative control group. The percentages of individuals in the other patient categories who were positive by these tests were between those obtained with the positive and negative controls. Of the positive controls, 26 were positive by both IgM assays, 9 were IgM positive only by indirect ELISA, and 8 were IgM positive only by IgM capture ELISA. A total of 70% of the positive control patients who were tested for O agglutinins by the Widal tube agglutination assay were positive; however, 29% of the negative control patients were also positive. The indirect IgG ELISA was the single most effective test for the serodiagnosis of typhoid fever in this population. PMID:2754002

Detection of anti-lactoferrin antibodies and anti-myeloperoxidase antibodies in autoimmune hepatitis: a retrospective study.

PubMed

Tan, Liming; Zhang, Yuhong; Peng, Weihua; Chen, Juanjuan; Li, Hua; Ming, Feng

2014-01-01

Anti-lactoferrin antibodies (ALA) and anti-myeloperoxidase antibodies (AMPA) are specific serological markers for autoimmune hepatitis (AIH). The project aimed to detect ALA and AMPA and explore their clinical significances in AIH patients. 59 AIH patients, 217 non AIH patients, and 50 healthy controls were enrolled in this study. ALA and AMPA were detected by ELISA. Antineutropil cytoplasmic antibodies (ANCA) and anti-smooth muscle antibodies (ASMA) were examined by indirect immunofluorescence. Antimitochondrial antibody M2 subtype (AMA-M2), anti-liver kidney microsomal antibody Type 1 (LKM1), anti-liver cytosol antibody Type 1 (LC1), and anti-soluble liver antigen/liver-pancreas antibodies (SLA/LP) were tested by immunoblot. The positivity for ALA was 18.6% in AIH group, only one patient in non-AIH group was positive for ALA; the positivity for AMPA was 59.3% in AIH group, with significant differences (P < 0.01) compared with other groups. The specificities for ALA and AMPA were 99.63% and 97.75%; the sensitivities were 18.64% and 59.32%; and the accuracy rates were 84.97% and 90.80%, respectively. A certain correlation was observed between ALA and SLA/LP, AMPA and ANCA, ASMA in AIH group. ALA and AMPA were associated with AIH, and had high clinical diagnostic value. Co-detection with other relative autoantibodies could play an important role in differential diagnosis of AIH.

Mercaptoethanol-resistant human serum antibodies reacting with endotoxin from Neisseria gonorrhoeae.

PubMed Central

Maeland, J A; Larsen, B

1975-01-01

Sera from fifty patients with gonorrhoea, thirty with non-specific urethritis, and eighty blood donors were treated with mercaptoethanol (ME) and examined by the indirect haemagglutination test for antibodies against endotoxin from gonococci. Erythrocytes sensitized with determinant a of endotoxin from Strains 8551, V, and VII, or determinant b from Strain V were used. The percentage of sera active in the haemagglutination test was much higher in the gonorrhoea group than in the controls. The geometric mean titre was also significantly higher in the gonorrhoea group. This applied for all four antigens used. Results obtained in an anti-globulin test indicated that the titre of ME-treated serum was determined by IgG antibodies against the endotoxin. Many sera had titres which varied according to the strain origin of the antigen used in the test. The sensitivity of tests for antibodies was increased by using endotoxin from several different strains of gonococci for the examination of each serum. A simplified procedure for determination of antibodies against endotoxin from different strains of gonococci was elaborated. PMID:48404

An experimental test for indirect benefits in Drosophila melanogaster

PubMed Central

Rundle, Howard D; Ödeen, Anders; Mooers, Arne Ø

2007-01-01

Background Despite much empirical attention, tests for indirect benefits of mate choice have rarely considered the major components of sexual and nonsexual offspring fitness relevant to a population. Here we use a novel experimental design to test for the existence of any indirect benefits in a laboratory adapted population of D. melanogaster. Our experiment compared the fitness (mating success, longevity, and productivity) of individuals possessing genomes that derived two generations previously from males that were either entirely successful (studs) or wholly unsuccessful (duds) at achieving mates in three subsequent rounds of mating trials. Results Males from the stud treatment were 30% more successful on average at securing mates than males from the dud treatment. In contrast, we found no difference between treatments in measures of productivity or of longevity when measured in a mixed-sex environment. In the absence of females, however, males in the stud treatment outlived males in the dud treatment. Conclusion Our results suggest that mating with successful males in this population provides an indirect benefit to females and that, at least in this environment, the benefit arises primarily through the production of more attractive male offspring. However, it is unclear whether this represents solely a traditional sexy sons benefit or whether there is an additional good genes component (with male offspring simply allocating their surplus condition to traits that enhance their mating success). The lack of any detectable differences in female fitness between the two treatments suggests the former, although the longevity advantage of males in the stud treatment when females were absent is consistent with the latter. Determining the effect of this indirect benefit on the evolution of female mate preferences (or resistance) will require comparable data on the direct costs of mating with various males, and an understanding of how these costs and benefits integrate

Antibody class capture assays for varicella-zoster virus.

PubMed Central

Forghani, B; Myoraku, C K; Dupuis, K W; Schmidt, N J

1984-01-01

Pooled monoclonal antibodies to varicella-zoster virus (VZV) were used as "detector" antibodies in a four-phase enzyme immunofluorescence assay for determination of immunoglobulin M (IgM), IgA, and IgG antibodies to VZV. Polyclonal antisera specific for heavy chains of human IgM, IgA, and IgG were employed as "capture" antibodies on the solid phase. The antibody class capture assay (ACCA) for VZV IgM antibody detected high titers of virus-specific IgM in all patients with varicella and in 5 of 10 zoster patients. VZV IgM antibody was not detected in patients with primary herpes simplex virus infections or in other individuals without active VZV infection, with one exception, a patient with encephalitis who had other serological findings compatible with a reactivated VZV infection. VZV-specific IgA and IgG antibody titers demonstrable by ACCA were compared with those measured by solid-phase indirect enzyme immunofluorescence assay (EIFA). VZV IgA antibody titers detected in patients with varicella and zoster were variable and could not be considered to be reliable markers of active VZV infection. IgA antibody titers detected by ACCA tended to be higher than those demonstrated by solid-phase indirect EIFA in varicella and zoster patients. VZV IgG antibody titers detected by ACCA in patients with varicella, and to a lesser extent in zoster patients, were as high as or higher than those demonstrated by solid-phase indirect EIFA. However, ACCA was totally insensitive in detecting VZV IgG antibody in individuals with past infections with VZV and would not be a suitable approach for determination of immunity status to VZV. PMID:6330163

Detection of antinuclear antibodies in SLE.

PubMed

Kumar, Yashwant; Bhatia, Alka

2014-01-01

The antinuclear antibodies (ANA) also known as antinuclear factors (ANF) are unwanted molecules which bind and destroy certain structures within the nucleus. In systemic lupus erythematosus (SLE), they are produced in excess; hence their detection in the blood of patients is important for diagnosis and monitoring of the disease. Several methods are available which can be used to detect ANA; nevertheless, indirect immunofluorescence antinuclear antibody test (IF-ANA) is considered a "reference method" for their detection. Though IF-ANA is relatively easier to perform, its interpretation requires considerable skill and experience. The chapter therefore is aimed to provide comprehensive details to readers, not only about its methodology but also the result interpretation and reporting aspects of IF-ANA.

Rhinovirus antibodies in an isolated Amazon Indian tribe.

PubMed

Thwing, C J; Arruda, E; Vieira Filho, J P; Castelo Filho, A; Gwaltney, J M

1993-06-01

In early 1985, the Parakana-Apiterewa, a small, primitive Indian tribe, was contacted in the southern Amazon Basin. The tribe was thought to have been totally isolated from civilization until recent development of their land. Blood specimens were collected in 1985, shortly after the discovery of the tribe, and analyzed for the presence of rhinovirus-neutralizing antibody to nine different immunotypes. Six to forty-seven percent of the serum samples tested contained antibody to at least one immunotype of rhinovirus. The prevalence of rhinovirus antibody in the Parakana-Apiterewa Indians was similar to that reported in United States populations, suggesting that there had been considerable direct or indirect contact in the past between tribe members and persons in the outside world.

Antinuclear antibody determination in a routine laboratory.

PubMed Central

Feltkamp, T E

1996-01-01

Pitfalls in the method for demonstrating antinuclear antibodies (ANA) by the indirect immunofluorescence technique are described and the use of international standard preparations outlined. Determination of the optimal border dilution dividing positive from negative results is discussed. Each laboratory is a unique setting; it must define its own method, which should rarely be changed. One should not rely on copying methods from other laboratories or commercial firms, but the reproducibility of the nuclear substrate, the conjugate, and other variables should be controlled daily by the use of a control serum which has been related to the WHO standard preparation for ANA of the homogeneous type. Since many sera contain mixtures of different ANA, the results of routine tests are best expressed in titres or expressions of the intensity of fluorescence. The ANA test using the immunofluorescence technique should be used as a screening method for other tests allowing a more defined interpretation of the ANA. Each laboratory should individually determine the border between positive and negative results. Therefore about 200 sera from local healthy controls equally distributed over sex and age, and 100 sera from local patients with definite SLE should be tested. Since the local clinicians should become acquainted with this border it should rarely be changed. Finally each laboratory should participate regularly in national and international quality control rounds, where sera known to be difficult to interpret are tested. The judgment of the organisers of these rounds should stimulate improvements in the participating laboratories. PMID:8984936

Frequency and significance of antibodies to liver/kidney microsome type 1 in adults with chronic active hepatitis.

PubMed

Czaja, A J; Manns, M P; Homburger, H A

1992-10-01

To assess the frequency of antibodies to liver/kidney microsome type 1 (anti-LKM1) in patients with chronic active hepatitis, 131 such patients were tested by an indirect immunofluorescence assay. Of 62 patients with type 1 autoimmune hepatitis, none were seropositive. In contrast, 3 of 11 patients with autoimmune hepatitis and antimitochondrial antibodies (27%) were seropositive for anti-LKM1. Each had responded to corticosteroid therapy, and retesting of sera confirmed that each had been misclassified as antimitochondrial antibody positive. None of the patients with chronic active hepatitis B (14 patients) or C (24 patients) had anti-LKM1. Similarly, none of the 20 patients with cryptogenic disease had these antibodies. It is concluded that anti-LKM1 is specific for type 2 autoimmune hepatitis and is infrequent in adult patients seen at a referral center in the United States for chronic active hepatitis. Anti-LKM1 reactivity may be misinterpreted as antimitochondrial antibody reactivity by indirect immunofluorescence. Chronic hepatitis B and C virus infections are not important stimuli for the production of anti-LKM1, and testing for anti-LKM 1 is unlikely to clarify the nature of cryptogenic disease.

[Isolation and immunology identification of spermagglutinating antibodies from human serum].

PubMed

Cibulka, J; Ulcova-Gallová, Z; Balvín, M; Bibková, K; Micanová, Z

2009-06-01

Isolation of spermagglutinating antibodies and their assesment. Retrospective study. Special consulting for reptoduction immunology, Department of Obstetrics and Gynecology, Charles University and Faculty Hospital, Plzen. Fractionation of serum samples by liquid exclusion chromatography, examination of full sera and their chromatographic fractions by Friberg teste (Tray Agglutination Test--TAT), indirect antiimmunoglobulin reaction test (i-MAR test) and by supplementar radial immunodiffusiona (RID). Isolation of spermagglutinating fractions of antisperm antibodies positive sera preserved spermagglutinating aktivity and confirmed great spermagglutinating potential of IgM. According to assesment of the presence of IgG and IgG we reported possible states of immunisation: actual immunisation with IgM activity, perpetual stimulation (IgG and IgM) and, finaly, anamnestic titres in IgG. These findings can help us to choose an optimal way of treatment. Excluding gel chromatography is suitable method for serum proteins fractionation, but not their identification--presence of antisperm antibodies does not affect the chromatographic spectrum, nor the RID patterns.

Diagnosis of ocular and glandular toxoplasmosis using the Indian ink immunoreaction.

PubMed

Safar, E H; Azab, M E; Osman, Z M

1984-01-01

The Indian ink immunoreaction (IIR) as a method for diagnosis of Toxoplasma infection has been evaluated. 68 sera from patients with suspected ocular and glandular toxoplasmosis and 30 control sera from normal individuals were tested using both indirect fluorescent antibody test (IFAT) and IIR. The test proved to be specific, simple and rapid especially for screening purposes.

Human oesophagus: a convenient antigenic substrate for the determination of anti-endomysium antibodies in the serological diagnosis of coeliac disease.

PubMed

Uibo, O; Lambrechts, A; Mascart-Lemone, F

1995-01-01

Immunoglobulin (Ig) A-class anti-endomysium antibodies are superior to other current antibody tests for detecting coeliac disease. We aimed to evaluate the suitability of human oesophagus for the determination of anti-endomysium antibodies. The specificity of monkey and human oesophageal tissue as antigenic substrate were compared using indirect immunofluorescence analysis. Overall, 159 individuals were studied: 56 patients with biopsy-proven coeliac disease (39 with active disease) and 103 controls. The patients' IgA-class anti-endomysium antibodies were compared using unfixed cryostat sections of human and monkey oesophagus. Indirect immunofluorescence analysis was performed with an initial serum sample dilution of 1:5, and if positive, the highest dilution yielding a positive reaction was reported. The anti-endomysium antibody test was positive in 38 out of 39 patients with active coeliac disease using monkey oesophagus (sensitivity 97%) and in all 39 patients with active coeliac disease using human oesophagus (sensitivity 100%). Ten out of 17 coeliac patients on a gluten-free diet had positive anti-endomysium antibodies using monkey oesophagus and 12 using human oesophagus as the antigenic substrate. This test was negative in all 103 controls using both substrates. Our study shows that human oesophageal tissue can be used instead of monkey tissue for determining anti-endomysium antibodies. Human tissue is a more sensitive antigenic substrate than monkey oesophagus and can be used to determine low titres of antibodies. Improving the diagnostic sensitivity of the anti-endomysium antibody test would make an important contribution to screening for coeliac disease.

Specific detection of Vibrio parahaemolyticus by fluorescence quenching immunoassay based on quantum dots.

PubMed

Wang, Ling; Zhang, Junxian; Bai, Haili; Li, Xuan; Lv, Pintian; Guo, Ailing

2014-07-01

In this study, anti-Vibrio parahaemolyticus polyclonal and monoclonal antibodies were prepared through intradermal injection immune and lymphocyte hybridoma technique respectively. CdTe quantum dots (QDs) were synthesized at pH 9.3, 98 °C for 1 h with stabilizer of 2.7:1. The fluorescence intensity was 586.499, and the yield was 62.43%. QD probes were successfully prepared under the optimized conditions of pH 7.4, 37 °C for 1 h, 250 μL of 50 mg/mL EDC · HCl, 150 μL of 4 mg/mL NHS, buffer system of Na2HPO4-citric acid, and 8 μL of 2.48 mg/mL polyclonal antibodies. As gold nanoparticles could quench fluorescence of quantum dots, the concentration of V. parahaemolyticus could be detected through measuring the reduction of fluorescence intensity in immune sandwich reaction composed of quantum dot probe, gold-labeled antibody, and the sample. For pure culture, fluorescence intensity of the system was proportional with logarithm concentration of antigen, and the correlation coefficient was 99.764%. The fluorescence quenching immunoassay based on quantum dots is established for the first time to detect Vibrio parahaemolyticus. This method may be used as rapid testing procedure due to its high simplicity and sensitivity.

Immunohistochemical detection of chlamydiae in formalin-fixed tissue sections: comparison of a monoclonal antibody with yolk derived antibodies (IgY).

PubMed

Kunz, U S; Pospischil, A; Paccaud, M F

1991-06-01

Immunohistological detection of chlamydiae in formalin-fixed and paraffin-embedded sections of various organs from several species is described. In a retrospective study, two antisera, a commercially available monoclonal murine antibody (IgMur) and vitelline immunoglobulins (IgY), extracted from the egg yolk of immunized hens, were compared and tested for their applicability under routine condition. Both antisera were applied to tissues from which chlamydiae had been isolated or in which the presence of chlamydiae had been suspected in specially stained sections. Antigen labelling was optimal with the monoclonal antibody. Vitelline immunoglobulins produced some unspecific reactions, especially in lung tissue sections. Because of the antigenic relationship between the vitelline antibodies and tissues of birds, IgY are not suitable for the detection of psittacosis on avian substrates, when using an indirect immunological method. Staining in other tissues e.g. intestine or placenta was of equal quality as that attained with monoclonal antibodies. Depending on the advantages and disadvantages in every individual case, one of the two antibodies may be chosen for further studies. Vitelline antibodies should be preferred with respect to animal welfare.

A comparison of the absorbed fluorescent treponemal antibody (FTA-ABS) test and other screening tests for treponemal disease in patients attending a venereal disease clinic.

PubMed

Wilkinson, A E; Scrimgeour, G; Rodin, P

1972-05-01

Screening tests-absorbed fluorescent treponemal (FTA-ABS), the Reiter protein complement-fixation (RPCFT), VDRL slide test, automated reagin-and cardiolipin Wassermann reaction-were carried out on 1922 consecutive new patients attending the Whitechapel Clinic over a three-month period.Taking the FTA-ABS test results as an index, the most efficient combination of conventional tests was found to be the RPCFT and automated reagin test. The cardiolipin WR proved to be under-sensitive and of little value compared with the other tests.Forty-two per cent of the 107 sera reactive in the FTA-ABS test were not detected by the RPCFT or ART tests. An assessment based on the TPI test results and clinical findings in these patients is presented. The scope and limitations of the FTA-ABS test as a screening procedure are discussed.

A capillary electrophoresis chip for the analysis of print and film photographic developing agents in commercial processing solutions using indirect fluorescence detection.

PubMed

Sirichai, S; de Mello, A J

2001-01-01

The separation and detection of both print and film developing agents (CD-3 and CD-4) in photographic processing solutions using chip-based capillary electrophoresis is presented. For simultaneous detection of both analytes under identical experimental conditions a buffer pH of 11.9 is used to partially ionise the analytes. Detection is made possible by indirect fluorescence, where the ions of the analytes displace the anionic fluorescing buffer ion to create negative peaks. Under optimal conditions, both analytes can be analyzed within 30 s. The limits of detection for CD-3 and CD-4 are 0.17 mM and 0.39 mM, respectively. The applicability of the method for the analysis of seasoned photographic processing developer solutions is also examined.

Generation of anti-azoxystrobin monoclonal antibodies from regioisomeric haptens functionalized at selected sites and development of indirect competitive immunoassays.

PubMed

Parra, Javier; Mercader, Josep V; Agulló, Consuelo; Abad-Somovilla, Antonio; Abad-Fuentes, Antonio

2012-02-17

Azoxystrobin is a modern strobilurin fungicide used around the world to combat prime diseases affecting highly valuable crops. Accordingly, residues of this chemical are frequently found in food, even though mostly under maximum tolerated levels. We herein describe the development of an indirect competitive immunoassay for the determination of azoxystrobin residues. A panel of monoclonal antibodies displaying subnanomolar affinity to azoxystrobin was generated using, as immunizing haptens in mice, four functionalized derivatives carrying the same spacer arm located at different rationally chosen positions. This collection of antibodies was thoroughly characterized with homologous and heterologous antigens, and the immunoassay consisting of monoclonal antibody AZo6#49 and the coating conjugate OVA-AZb6, which displayed an IC(50) value of 0.102 μg L(-1) and a LOD of 0.017 μg L(-1), was eventually optimized. The response to different pH and ionic strength conditions of the specific assay was studied using a biparametric approach. In addition, the influence of Tween 20 and organic solvents over the assay parameters was also evaluated. After optimization, the developed immunochemical assay was applied to the analysis of azoxystrobin in spiked juices of relevant fruits and vegetables, showing excellent recoveries between 2 and 500 μg L(-1). Copyright © 2011 Elsevier B.V. All rights reserved.

MP-4 Contributes to Snake Venom Neutralization by Mucuna pruriens Seeds through an Indirect Antibody-mediated Mechanism*

PubMed Central

Kumar, Ashish; Gupta, Chitra; Salunke, Dinakar M.

2016-01-01

Mortality due to snakebite is a serious public health problem, and available therapeutics are known to induce debilitating side effects. Traditional medicine suggests that seeds of Mucuna pruriens can provide protection against the effects of snakebite. Our aim is to identify the protein(s) that may be important for snake venom neutralization and elucidate its mechanism of action. To this end, we have identified and purified a protein from M. pruriens, which we have named MP-4. The full-length polypeptide sequence of MP-4 was obtained through N-terminal sequencing of peptide fragments. Sequence analysis suggested that the protein may belong to the Kunitz-type protease inhibitor family and therefore may potentially neutralize the proteases present in snake venom. Using various structural and biochemical tools coupled with in vivo assays, we are able to show that MP-4 does not afford direct protection against snake venom because it is actually a poor inhibitor of serine proteases. Further experiments showed that antibodies generated against MP-4 cross-react with the whole venom and provide protection to mice against Echis carinatus snake venom. This study shows that the MP-4 contributes significantly to the snake venom neutralization activity of M. pruriens seeds through an indirect antibody-mediated mechanism. PMID:26987900

Comparing Direct versus Indirect Measures of the Pedagogical Effectiveness of Team Testing

ERIC Educational Resources Information Center

Bacon, Donald R.

2011-01-01

Direct measures (tests) of the pedagogical effectiveness of team testing and indirect measures (student surveys) of pedagogical effectiveness of team testing were collected in several sections of an undergraduate marketing course with varying levels of the use of team testing. The results indicate that although students perceived team testing to…

Test-Potentiated Learning: Distinguishing Between Direct and Indirect Effects of Tests

PubMed Central

Arnold, Kathleen M.; McDermott, Kathleen B.

2013-01-01

The facilitative effect of retrieval practice, or testing, on the probability of later retrieval has been the focus of much recent empirical research. A lesser-known benefit of retrieval practice is that it may also enhance the ability of a learner to benefit from a subsequent restudy opportunity. This facilitative effect of retrieval practice on subsequent encoding is known as test-potentiated learning. Thus far, however, the literature has not isolated the indirect effect of retrieval practice on subsequent memory (via enhancing the effectiveness of restudy) from the direct effects of retrieval on subsequent memory. The experiment presented here uses conditional probability to disentangle test-potentiated learning from the direct effects of retrieval practice. The results indicate that unsuccessful retrieval attempts enhance the effectiveness of subsequent restudy, demonstrating that tests do potentiate subsequent learning. PMID:22774852

Testing Eurasian wild boar piglets for serum antibodies against Mycobacterium bovis.

PubMed

Che' Amat, A; González-Barrio, D; Ortiz, J A; Díez-Delgado, I; Boadella, M; Barasona, J A; Bezos, J; Romero, B; Armenteros, J A; Lyashchenko, K P; Venteo, A; Rueda, P; Gortázar, C

2015-09-01

Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (n=53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account. Copyright © 2015 Elsevier B.V. All rights reserved.

How to use … the Monospot and other heterophile antibody tests.

PubMed

Marshall-Andon, Tess; Heinz, Peter

2017-08-01

Epstein-Barr virus (EBV) is a highly prevalent virus, transmitted via saliva, which often causes asymptomatic infection in children but frequently results in infectious mononucleosis in adolescents. Heterophile antibody tests, including the Monospot test, are red cell or latex agglutination assays, which detect antired cell antibodies produced as part of a polyclonal antibody response occurring during EBV infection. Heterophile antibody tests are rapid, cheap and specific tests that can be performed from the onset of symptoms of infectious mononucleosis. In adolescents, heterophile antibody tests have high specificity and sensitivity in the diagnosis of primary acute EBV infection. However, the tests have low sensitivity and low negative predictive value in young children and are not useful under the age of 4. Heterophile tests may be positive in other viral infections, autoimmune disease and haematological malignancies, but do not appear to be positive in primary bacterial infection. Virus-specific serology is required in children under the age of 4 or if an older child is heterophile negative. Virus-specific serology allows diagnosis and the pattern of positivity and negativity enables the clinician to stage the EBV infection. Virus-specific serology appears to have better sensitivity in young children, but there is cross-reaction with other herpesvirus infections, a longer turnaround time and it is more expensive to perform. Further research is needed to establish which children benefit from and hence require testing for heterophile antibodies, the cost-effectiveness of EBV investigations and whether heterophile titres have predictive value for the severity of infection and the likelihood of complications. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

PubMed

Blagojević Zagorac, Gordana; Mahmutefendić, Hana; Maćešić, Senka; Karleuša, Ljerka; Lučin, Pero

2017-03-01

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Confocal fluorescence microscopy in a murine model of microdissection testicular sperm extraction to improve sperm retrieval.

PubMed

Smith, Ryan P; Lowe, Greg J; Kavoussi, Parviz K; Steers, William D; Costabile, Raymond A; Herr, John C; Shetty, Jagathpala; Lysiak, Jeffrey J

2012-05-01

Microdissection testicular sperm extraction markedly improves the sperm retrieval rates in men with nonobstructive azoospermia. However, localizing sperm foci can be time-consuming and it is not always successful. Fiberoptic confocal fluorescent microscopy offers the advantage of rapid in vivo detection of fluorescently labeled sperm in the seminiferous tubules. After establishing the feasibility of fiberoptic confocal fluorescent microscopy to identify antibody labeled sperm in vivo C57/B6 mice underwent intraperitoneal injection of busulfan to induce azoospermia. During spermatogenesis reestablishment at approximately 16 weeks the mice were anesthetized and the testes were delivered through a low midline incision. Fluorescein isothiocyanate labeled antibody to intra-acrosomal protein Hs-14 was injected retrograde into a single murine rete testis. The testes were imaged in vivo with fiberoptic confocal fluorescent microscopy and sperm foci were detected. The respective seminiferous tubules were excised and squash prepared for immunofluorescence microscopy. Sperm foci were identified in the testis injected with fluorescently tagged antibody by in vivo fiberoptic confocal fluorescence microscopy. The contralateral control testis of each mouse showed no specific signal. Immunofluorescence microscopy of the excised tubules provided morphological confirmation of the presence of labeled sperm with an absence in controls. Findings were consistent in the feasibility portion of the study and in the busulfan model of nonobstructive azoospermia. Fiberoptic confocal fluorescent microscopy was feasible during microdissection testicular sperm extraction in an azoospermic mouse model to identify fluorescently labeled sperm in vivo. Translation to the clinical setting could decrease operative time and improve the sperm harvest rate. Copyright © 2012 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

Detection of anthrax protective antigen (PA) using europium labeled anti-PA monoclonal antibody and time-resolved fluorescence ◊

PubMed Central

Stoddard, Robyn A.; Quinn, Conrad P.; Schiffer, Jarad M.; Boyer, Anne E.; Goldstein, Jason; Bagarozzi, Dennis A.; Soroka, Stephen D.; Dauphin, Leslie A.; Hoffmaster, Alex R.

2015-01-01

Inhalation anthrax is a rare but acute infectious disease following adsorption of Bacillus anthracis spores through the lungs. The disease has a high fatality rate if untreated, but early and correct diagnosis has a significant impact on case patient recovery. The early symptoms of inhalation anthrax are, however, non-specific and current anthrax diagnostics are primarily dependent upon culture and confirmatory real-time PCR. Consequently, there may be a significant delay in diagnosis and targeted treatment. Rapid, culture-independent diagnostic tests are therefore needed, particularly in the context of a large scale emergency response. The aim of this study was to evaluate the ability of monoclonal antibodies to detect anthrax toxin proteins that are secreted early in the course of B. anthracis infection using a time-resolved fluorescence (TRF) immunoassay. We selected monoclonal antibodies that could detect protective antigen (PA), as PA83 and also PA63 and LF in the lethal toxin complex. The assay reliable detection limit (RDL) was 6.63 × 10−6 μM (0.551 ng/ml) for PA83 and 2.51 × 10−5 μM (1.58 ng/ml) for PA63. Despite variable precision and accuracy of the assay, PA was detected in 9 out of 10 sera samples from anthrax confirmed case patients with cutaneous (n=7), inhalation (n=2), and gastrointestinal (n=1) disease. Anthrax Immune Globulin (AIG), which has been used in treatment of clinical anthrax, interfered with detection of PA. This study demonstrates a culture-independent method of diagnosing anthrax through use of monoclonal antibodies to detect PA and LF in the lethal toxin complex. PMID:24857756

Polyvalent heat-killed antigen for the diagnosis of infection with Legionella pneumophila.

PubMed Central

Fallon, R J; Abraham, W H

1982-01-01

A polyvalent antigen composed of heat-killed agar-grown Legionella pneumophila serogroups 1-4 suspended in a suspension of yolk-sac from embryonated hens' eggs has been examined for use in the indirect fluorescent antibody test for Legionella infection. The serological response detected by monovalent antigen correlated well with that detected by polyvalent antigen. The use of polyvalent antigen forms a useful screening test for the detection of antibody to L pneumophila, but positive results must be confirmed by test using monovalent antigen. PMID:7042762

Passive haemagglutination test for antibodies against rabies virus*

PubMed Central

Gough, P. M.; Dierks, R. E.

1971-01-01

All the procedures now available for the measurement of rabies virus antibodies in serum have certain disadvantages. The serum neutralization test (SN), whether carried out by assay in mice or by the plaque-reduction technique, requires several days before the titrations are completed, necessitates special facilities for keeping large numbers of animals and tissue-culture plates, and is relatively expensive. A complement-fixation test is very insensitive, giving low titres in comparison with SN tests, and a haemagglutination-inhibition procedure is complicated by the presence of nonspecific reactions. A rabies passive haemagglutination technique (RPHA), developed to overcome many of these problems, is described. Titres obtained with human sera by the RPHA procedure correlated well with those obtained by SN tests. Both IgG and IgM classes of antibodies were measured by the RPHA procedure; however, it appeared to be more sensitive for detecting IgM than was the SN test and, therefore, gave higher titres for this class of immunoglobulins. PMID:5317009

Evaluation of ID-PaGIA syphilis antibody test.

PubMed

Naaber, Paul; Makoid, Ene; Aus, Anneli; Loivukene, Krista; Poder, Airi

2009-01-01

Laboratory diagnosis of syphilis is usually accomplished by serology. There are currently a large number of different commercial treponemal tests available that vary in format, sensitivity and specificity. To evaluate the ID-PaGIA Syphilis Antibody Test as an alternative to other specific treponemal tests for primary screening or confirmation of diagnosis. Serum samples from healthy adults (n = 100) were used for detection of specificity of ID-PaGIA. To evaluate sensitivity of ID-PaGIA serum samples (n = 101) from patients with confirmed or suspected syphilis were tested for syphilis antibodies with FTA-Abs IgM, ID-PaGIA, ELISA IgM and TPHA tests. No false-positive results were found with ID-PaGIA. Sensitivity of various treponemal tests was the following: FTA-Abs IgM: 95.5%, ID-PaGIA and ELISA IgM: 94%, and TPHA 75%. The positive and negative predictive values of ID-PaGIA were 100 and 89.5%, respectively. Compared with other treponemal tests ID-PaGIA has excellent sensitivity and specificity.

Fluorescent IgG fusion proteins made in E. coli

PubMed Central

Luria, Yael; Raichlin, Dina; Benhar, Itai

2012-01-01

Antibodies are among the most powerful tools in biological and biomedical research and are presently the fastest growing category of new bio-pharmaceutics. The most common format of antibody applied for therapeutic, diagnostic and analytical purposes is the IgG format. For medical applications, recombinant IgGs are made in cultured mammalian cells in a process that is too expensive to be considered for producing antibodies for diagnostic and analytical purposes. Therefore, for such purposes, mouse monoclonal antibodies or polyclonal sera from immunized animals are used. While looking for an easier and more rapid way to prepare full-length IgGs for therapeutic purposes, we recently developed and reported an expression and purification protocol for full-length IgGs, and IgG-based fusion proteins in E. coli, called “Inclonals.” By applying the Inclonals technology, we could generate full-length IgGs that are genetically fused to toxins. The aim of the study described herein was to evaluate the possibility of applying the “Inclonals” technology for preparing IgG-fluorophore fusion proteins. We found that IgG fused to the green fluorescent proteins enhanced GFP (EGFP) while maintaining functionality in binding, lost most of its fluorescence during the refolding process. In contrast, we found that green fluorescent Superfolder GFP (SFGFP)-fused IgG and red fluorescent mCherry-fused IgG were functional in antigen binding and maintained fluorescence intensity. In addition, we found that we can link several SFGFPs in tandem to each IgG, with fluorescence intensity increasing accordingly. Fluorescent IgGs made in E. coli may become attractive alternatives to monoclonal or polyclonal fluorescent antibodies derived from animals. PMID:22531449

Monoclonal antibody against human ovarian tumor-associated antigens.

PubMed

Poels, L G; Peters, D; van Megen, Y; Vooijs, G P; Verheyen, R N; Willemen, A; van Niekerk, C C; Jap, P H; Mungyer, G; Kenemans, P

1986-05-01

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.

Development, characterization, and lethal effect of monoclonal antibodies against hemocytes in an adult female tick, Ornithodoros moubata (Acari: Argasidae).

PubMed

Matsuo, T; Tsukamoto, D; Inoue, N; Fujisaki, K

2003-12-01

In the present study, 19 monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established, and the reactivity of the hemocytes to these mAbs was examined by an indirect fluorescent antibody test (IFAT), Western blot and immunoprecipitation analyses. It was shown that the reactivities of the hemocytes to the mAbs varied among morphologically similar hemocyte types, and most mAbs produced in the present study showed the multiple band reactivity. However, the presence of shared epitopes among peptide subunits of the same protein or entirely different proteins are not common, so their reactivity could not be explained in detail. These results suggest that there are morphologically similar but functionally differentiated hemocytes. Therefore, in addition to morphological classification, the molecular-based classification of the hemocytes is also required. In order to assess the lethal effect of blood meal containing each mAb, artificial feeding was performed. The OmHC 31 showed the strongest lethal effect on adult female O. moubata. In conclusion, anti-hemocyte mAbs produced in this study are useful not only for the immunological classification of hemocytes but also for the immunological control of the tick.

Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.

PubMed

Liao-Chan, Sindy; Daine-Matsuoka, Barbara; Heald, Nathan; Wong, Tiffany; Lin, Tracey; Cai, Allen G; Lai, Michelle; D'Alessio, Joseph A; Theunissen, Jan-Willem

2015-01-01

Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.

Tailoring Antibody Testing and How to Use it in the Calculated Panel Reactive Antibody Era: The Northwestern University Experience

PubMed Central

Tambur, Anat R.; Leventhal, Joseph; Kaufman, Dixon B.; Friedewald, John; Miller, Joshua; Abecassis, Michael M.

2014-01-01

Background Patients with human leukocyte antigen antibodies constitute a significantly disadvantaged population among those awaiting renal transplantation. We speculated that more understanding of the patients’ antibody makeup would allow a more “immunologic” evaluation of crossmatch data, facilitate the use of virtual crossmatch (XM), and lead to more transplantability of these patients. Methods We retrospectively compared the transplantability and transplant outcome of two consecutive patient populations transplanted in our center. Group I (n=374) was evaluated using solid-phase base testing for determination of percentage panel reactive antibody (“PRA screen”) with limited antibody identification testing. Group II (n=333) was tested in a more comprehensive manner with major emphasis on antibody identification, antibody strength assignment, and the use of pronase for crossmatch. Results Given this approach, 49% (166/333) of the transplanted patients in group II were sensitized compared with 40% (150/374) of the recipients in group I; P=0.012. Transplant outcome at 1-year posttransplant was similar in both groups. Conclusions We conclude that comprehensive evaluation of human leukocyte antigen sensitization and application of immunologic in analyzing compatibility between donor and recipient can increase the transplantability of sensitized patients while maintaining similar outcome. Our approach is in line with United Network for Organ Sharing new guidelines for calculated panel reactive antibody and virtual XM analysis. We hope this report will prompt additional transplant programs to consider how they will use the new United Network for Organ Sharing algorithms. PMID:18946342

MEMS reagent and sample handling procedure: Feasibility of viral antibody detection by passive immune agglutination

NASA Technical Reports Server (NTRS)

Bailey, G. D.; Tenoso, H. J.

1975-01-01

An attempt was made to develop a test requiring no preadsorption steps for the assessment of antibodies to rubella and mumps viruses using the passive immune agglutination (PIA) method. Both rubella and mumps antigens and antibodies were prepared. Direct PIA tests, using rubella antigen-coated beads, and indirect PIA tests, using rubella antibody-coated beads, were investigated. Attempts, using either method, were unsuccessful. Serum interference along with nonspecific agglutination of beads by the rubella antigen resulted in no specific response under the test conditions investigated. A new, highly sensitive approach, the enzyme immunoassay (EIA) test system, is recommended to overcome the nonspecificity. This system is a logical outgrowth of some of the solid phase work done on MEMS and represents the next generation tests system that can be directly applied to early disease detection and monitoring.

Rapid, whole blood diagnostic test for detecting anti-hantavirus antibody in rats.

PubMed

Amada, Takako; Yoshimatsu, Kumiko; Yasuda, Shumpei P; Shimizu, Kenta; Koma, Takaaki; Hayashimoto, Nobuhito; Gamage, Chandika D; Nishio, Sanae; Takakura, Akira; Arikawa, Jiro

2013-10-01

Hantavirus is a causative agent of rodent-borne viral zoonoses, hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome. Seoul virus (SEOV) is a causative agent of urban and laboratory rat-associated HFRS worldwide. Surveillance of rodents has been done mainly by serological detection of hantavirus-specific antibodies by enzyme linked immunosorbent assay (ELISA) and immunofluorescent antibody assay (IFA). An immunochromatographic (ICG) test was developed with the N-terminal 103 amino acids of nucleocapsid protein of Hantaan virus expressed by Escherichia coli as an antigen to detect IgG antibody specific to hantavirus in sera from Rattus sp. animals. Antibody-detecting sensitivity of the ICG test was the same as that of ELISA and about 100-times higher than that of IFA. Overall sensitivities and specificities of the ICG test in comparison to ELISA and IFA for sera from 192 urban rats and 123 laboratory rats were 99.3% and 100%, respectively. Diluted whole blood samples without separation could be used for the ICG test. The ICG test enabled detection of antibodies to SEOV, Hantaan, Dobrava/Belgrade, and Thailand viruses, which are causative agents of HFRS throughout Eurasia. The ICG test is a rapid, simple and safe method for diagnosis of SEOV infection in rats. Copyright © 2013 Elsevier B.V. All rights reserved.

Quantum dot immunoassays in renewable surface column and 96-well plate formats for the fluorescence detection of Botulinum neurotoxin using high-affinity antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Warner, Marvin G.; Grate, Jay W.; Tyler, Abby J.

2009-09-01

A fluorescence sandwich immunoassay using high affinity antibodies and quantum dot (QD) reporters has been developed for detection of botulinum toxin serotype A (BoNT/A). For the development of the assay, a nontoxic recombinant fragment of the holotoxin (BoNT/A-HC-fragment) has been used as a structurally valid simulant for the full toxin molecule. The antibodies used, AR4 and RAZ1, bind to nonoverlapping epitopes present on both the full toxin and on the recombinant fragment. In one format, the immunoassay is carried out in a 96-well plate with detection in a standard plate reader. Detection down to 31 pM of the BoNT/Hc-fragment wasmore » demonstrated with a total incubation time of 3 hours, using AR4 as the capture antibody and QD-coupled RAZ1 as the reporter. In a second format, the AR4 capture antibody was coupled to Sepharose beads, and the immunochemical reactions were carried out in microcentrifuge tubes with an incubation time of 1 hour. These beads were subsequently captured and concentrated in a rotating rod “renewable surface” flow cell as part of a sequential injection fluidic system. This flow cell was equipped with a fiber optic system for fluorescence measurements. In PBS buffer solution matrix, the BoNT/A-HC-fragment was detected to concentrations as low as 5 pM using the fluidic measurement approach.« less

Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging.

PubMed

Wolak, Daniel J; Pizzo, Michelle E; Thorne, Robert G

2015-01-10

Antibody-based therapeutics exhibit great promise in the treatment of central nervous system (CNS) disorders given their unique customizable properties. Although several clinical trials have evaluated therapeutic antibodies for treatment of CNS disorders, success to date has likely been limited in part due to complex issues associated with antibody delivery to the brain and antibody distribution within the CNS compartment. Major obstacles to effective CNS delivery of full length immunoglobulin G (IgG) antibodies include transport across the blood-brain and blood-cerebrospinal fluid barriers. IgG diffusion within brain extracellular space (ECS) may also play a role in limiting central antibody distribution; however, IgG transport in brain ECS has not yet been explored using established in vivo methods. Here, we used real-time integrative optical imaging to measure the diffusion properties of fluorescently labeled, non-targeted IgG after pressure injection in both free solution and in adult rat neocortex in vivo, revealing IgG diffusion in free medium is ~10-fold greater than in brain ECS. The pronounced hindered diffusion of IgG in brain ECS is likely due to a number of general factors associated with the brain microenvironment (e.g. ECS volume fraction and geometry/width) but also molecule-specific factors such as IgG size, shape, charge and specific binding interactions with ECS components. Co-injection of labeled IgG with an excess of unlabeled Fc fragment yielded a small yet significant increase in the IgG effective diffusion coefficient in brain, suggesting that binding between the IgG Fc domain and endogenous Fc-specific receptors may contribute to the hindered mobility of IgG in brain ECS. Importantly, local IgG diffusion coefficients from integrative optical imaging were similar to those obtained from ex vivo fluorescence imaging of transport gradients across the pial brain surface following controlled intracisternal infusions in anesthetized animals. Taken

Probing the extracellular diffusion of antibodies in brain using in vivo integrative optical imaging and ex vivo fluorescence imaging

PubMed Central

Wolak, Daniel J.; Pizzo, Michelle E.; Thorne, Robert G.

2014-01-01

Antibody-based therapeutics exhibit great promise in the treatment of central nervous system (CNS) disorders given their unique customizable properties. Although several clinical trials have evaluated therapeutic antibodies for treatment of CNS disorders, success to date has likely been limited in part due to complex issues associated with antibody delivery to the brain and antibody distribution within the CNS compartment. Major obstacles to effective CNS delivery of full length immunoglobulin G (IgG) antibodies include transport across the blood-brain and blood-cerebrospinal fluid barriers. IgG diffusion within brain extracellular space (ECS) may also play a role in limiting central antibody distribution; however, IgG transport in brain ECS has not yet been explored using established in vivo methods. Here, we used real-time integrative optical imaging to measure the diffusion properties of fluorescently labeled, non-targeted IgG after pressure injection in both free solution and in adult rat neocortex in vivo, revealing IgG diffusion in free medium is ~10-fold greater than in brain ECS. The pronounced hindered diffusion of IgG in brain ECS is likely due to a number of general factors associated with the brain microenvironment (e.g. ECS volume fraction and geometry/width) but also molecule-specific factors such as IgG size, shape, charge and specific binding interactions with ECS components. Co-injection of labeled IgG with an excess of unlabeled Fc fragment yielded a small yet significant increase in the IgG effective diffusion coefficient in brain, suggesting that binding between the IgG Fc domain and endogenous Fc-specific receptors may contribute to the hindered mobility of IgG in brain ECS. Importantly, local IgG diffusion coefficients from integrative optical imaging were similar to those obtained from ex vivo fluorescence imaging of transport gradients across the pial brain surface following controlled intracisternal infusions in anesthetized animals. Taken

Neutralizing Antibody Response in Dogs and Cats Inoculated with Commercial Inactivated Rabies Vaccines

PubMed Central

SHIRAISHI, Rikiya; NISHIMURA, Masaaki; NAKASHIMA, Ryuji; ENTA, Chiho; HIRAYAMA, Norio

2013-01-01

ABSTRACT In Japan, the import quarantine regulation against rabies has required from 2005 that dogs and cats should be inoculated with the rabies vaccine and that the neutralizing antibody titer should be confirmed to be at least 0.5 international units (IU)/ml. The fluorescent antibody virus neutralization (FAVN) test is used as an international standard method for serological testing for rabies. To achieve proper immunization of dogs and cats at the time of import and export, changes in the neutralizing antibody titer after inoculation of the rabies vaccine should be understood in detail. However, few reports have provided this information. In this study, we aimed to determine evaluated, such changes by using sera from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled the necessary titer of neutralizing antibodies to be maintained in the long term. However, inappropriate timing of blood sampling after vaccination could result in insufficient detected levels of neutralizing antibodies. PMID:24389741

Neutralizing antibody response in dogs and cats inoculated with commercial inactivated rabies vaccines.

PubMed

Shiraishi, Rikiya; Nishimura, Masaaki; Nakashima, Ryuji; Enta, Chiho; Hirayama, Norio

2014-04-01

In Japan, the import quarantine regulation against rabies has required from 2005 that dogs and cats should be inoculated with the rabies vaccine and that the neutralizing antibody titer should be confirmed to be at least 0.5 international units (IU)/ml. The fluorescent antibody virus neutralization (FAVN) test is used as an international standard method for serological testing for rabies. To achieve proper immunization of dogs and cats at the time of import and export, changes in the neutralizing antibody titer after inoculation of the rabies vaccine should be understood in detail. However, few reports have provided this information. In this study, we aimed to determine evaluated, such changes by using sera from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled the necessary titer of neutralizing antibodies to be maintained in the long term. However, inappropriate timing of blood sampling after vaccination could result in insufficient detected levels of neutralizing antibodies.

MP-4 Contributes to Snake Venom Neutralization by Mucuna pruriens Seeds through an Indirect Antibody-mediated Mechanism.

PubMed

Kumar, Ashish; Gupta, Chitra; Nair, Deepak T; Salunke, Dinakar M

2016-05-20

Mortality due to snakebite is a serious public health problem, and available therapeutics are known to induce debilitating side effects. Traditional medicine suggests that seeds of Mucuna pruriens can provide protection against the effects of snakebite. Our aim is to identify the protein(s) that may be important for snake venom neutralization and elucidate its mechanism of action. To this end, we have identified and purified a protein from M. pruriens, which we have named MP-4. The full-length polypeptide sequence of MP-4 was obtained through N-terminal sequencing of peptide fragments. Sequence analysis suggested that the protein may belong to the Kunitz-type protease inhibitor family and therefore may potentially neutralize the proteases present in snake venom. Using various structural and biochemical tools coupled with in vivo assays, we are able to show that MP-4 does not afford direct protection against snake venom because it is actually a poor inhibitor of serine proteases. Further experiments showed that antibodies generated against MP-4 cross-react with the whole venom and provide protection to mice against Echis carinatus snake venom. This study shows that the MP-4 contributes significantly to the snake venom neutralization activity of M. pruriens seeds through an indirect antibody-mediated mechanism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

An influenza A virus agglutination test using antibody-like polymers.

PubMed

Sukjee, Wannisa; Thitithanyanont, Arunee; Wiboon-Ut, Suwimon; Lieberzeit, Peter A; Paul Gleeson, M; Navakul, Krongkaew; Sangma, Chak

2017-10-01

Antibodies are commonly used in diagnostic routines to identify pathogens. The testing protocols are relatively simple, requiring a certain amount of a specific antibody to detect its corresponding pathogen. Antibody functionality can be mimicked by synthesizing molecularly imprinted polymers (MIPs), i.e. polymers that can selectively recognize a given template structure. Thus, MIPs are sometimes termed 'plastic antibody (PA)'. In this study, we have synthesized new granular MIPs using influenza A virus templates by precipitation polymerization. The selective binding of influenza A to the MIP particles was assessed and subsequently contrasted with other viruses. The affinities of influenza A virus towards the MIP was estimated based on an agglutination test by measuring the amount of influenza subtypes absorbed onto the MIPs. The MIPs produced using the H1N1 template showed specific reactivity to H1N1 while those produced using H5N1 and H3N2 templates showed cross-reactivity.

[Analysis on willingness to pay for HIV antibody saliva rapid test and related factors].

PubMed

Li, Junjie; Huo, Junli; Cui, Wenqing; Zhang, Xiujie; Hu, Yi; Su, Xingfang; Zhang, Wanyue; Li, Youfang; Shi, Yuhua; Jia, Manhong

2015-02-01

To understand the willingness to pay for HIV antibody saliva rapid test and its influential factors among people seeking counsel and HIV test, STD clinic patients, university students, migrant people, female sex workers (FSWs), men who have sex with men (MSM) and injecting drug users (IDUs). An anonymous questionnaire survey was conducted among 511 subjects in the 7 groups selected by different sampling methods, and 509 valid questionnaires were collected. The majority of subjects were males (54.8%) and aged 20-29 years (41.5%). Among the subjects, 60.3% had education level of high school or above, 55.4% were unmarried, 37.3% were unemployed, 73.3% had monthly expenditure <2 000 Yuan RMB, 44.2% had received HIV test, 28.3% knew HIV saliva test, 21.0% were willing to receive HIV saliva test, 2.0% had received HIV saliva test, only 1.0% had bought HIV test kit for self-test, and 84.1% were willing to pay for HIV antibody saliva rapid test. Univariate logistic regression analysis indicated that subject group, age, education level, employment status, monthly expenditure level, HIV test experience and willingness to receive HIV saliva test were correlated statistically with willingness to pay for HIV antibody saliva rapid test. Multivariate logistic regression analysis showed that subject group and monthly expenditure level were statistically correlated with willingness to pay for HIV antibody saliva rapid test. The willingness to pay for HIV antibody saliva rapid test and acceptable price of HIV antibody saliva rapid test varied in different areas and populations. Different populations may have different willingness to pay for HIV antibody saliva rapid test;the affordability of the test could influence the willingness to pay for the test.

Development and testing of monoclonal antibody-based rapid immunodiagnostic test kits for direct detection of Vibrio cholerae O139 synonym Bengal.

PubMed

Hasan, J A; Huq, A; Nair, G B; Garg, S; Mukhopadhyay, A K; Loomis, L; Bernstein, D; Colwell, R R

1995-11-01

We report on the development and testing of two monoclonal antibody-based rapid immunodiagnostic test kits, BengalScreen, a coagglutination test, and Bengal DFA, a direct fluorescent-antibody test, for direct detection of Vibrio cholerae O139 synonym Bengal in clinical and environmental specimens. The BengalScreen test requires less than 5 min to complete and can be used in the field. Bengal DFA, being more sensitive than BengalScreen, requires only one reagent and less than 20 min for detection and enumeration of V. cholerae O139 synonym Bengal. In tests for specificity, all 40 strains of V. cholerae O139 reacted with both test kits, whereas 157 strains of heterologous species examined did not, yielding 100% specificity in this study. A field trial was conducted in with both BengalScreen and Bengal DFA, and the results were compared with those obtained by conventional culture methods. BengalScreen demonstrated a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 94%. Results obtained by Bengal DFA, on the other hand, were 100% sensitive and 100% specific and yielded 100% positive and negative predictive values compared with culture methods. In a second evaluation, 93 stool specimens from Mexico that were negative for V. cholerae O139 by culture were also tested with both the BengalScreen and Bengal DFA kits. None of the 93 specimens were positive for V. cholerae O139 by both tests. A concentration method was optimized for screening of environmental water samples for V. cholerae O139 synonym Bengal with rapid test kits. BengalScreen results were unequivocally positive when water samples contained at least 2.0 x 10(3) CFU/ml, whereas Bengal DFA demonstrated an unequivocally positive reaction when the water sample contained at least 1.5 x 10(2) CFU/ml. When Bengal DFA was compared with conventional culture methods for enumeration of V. cholerae O139 synonym Bengal organisms, no difference was observed.

Comparison of the PRNT and an immune fluorescence assay in yellow fever vaccinees receiving immunosuppressive medication.

PubMed

Wieten, Rosanne W; Jonker, Emile F F; Pieren, Daan K J; Hodiamont, Caspar J; van Thiel, Pieter P A M; van Gorp, Eric C M; de Visser, Adriëtte W; Grobusch, Martin P; Visser, Leo G; Goorhuis, Abraham

2016-03-04

The 17D-yellow fever (YF) vaccination is considered contraindicated in immune-compromised patients; however, accidental vaccination occurs. In this population, measuring the immune response is useful in clinical practice. In this study we compare two antibody tests (the Immune Fluorescence Assay and the Plaque Reduction Neutralization Test) in a group of Dutch immune-compromised travellers with a median of 33 days (IQR [28-49]) after primary YF vaccination. We collected samples of 15 immune-compromised vaccinees vaccinated with the 17D yellow fever vaccine between 2004 and 2012. All samples measured in the plaque reduction neutralization test yielded positive results (>80% virus neutralization with a 1:10 serum dilution). Immune Fluorescence Assay sensitivity was 28% (95% CI [0.12-0.49]). No adverse events were reported. All immune-compromised patients mounted an adequate response with protective levels of virus neutralizing antibodies to the 17-D YF vaccine. No adverse effects were reported. Compared to the plaque reduction neutralization test, the sensitivity of the Immune Fluorescence Assay test was low. Further research is needed to ascertain that 17D vaccination in immune-compromised patients is safe. Copyright © 2016 Elsevier Ltd. All rights reserved.

Seroprevalence of Ehrlichia ruminantium antibodies and its associated risk factors in indigenous goats of South Africa.

PubMed

Mdladla, Khanyisile; Dzomba, Edgar F; Muchadeyi, Farai C

2016-03-01

The present study investigated the seroprevalence of antibodies to Ehrlichia ruminantium and the associated risk factors in goats from five different farming provinces of South Africa. Sera collected from 686 goats of the commercial meat type (n=179), mohair type (n=9), non-descript indigenous goats from Eastern Cape (n=56), KwaZulu-Natal (n=209), Limpopo (n=111), North West (n=61) and Northern Cape (n=11) provinces and a feral Tankwa goat (n=50) were tested for the presence of immunoglobulin G (IgG) antibodies to antigens of E. ruminantium using the indirect fluorescent-antibody test (IFAT). Fifty two percent of these goats had ticks. The overall seroprevalence of antibodies to E. ruminantium was 64.87% (445/686) with the highest seroprevalence reported for Limpopo (95.50%) and lowest for Northern Cape (20.29%). Highest seroprevalence for antibodies to E. ruminantium was observed in goats from endemic regions (76.09%), and from smallholder production systems (89.54%). High seroprevalence was also observed in non-descript indigenous goats (85.04%), adult goat (69.62%), in does (67.46%) and goats infested with ticks (85.79%). The logistic model showed a gradient of increasing risk for commercial meat type Savanna (OR=3.681; CI=1.335-10.149) and non-descript indigenous (OR=3.466; CI=1.57-7.645) compared to Boer goats and for goats from the smallholder production system (OR=2.582; CI=1.182-5.639) and those with ticks (OR=3.587; CI=2.105-6.112). Results from this study showed that E. ruminantium infections were prevalent but were widely and unevenly distributed throughout South Africa. Findings from the study facilitate identification and mapping of risk areas for heartwater and its endeminicity in South Africa and should be taken into consideration for future disease control strategies and local goat improvement programs. Copyright © 2016 Elsevier B.V. All rights reserved.

21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

Code of Federal Regulations, 2010 CFR

2010-04-01

...) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems...

Accuracy estimation of an indirect ELISA for the detection of West Nile Virus antibodies in wild birds using a latent class model.

PubMed

Tamba, Marco; Caminiti, Antonino; Prosperi, Alice; Desprès, Philippe; Lelli, Davide; Galletti, Giorgio; Moreno, Ana; Paternoster, Giulia; Santi, Annalisa; Licata, Elio; Lecollinet, Sylvie; Gelmini, Luca; Rugna, Gianluca; Procopio, Anna; Lavazza, Antonio

2017-10-01

West Nile virus (WNV) and Usutu virus (USUV), genus Flavivirus, are members of the Japanese encephalitis virus antigenic complex, and are maintained primarily in an enzootic cycle between mosquitoes and birds. WNV is zoonotic, and poses a threat to public health, especially in relation to blood transfusion. Serosurveillance of wild birds is suitable for early detection of WNV circulation, although concerns remain to be addressed as regards i) the type of test used, whether ELISA, virus neutralization test (VNT), plaque reduction neutralization test (PRNT), ii) the reagents (antigens, revealing antibodies), iii) the different bird species involved, and iv) potential cross-reactions with other Flaviviruses, such as USUV. The authors developed an indirect IgG ELISA with pan-avian specificity using EDIII protein as antigen and a monoclonal antibody (mAb 1A3) with broad reactivity for avian IgG. A total of 140 serum samples were collected from juvenile European magpies (Pica pica) in areas where both WNV and USUV were co-circulating. The samples were then tested using this in-house ELISA and VNT in parallel. Estimation of test accuracy was performed using different Bayesian two latent class models. At a cut-off set at an optical density percentage (OD%) of 15, the ELISA showed a posterior median of diagnostic sensitivity (DSe) of 88% (95%PCI: 73-99%) and a diagnostic specificity (DSp) of 86% (95%PCI: 68-99%). At this cut-off, ELISA and VNT (cut-off 1/10) performances were comparable: DSe=91% (95%PCI: 79-99%), and DSp=77% (95%PCI: 59-98%). With the cut-off increased to 30 OD%, the ELISA DSe dropped to 78% (95%PCI: 52-99%), and the DSp rose to 94% (95%PCI: 83-100%). In field conditions, the cut-off that yields the best accuracy for the ELISA appears to correspond to 15 OD%. In areas where other Flaviviruses are circulating, however, it might be appropriate to raise the cut-off to 30 OD% in order to achieve higher specificity and reduce the detection of seropositive birds

Highly sensitive detection of target molecules using a new fluorescence-based bead assay

NASA Astrophysics Data System (ADS)

Scheffler, Silvia; Strauß, Denis; Sauer, Markus

2007-07-01

Development of immunoassays with improved sensitivity, specificity and reliability are of major interest in modern bioanalytical research. We describe the development of a new immunomagnetic fluorescence detection (IM-FD) assay based on specific antigen/antibody interactions and on accumulation of the fluorescence signal on superparamagnetic PE beads in combination with the use of extrinsic fluorescent labels. IM-FD can be easily modified by varying the order of coatings and assay conditions. Depending on the target molecule, antibodies (ABs), entire proteins, or small protein epitopes can be used as capture molecules. The presence of target molecules is detected by fluorescence microscopy using fluorescently labeled secondary or detection antibodies. Here, we demonstrate the potential of the new assay detecting the two tumor markers IGF-I and p53 antibodies in the clinically relevant concentration range. Our data show that the fluorescence-based bead assay exhibits a large dynamic range and a high sensitivity down to the subpicomolar level.

Ma2 antibodies: an evaluation of commercially available detection methods.

PubMed

Johannis, Wibke; Renno, Joerg H; Wielckens, Klaus; Voltz, Raymond

2011-01-01

Ma2 antibodies belong to the onconeuronal antibodies which define a "definite" paraneoplastic neurological syndrome (PNS). Because of the clinical relevance, use of two separate methods (indirect immunofluorescence technique--IFT--and immunoblot) is advocated; however, with an increasing number of commercially available assay systems, usually only one assay is performed. We compared IFT and three commercially available immunoblots (ravo Diagnostika, Euroimmun, Milenia Biotec) on sera from 35 patients with clinically suspected PNS. 17 were Ma2 antibody associated as defined by consensus result (showing positive reactivity in 2 assays), 18 were Ma2 antibody negative controls. Sensitivity/specificity for single assays were for IFT 94%/94%, for ravo Diagnostika PNS blot 88%/100%, for Euroimmun Neuronal Antigens Profile blot 100%/89%, and for Milenia Biotec MTR blot 94%/100%. Our data confirm, although all tests performed well, a combination of 2 independent assays is still advisable for Ma2 antibody detection in order to achieve higher sensitivity and specificity rates.

Analysis of erythritol in foods by polyclonal antibody-based indirect competitive ELISA.

PubMed

Sreenath, Kundimi; Venkatesh, Yeldur P

2008-05-01

Sugar alcohols are widely used as food additives and drug excipients. Erythritol (INS 968) is an important four-carbon sugar alcohol in the food industry. Erythritol occurs naturally in certain fruits, vegetables, and fermented foods. Currently, HPLC and GC methods are in use for the quantification of erythritol in natural/processed foods. However, an immunoassay for erythritol has not been developed so far. We have utilized affinity-purified erythritol-specific antibodies generated earlier [9] to develop an indirect competitive ELISA. With erythritol-BSA conjugate (54 mol/mol; 100 ng/well) as the coating antigen, a calibration curve was prepared using known amounts of standard meso-erythritol (0.1-100,000 ng) in the immunoassay. Watermelon (Citrullus lanatus) and red wine were selected as the food sources containing meso-erythritol. The amount of meso-erythritol was calculated as 2.36 mg/100 g fresh weight of watermelon and 206.7 mg/L of red wine. The results obtained from the immunoassay are in close agreement with the reported values analyzed by HPLC and GC (22-24 mg/kg in watermelon and 130-300 mg/L in red wine). The recovery analyses showed that added amounts of meso-erythritol were recovered fairly accurately with recoveries of 86-105% (watermelon) and 85-93.3% (red wine). The method described here for erythritol is the first report of an immunoassay for a sugar alcohol.

Investigation of Antigen-Antibody Interactions of Sulfonamides with a Monoclonal Antibody in a Fluorescence Polarization Immunoassay Using 3D-QSAR Models

PubMed Central

Wang, Zhanhui; Kai, Zhenpeng; Beier, Ross C.; Shen, Jianzhong; Yang, Xinling

2012-01-01

A three-dimensional quantitative structure-activity relationship (3D-QSAR) model of sulfonamide analogs binding a monoclonal antibody (MAbSMR) produced against sulfamerazine was carried out by Distance Comparison (DISCOtech), comparative molecular field analysis (CoMFA), and comparative molecular similarity indices analysis (CoMSIA). The affinities of the MAbSMR, expressed as Log10IC50, for 17 sulfonamide analogs were determined by competitive fluorescence polarization immunoassay (FPIA). The results demonstrated that the proposed pharmacophore model containing two hydrogen-bond acceptors, two hydrogen-bond donors and two hydrophobic centers characterized the structural features of the sulfonamides necessary for MAbSMR binding. Removal of two outliers from the initial set of 17 sulfonamide analogs improved the predictability of the models. The 3D-QSAR models of 15 sulfonamides based on CoMFA and CoMSIA resulted in q2 cv values of 0.600 and 0.523, and r2 values of 0.995 and 0.994, respectively, which indicates that both methods have significant predictive capability. Connolly surface analysis, which mainly focused on steric force fields, was performed to complement the results from CoMFA and CoMSIA. This novel study combining FPIA with pharmacophore modeling demonstrates that multidisciplinary research is useful for investigating antigen-antibody interactions and also may provide information required for the design of new haptens. PMID:22754368

Prevalence of antinuclear and anti-erythrocyte antibodies in healthy cats.

PubMed

Abrams-Ogg, Anthony C G; Lim, Sophia; Kocmarek, Helen; Ho, Kim; Blois, Shauna L; Shewen, Patricia E; Wood, R Darren; Bienzle, Dorothee

2018-03-01

Positive antinuclear antibody and direct antiglobulin tests support diagnoses such as systemic lupus erythematosus and immune-mediated anemia, respectively. Positive tests may occur in cats, but the prevalence of positive results in healthy cats is not well known. The study's purpose was to determine prevalences of positive antinuclear antibody and direct antiglobulin tests in healthy cats. Antinuclear antibody titers were measured by indirect immunofluorescence, and anti-erythrocyte antibodies were measured by the microtitration direct antiglobulin test at 37, 23, and 4°C in 61 client-owned and 28 facility-owned cats. Differences between the 2 groups were examined using chi-squared tests. For the antinuclear antibody tests, 70% of client-owned cats were negative, 10% had weak titers (1:40-1:80), and 20% had strong titers (1:160-1:320). Facility-owned cats had significantly fewer positive titers with 96% negative and one positive (1:8). For the antiglobulin test at 37°C, 93% of all cats were negative, 2 cats in each group were positive at low dilutions (1:2), and 2 client-owned cats were transiently positive at high dilutions (≥ 1:2048). At 23°C, 90% of all cats were negative, and 2 client-owned and 5 facility-owned cats were positive at low dilutions (1:2-1:8). At 4°C, 67% of client-owned cats had invalid results (negative control well agglutination), and 33% had negative results, while of facility-owned cats 14% had invalid results, 14% had agglutination at low dilutions, and 72% were negative. Healthy cats may have positive antinuclear antibody and direct antiglobulin tests, but the prevalence of strong reactions is low. © 2018 American Society for Veterinary Clinical Pathology.

Immunocytochemical methods to study the distribution of Orientia tsutsugamushi in Leptotrombidium (Acari: Trombiculidae) chiggers.

PubMed

Myint, K S; Linthicum, K J; Tanskul, P; Lerdthusnee, K; Vaughn, D W; Manomuth, C; Mongkolsirichaikul, D; Hansukjariya, P; Hastriter, M W

1998-07-01

Immunocytochemical methods were developed and tested for their ability to detect the distribution of Orientia tsutsugamushi in paraffin sections of adult chiggers (Leptotrombidium imphalum Vercammen-Grandjean & Langston). Rickettsial antigen was detected by application of a simple direct or amplified immunocytochemistry procedure and an indirect immunofluorescent procedure. In the direct procedure alkaline phosphatase conjugation to the mouse polyclonal antibody to the Karp strain was followed by the HistoMark Red test system to detect rickettsial antigen. The amplification procedure used a similar method but used an unlabeled primary antibody followed by secondary biotinylated antimouse IgG, streptavidin-alkaline phosphatase, and the HistoMark Red test system. The immunofluorescent procedure included a biotinylated secondary antibody followed by addition of a streptavidin-FITC conjugate. Specific tissue tropisms in infected chiggers were observed in the salivary glands, nervous tissue, and ovaries of adult female mites in all procedures; however, nonspecific fluorescence of the chigger limited definitive identification of tissue tropisms with the indirect immunofluorescent procedure.

Fluorescence Immunofiltration Assay of Brucella Melitensis.

DTIC Science & Technology

1995-01-01

second urease -labelled antibody directed against fluorescein. The assay system is useful for measuring protein, virus and bacteria in aqueous...binding site for the signal-generating urease -labelled antibody, it is a highly fluorescent molecule and has signal-generating capacity of its own

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Prevalence and implications of feline coronavirus infections of captive and free-ranging cheetahs (Acinonyx jubatus).

PubMed Central

Heeney, J L; Evermann, J F; McKeirnan, A J; Marker-Kraus, L; Roelke, M E; Bush, M; Wildt, D E; Meltzer, D G; Colly, L; Lukas, J

1990-01-01

The extent and progression of exposure to feline infectious peritonitis (FIP) virus in the cheetah, Acinonyx jubatus, was monitored by a world-wide serological survey with indirect fluorescent antibody titers to coronavirus. The indirect fluorescent antibody assay was validated by Western blots, which showed that all indirect fluorescent antibody-positive cheetah sera detected both domestic cat and cheetah coronavirus structural proteins. There was a poor correlation between indirect fluorescent antibody results and the presence of coronaviruslike particles in cheetah feces, suggesting that electron microscopic detection of shed particles may not be an easily interpreted diagnostic parameter for FIP disease. Low, but verifiable (by Western blots [immunoblots]) antibody titers against coronavirus were detected in eight free-ranging cheetahs from east Africa as well as from captive cheetahs throughout the world. Of 20 North American cheetah facilities screened, 9 had cheetahs with measurable antibodies to feline coronavirus. Five facilities showed patterns of an ongoing epizootic. Retrospective FIP virus titers of an FIP outbreak in a cheetah-breeding facility in Oregon were monitored over a 5-year period and are interpreted here in terms of clinical disease progression. During that outbreak the morbidity was over 90% and the mortality was 60%, far greater than any previously reported epizootic of FIP in any cat species. Age of infection was a significant risk factor in this epizootic, with infants (less than 3 months old) displaying significantly higher risk for mortality than subadults or adults. Based upon these observations, empirical generalizations are drawn which address epidemiologic concerns for cheetahs in the context of this lethal infectious agent. Images PMID:2157864

Evaluation of the molecular recognition of monoclonal and polyclonal antibodies for sensitive detection of 2,4,6-trinitrotoluene (TNT) by indirect competitive surface plasmon resonance immunoassay.

PubMed

Shankaran, Dhesingh Ravi; Kawaguchi, Toshikazu; Kim, Sook Jin; Matsumoto, Kiyoshi; Toko, Kiyoshi; Miura, Norio

2006-11-01

Detection of TNT is an important environmental and security concern all over the world. We herein report the performance and comparison of four immunoassays for rapid and label-free detection of 2,4,6-trinitrotoluene (TNT) based on surface plasmon resonance (SPR). The immunosensor surface was constructed by immobilization of a home-made 2,4,6-trinitrophenyl-keyhole limpet hemocyanin (TNPh-KLH) conjugate onto an SPR gold surface by simple physical adsorption within 10 min. The immunoreaction of the TNPh-KLH conjugate with four different antibodies, namely, monoclonal anti-TNT antibody (M-TNT Ab), monoclonal anti-trinitrophenol antibody (M-TNP Ab), polyclonal anti-trinitrophenyl antibody (P-TNPh Ab), and polyclonal anti-TNP antibody (P-TNP Ab), was studied by SPR. The principle of indirect competitive immunoreaction was employed for quantification of TNT. Among the four antibodies, the P-TNPh Ab prepared by our group showed highest sensitivity with a detection limit of 0.002 ng/mL (2 ppt) TNT. The lowest detection limits observed with other commercial antibodies were 0.008 ng/mL (8 ppt), 0.25 ng/mL (250 ppt), and 40 ng/mL (ppb) for M-TNT Ab, P-TNP Ab, and M-TNP Ab, respectively, in the similar assay format. The concentration of the conjugate and the antibodies were optimized for use in the immunoassay. The response time for an immunoreaction was 36 s and a single immunocycle could be done within 2 min, including the sensor surface regeneration using pepsin solution. In addition to the quantification of TNT, all immunoassays were evaluated for robustness and cross-reactivity towards several TNT analogs.

Design of a single-step immunoassay principle based on the combination of an enzyme-labeled antibody release coating and a hydrogel copolymerized with a fluorescent enzyme substrate in a microfluidic capillary device.

PubMed

Wakayama, Hideki; Henares, Terence G; Jigawa, Kaede; Funano, Shun-ichi; Sueyoshi, Kenji; Endo, Tatsuro; Hisamoto, Hideaki

2013-11-21

A combination of an enzyme-labeled antibody release coating and a novel fluorescent enzyme substrate-copolymerized hydrogel in a microchannel for a single-step, no-wash microfluidic immunoassay is demonstrated. This hydrogel discriminates the free enzyme-conjugated antibody from an antigen-enzyme-conjugated antibody immunocomplex based on the difference in molecular size. A selective and sensitive immunoassay, with 10-1000 ng mL(-1) linear range, is reported.

Production and characterization of monoclonal antibodies against ochratoxin B.

PubMed

Heussner, Alexandra H; Moeller, Ines; Day, Billy W; Dietrich, Daniel R; O'Brien, Evelyn

2007-05-01

Monoclonal antibodies against ochratoxin B (OTB) were generated by immunizing Balb/c mice with OTB conjugated to keyhole limpet hemocyanin (KLH) via carbodiimide reactions with CHMC and EDAC. A stable hybridoma cell line 2F1.E10 was produced by fusion of murine splenocytes and myeloma cells. The obtained antibodies were characterized using an indirect competitive ELISA. The detection limit was calculated (27+/-2 nM OTB) and 50% binding inhibition was reached at 500 nM free OTB. A low cross-reactivity to ochratoxin A (OTA) of 3.3% and no cross-reactivities to either coumarin or DL-phenylalanine were observed, suggesting a highly specific OTB antibody. The antibody type was identified as IgG class 1 with the light chain being of the kappa configuration. These antibodies can be used in an indirect competitive ELISA to detect OTB in the nanomolar to micromolar concentration range and may be useful for the analysis of contaminated food items.

Clover-Tagged Porcine Reproductive and Respiratory Syndrome Virus Infectious Clones for Rapid Detection of Virus Neutralizing Antibodies.

PubMed

Huang, Baicheng; Xiao, Xia; Xue, Biyun; Zhou, En-Min

2018-06-24

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a widespread disease that affects domestic pigs of all ages. Accurate and rapid detection of PRRSV specific neutralizing antibodies levels in a pig herd is beneficial for the evaluation of the herd's immunity to combat the specific viral infection. However, the current methods for viral detection, including fluorescent focus neutralization (FFN) and cytopathic effect (CPE) reduction neutralizing assays, are subjective and time-consuming. Therefore, a Clover-tagged PRRSV virus neutralization assay were developed that instrumentally measures the fluorescence signal of Clover stably expressing by a PRRSV infectious clone for at least 10 passages. Herein, the results showed that the proposed Clover-tagged PRRSV neutralization assay is reliable using instrumental measurements of the fluorescence signal of Clover and allows for rapid detection of neutralizing antibodies against PRRSV. The assay was evaluated by testing swine sera from experimental and field samples, and comparisons were made with the traditional FFN and CPE reduction assays. These results suggest that the Clover-tagged PRRSV infectious clone offers a fast and reliable testing method for neutralizing antibodies and could permit high-throughput screening of new antiviral agents. Copyright © 2018. Published by Elsevier B.V.

Testing the optimal defence hypothesis for two indirect defences: extrafloral nectar and volatile organic compounds

PubMed Central

Radhika, Venkatesan; Kost, Christian; Bartram, Stefan; Heil, Martin

2008-01-01

Many plants respond to herbivory with an increased production of extrafloral nectar (EFN) and/or volatile organic compounds (VOCs) to attract predatory arthropods as an indirect defensive strategy. In this study, we tested whether these two indirect defences fit the optimal defence hypothesis (ODH), which predicts the within-plant allocation of anti-herbivore defences according to trade-offs between growth and defence. Using jasmonic acid-induced plants of Phaseolus lunatus and Ricinus communis, we tested whether the within-plant distribution pattern of these two indirect defences reflects the fitness value of the respective plant parts. Furthermore, we quantified photosynthetic rates and followed the within-plant transport of assimilates with 13C labelling experiments. EFN secretion and VOC emission were highest in younger leaves. Moreover, the photosynthetic rate increased with leaf age, and pulse-labelling experiments suggested transport of carbon to younger leaves. Our results demonstrate that the ODH can explain the within-plant allocation pattern of both indirect defences studied. PMID:18493790

Evaluation of a monoclonal antibody-based rapid immunochromatographic test for direct detection of rabies virus in the brain of humans and animals.

PubMed

Ahmed, Kamruddin; Wimalaratne, Omala; Dahal, Narapati; Khawplod, Pakamatz; Nanayakkara, Susilakanthi; Rinzin, Karma; Perera, Devika; Karunanayake, Dushantha; Matsumoto, Takashi; Nishizono, Akira

2012-04-01

Rabies diagnosis uses a direct fluorescent antibody test (FAT) that is difficult, costly, and time-consuming, and requires trained personnel. We developed a rapid immunochromatographic test (RICT) for the diagnosis of rabies. The efficacy of the RICT was compared with that of the FAT. Brain samples were collected from humans, dogs, cats, and other animals in Sri Lanka (n = 248), Bhutan (n = 27), and Thailand (n = 228). The sensitivity (0.74-0.95), specificity (0.98-1.0), positive predictive value (0.98-1.0), negative predictive value (0.75-0.97), accuracy (0.91-0.98), and kappa measure of agreement (0.79-0.93) were all satisfactory for animal samples and samples preserved in 50% glycerol saline solution. Because the RICT showed high sensitivity but low specificity with human brain samples, it is unsuitable for confirming rabies in humans. No amino acid substitutions were found in the antibody attachment sites of the nucleoprotein gene with FAT-positive, RICT-negative samples. The RICT is reliable, user friendly, rapid, robust, and can be used in laboratories with a modest infrastructure.

Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins.

PubMed

Vásquez-Garzón, Verónica R; Rouimi, Patrick; Jouanin, Isabelle; Waeg, Georg; Zarkovic, Neven; Villa-Treviño, Saul; Guéraud, Françoise

2012-05-01

Among disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Carbonylation of proteins can occur directly or indirectly through the adduction of lipid oxidation products. In this study, three classical and easy-to-perform techniques to detect direct or indirect carbonylation of proteins were compared. A model protein apomyoglobin and a complex mixture of rat liver cytosolic proteins were exposed to cumene hydroperoxide oxidation or adduction to the lipid peroxidation product 4-hydroxynonenal in order to test direct or indirect carbonylation, respectively. The technique using a specific anti-4-hydroxynonenal-histidine adduct antibody was effective to detect in vitro modification of model apomyoglobin and cytosolic proteins by 4-hydroxynonenal but not by direct carbonylation which was achieved by techniques using biotin-coupled hydrazide or dinitrophenylhydrazine derivatization of carbonyls. Sequential use of these methods enabled the detection of both direct and indirect carbonyl modification in proteins, although constitutively biotinylated proteins were detected by biotin-hydrazide. Although rather classical and efficient, methods for carbonyl detection on proteins in oxidative stress studies may be biased by some artifactual detections and complicated by proteins multimerizations. The use of more and more specific available antibodies is recommended to complete detection of lipid peroxidation product adducts on proteins.

Radioimmunotherapy of MCF7 breast cancer cell line with 131I-PR81 monoclonal antibody against MUC1: comparison of direct and indirect radioiodination methods.

PubMed

Mohammadnejad, Javad; Rasaee, Mohammad Javad; Babaei, Mohammad Hosein; Paknejad, Malihe; Hasan, Zahir Mohammad; Salouti, Mojtaba; Gandomkar, Mostafa; Sadri, Keyvan

2010-01-01

PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to compare the two labeling methods (direct and indirect radioiodination) for application of this antibody against MUC1 as a radioimmunotherapeutical agent.Monoclonal antibody (PR81) against the tandem repeat of the core protein (MUC1) was prepared, characterized, purified, and labeled with 131I using the direct (chloramin-T) and indirect (Fmoc-D-Tyr (tBu)-D-Tyr (tBu)-D-Lys (Boc)-OH (YYK) attached to N-hydroxysuccinimide as a linker between PR81 and 131I) methods. The immunoreactivity of 131I-PR81 and 131I-TP-PR81 complexes with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of 131I-PR81 and 131I-YYK-peptide-PR81 complexes in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the 131I-PR81 and 131I- YYK-peptide -PR81 complexes was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The labeling efficiency was determined by measuring the percentage recovery of radioactivity in the final product relative to the initial activity in the shipment vial, was found to be 59.9% +/- 7.9% for direct and 50% +/- 3.2% for indirect methods. 131I-PR81 and 131I- YYK- peptide -PR81 complexes showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled products in human serum which was measured by thin layer chromatography (TLC) was found to be more than 50% over 24 hr for 131I-PR81 and 70% for 131I- YYK-peptide -PR81 complexes. Cell toxicity and in vitro internalization studies showed that the 131I-PR81 and 131I- YYK-peptide -PR81 complexes inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to 40

Is there a role for antibody testing in the diagnosis of invasive candidiasis?

PubMed

Quindós, Guillermo; Moragues, María Dolores; Pontón, José

2004-03-01

During the last decades, the use of antibody tests for the diagnosis of invasive mycoses has declined as a consequence of the general belief that they are insensitive and non-specific. However, there is a clear evidence that antibodies can be detected in highly immunodeficient patients (such as bone marrow transplant recipients), and that those antibodies are useful for the diagnosis. Antibody tests are currently in use as diagnostic tools for some primary mycoses, such as the endemic mycoses, aspergilloma, allergic bronchopulmonary aspergilosis and sporothrichosis. For invasive candidiasis, diagnostic methods must differentiate Candida colonization of mucous membranes or superficial infection from tissue invasion by this microorganism. Substantial progress has been made in diagnosis of invasive candidiasis with the development of a variety of methods for the detection of antibodies and antigens. However, no single test has found widespread clinical use and there is a consensus that diagnosis based on a single specimen lacks sensitivity. It is necessary to test sequential samples taken while the patient is at greatest risk for developing invasive candidiasis to optimize the diagnosis. Results obtained from a panel of diagnostic tests in association with clinical aspects will likely be the most useful strategy for early diagnosis and therapy.

Bioluminescent Antibodies for Point‐of‐Care Diagnostics

PubMed Central

Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto

2017-01-01

Abstract We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no‐wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP‐tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max>500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. PMID:28510347

Staphylococcal enterotoxin-specific IgE antibodies in atopic dermatitis.

PubMed

Ide, Fumihito; Matsubara, Tomoyo; Kaneko, Miho; Ichiyama, Takashi; Mukouyama, Tokuko; Furukawa, Susumu

2004-06-01

The authors clarified the clinical significance of the measurement of serum concentrations of specific IgE antibodies to staphylococcal enterotoxin (SE) A- and SEB in atopic dermatitis (AD). The serum concentrations of SEA- and SEB-specific IgE antibodies in 140 pediatric patients with AD were measured with an immuno CAP -radioallergosorbent test system (RAST). To check the cross-reaction of specific IgE antibodies to SEA/SEB and other allergens, the CAP RAST fluorescent enzyme immunoassay inhibition test was performed. Forty-seven patients (33.6%) tested positive for either SEA- or SEB-specific IgE antibodies. School children showed higher positive rates of SEA/SEB-specific IgE antibodies than infants or young children. The patients with severe AD and those with exacerbation of symptoms in summer, had higher positive rates of SEA/SEB-specific IgE antibodies than patients with mild AD or those with exacerbation in winter. In addition, the positive rates of specific IgE antibodies to both dog-dander and cat-dander were higher in patients with positive SEA/SEB-specific IgE antibodies than in patients with negative ones. No cross-reactions occurred among specific IgE antibodies to SEA/SEB and dog/cat dander with one patient's serum, which had positive IgE-specific antibodies against cat/dog dander and SEA/SEB. The positive rate of SEA/SEB-specific IgE antibodies in the patients with dogs and/or cats as pets was 48.4%, which was higher than in those with no pets. Atopic dermatitis patients who exhibit high positive rates of SEA/SEB-specific IgE antibodies were found to be school children, severe cases, cases with high serum concentrations of total IgE, cases with exacerbation in summer, and cases with dogs and/or cats as pets. The measurement of serum concentrations of specific IgE antibodies to SEA and SEB, thus has some value for evaluating AD patients.

West Nile virus--neutralizing antibodies in humans in Greece.

PubMed

Papa, Anna; Perperidou, Parthena; Tzouli, Anisa; Castilletti, Concetta

2010-10-01

Serum samples collected during March-May 2007 from 392 residents of Imathia prefecture, Northern Greece, were tested by indirect immunofluorescence assay and enzyme-linked immunosorbent assay for IgG antibodies against West Nile virus (WNV). Microneutralization assay was applied in six positive samples. Four (4/392, 1.02%) were found positive for WNV-neutralizing antibodies. None of the positive individuals had a history of travel in endemic area or flavivirus vaccination, suggesting that WNV, or an antigenically related flavivirus, circulates in an endemic sylvatic cycle, at least locally, in rural areas in Greece. Human, animal, and vector surveillance systems have to be implemented to provide an early detection of WNV activity in Greece.

Monoclonal antibody-tagged receptor-targeted contrast agents for detection of cancers

NASA Astrophysics Data System (ADS)

Soukos, N. S.; Hamblin, Michael R.; Deutsch, Thomas F.; Hasan, Tayyaba

2001-07-01

Oral cancer and precancer overexpress the epidermal growth factor receptor (EGFR) and monoclonal antibodies against EGFR coupled to photoactive dyes may have a potential both as a diagnostic and treatment modalities for oral premalignancy. We asked whether an anti-EGFR mab (C225) conjugated with the fluorescence dye indocyanine Cy5.5 could detect dysplastic changes in the hamster cheek pouch carcinogenesis model. Secondly, we tested whether the same antibody conjugated with the photosensitizer chlorin (e6) could be used together with illumination to reduce levels of expression of EGFR as evaluated by the immunophotodetection procedure. Increased fluorescence appeared to correlate with development of premalignancy when the C225-Cy5.5 conjugate was used. Areas with increased fluorescence signal were found in carcinogen-treated but clinically normal cheek pouches, that revealed dysplastsic changes by histology. The immunophotodetection procedure was carried out after photoummunotherapy with the C225-ce6 conjugate, and showed a significant reduction in fluorescence in the illuminated compared to the non-illuminated areas in the carcinogen- treated but not the normal cheek pouch. The results demonstrate that the use of anti-EGFR Mab targeted photoactive dyes may serve as a feedback controlled optical diagnosis and therapy procedure for oral premalignant lesions.

Practical diagnostic testing for human immunodeficiency virus.

PubMed Central

Jackson, J B; Balfour, H H

1988-01-01

Since the discovery of human immunodeficiency virus (HIV) as the causative agent of acquired immunodeficiency syndrome in 1983, there has been a proliferation of diagnostic tests. These assays can be used to detect the presence of HIV antibody, HIV antigen, HIV ribonucleic and deoxyribonucleic acids, and HIV reverse transcriptase. Enzyme-linked immunosorbent assays, Western blot, radioimmunoprecipitation assays, indirect immunofluorescence assays, reverse transcriptase assays, and several molecular hybridization techniques are currently available. Enzyme-linked immunosorbent, Western blot, and indirect immunofluorescence assays for HIV antibody are very sensitive, specific, and adaptable to most laboratories. An enzyme-linked immunosorbent assay for HIV antigen is also readily adaptable to most laboratories and will be commercially available soon. While the other assays are more tedious, they are valuable confirmatory tests and are suitable for reference laboratories. The biohazards of performing HIV testing can be minimized with proper biosafety measures. Images PMID:3060241

Simultaneous multicolor detection system of the single-molecular microbial antigen by total internal reflection fluorescence microscopy with fluorescent nanocrystal quantum dots

NASA Astrophysics Data System (ADS)

Hoshino, Akiyoshi; Fujioka, Kouki; Yamamoto, Mayu; Manabe, Noriyoshi; Yasuhara, Masato; Suzuki, Kazuo; Yamamoto, Kenji

2005-11-01

Immunological diagnostic methods have been widely performed and showed high performance in molecular and cellular biology, molecular imaging, and medical diagnostics. We have developed novel methods for the fluorescent labeling of several antibodies coupled with fluorescent nanocrystals QDs. In this study we demonstrated that two bacterial toxins, diphtheria toxin and tetanus toxin, were detected simultaneously in the same view field of a cover slip by using directly QD-conjugated antibodies. We have succeeded in detecting bacterial toxins by counting luminescent spots on the evanescent field with using primary antibody conjugated to QDs. In addition, each bacterial toxin in the mixture can be separately detected by single excitation laser with emission band pass filters, and simultaneously in situ pathogen quantification was performed by calculating the luminescent density on the surface of the cover slip. Our results demonstrate that total internal reflection fluorescence microscopy (TIRFM) enables us to distinguish each antigen from mixed samples and can simultaneously quantitate multiple antigens by QD-conjugated antibodies. Bioconjugated QDs could have great potentialities for in practical biomedical applications to develop various high-sensitivity detection systems.

Immunofluorescent studies on human spermatozoa. III. Immunoglobulin classes of human spermatozoal antibodies

PubMed Central

Hansen, K. Brogaard

1972-01-01

Antibodies in human sera against four different antigens of human spermatozoa discovered by means of an indirect two-layer immunofluorescence technique (IFT) were characterized by determination of the class of immunoglobulins to which they belonged. A three-layer IFT using monospecific antisera against human IgG, IgA or IgM as the second layer was employed together with fractionation of sera on Sephadex G-200 or DEAE-cellulose followed by testing of the concentrated pools in a two-layer IFT. The study revealed that antibodies against the antigen in the front part of the acrosome were primarily IgM and those against the antigen in the tail primarily IgG. Antibodies against antigens in the equatorial segment and the postnuclear cap showed a varying predominance of these two immunoglobulins. Spermatozoal antibody as IgA was found only in small amounts. PMID:4558409

Fluorescence correlation spectroscopy study of the complexation of DNA hybrids, IgG antibody, and a chimeric protein of IgG-binding ZZ domains fused with a carbohydrate binding module.

PubMed

Rosa, A M M; Prazeres, D M F; Paulo, P M R

2017-06-28

Fluorescence correlation spectroscopy (FCS) was used to characterize the molecular interactions between the four components of a DNA recognition system. A fluorescent DNA probe was used to assess: (i) the hybridization with a complementary biotin-labeled target, (ii) the complexation of the resulting hybrid and an anti-biotin antibody, and (iii) the binding of the latter complex to a ZZ-CBM fusion protein that combines small synthetic IgG Fc-binding Z domains with a carbohydrate binding module (CBM). These binding interactions were monitored by exposing the fluorescent DNA probe to different amounts and combinations of the other molecules in solution. Through the analysis of FCS autocorrelation curves, an association constant (K a ) of 2.9 × 10 7 M -1 was estimated for DNA·DNA hybridization, and the presence of (non-) complementary target DNA in solution could be discriminated. The specific capture of biotinylated DNA hybrids by anti-biotin IgG was verified, with an apparent K a of 2.5 × 10 6 M -1 . The increment in the diffusion time measured when the DNA·DNA:antibody complexes were in contact with the ZZ-CBM fusion protein suggested that the binding occurs at a stoichiometric ratio of DNA/antibody complex to fusion larger than 1 : 1. The FCS-derived information obtained is useful to gain insight into molecular interactions involved in diagnostic assays.

Effect of pressure on interactions of anti-fluorescent probe monoclonal antibody with a ligand and inhibitors

NASA Astrophysics Data System (ADS)

Nishimoto, M.; Goto, M.; Tamai, N.; Nagamune, H.; Kaneshina, S.; Matsuki, H.

2010-03-01

Interactions of anti-fluorescent probe monoclonal antibody (immunoglobulin G (IgG)-49) with a ligand (fluorescein (FL)) and three kinds of inhibitors (1-tetradecanol (C14OH), 1-tetradecanoic acid (C13COOH) and 5-aminofluorescein (5-FLNH2)) under high pressure were examined by methods of fluorescence spectroscopy. Pressure promoted the dissociation between FL and IgG-49 from the complex. The standard volume changes of the dissociation became negative, hence, the binding of FL to IgG-49 expands the volume of the complex. The volume expansion may be closely related to the large hydrophobicity around binding sites of FL in the IgG-49 molecule. Further, the standard volume changes of IgG-49 for the inhibitor binding, which were calculated from the Johnson-Eyring plots, became all negative. The volume change for 5-FLNH2 was smaller than those for C14OH and C13COOH. This means that the volume of IgG-49 shrinks by the addition of the inhibitors in contrast with the FL binding. The differences among inhibitors are attributable to the differences in interaction modes to IgG-49 among them.

Bioluminescent Antibodies for Point-of-Care Diagnostics.

PubMed

Xue, Lin; Yu, Qiuliyang; Griss, Rudolf; Schena, Alberto; Johnsson, Kai

2017-06-12

We introduce a general method to transform antibodies into ratiometric, bioluminescent sensor proteins for the no-wash quantification of analytes. Our approach is based on the genetic fusion of antibody fragments to NanoLuc luciferase and SNAP-tag, the latter being labeled with a synthetic fluorescent competitor of the antigen. Binding of the antigen, here synthetic drugs, by the sensor displaces the tethered fluorescent competitor from the antibody and disrupts bioluminescent resonance energy transfer (BRET) between the luciferase and fluorophore. The semisynthetic sensors display a tunable response range (submicromolar to submillimolar) and large dynamic range (ΔR max >500 %), and they permit the quantification of analytes through spotting of the samples onto paper followed by analysis with a digital camera. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Plasmonically amplified fluorescence bioassay with microarray format

NASA Astrophysics Data System (ADS)

Gogalic, S.; Hageneder, S.; Ctortecka, C.; Bauch, M.; Khan, I.; Preininger, Claudia; Sauer, U.; Dostalek, J.

2015-05-01

Plasmonic amplification of fluorescence signal in bioassays with microarray detection format is reported. A crossed relief diffraction grating was designed to couple an excitation laser beam to surface plasmons at the wavelength overlapping with the absorption and emission bands of fluorophore Dy647 that was used as a label. The surface of periodically corrugated sensor chip was coated with surface plasmon-supporting gold layer and a thin SU8 polymer film carrying epoxy groups. These groups were employed for the covalent immobilization of capture antibodies at arrays of spots. The plasmonic amplification of fluorescence signal on the developed microarray chip was tested by using interleukin 8 sandwich immunoassay. The readout was performed ex situ after drying the chip by using a commercial scanner with high numerical aperture collecting lens. Obtained results reveal the enhancement of fluorescence signal by a factor of 5 when compared to a regular glass chip.

Investigation of neutron radiation effects on polyclonal antibodies (IgG) and fluorescein dye for astrobiological applications.

PubMed

Le Postollec, A; Coussot, G; Baqué, M; Incerti, S; Desvignes, I; Moretto, P; Dobrijevic, M; Vandenabeele-Trambouze, O

2009-09-01

Detecting life in the Solar System is one of the great challenges of new upcoming space missions. Biochips have been proposed as a way to detect organic matter on extraterrestrial objects. A biochip is a miniaturized device composed of biologically sensitive systems, such as antibodies, which are immobilized on a slide. In the case of in situ measurements, the main concern is to ensure the survival of the antibodies under space radiation. Our recent computing simulation of cosmic ray interactions with the martian environment shows that neutrons are one of the dominant species at soil level. Therefore, we have chosen, in a first approach, to study antibody resistance to neutrons by performing irradiation experiments at the Applications Interdisciplinaires des Faisceaux d'Ions en Région Aquitaine (AIFIRA) platform, a French ion beam facility at the Centre d'Etudes Nucléaires de Bordeaux-Gradignan in Bordeaux. Antibodies and fluorescent dyes, freeze-dried and in buffer solution, were irradiated with 0.6 MeV and 6 MeV neutrons. Sample analyses demonstrated that, in the conditions tested, antibody recognition capability and fluorescence dye intensity are not affected by the neutrons.

Development of a new rabbit monoclonal antibody and its based competitive indirect enzyme-linked immunosorbent assay for rapid detection of sulfonamides.

PubMed

Liu, Na; Han, Zheng; Lu, Lei; Wang, Lin; Ni, Geng; Zhao, Zhihui; Wu, Aibo; Zheng, Xiaodong

2013-02-01

Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody (RabMAb) against sulfonamides (SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay (ELISA) was then developed and applied to real sample analysis. A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration (IC(50)) values for four SAs were all below 10 ng mL(-1) , with 0.68 ng mL(-1) sulfathiazole (STZ), 1.11 ng mL(-1) sulfadiazine (SD), 1.15 ng mL(-1) sulfapyridine (SP) and 5.27 ng mL(-1) sulfamethoxazole (SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs (STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses. © 2012 Society of Chemical Industry.

The Fluorescent Antibody Technique in the Diagnosis of Equine Rhinopneumonitis Virus Abortion

PubMed Central

Smith, I. M.; Girard, A.; Corner, A. H.; Mitchell, D.

1972-01-01

Using two known positive equine viral rhinopneumonitis (EVR) sera, conjugates were prepared with fluorescein isothiocyanate and tested for specificity using EVR infected tissue culture cells. The conjugate was then applied to selected tissues from 32 aborted fetuses and foals submitted during a natural outbreak of EVR. Antigen was detected in various tissues by immunofluorescence in 20 cases (62.5%). In 24 cases bovine fetal kidney cell monolayers were inoculated with a pool of lung and liver and EVR virus was isolated from 15 (62.5%). Histological examination of various tissues from 29 cases resulted in the diagnosis of EVR in 19 (65.5%), based upon the presence of focal areas of necrosis and intranuclear inclusion bodies. Correlation of results was not obtained in two cases. One was diagnosed positive histologically and negative on fluorescence, the other was negative histologically and by virus isolation but showed fluorescence. The distribution of fluorescence in various infected fetal tissues indicated that the combined examination of lung and thymus gland was most likely to provide a positive diagnosis. ImagesFig. 1.Fig. 2. PMID:4114979

Microfluidic antibody arrays for simultaneous cell separation and stimulus.

PubMed

Liu, Yan; Germain, Todd; Pappas, Dimitri

2014-12-01

A microfluidic chip containing stamped antibody arrays was developed for simultaneous cell separation and drug testing. Poly(dimethyl siloxane) (PDMS) stamping was used to deposit antibodies in a microfluidic channel, forming discrete cell-capture regions on the surface. Cell mixtures were then introduced, resulting in the separation of cells when specific antibodies were used. Anti-CD19 antibody regions resulted in 94 % capture purity for CD19+ Ramos cells. An antibody that captures multiple cell types, for example anti-CD71, can also be used to capture several cell types simultaneously. Cells could also be loaded onto the arrays with spatial control using laminar streams. Both Ramos B cells and HuT 78 T cells were isolated in the chip and exposed to staurosporine in the same channel. Both cell lines had similar responses to the drug, with 2-10 % of cells remaining viable after 20 h of drug treatment, depending on cell type. The chip can also be used to analyze the efficacy of antibody therapy against cancer cells. Anti-CD95 was deposited on the surface and used for simultaneous cell capture and apoptosis induction via the extrinsic pathway. Cells captured on anti-CD95 surfaces had significant viability loss (15 % viability after 24 h) when compared with a control anti-CD71 antibody (81 % viability after 24 h). This chip can be used for a variety of cell separation and/or drug testing studies, enabling researchers to isolate cells and test them against different anti-cancer compounds and to follow cell response using fluorescence or other readout methods.