[International classification of various types of monoclonal antibodies].

PubMed

Scheen, A J

2009-01-01

Significant advances in the development of monoclonal antibodies ("mabs") have been acknowledged during the last two decades. Successive developments led to the marketing of murine antibodies ("o-mab" first, followed by chimeric antibodies ("xi-mab"), humanised antibodies ("zu-mab") and, finally, human monoclonal antibodies ("u-mab"). In order to facilitate the distinction between the various monoclonal antibodies used in clinical practice, an international nomenclature has been proposed with the use of a specific suffix corresponding to the origine/source of "mabs" preceded by an infix referring to the medicine's target. The efforts in developing new types of monoclonal antibodies aimed at improving their pharmacokinetics (longer half-life), pharmacodynamics (better efficacy because of stronger affinity to human receptor), and safety profile (less antigenic and immunogenic reactions). These progresses could be obtained thanks to the remarkable development of molecular biotechnology.

Antibody-mediated immune suppression is improved when blends of anti-RBC monoclonal antibodies are used in mice.

PubMed

Bernardo, Lidice; Amash, Alaa; Marjoram, Danielle; Lazarus, Alan H

2016-08-25

Although the prevention of hemolytic disease of the fetus and newborn is highly effective using polyclonal anti-D, a recombinant alternative is long overdue. Unfortunately, anti-D monoclonal antibodies have been, at best, disappointing. To determine the primary attribute defining an optimal antibody, we assessed suppression of murine red blood cell (RBC) immunization by single-monoclonal antibodies vs defined blends of subtype-matched antibodies. Allogeneic RBCs expressing the HOD antigen (hen egg lysozyme [HEL]-ovalbumin-human transmembrane Duffy(b)) were transfused into naïve mice alone or together with selected combinations of HEL-specific antibodies, and the resulting suppressive effect was assessed by evaluating the antibody response. Polyclonal HEL antibodies dramatically inhibited the antibody response to the HOD antigen, whereas single-monoclonal HEL antibodies were less effective despite the use of saturating doses. A blend of monoclonal HEL-specific antibodies reactive with different HEL epitopes significantly increased the suppressive effect, whereas a blend of monoclonal antibodies that block each other's binding to the HEL protein did not increase suppression. In conclusion, these data show that polyclonal antibodies are superior to monoclonal antibodies at suppressing the immune response to the HOD cells, a feature that can be completely recapitulated using monoclonal antibodies to different epitopes. © 2016 by The American Society of Hematology.

Synergistic protection of mice against plague with monoclonal antibodies specific for the F1 and V antigens of Yersinia pestis.

PubMed

Hill, Jim; Copse, Catherine; Leary, Sophie; Stagg, Anthony J; Williamson, E Diane; Titball, Richard W

2003-04-01

Monoclonal antibodies specific for Yersinia pestis V antigen and F1 antigen, administered singly or in combination, protected mice in models of bubonic and pneumonic plague. Antibodies showed synergy when administered prophylactically and as a therapy 48 h postinfection. Monoclonal antibodies therefore have potential as a treatment for plague.

Monoclonal antibodies with group specificity toward sulfonamides: selection of hapten and antibody selectivity.

PubMed

Wang, Zhanhui; Beier, Ross C; Sheng, Yajie; Zhang, Suxia; Jiang, Wenxiao; Wang, Zhaopeng; Wang, Jin; Shen, Jianzhong

2013-05-01

Immunoassays based on the current available antibodies for large multi-sulfonamide screening programs have suffered from high selectivity for individual sulfonamides and a wide range of selectivities for different sulfonamides. In this study, five synthesized haptens, HS, BS, CS, SA10, and TS and two sulfonamides, SG and SMX were used as haptens, which may or may not contain a ring structure at the N1 position of the sulfonamides, were selected to evaluate the effectiveness for producing group-specific monoclonal antibodies (MAbs). Mice immunized with three different two-ring haptens were used for hybridoma production, which resulted in three unique MAbs recognizing 10, 13, and 15 sulfonamides showing 50 % inhibition (IC50) at concentrations below 100 ng mL(-1). MAb 4D11 derived from one novel immunizing hapten could recognize 12 sulfonamides with IC50 values ranging from 1.2 to 12.4 ng mL(-1), almost within 1 order of magnitude. These produced MAbs show lower IC50 values in addition to significantly improved group specificity compared with previously generated MAbs. This study clearly indicates that the careful selection of the immunizing hapten has an important effect on the specificity of the generated antibodies.

Uses of monoclonal antibody 8H9

DOEpatents

Cheung, Nai-Kong V.

2013-04-09

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

Uses of monoclonal antibody 8H9

DOEpatents

Cheung, Nai-Kong V.

2010-06-22

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides different uses of the monoclonal antibody 8H9 or its derivative.

Uses of monoclonal antibody 8H9

DOEpatents

Cheung, Nai-Kong V

2013-08-06

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

Uses of monoclonal antibody 8H9

DOEpatents

Cheung, Nai-Kong V.

2010-06-15

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also provides an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.

Uses of monoclonal antibody 8H9

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

Cheung, Nai-Kong V.

This invention provides a composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a suitable carrier. This invention provides a pharmaceutical composition comprising an effective amount of monoclonal antibody 8H9 or a derivative thereof and a pharmaceutically acceptable carrier. This invention also provides an antibody other than the monoclonal antibody 8H9 comprising the complementary determining regions of monoclonal antibody 8H9 or a derivative thereof, capable of binding to the same antigen as the monoclonal antibody 8H9. This invention provides a substance capable of competitively inhibiting the binding of monoclonal antibody 8H9. This invention also providesmore » an isolated scFv of monoclonal antibody 8H9 or a derivative thereof. This invention also provides the 8H9 antigen. This invention also provides a method of inhibiting the growth of tumor cells comprising contacting said tumor cells with an appropriate amount of monoclonal antibody 8H9 or a derivative thereof.« less

Generation of HER2 monoclonal antibodies using epitopes of a rabbit polyclonal antibody.

PubMed

Hu, Francis Jingxin; Uhlen, Mathias; Rockberg, Johan

2014-01-25

One of the issues in using polyclonal antibodies is the limited amount of reagent available from an immunisation, leading to batch-to-batch variation and difficulties in obtaining the same antibody performance when the same antigen is re-immunised into several separate animals. This led to the development of hybridoma technology allowing, at least theoretically, for an unlimited production of a specific binder. Nevertheless, polyclonal antibodies are widely used in research and diagnostics and there exists a need for robust methods to convert a polyclonal antibody with good binding performance into a renewable monoclonal with identical or similar binding specificity. Here we have used precise information regarding the functional recognition sequence (epitope) of a rabbit polyclonal antibody with attractive binding characteristics as the basis for generation of a renewable mouse monoclonal antibody. First, the original protein fragment antigen was used for immunisation and generation of mouse hybridoma, without obtaining binders to the same epitope region. Instead a peptide designed using the functional epitope and structural information was synthesised and used for hybridoma production. Several of the monoclonal antibodies generated were found to have similar binding characteristics to those of the original polyclonal antibody. These monoclonal antibodies detected native HER2 on cell lines and were also able to stain HER2 in immunohistochemistry using xenografted mice, as well as human normal and cancer tissues. Copyright © 2013 Elsevier B.V. All rights reserved.

Therapeutic Recombinant Monoclonal Antibodies

ERIC Educational Resources Information Center

Bakhtiar, Ray

2012-01-01

During the last two decades, the rapid growth of biotechnology-derived techniques has led to a myriad of therapeutic recombinant monoclonal antibodies with significant clinical benefits. Recombinant monoclonal antibodies can be obtained from a number of natural sources such as animal cell cultures using recombinant DNA engineering. In contrast to…

Novel anti-Sialyl-Tn monoclonal antibodies and antibody-drug conjugates demonstrate tumor specificity and anti-tumor activity.

PubMed

Prendergast, Jillian M; Galvao da Silva, Ana Paula; Eavarone, David A; Ghaderi, Darius; Zhang, Mai; Brady, Dane; Wicks, Joan; DeSander, Julie; Behrens, Jeff; Rueda, Bo R

Targeted therapeutics that can differentiate between normal and malignant tumor cells represent the ideal standard for the development of a successful anti-cancer strategy. The Sialyl-Thomsen-nouveau antigen (STn or Sialyl-Tn, also known as CD175s) is rarely seen in normal adult tissues, but it is abundantly expressed in many types of human epithelial cancers. We have identified novel antibodies that specifically target with high affinity the STn glycan independent of its carrier protein, affording the potential to recognize a wider array of cancer-specific sialylated proteins. A panel of murine monoclonal anti-STn therapeutic antibodies were generated and their binding specificity and efficacy were characterized in vitro and in in vivo murine cancer models. A subset of these antibodies were conjugated to monomethyl auristatin E (MMAE) to generate antibody-drug conjugates (ADCs). These ADCs demonstrated in vitro efficacy in STn-expressing cell lines and significant tumor growth inhibition in STn-expressing tumor xenograft cancer models with no evidence of overt toxicity.

Generation of Murine Monoclonal Antibodies by Hybridoma Technology.

PubMed

Holzlöhner, Pamela; Hanack, Katja

2017-01-02

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

Understanding the Cellular Function of TRPV2 Channel through Generation of Specific Monoclonal Antibodies

PubMed Central

Cohen, Matthew R.; Huynh, Kevin W.; Cawley, Daniel; Moiseenkova-Bell, Vera Y.

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca2+-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function. PMID:24392006

Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies.

PubMed

Cohen, Matthew R; Huynh, Kevin W; Cawley, Daniel; Moiseenkova-Bell, Vera Y

2013-01-01

Transient receptor potential vanilloid 2 (TRPV2) is a Ca(2+)-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function.

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.

Screening for epitope specificity directly on culture supernatants in the early phase of monoclonal antibody production by an ELISA with biotin-labeled antigen.

PubMed

Andersen, Ditte C; Jensen, Charlotte H; Gregersen, Annemette; Brandt, Jette; Kliem, Anette; Skjødt, Karsten; Koch, Claus; Teisner, Børge

2004-01-01

This report describes an assay for comparison of epitope specificity in groups of monoclonal antibodies against a given antigen. The only prerequisite is the biotin-labeled antigen. One of the monoclonal antibodies is captured onto a plastic surface via a rabbit anti-mouse Ig, and the other preincubated with biotinylated antigen. When the two antibodies react with the same epitope subsequent binding of the biotin-labeled antigen is abolished (inhibition). In the cases where no inhibition was observed, the two antibodies were considered to react with distinct, independent epitopes. The obvious advantages using this assay, are that it can be performed directly on culture supernatants in the early phase of monoclonal antibody production, and also works for antigens with repetitive epitopes. Moreover, the bonus effect, i.e., a signal in excess of the reference signal when sets of monoclonal antibodies with different epitope specificity are compared, gives a relative measure of affinity.

The therapeutic monoclonal antibody market

PubMed Central

Ecker, Dawn M; Jones, Susan Dana; Levine, Howard L

2015-01-01

Since the commercialization of the first therapeutic monoclonal antibody product in 1986, this class of biopharmaceutical products has grown significantly so that, as of November 10, 2014, forty-seven monoclonal antibody products have been approved in the US or Europe for the treatment of a variety of diseases, and many of these products have also been approved for other global markets. At the current approval rate of ∼ four new products per year, ∼70 monoclonal antibody products will be on the market by 2020, and combined world-wide sales will be nearly $125 billion. PMID:25529996

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.

Monoclonal antibody form and function: manufacturing the right antibodies for treating drug abuse.

PubMed

Peterson, Eric; Owens, S Michael; Henry, Ralph L

2006-05-26

Drug abuse continues to be a major national and worldwide problem, and effective treatment strategies are badly needed. Antibodies are promising therapies for the treatment of medical problems caused by drug abuse, with several candidates in preclinical and early clinical trials. Monoclonal antibodies can be designed that have customized affinity and specificity against drugs of abuse, and because antibodies can be designed in various forms, in vivo pharmacokinetic characteristics can be tailored to suit specific clinical applications (eg, long-acting for relapse prevention, or short-acting for overdose). Passive immunization with antibodies against drugs of abuse has several advantages over active immunization, but because large doses of monoclonal antibodies may be needed for each patient, efficient antibody production technology is essential. In this minireview we discuss some of the antibody forms that may be effective clinical treatments for drug abuse, as well as several current and emerging production systems that could bridge the gap from discovery to patient use.

Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.

1994-01-01

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples.

Monoclonal antibodies and recombinant immunoglobulins for the treatment of multiple sclerosis.

PubMed

Gensicke, Henrik; Leppert, David; Yaldizli, Özgür; Lindberg, Raija L P; Mehling, Matthias; Kappos, Ludwig; Kuhle, Jens

2012-01-01

Multiple sclerosis (MS) is an inflammatory and degenerative disease leading to demyelination and axonal damage in the CNS. Autoimmunity plays a central role in MS pathogenesis. Per definition, monoclonal antibodies are recombinant biological compounds with a well defined target, thus carrying the promise of targeting pathogenic cells or molecules with high specificity, avoiding undesired off-target effects. Natalizumab was the first monoclonal antibody to be approved for the treatment of MS. Several other monoclonal antibodies are in development and have demonstrated promising efficacy in phase II studies. They can be categorized according to their mode of action into compounds targeting (i) leukocyte migration into the CNS (natalizumab); (ii) cytolytic antibodies (rituximab, ocrelizumab, ofatumumab, alemtuzumab); or (iii) antibodies and recombinant proteins targeting cytokines and chemokines and their receptors (daclizumab, ustekinumab, atacicept, tabalumab [Ly-2127399], secukinumab [AIN457]). In this review, we discuss the specific molecular targets, clinical efficacy and safety of these compounds and discuss criteria to anticipate the position of monoclonal antibodies in the diversifying armamentarium of MS therapy in the coming years.

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells.

PubMed

Meng, Weixu; Li, Leike; Xiong, Wei; Fan, Xuejun; Deng, Hui; Bett, Andrew J; Chen, Zhifeng; Tang, Aimin; Cox, Kara S; Joyce, Joseph G; Freed, Daniel C; Thoryk, Elizabeth; Fu, Tong-Ming; Casimiro, Danilo R; Zhang, Ningyan; A Vora, Kalpit; An, Zhiqiang

2015-01-01

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.

Monoclonal antibodies to cyclodiene insecticides and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.

1994-08-02

Methods are described for making specific monoclonal antibodies useful for detection of cyclodienes in foods and environmental samples. Monoclonal antibodies specifically reactive with cyclodienes can detect accumulated pesticides in food, tissue or environmental samples. Extraction and preparation of organic samples for immunoassay in a polar-nonpolar reaction medium permits detection of halogenated organic ring structures at concentrations in samples. 13 figs.

[Development of ELISA on the basis of monoclonal antibodies for detecting specific activity of the vaccine against hemorrhagic fever with renal syndrome].

PubMed

Dzagurova, T K; Solopova, O N; Sveshnikov, P G; Korotina, N A; Balovneva, M V; Leonovich, O A; Varlamov, N E; Malkin, G A; Sotskova, S E; Tkachenko, E A

2013-01-01

The monoclonal antibodies to Puumala, Dobrava, Hantaan, and Seoul hantaviruses were obtained using mice. The viruses were known to cause HFRS, and two variants of ELISA were designed. First, Hanta-PUU variant, was constructed using monoclonal antibodies to Puumala virus envelope glycoprotein (G(N):G(C)) for detecting only Puumala virus antigen. The second, Hanta-N variant, was constructed using monoclonal antibodies to Dobrava and Puumala nucleocapsid proteins for detecting four above mentioned hantaviruses. Both Hanta-PUU and Hanta-N assays were reliable in detecting specific hantavirus antigens and the immunogenecity of hantavirus vaccines.

Chimeric Antigen Receptor (CAR)-Specific Monoclonal Antibody to Detect CD19-Specific T Cells in Clinical Trials

PubMed Central

Jena, Bipulendu; Maiti, Sourindra; Huls, Helen; Singh, Harjeet; Lee, Dean A.; Champlin, Richard E.; Cooper, Laurence J. N.

2013-01-01

Clinical trials targeting CD19 on B-cell malignancies are underway with encouraging anti-tumor responses. Most infuse T cells genetically modified to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of a CD19-specific mouse monoclonal antibody (mAb, clone FMC63). We describe a novel anti-idiotype monoclonal antibody (mAb) to detect CD19-specific CAR+ T cells before and after their adoptive transfer. This mouse mAb was generated by immunizing with a cellular vaccine expressing the antigen-recognition domain of FMC63. The specificity of the mAb (clone no. 136.20.1) was confined to the scFv region of the CAR as validated by inhibiting CAR-dependent lysis of CD19+ tumor targets. This clone can be used to detect CD19-specific CAR+ T cells in peripheral blood mononuclear cells at a sensitivity of 1∶1,000. In clinical settings the mAb is used to inform on the immunophenotype and persistence of administered CD19-specific T cells. Thus, our CD19-specific CAR mAb (clone no. 136.20.1) will be useful to investigators implementing CD19-specific CAR+ T cells to treat B-lineage malignancies. The methodology described to develop a CAR-specific anti-idiotypic mAb could be extended to other gene therapy trials targeting different tumor associated antigens in the context of CAR-based adoptive T-cell therapy. PMID:23469246

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

PubMed

Worobec, E A; Shastry, P; Smart, W; Bradley, R; Singh, B; Paranchych, W

1983-09-01

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types.

Monoclonal antibodies against colonization factor antigen I pili from enterotoxigenic Escherichia coli.

PubMed Central

Worobec, E A; Shastry, P; Smart, W; Bradley, R; Singh, B; Paranchych, W

1983-01-01

Hybridomas secreting monoclonal antibodies directed against intact colonization factor antigen I pili have been produced by the fusion of spleen cells from immunized BALB/c mice with NS1/SP2 myeloma cells. The four monoclones with the highest antibody titer, as detected by enzyme-linked immunosorbant assay (ELISA), were chosen for antibody amplification by production of mouse ascitic fluid. These four were examined for antibody specificity by ELISA and immunoblot assays, using six different pilus types. Three of the four monoclonal isolates were specific for only colonization factor antigen I pili in both assays, whereas the remaining isolate showed a distinct cross-reactivity with K99 pili in the ELISA assay but not in immunoblot analysis. These results indicate that this monoclone may be recognizing a common structural element between the two adhesive pilus types. Images PMID:6136463

In vivo targeting of dead tumor cells in a murine tumor model using a monoclonal antibody specific for the La autoantigen.

PubMed

Al-Ejeh, Fares; Darby, Jocelyn M; Pensa, Katherine; Diener, Kerrilyn R; Hayball, John D; Brown, Michael P

2007-09-15

To investigate the potential of the La-specific monoclonal antibody (mAb) 3B9 as an in vivo tumor-targeting agent. The murine EL4 lymphoma cell line was used for in vitro studies and the EL4 model in which apoptosis was induced with cyclophosphamide and etoposide was used for in vivo studies. In vitro studies compared 3B9 binding in the EL4 cell with that in its counterpart primary cell type of the thymocyte. For in vivo studies, 3B9 was intrinsically or extrinsically labeled with carbon-14 or 1,4,7,10-tetra-azacylododecane-N,N',N'',N''''-tetraacetic acid-indium-111, respectively, and biodistribution of the radiotracers was investigated in EL4 tumor-bearing mice, which were treated or not with chemotherapy. La-specific 3B9 mAb bound EL4 cells rather than thymocytes, and binding was detergent resistant. 3B9 binding to dead EL4 cells in vitro was specific, rapid, and saturable. Significantly, more 3B9 bound dead EL4 tumor explant cells after host mice were treated with chemotherapy, which suggested that DNA damage induced 3B9 binding. Tumor binding of 3B9 in vivo was antigen specific and increased significantly after chemotherapy. Tumor accumulation of 3B9 peaked at approximately 50% of the injected dose per gram of tumor 72 h after chemotherapy and correlated with increased tumor cell death. Tumor/organ ratios of 3B9 biodistribution, which included the tumor/blood ratio, exceeded unity 48 or more hours after chemotherapy. La-specific mAb selectively targeted dead tumor cells in vivo, and targeting was augmented by cytotoxic chemotherapy. This novel cell death radioligand may be useful both for radioimmunoscintigraphy and radioimmunotherapy.

Antibody immunoprophylaxis and immunotherapy for influenza virus infection: Utilization of monoclonal or polyclonal antibodies?

PubMed

Berry, Cassandra M

2018-03-04

Control programs for emerging influenza are in urgent need of novel therapeutic strategies to mitigate potentially devastating threats from pathogenic strains with pandemic potential. Current vaccines and antivirals have inherent limitations in efficacy, especially with rapid evolutionary changes of influenza viruses. Antibody-based antiviral protection harnesses the natural power of the immune system. Antibodies present prophylactic and therapeutic intervention options for prevention and control of influenza, especially for at-risk populations. Specific monoclonal antibodies are well defined in purity and initial efficacy but polyclonal antibodies are easier to scale-up and cost-effective with long-term efficacy, using batches with broadly neutralizing properties against influenza variants. This review presents the pros and cons of monoclonal versus polyclonal antibody therapy for influenza.

Study of blood group B antigen with a specific monoclonal antibody (anti-B, b-183).

PubMed Central

Rouger, P; Edelman, L; Doinel, C; Reviron, J; Salmon, C; Bach, J F

1983-01-01

A murine anti-B monoclonal antibody was obtained by the hybridoma technique. This antibody called anti-B (b-183) is of IgM nature; it is capable of agglutinating normal B, B3, Bx, cis AB and some acquired B red cells. Its association constant is 1.1 X 10(8) l/mol, and appears high compared to those of the monoclonal anti-A. This monoclonal anti-B was used to determine the number of B sites on B3 and Bx red cells. PMID:6840810

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.

Quality control of murine monoclonal antibodies using isoelectric focusing affinity immunoblot analysis

NASA Technical Reports Server (NTRS)

Hamilton, Robert G.; Rodkey, L. Scott; Reimer, Charles B.

1987-01-01

The quality control of murine hybridoma secretory products has been performed using two approaches for isoelectric focusing affinity immunoblot analysis: (1) a method in which antigen-coated nitrocellulose is placed on top of an acrylamide gel containing isoelectrically focused ascites to bind the antigen specific monoclonal antibody; and (2) a method in which focused ascite proteins were passively blotted onto nitrocellulose and specific monoclonal antibodies were detected with enzyme-conjugated antigen. Analysis by both methods of batches of ascites containing antihuman IgG antibodies that were produced by six hybridomas permitted effective monitoring of immunoreactive antibodies for pI microheterogeneity.

Fine mapping of HIV-1 Nef-epitopes by monoclonal antibodies.

PubMed

Siakkou, H; Jahn, S; Kienzle, N; Ulrich, R; Grötzinger, C; Schneider, T; Kohleisen, B; Pauli, G; Spohn, R; Jung, G

1993-01-01

A panel of newly isolated murine monoclonal antibodies is described which are specific for the Nef protein of the human immunodeficiency virus type 1 (HIV-1). Epitope mapping using recombinant Nef-related proteins, synthetic peptides and lipopeptides showed 3 independent antigenic determinants located within the regions of amino acids 83-93, 175-190 and 86-166 of the Nef protein. None of the monoclonal antibodies reacted with recombinant Nef proteins of HIV-2.

Human tumor xenografts in mouse as a model for evaluating therapeutic efficacy of monoclonal antibodies or antibody-drug conjugate targeting receptor tyrosine kinases.

PubMed

Feng, Liang; Wang, Wei; Yao, Hang-Ping; Zhou, Jianwei; Zhang, Ruiwen; Wang, Ming-Hai

2015-01-01

Targeting receptor tyrosine kinases by therapeutic monoclonal antibodies and antibody-drug conjugates has met with tremendous success in clinical oncology. Currently, numerous therapeutic monoclonal antibodies are under preclinical development. The potential for moving candidate antibodies into clinical trials relies heavily on therapeutic efficacy validated by human tumor xenografts in mice. Here we describe methods used to determine therapeutic efficacy of monoclonal antibodies or antibody-drug conjugates specific to human receptor tyrosine kinase using human tumor xenografts in mice as the model. The end point of the study is to determine whether treatment of tumor-bearing mice with a monoclonal antibody or antibody-drug conjugates results in significant delay of tumor growth.

Passive vaccination with a human monoclonal antibody: generation of antibodies and studies for efficacy in Bacillus anthracis infections.

PubMed

vor dem Esche, Ulrich; Huber, Maria; Zgaga-Griesz, Andrea; Grunow, Roland; Beyer, Wolfgang; Hahn, Ulrike; Bessler, Wolfgang G

2011-07-01

A major difficulty in creating human monoclonal antibodies is the lack of a suitable myeloma cell line to be used for fusion experiments. In order to create fully human monoclonal antibodies for passive immunization, the human mouse heteromyeloma cell line CB-F7 was evaluated. Using this cell line, we generated human monoclonal antibodies against Bacillus anthracis toxin components. Antibodies against protective antigen (PA) and against lethal factor (LF) were obtained using peripheral blood lymphocytes (PBLs) from persons vaccinated with the UK anthrax vaccine. PBL were fused with the cell line CB-F7. We obtained several clones producing PA specific Ig and one clone (hLF1-SAN) producing a monoclonal antibody (hLF1) directed against LF. The LF binding antibody was able to neutralize Anthrax toxin activity in an in vitro neutralization assay, and preliminary in vivo studies in mice also indicated a trend towards protection. We mapped the epitope of the antibody binding to LF by dot blot analysis and ELIFA using 80 synthetic LF peptides of 20 amino acid lengths with an overlapping range of 10 amino acids. Our results suggest the binding of the monoclonal antibody to the peptide regions 121-150 or 451-470 of LF. The Fab-fragment of the antibody hLF1 was cloned in Escherichia coli and could be useful as part of a fully human monoclonal antibody for the treatment of Anthrax infections. In general, our studies show the applicability of the CB-F7 line to create fully human monoclonal antibodies for vaccination. Copyright © 2010 Elsevier GmbH. All rights reserved.

Fully human monoclonal antibodies from antibody secreting cells after vaccination with Pneumovax®23 are serotype specific and facilitate opsonophagocytosis.

PubMed

Smith, Kenneth; Muther, Jennifer J; Duke, Angie L; McKee, Emily; Zheng, Nai-Ying; Wilson, Patrick C; James, Judith A

2013-05-01

B lymphocyte memory generates antibody-secreting cells (ASCs) that represent a source of protective antibodies that may be exploited for therapeutics. Here we vaccinated four donors with Pneumovax®23 and produced human monoclonal antibodies (hmAbs) from ASCs. We have cloned 137 hmAbs and the specificities of these antibodies encompass 19 of the 23 serotypes in the vaccine, as well as cell wall polysaccharide (CWPS). Although the majority of the antibodies are serotype specific, 12% cross-react with two serotypes. The Pneumovax®23 ASC antibody sequences are highly mutated and clonal, indicating an anamnestic response, even though this was a primary vaccination. Hmabs from 64% of the clonal families facilitate opsonophagocytosis. Although 9% of the total antibodies bind to CWPS impurity in the vaccine, none of these clonal families showed opsonophagocytic activity. Overall, these studies have allowed us to address unanswered questions in the field of human immune responses to polysaccharide vaccines, including the cross-reactivity of individual antibodies between serotypes and the percentage of antibodies that are protective after vaccination with Pneumovax®23. Copyright © 2012 Elsevier GmbH. All rights reserved.

Monoclonal antibody technologies and rapid detection assays

USDA-ARS?s Scientific Manuscript database

Novel methodologies and screening strategies will be outlined on the use of hybridoma technology for the selection of antigen specific monoclonal antibodies. The development of immunoassays used for diagnostic detection of prions and bacterial toxins will be discussed and examples provided demonstr...

Endotoxin reduces specific pulmonary uptake of radiolabeled monoclonal antibody to angiotensin-converting enzyme

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

Muzykantov, V.R.; Puchnina, E.A.; Atochina, E.N.

The biodistribution of radiolabeled monoclonal antibody (Mab) to angiotensin-converting enzyme (ACE) was examined in normal and endotoxin-treated rats. Endotoxin administration at a dose of 4 mg/kg induced mild or middle pulmonary edema. The ACE activity in lung homogenate remained virtually unchanged, while the activity of serum ACE increased 15 hr after endotoxin infusion. In normal rats, anti-ACE Mab accumulates specifically in the lung after i.v. injection. Endotoxin injection induces reduction of specific pulmonary uptake of this antibody. Even in non-edematous endotoxemia, the accumulation of anti-ACE Mab antibody (Mab 9B9) decreased from 19.02 to 11.91% of ID/g of tissue without anymore » change in accumulation of control nonspecific IgG. The antibody distribution in other organs and its blood level were almost the same as in the control. In a case of endotoxemia accompanied by increased microvascular permeability, the lung accumulation of Mab 9B9 was reduced to 9.17% of ID/g of tissue, while the accumulation of nonspecific IgG increased to 1.44% versus 0.89% in the control.« less

Monoclonal antibodies against the rat liver glucocorticoid receptor.

PubMed Central

Okret, S; Wikström, A C; Wrange, O; Andersson, B; Gustafsson, J A

1984-01-01

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR. Images PMID:6200880

Characterization of a monoclonal antibody that specifically inhibits triosephosphate isomerase activity of Taenia solium.

PubMed

Víctor, Sanabria-Ayala; Yolanda, Medina-Flores; Araceli, Zavala-Carballo; Lucía, Jiménez; Abraham, Landa

2013-08-01

In the present study, we obtained and characterized partially a monoclonal antibody (4H11D10B11 mAb) against triosephosphate isomerase from Taenia solium (TTPI). This antibody recognized the enzyme by both ELISA and western blot and was able to inhibit its enzymatic activity in 74%. Moreover, the antigen-binding fragments (Fabs), products of digestion of the monoclonal antibody with papain, retained almost the same inhibitory effect. We determined the binding site by ELISA; synthetic peptides containing sequences from different non-conserved regions of the TTPI were confronted to the 4H11D10B11 mAb. The epitope recognized by the monoclonal antibody was located on peptide TTPI-56 (ATPAQAQEVHKVVRDWIRKHVDAGIADKARI), and an analysis of mimotopes, obtained with the 4H11D10B11 mAb, suggests that the epitope spans the sequence WIRKHVDAGIAD, residues 193-204 of the enzyme. This epitope is located within helix 6, next to loop 6, an essential active loop during catalysis. The antibody did not recognize triosephosphate isomerase from man and pig, definitive and intermediary hosts of T. solium, respectively. Furthermore, it did not bind to the catalytic site, since kinetic analysis demonstrated that inhibition had a non-competitive profile. Copyright © 2013 Elsevier Inc. All rights reserved.

Monoclonal antibodies for the measurement of class-specific antibody responses in the green turtle, Chelonia mydas.

PubMed

Herbst, L H; Klein, P A

1995-06-01

Monoclonal antibodies (Mabs) were developed against the known immunoglobulin classes of the green turtle, Chelonia mydas. Plasma protein fractions enriched for 5.7S IgY, 7S IgY, and IgM turtle immunoglobulins were used to immunize Balb/c mice for hybridoma production and for hybridoma screening. Fifteen hybridomas produced Mabs with specificity for turtle immunoglobulins and for affinity purified dinitrophenol (DNP) specific turtle antibodies. Three Mabs specific for either turtle 5.7S IgY heavy chain (HL814), 7S IgY heavy chain (HL857), or IgM heavy chain (HL846) were purified and used in an enzyme-linked immunosorbent assay (ELISA) to measure antibody responses in two turtles immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) over a 10 month period. In both turtles the 7S IgY antibody response developed within 5 weeks of the first inoculation and remained high over the following 9 months. The 5.7S IgY antibody response was detected in one turtle at 3-4 months and in the other at 8 months, and reached high levels in both individuals by 10 months. The IgM responses were difficult to interpret. One turtle had pre-inoculation anti-DNP IgM antibody in its plasma and the other developed only a weak, transient response at about 4 months. The class-specific antibody activity in immune turtle plasma could be strongly inhibited by soluble DNP or by rabbit anti-DNP specific antiserum, showing that these antibody responses were directed predominantly to the DNP hapten on the DNP-BSA antigen. Antibody responses to the BSA carrier could not be detected in either turtle over the course of the immunization. Mab HL814, specific for an epitope on the 5.7S green turtle immunoglobulin heavy chain, will be useful for characterizing the molecular relationships of 5.7S, 7S and IgM heavy chains and the role of 5.7S antibody in humoral immunity in this species. All anti-turtle Ig Mabs were screened against the plasma globulins of Loggerhead (Caretta caretta), Olive

Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

PubMed

Liu, Yuxiu; Hao, Liying; Li, Xiangdong; Wang, Linxiao; Zhang, Jianpo; Deng, Junhua; Tian, Kegong

2017-06-01

Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Idiotypic Cascades in Cancer Patients Treated with Monoclonal Antibody CO17-1A

NASA Astrophysics Data System (ADS)

Wettendorff, Martine; Iliopoulos, Dimitrios; Tempero, Margaret; Kay, David; Defreitas, Elaine; Koprowski, Hilary; Herlyn, Dorothee

1989-05-01

We have previously shown that gastrointestinal cancer patients treated with monoclonal antibody CO17-1A (Ab1) developed anti-idiotypic antibodies (Ab2) to the Ab1. We now demonstrate that patients produce anti-anti-idiotypic antibodies (Ab3) to their autologous Ab2. Ab3 were demonstrated in culture supernatants of peripheral blood mononuclear cells from five Ab1-treated patients after stimulation of the cells with heterologous Ab2 that functionally mimicked the tumor antigen (Ag) defined by Ab1 and immunologically cross reacted with the patients' Ab2. Ab3 shared idiotopes with Ab1 and were Ab1-like in their binding specificities to tumor cells, Ag, and Ab2. Such antibodies were also elicited by stimulating cells with Ag. However, they were not produced by stimulating posttreatment mononuclear cells with control proteins or by stimulating pretreatment cells with either Ag or Ab2. Our results demonstrate idiotypic cascades in cancer patients treated with monoclonal antibody. Ag-specific Ab3 responses may underlie delayed clinical responses often observed in cancer patients treated with monoclonal antibodies of various specificities.

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

NASA Astrophysics Data System (ADS)

Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Discovery of functional monoclonal antibodies targeting G-protein-coupled receptors and ion channels.

PubMed

Wilkinson, Trevor C I

2016-06-15

The development of recombinant antibody therapeutics is a significant area of growth in the pharmaceutical industry with almost 50 approved monoclonal antibodies on the market in the US and Europe. Despite this growth, however, certain classes of important molecular targets have remained intractable to therapeutic antibodies due to complexity of the target molecules. These complex target molecules include G-protein-coupled receptors and ion channels which represent a large potential target class for therapeutic intervention with monoclonal antibodies. Although these targets have typically been addressed by small molecule approaches, the exquisite specificity of antibodies provides a significant opportunity to provide selective modulation of these target proteins. Given this opportunity, substantial effort has been applied to address the technical challenges of targeting these complex membrane proteins with monoclonal antibodies. In this review recent progress made in the strategies for discovery of functional monoclonal antibodies for these challenging membrane protein targets is addressed. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Class-specific effector functions of therapeutic antibodies.

PubMed

Pascal, Virginie; Laffleur, Brice; Cogné, Michel

2012-01-01

Physiology usually combines polyclonal antibodies of multiple classes in a single humoral response. Beyond their common ability to bind antigens, these various classes of human immunoglobulins carry specific functions which can each serve specific goals. In many cases, the function of a monoclonal therapeutic antibody may thus be modulated according to the class of its constant domains. Depending on the immunoglobulin class, different functional assays will be used in order to evaluate the functional activity of a monoclonal antibody.

Combining Phage and Yeast Cell Surface Antibody Display to Identify Novel Cell Type-Selective Internalizing Human Monoclonal Antibodies.

PubMed

Bidlingmaier, Scott; Su, Yang; Liu, Bin

2015-01-01

Using phage antibody display, large libraries can be generated and screened to identify monoclonal antibodies with affinity for target antigens. However, while library size and diversity is an advantage of the phage display method, there is limited ability to quantitatively enrich for specific binding properties such as affinity. One way of overcoming this limitation is to combine the scale of phage display selections with the flexibility and quantitativeness of FACS-based yeast surface display selections. In this chapter we describe protocols for generating yeast surface antibody display libraries using phage antibody display selection outputs as starting material and FACS-based enrichment of target antigen-binding clones from these libraries. These methods should be widely applicable for the identification of monoclonal antibodies with specific binding properties.

Monoclonal antibodies against trophectoderm-specific markers during mouse blastocyst formation.

PubMed Central

Brûlet, P; Babinet, C; Kemler, R; Jacob, F

1980-01-01

Two-dimensional gel electrophoresis has allowed the detection of proteins characteristic of inner cell mass and trophectoderm in mouse blastocyst. Certain of the proteins characterizing trophectoderm copurify with intermediate filaments from trophectoderm and a trophoblastoma cell line. A monoclonal antibody prepared against proteins of these intermediate filaments labels a filament network in trophectoderm but not in inner cell mass cells. Images PMID:6933460

Generation of cell type-specific monoclonal antibodies for the planarian and optimization of sample processing for immunolabeling.

PubMed

Forsthoefel, David J; Waters, Forrest A; Newmark, Phillip A

2014-12-21

Efforts to elucidate the cellular and molecular mechanisms of regeneration have required the application of methods to detect specific cell types and tissues in a growing cohort of experimental animal models. For example, in the planarian Schmidtea mediterranea, substantial improvements to nucleic acid hybridization and electron microscopy protocols have facilitated the visualization of regenerative events at the cellular level. By contrast, immunological resources have been slower to emerge. Specifically, the repertoire of antibodies recognizing planarian antigens remains limited, and a more systematic approach is needed to evaluate the effects of processing steps required during sample preparation for immunolabeling. To address these issues and to facilitate studies of planarian digestive system regeneration, we conducted a monoclonal antibody (mAb) screen using phagocytic intestinal cells purified from the digestive tracts of living planarians as immunogens. This approach yielded ten antibodies that recognized intestinal epitopes, as well as markers for the central nervous system, musculature, secretory cells, and epidermis. In order to improve signal intensity and reduce non-specific background for a subset of mAbs, we evaluated the effects of fixation and other steps during sample processing. We found that fixative choice, treatments to remove mucus and bleach pigment, as well as methods for tissue permeabilization and antigen retrieval profoundly influenced labeling by individual antibodies. These experiments led to the development of a step-by-step workflow for determining optimal specimen preparation for labeling whole planarians as well as unbleached histological sections. We generated a collection of monoclonal antibodies recognizing the planarian intestine and other tissues; these antibodies will facilitate studies of planarian tissue morphogenesis. We also developed a protocol for optimizing specimen processing that will accelerate future efforts to

[New method for analyzing pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies].

PubMed

Zhao, Yan; Qu, Hui-Hua; Wang, Qing-Guo

2013-09-01

Study on pharmacodynamic material basis of traditional Chinese medicines is one of the key issues for the modernization of traditional Chinese medicine. Having introduced the monoclonal antibody technology into the study on pharmacodynamic material basis of traditional Chinese medicines, the author prepared the immunoaffinity chromatography column by using monoclonal antibodies in active components of traditional Chinese medicines, so as to selectively knock out the component from herbs or traditional Chinese medicine compounds, while preserving all of the other components and keeping their amount and ratio unchanged. A comparative study on pharmacokinetics and pharmacodynamics was made to explicitly reveal the correlation between the component and the main purpose of traditional Chinese medicines and compounds. The analysis on pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies is a new method for study pharmacodynamic material basis in line with the characteristics of traditional Chinese medicines. Its results can not only help study material basis from a new perspective, but also help find the modern scientific significance in single herb or among compounds of traditional Chinese medicines.

Evaluation of oriented lysozyme immobilized with monoclonal antibody

NASA Astrophysics Data System (ADS)

Aoyagi, Satoka; Okada, Keigo; Shigyo, Ayako; Man, Naoki; Karen, Akiya

2008-12-01

The orientation of a lysozyme immobilized with a monoclonal antibody was evaluated based on determination of the uppermost surface structure using time-of-flight secondary ion mass spectrometry (TOF-SIMS). Specific peaks of the oriented lysozyme immobilized with monoclonal anti-lysozyme antibody were obtained in comparison with reference samples, non-oriented immobilized lysozyme and immobilized anti-lysozyme antibody. All samples were freeze-dried before TOF-SIMS measurement, and then each sample was measured using TOF-SIMS with a bismuth cluster ion source. TOF-SIMS spectra were analyzed to select peaks specific to the oriented immobilized lysozyme as well as to identify their chemical formula and ensemble of amino acids. The possible chemical formulae of the lysozyme fragments were then investigated with an element matching program and a residue matching program. The results from TOF-SIMS spectra analysis were compared to the amino acid sequence of the lysozyme and its three-dimensional structure registered in the protein data bank. Finally, the fragment-ion-generating regions of the oriented immobilized lysozyme were determined based on the suggested residues and the three-dimensional structure.

Structural isoforms of the circadian neuropeptide PDF expressed in the optic lobes of the cricket Gryllus bimaculatus: immunocytochemical evidence from specific monoclonal antibodies.

PubMed

Honda, Takeshi; Matsushima, Ayami; Sumida, Kazunori; Chuman, Yoshiro; Sakaguchi, Kazuyasu; Onoue, Hitoshi; Meinertzhagen, Ian A; Shimohigashi, Yasuyuki; Shimohigashi, Miki

2006-11-20

Pigment-dispersing factor (PDF) is an 18-mer peptide that acts as a principal neurotransmitter of the insect circadian clock. Our previous study, utilizing anti-Uca beta-PDH polyclonal antibody (pAb) to immunolabel the optic lobe of the cricket Gryllus bimaculatus, suggested the existence of an alternative PDF-like peptide in the outer cells of the first neuropile, or lamina (La), which were much less immunoreactive than the inner cells of the second neuropile, the medulla (Me). To obtain structural information about such a PDF-like peptide, we prepared 10 anti-Gryllus PDF monoclonal (mAb) and pAb antibodies and analyzed their detailed epitope specificities. The PDFMe and PDFLa inner cells and their axonal projections were clearly immunoreactive to all these antibodies, revealing the widespread immunocytochemical organization of the PDF system in the optic lobe, as seen previously with anti-Uca beta-PDH pAb and anti-Gryllus PDF mAb, the epitope structures of which were also clarified in this study. The lamina outer cells, which we found lacked a target pdf mRNA, displayed specific immunoreactivities, indicating that the cells contain a distinct PDF-like peptide possessing both N- and C-terminal structures. These cells were not immunolabeled by some other monoclonal antibodies, however, implying that the PDFLa outer cells have a PDF isoform peptide devoid of Asn at positions 6 and 16. This isoform was also identified in a varicose arborization in the lamina. These results suggest not only the structure of the peptide, but also the possibility of additional functions of this novel PDF isoform.

Large Scale Generation and Characterization of Anti-Human CD34 Monoclonal Antibody in Ascetic Fluid of Balb/c Mice

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Kazemi, Tohid; Aghebati Maleki, Ali; Sineh sepehr, Koushan

2013-01-01

Purpose: Monoclonal antibodies or specific antibodies are now an essential tool of biomedical research and are of great commercial and medical value. The purpose of this study was to produce large scale of monoclonal antibody against CD34 in order to diagnostic application in leukemia and purification of human hematopoietic stem/progenitor cells. Methods: For large scale production of monoclonal antibody, hybridoma cells that produce monoclonal antibody against human CD34 were injected into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. 5 ml ascitic fluid was harvested from each mouse in two times. Evaluation of mAb titration was assessed by ELISA method. The ascitic fluid was examined for class and subclasses by ELISA mouse mAb isotyping Kit. mAb was purified from ascitic fluid by affinity chromatography on Protein A-Sepharose. Purity of monoclonal antibody was monitored by SDS -PAGE and the purified monoclonal antibody was conjugated with FITC. Results: Monoclonal antibodies with high specificity and sensitivity against human CD34 by hybridoma technology were prepared. The subclass of antibody was IgG1 and its light chain was kappa. Conclusion: The conjugated monoclonal antibody could be a useful tool for isolation, purification and characterization of human hematopoietic stem cells. PMID:24312838

Viral Epitopes and Monoclonal Antibodies: Isolation of Blocking Antibodies that Inhibit Virus Neutralization

NASA Astrophysics Data System (ADS)

Massey, Richard J.; Schochetman, Gerald

1981-07-01

The inability of pathogenic animal viruses to be completely neutralized by antibodies can lead to chronic viral infections in which infectious virus persists even in the presence of excess neutralizing antibody. A mechanism that results in this nonneutralized fraction of virus was defined by the topographical relationships of viral epitopes identified with monoclonal antibodies wherein monoclonal antibodies bind to virus and sterically block the binding of neutralizing antibodies.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

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

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

In-situ Detection of Squalane in Sedimentary Organic Matter Using Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Bailey, J. V.; Corsetti, F. A.; Moldowan, J. M.; Fago, F.; Caron, D.

2008-12-01

Sedimentary geolipids can serve as powerful tools for reconstructing ancient ecosystems, but only if investigators can demonstrate that the hydrocarbons are indigenous to their host rocks. The association of molecules with primary sedimentary fabrics could indicate a syngenetic relationship. However, traditional biomarker analyses require extraction from large quantities of powdered rock, confounding detailed spatial correlations. Biological studies commonly use antibodies as extremely sensitive molecular probes. When coupled with fluorescent labels, antibodies allow for the visual localization of molecules. Here we show that monoclonal antibodies that bind specifically to geolipid compounds can be used for in situ detection and labeling of such compounds in mineral-bound organic macerals. Monoclonal antibodies to squalene, produced for human health studies, also react with the geolipid, squalane. We show that squalene antibodies do not react with other common sedimentary hydrocarbons. We also show that squalane antibodies bind specifically to isolated organic-rich lamina in Eocene-age, squalane-containing rocks. These results suggest that squalane is confined to discrete organo-sedimentary fabrics within those rocks, providing evidence for its syngeneity. The chemical similarity of squalane to other sedimentary hydrocarbons hints at the potential for developing monoclonal antibodies to a variety of biomarkers that could then be localized in rocks, sediments, and extant cells.

HIV-1-Specific IgA Monoclonal Antibodies from an HIV-1 Vaccinee Mediate Galactosylceramide Blocking and Phagocytosis

PubMed Central

2018-01-01

ABSTRACT Vaccine-elicited humoral immune responses comprise an array of antibody forms and specificities, with only a fraction contributing to protective host immunity. Elucidation of antibody effector functions responsible for protective immunity against human immunodeficiency virus type 1 (HIV-1) acquisition is a major goal for the HIV-1 vaccine field. Immunoglobulin A (IgA) is an important part of the host defense against pathogens; however, little is known about the role of vaccine-elicited IgA and its capacity to mediate antiviral functions. To identify the antiviral functions of HIV-1-specific IgA elicited by vaccination, we cloned HIV-1 envelope-specific IgA monoclonal antibodies (MAbs) by memory B cell cultures from peripheral blood mononuclear cells from an RV144 vaccinee and produced two IgA clonal cell lines (HG129 and HG130) producing native, nonrecombinant IgA MAbs. The HG129 and HG130 MAbs mediated phagocytosis by monocytes, and HG129 blocked HIV-1 Env glycoprotein binding to galactosylceramide, an alternative HIV-1 receptor. These findings elucidate potential antiviral functions of vaccine-elicited HIV-1 envelope-specific IgA that may act to block HIV-1 acquisition at the portal of entry by preventing HIV-1 binding to galactosylceramide and mediating antibody Fc receptor-mediated virion phagocytosis. Furthermore, these findings highlight the complex and diverse interactions of vaccine-elicited IgA with pathogens that depend on IgA fine specificity and form (e.g., multimeric or monomeric) in the systemic circulation and mucosal compartments. IMPORTANCE Host-pathogen interactions in vivo involve numerous immune mechanisms that can lead to pathogen clearance. Understanding the nature of antiviral immune mechanisms can inform the design of efficacious HIV-1 vaccine strategies. Evidence suggests that both neutralizing and nonneutralizing antibodies can mediate some protection against HIV in animal models. Although numerous studies have characterized the

Monoclonal antibodies for chronic pain: A practical review of mechanisms and clinical applications

PubMed Central

Yeh, Ju-Fen; Akinci, Aysen; Al Shaker, Mohammed; Chang, Ming Hong; Danilov, Andrei; Guillen, Rocio; Johnson, Kirk W; Kim, Yong-Chul; Skljarevski, Vladimir; Dueñas, Héctor J; Tassanawipas, Warat

2017-01-01

Context Monoclonal antibodies are being investigated for chronic pain to overcome the shortcomings of current treatment options. Objective To provide a practical overview of monoclonal antibodies in clinical development for use in chronic pain conditions, with a focus on mechanisms of action and relevance to specific classes. Methods Qualitative review using a systematic strategy to search for randomized controlled trials, systematic and nonsystematic (narrative) reviews, observational studies, nonclinical studies, and case reports for inclusion. Studies were identified via relevant search terms using an electronic search of MEDLINE via PubMed (1990 to June 2017) in addition to hand-searching reference lists of retrieved systematic and nonsystematic reviews. Results Monoclonal antibodies targeting nerve growth factor, calcitonin gene-related peptide pathways, various ion channels, tumor necrosis factor-α, and epidermal growth factor receptor are in different stages of development. Mechanisms of action are dependent on specific signaling pathways, which commonly involve those related to peripheral neurogenic inflammation. In clinical studies, there has been a mixed response to different monoclonal antibodies in several chronic pain conditions, including migraine, neuropathic pain conditions (e.g., diabetic peripheral neuropathy), osteoarthritis, chronic back pain, ankylosing spondylitis, and cancer. Adverse events observed to date have generally been mild, although further studies are needed to ensure safety of monoclonal antibodies in early stages of development, especially where there is an overlap with non-pain-related pathways. High acquisition cost remains another treatment limitation. Conclusion Monoclonal antibodies for chronic pain have the potential to overcome the limitations of current treatment options, but strategies to ensure their appropriate use need to be determined. PMID:29056066

Two monoclonal antibodies specific for different epitopes within the amino-terminal region of F pilin.

PubMed Central

Frost, L S; Lee, J S; Scraba, D G; Paranchych, W

1986-01-01

Two murine monoclonal antibodies (JEL 92 and 93) specific for adjacent epitopes on F pilin were purified and characterized. JEL 93 immunoglobulin G (IgG) and its Fab fragments were specific for the amino-terminal region and were completely reactive with a synthetic peptide representing the first eight amino acids of F pilin. The acetyl group was demonstrated to be an important part of the epitope, since an unacetylated version of the amino-terminal peptide was 100-fold less reactive with JEL 93 IgG. JEL 92 IgG reacted with the region of F pilin surrounding Met-9, represented by a tryptic peptide derived from the first 17 amino acids. This reactivity was completely abolished by cleavage of the peptide with cyanogen bromide. As shown by electron microscopy, both monoclonal antibodies bound to a vesiclelike structure at one end of purified free pili and did not bind to the sides of the pili, nor did they appear to bind to the tip. When sonication was used to break pili into shorter fragments, the number of binding sites for JEL 92 but not JEL 93 IgG increased as measured by a competitive enzyme-linked immunosorbent assay. Images PMID:2428808

Specific antibody for pesticide residue determination produced by antibody-pesticide complex

USDA-ARS?s Scientific Manuscript database

A new method for specific antibody production was developed using antibody (Ab)-pesticide complex as a unique immunogen. Parathion (PA) was the targeted pesticide, and rabbit polyclonal antibody (Pab) and mouse monoclonal antibody (Mab) were used as carrier proteins. The Ab-PA complexes were genera...

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

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

Bidart, J.M.; Troalen, F.; Salesse, R.

1987-06-25

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptidesmore » spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.« less

Mass-Production and Characterization of Anti-CD20 Monoclonal Antibody in Peritoneum of Balb/c Mice

PubMed Central

Sineh sepehr, Koushan; Baradaran, Behzad; Majidi, Jafar; Abdolalizadeh, Jalal; Aghebati, leili; Zare Shahneh, Fatemeh

2013-01-01

Purpose: Monoclonal antibodies are important tools are used in basic research as well as, in diagnosis, imaging and treatment of immunodeficiency diseases, infections and cancers. The purpose of this study was to produce large scale of monoclonal antibody against CD20 in order to diagnostic application in leukemia and lymphomas disorders. Methods: Hybridoma cells that produce monoclonal antibody against human CD20 were administered into the peritoneum of the Balb/c mice which have previously been primed with 0.5 ml Pristane. After twelve days, approximately 7 ml ascetic fluid was harvested from the peritoneum of each mouse. Evaluation of mAb titration was assessed by ELISA method. In the present study, we describe a protocol for large scale production of MAbs. Results: We prepared monoclonal antibodies (mAbs) with high specificity and sensitivity against human CD20 by hybridoma method and characterized them by ELISA. The subclass of antibody was IgG2a and its light chain was kappa. Ascetic fluid was purified by Protein-A Sepharose affinity chromatography and the purified monoclonal antibody was conjugated with FITC and Immunofluorescence was done for confirming the specific binding. Conclusion: The conjugated monoclonal antibody could have application in diagnosis B-cell lymphomas, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells. PMID:24312821

Rapid Multiplex Assay for Serotyping Pneumococci with Monoclonal and Polyclonal Antibodies

PubMed Central

Yu, Jigui; Lin, Jisheng; Benjamin, William H.; Waites, Ken B.; Lee, Che-hung; Nahm, Moon H.

2005-01-01

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy. PMID:15634965

[Monoclonal antibodies in diagnosis of acute leukemias].

PubMed

Krawczyńska, A; Robak, T

1996-01-01

Immunophenotyping has become an essential component for the study of acute myeloblastic (AML) and lymphoblastic (ALL) leukaemias. The recent development of highly specific monoclonal antibodies (Mc Ab) to differentiation antigens (CD) of haematopoetic cells have made it readily available to clinical laboratories in most major hospitals. Immunophenotyping complements standard morphology by providing information on lineage, stage of differentiation and clonality. In addition some of the flow cytometry findings have independent prognostic significance. Monoclonal antibodies useful in defining lineage (B-cell versus T-cell) and stages of differentiation of ALL. It can be also used in identifying characteristic feature of AML and aiding in lineage determination in acute leukaemias that are morphologically undifferentiated. Surface immunophenotyping is especially helpful for recognizing mixed lineage acute leukaemia and diagnosing certain rare entities such as erythroleukaemia (M6), acute megakaryocytic leukaemia (M7) and minimally differentiation acute myeloid leukaemia.

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

PubMed

Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-11-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients.

Monitoring multiple myeloma patients treated with daratumumab: teasing out monoclonal antibody interference.

PubMed

McCudden, Christopher; Axel, Amy E; Slaets, Dominique; Dejoie, Thomas; Clemens, Pamela L; Frans, Sandy; Bald, Jaime; Plesner, Torben; Jacobs, Joannes F M; van de Donk, Niels W C J; Moreau, Philippe; Schecter, Jordan M; Ahmadi, Tahamtan; Sasser, A Kate

2016-06-01

Monoclonal antibodies are promising anti-myeloma treatments. As immunoglobulins, monoclonal antibodies have the potential to be identified by serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE). Therapeutic antibody interference with standard clinical SPE and IFE can confound the use of these tests for response assessment in clinical trials and disease monitoring. To discriminate between endogenous myeloma protein and daratumumab, a daratumumab-specific immunofixation electrophoresis reflex assay (DIRA) was developed using a mouse anti-daratumumab antibody. To evaluate whether anti-daratumumab bound to and shifted the migration pattern of daratumumab, it was spiked into daratumumab-containing serum and resolved by IFE/SPE. The presence (DIRA positive) or absence (DIRA negative) of residual M-protein in daratumumab-treated patient samples was evaluated using predetermined assessment criteria. DIRA was evaluated for specificity, limit of sensitivity, and reproducibility. In all of the tested samples, DIRA distinguished between daratumumab and residual M-protein in commercial serum samples spiked with daratumumab and in daratumumab-treated patient samples. The DIRA limit of sensitivity was 0.2 g/L daratumumab, using spiking experiments. Results from DIRA were reproducible over multiple days, operators, and assays. The anti-daratumumab antibody was highly specific for daratumumab and did not shift endogenous M-protein. As the treatment of myeloma evolves to incorporate novel monoclonal antibodies, additional solutions will be needed for clinical monitoring of patient responses to therapeutic regimens. In the interim, assays such as DIRA can inform clinical outcomes by distinguishing daratumumab from endogenous M-protein by IFE.

Production and characterization of murine monoclonal antibody against synthetic peptide of CD34.

PubMed

Maleki, Leili Aghebati; Majidi, Jafar; Baradaran, Behzad; Abdolalizadeh, Jalal; Akbari, Aliakbar Movassaghpour

2013-01-01

The treatment of hematologic malignancies and immunodeficiency diseases are offered by hematopoietic stem cells (HSCs) as a unique self-renewal and differentiation source which most commonly is selected by CD34 surface marker for HSC. The purpose of this study was to develop and characterize monoclonal antibody against CD34 antigen for detection of hematopoietic stem cells. Balb/c mice were immunized with two synthetic peptides of CD34 and Spleen cells were fused with SP2/0.Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by limiting dilution. Large scale of monoclonal antibodies was produced by mouse ascites production of mAb (in vivo) method. Monoclonal antibody was purified by chromatography. Then reactivity of these antibodies was evaluated in different immunological assays including ELISA, immunofluorescence (IF), western blot (WB) and flowcytometry. In this study, between five positive clone wells, two clones were chosen for limiting dilution. Limiting dilution product was one monoclone (3-D5 monoclone) with absorbance about 2. Isotype of this mAb was identified as IgG1 class with Kappa (κ) light chain. This antibody is highly specific and functional in biomedical applications such as ELISA, flowcytometry, immunofluorescence, and western blot assays.

Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies.

PubMed

Ulaeto, David O; Hutchinson, Alistair P; Nicklin, Stephen

2015-08-01

Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites.

Sub-Nanogram Detection of RDX Explosive by Monoclonal Antibodies

PubMed Central

Hutchinson, Alistair P.; Nicklin, Stephen

2015-01-01

Polyclonal and monoclonal antibodies were raised to protein carrier molecules haptenized with RDX, a major component of many plastic explosives including Semtex. Sera from immunized mice detected RDX protein conjugates in standard ELISA. Clonally purified monoclonal antibodies had detection limits in the sub-ng/mL range for underivatized RDX in competition ELISA. The monoclonal antibodies are not dependent on the presence of taggants added during the manufacturing process, and are likely to have utility in the detection of any explosive containing RDX, or RDX contamination of environmental sites. PMID:26252765

Biodistribution mechanisms of therapeutic monoclonal antibodies in health and disease.

PubMed

Tabrizi, Mohammad; Bornstein, Gadi Gazit; Suria, Hamza

2010-03-01

The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

Monoclonal Antibody Therapy for Advanced Neuroblastoma

Cancer.gov

NCI is sponsoring two clinical trials of a monoclonal antibody called ch14.18, in combination with other drugs, to see if the antibody may be helpful for children or young adults (up to age 21) with relapsed or refractory neuroblastoma.

[Batch release of immunoglobulin and monoclonal antibody products].

PubMed

Gross, S

2014-10-01

The Paul-Ehrlich Institute (PEI) is an independent institution of the Federal Republic of Germany responsible for performing official experimental batch testing of sera. The institute decides about the release of each batch and performs experimental research in the field. The experimental quality control ensures the potency of the product and also the absence of harmful impurities. For release of an immunoglobulin batch the marketing authorization holder has to submit the documentation of the manufacture and the results of quality control measures together with samples of the batch to the PEI. Experimental testing is performed according to the approved specifications regarding the efficacy and safety. Since implementation of the 15th German drug law amendment, the source of antibody is not defined anymore. According to § 32 German drug law, all batches of sera need to be released by an official control laboratory. Sera are medicinal products, which contain antibodies, antibody fragments or fusion proteins with a functional antibody portion. Therefore, all batches of monoclonal antibodies and derivatives must also be released by the PEI and the marketing authorization holder has to submit a batch release application. Under certain circumstances a waiver for certain products can be issued with regard to batch release. The conditions for such a waiver apply to the majority of monoclonal antibodies.

Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.

PubMed

Deckers, Susanne; Braren, Ingke; Greunke, Kerstin; Meyer, Nadine; Rühl, Dana; Bredehorst, Reinhard; Spillner, Edzard

2009-01-01

Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

Monoclonal antibody against Porphyromonas (Bacteroides) endodontalis lipopolysaccharide and application of the antibody for direct identification of the species.

PubMed Central

Hanazawa, S; Sagiya, T; Kitami, H; Ohta, K; Nishikawa, H; Kitano, S

1991-01-01

The aim of the present study was to develop a monoclonal antibody that recognizes the shared antigen of Porphyromonas endodontalis so that we could use the antibody in direct identification and detection of P. endodontalis in infectious material from apical periodontal patients. We established a hybridoma cell line producing monoclonal antibody (BEB5) specific for P. endodontalis. BEB5 antibody reacted with all of the P. endodontalis strains tested, but not with any of the other black-pigmented Porphyromonas and Bacteroides spp. The antibody reacted specifically with the lipopolysaccharide (LPS) of three P. endodontalis strains of different serotypes (O1K1, O1K2, and O1K-). Western blotting (immunoblotting) analysis confirmed the specificity of the antibody to these LPSs, because the antibody recognized the typical "repetitive ladder" pattern characteristic of LPS on sodium dodecyl sulfate-polyacrylamide electrophoretic gels. These observations demonstrate that P. endodontalis LPS is the shared antigen of this species. The antibody can specifically identify P. endodontalis on nitrocellulose membrane blots of bacterial colonies grown on agar. The antibody is also capable of directly detecting the presence of P. endodontalis in infectious material by immunoslot blot assay. These results indicate that LPS is the shared antigen of P. endodontalis and that BEB5 antibody against LPS is a useful one for direct identification and detection of the organisms in samples from apical periodontal patients. Images PMID:1774262

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus).

PubMed

Wellehan, James F X; Green, Linda G; Duke, Diane G; Bootorabi, Shadi; Heard, Darryl J; Klein, Paul A; Jacobson, Elliott R

2009-09-01

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.

A high affinity monoclonal antibody recognizing the light chain of human coagulating factor VII.

PubMed

Sarial, Sheila; Asadi, Farzad; Jeddi-Tehrani, Mahmood; Hadavi, Reza; Bayat, Ali Ahmad; Mahmoudian, Jafar; Taghizadeh-Jahed, Masoud; Shokri, Fazel; Rabbani, Hodjattallah

2012-12-01

Factor VII (FVII) is a serine protease-coagulating element responsible for the initiation of an extrinsic pathway of clot formation. Here we generated and characterized a high affinity monoclonal antibody that specifically recognizes human FVII. Recombinant human FVII (rh-FVII) was used for the production of a monoclonal antibody using BALB/c mice. The specificity of the antibody was determined by Western blot using plasma samples from human, mouse, sheep, goat, bovine, rabbit, and rat. Furthermore, the antibody was used to detect transiently expressed rh-FVII in BHK21 cell line using Western blot and sandwich ELISA. A mouse IgG1 (kappa chain) monoclonal antibody clone 1F1-B11 was produced against rh-FVII. The affinity constant (K(aff)) of the antibody was calculated to be 6.4×10(10) M(-1). The antibody could specifically recognize an epitope on the light chain of hFVII, with no reactivity with factor VII from several other animals. In addition, transiently expressed rh-FVII in BHK21 cells was recognized by 1F1-B11. The high affinity as well as the specificity of 1F1-B11 for hFVII will facilitate the affinity purification of hFVII and also production of FVII deficient plasma and minimizes the risk of bovine FVII contamination when fetal bovine serum-supplemented media are used for production and subsequent purification of rh-FVII.

Identification of a vesicular-arbuscular mycorrhizal fungus by using monoclonal antibodies in an enzyme-linked immunosorbent assay.

PubMed

Wright, S F; Morton, J B; Sworobuk, J E

1987-09-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains.

Development of VHH antibodies against dengue virus type 2 NS1 and comparison with monoclonal antibodies for use in immunological diagnosis.

PubMed

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection.

Development of VHH Antibodies against Dengue Virus Type 2 NS1 and Comparison with Monoclonal Antibodies for Use in Immunological Diagnosis

PubMed Central

Fatima, Aneela; Wang, Haiying; Kang, Keren; Xia, Liliang; Wang, Ying; Ye, Wei; Wang, Jufang; Wang, Xiaoning

2014-01-01

The possibility of using variable domain heavy-chain antibodies (VHH antibodies) as diagnostic tools for dengue virus (DENV) type 2 NS1 protein was investigated and compared with the use of conventional monoclonal antibodies. After successful expression of DENV type 2 NS1 protein, the genes of VHH antibodies against NS1 protein were biopanned from a non-immune llama library by phage display. VHH antibodies were then expressed and purified from Escherichia coli. Simultaneously, monoclonal antibodies were obtained by the conventional route. Sequence analysis of the VHH antibodies revealed novel and long complementarity determining regions 3 (CDR3). Epitope mapping was performed via a phage display peptide library using purified VHH and monoclonal antibodies as targets. Interestingly, the same region of NS1, which comprises amino acids 224HWPKPHTLW232, was conserved for both kinds of antibodies displaying the consensus motif histidine-tryptophan-tryptophan or tryptophan-proline-tryptophan. The two types of antibodies were used to prepare rapid diagnostic kits based on immunochromatographic assay. The VHH antibody immobilized rapid diagnostic kit showed better sensitivity and specificity than the monoclonal antibody immobilized rapid diagnostic kit, which might be due to the long CDR3 regions of the VHH antibodies and their ability to bind to the pocket and cleft of the targeted antigen. This demonstrates that VHH antibodies are likely to be an option for developing point-of-care tests against DENV infection. PMID:24751715

Monoclonal antibodies and method for detecting dioxins and dibenzofurans

DOEpatents

Vanderlaan, Martin; Stanker, Larry H.; Watkins, Bruce E.; Bailey, Nina R.

1989-01-01

Compositions of matter are described which include five monoclonal antibodies that react with dioxins and dibenzofurans, and the five hybridomas that produce these monoclonal antibodies. In addition, a method for the use of these antibodies in a sensitive immunoassay for dioxins and dibenzofurans is given, which permits detection of these pollutants in samples at concentrations in the range of a few parts per billion.

Human monoclonal antibodies: the residual challenge of antibody immunogenicity.

PubMed

Waldmann, Herman

2014-01-01

One of the major reasons for seeking human monoclonal antibodies has been to eliminate immunogenicity seen with rodent antibodies. Thus far, there has yet been no approach which absolutely abolishes that risk for cell-binding antibodies. In this short article, I draw attention to classical work which shows that monomeric immunoglobulins are intrinsically tolerogenic if they can be prevented from creating aggregates or immune complexes. Based on these classical studies two approaches for active tolerization to therapeutic antibodies are described.

Antibody-Directed Cytotoxic Agents: Use of Monoclonal Antibody to Direct the Action of Toxin A Chains to Colorectal Carcinoma Cells

NASA Astrophysics Data System (ADS)

Gilliland, D. Gary; Steplewski, Zenon; Collier, R. John; Mitchell, Kenneth F.; Chang, Tong H.; Koprowski, Hilary

1980-08-01

We have constructed cell-specific cytotoxic agents by covalently coupling the A chain from diphtheria toxin or ricin toxin to monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen. Antibody 1083-17-1A was modified by attachment of 3-(2-pyridyldithio)propionyl or cystaminyl groups and then treated with reduced A chain to give disulfide-linked conjugates that retained the original binding specificity of the antibody moiety. The conjugates showed cytotoxic activity for colorectal carcinoma cells in culture, but were not toxic in the same concentration range for a variety of cell lines that lacked the antigen. Under defined conditions virtually 100% of antigen-bearing cultured cells were killed, whereas cells that lacked the antigen were not affected. Conjugates containing toxin A chains coupled to monoclonal antibodies may be useful in studying functions of various cell surface components and, possibly, as tumor-specific therapeutic agents.

Mechanisms of monoclonal antibody stabilization and release from silk biomaterials

PubMed Central

Guziewicz, Nicholas A.; Massetti, Andrew J.; Perez-Ramirez, Bernardo J.; Kaplan, David L.

2013-01-01

The availability of stabilization and sustained delivery systems for antibody therapeutics remains a major clinical challenge, despite the growing development of antibodies for a wide range of therapeutic applications due to their specificity and efficacy. A mechanistic understanding of protein-matrix interactions is critical for the development of such systems and is currently lacking as a mode to guide the field. We report mechanistic insight to address this need by using well-defined matrices based on silk gels, in combination with a monoclonal antibody. Variables including antibody loading, matrix density, charge interactions, hydrophobicity and water access were assessed to clarify mechanisms involved in the release of antibody from the biomaterial matrix. The results indicate that antibody release is primarily governed by hydrophobic interactions and hydration resistance, which are controlled by silk matrix chemistry, peptide domain distribution and protein density. Secondary ionic repulsions are also critical in antibody stabilization and release. Matrix modification by free methionine incorporation was found to be an effective strategy for mitigating encapsulation induced antibody oxidation. Additionally, these studies highlight a characterization approach to improve the understanding and development of other protein sustained delivery systems, with broad applicability to the rapidly developing monoclonal antibody field. PMID:23859659

Generation of monoclonal antibodies specific for cell surface molecules expressed on early mouse endoderm.

PubMed

Gadue, Paul; Gouon-Evans, Valerie; Cheng, Xin; Wandzioch, Ewa; Zaret, Kenneth S; Grompe, Markus; Streeter, Philip R; Keller, Gordon M

2009-09-01

The development of functional cell populations such as hepatocytes and pancreatic beta cells from embryonic stem cell (ESC) is dependent on the efficient induction of definitive endoderm early in the differentiation process. To monitor definitive endoderm formation in mouse ESC differentiation cultures in a quantitative fashion, we generated a reporter cell line that expresses human CD25 from the Foxa3 locus and human CD4 from the Foxa2 locus. Induction of these reporter ESCs with high concentrations of activin A led to the development of a CD25-Foxa3+CD4-Foxa2+ population within 4-5 days of culture. Isolation and characterization of this population showed that it consists predominantly of definitive endoderm that is able to undergo hepatic specification under the appropriate conditions. To develop reagents that can be used for studies on endoderm development from unmanipulated ESCs, from induced pluripotent stem cells, and from the mouse embryo, we generated monoclonal antibodies against the CD25-Foxa3+CD4-Foxa2+ population. With this approach, we identified two antibodies that react specifically with endoderm from ESC cultures and from the early embryo. The specificity of these antibodies enables one to quantitatively monitor endoderm development in ESC differentiation cultures, to study endoderm formation in the embryo, and to isolate pure populations of culture- or embryo-derived endodermal cells.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, R.H.; Vanderlaan, M.; Bigbee, W.L.; Stanker, L.H.; Branscomb, E.W.; Grabske, R.J.

1984-11-29

The present invention provides monoclonal antibodies specific to and distinguishing between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype. 4 figs.

Monoclonal antibodies to human hemoglobin S and cell lines for the production thereof

DOEpatents

Jensen, Ronald H.; Vanderlaan, Martin; Bigbee, William L.; Stanker, Larry H.; Branscomb, Elbert W.; Grabske, Robert J.

1988-01-01

The present invention provides monoclonal antibodies specific to and distinguish between hemoglobin S and hemoglobin A and methods for their production and use. These antibodies are capable of distinguishing between two hemoglobin types which differ from each other by only a single amino acid residue. The antibodies produced according to the present method are useful as immunofluorescent markers to enumerate circulating red blood cells which have the property of altered expression of the hemoglobin gene due to somatic mutation in stem cells. Such a measurement is contemplated as an assay for in vivo cellular somatic mutations in humans. Since the monoclonal antibodies produced in accordance with the instant invention exhibit a high degree of specificity to and greater affinity for hemoglobin S, they are suitable for labeling human red blood cells for flow cytometric detection of hemoglobin genotype.

Survey of Latin American Neuroimmunologists on the Treatment of Multiple Sclerosis with Monoclonal Antibodies.

PubMed

Fragoso, Yara Dadalti

2015-01-01

Natalizumab and alemtuzumab are monoclonal antibodies approved for the treatment of relapsing-remitting multiple sclerosis (RRMS). A third monoclonal antibody, daclizumab, should soon become another alternative for RRMS therapy. A group of 26 doctors working at specific MS Units in seven different Latin American countries participated in the present study. All 26 neurologists had experience with natalizumab for the treatment of MS and were willing to discuss strategies for improving this treatment. Most neurologists had no confidence in starting a patient on natalizumab and alemtuzumab, which are new and efficient drugs approved by North American, European and most Latin American health agencies. The Latin American specialists felt they were not properly informed on daclizumab. Specific pharmacovigilance programs for each of these monoclonal antibodies were considered very important by the neurologists, who were also willing to discuss these therapeutic options with peers from other countries.

Impact of heat processing on the detection of the major shellfish allergen tropomyosin in crustaceans and molluscs using specific monoclonal antibodies.

PubMed

Kamath, Sandip D; Abdel Rahman, Anas M; Komoda, Toshikazu; Lopata, Andreas L

2013-12-15

The major heat-stable shellfish allergen, tropomyosin, demonstrates immunological cross-reactivity, making specific differentiation of crustaceans and molluscs for food labelling very difficult. The aim of this study was to evaluate the application of allergen-specific monoclonal antibodies in differential detection of shellfish-derived tropomyosin in 11 crustacean and 7 mollusc species, and to study the impact of heating on its detection. Cross-reactive tropomyosin was detected in all crustacean species, with partial detection in molluscs: mussels, scallops and snails but none in oyster, octopus and squid. Furthermore, we have demonstrated that heating of shellfish has a profound effect on tropomyosin detection. This was evident by the enhanced recognition of multiple tropomyosin variants in the analysed shellfish species. Specific monoclonal antibodies, targetting the N-terminal region of tropomyosin, must therefore be developed to differentiate tropomyosins in crustaceans and molluscs. This can help in correct food labelling practices and thus protection of consumers. Copyright © 2013 Elsevier Ltd. All rights reserved.

Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies

PubMed Central

Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

2015-01-01

Background: Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. Methods: To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Results: Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Conclusion: Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage. PMID:26605008

Preliminary Assessment of Various Additives on the Specific Reactivity of Anti- rHBsAg Monoclonal Antibodies.

PubMed

Yazdani, Yaghoub; Mohammadi, Saeed; Yousefi, Mehdi; Shokri, Fazel

2015-01-01

Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies (mAbs) against recombinant HBsAg (rHBsAg) epitopes. To estimate the protective effect of different additives on the stability of antibody against conformational epitopes (S3 antibody) and linear epitopes (S7 and S11 antibodies) of rHBsAg, heat shock at 37°C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs (S3) against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage.

A monoclonal antibody recognizes undifferentiation-specific carbohydrate moieties expressed on cell surface of the human dental pulp cells.

PubMed

Kang, Kyung-Jung; Ko, Seon-Yle; Ryu, Chun-Jeih; Jang, Young-Joo

2017-05-01

Human dental pulp cells are obtained from dental pulp tissue, and have the ability to form dentin and a pulp-like complex. Although adult stem cells have been identified from the primary culture by using specific cell surface markers, the identity of surface markers for the purification of stem cells within the dental pulp population are still unclear. Previously, we had constructed monoclonal antibodies against the undifferentiated cell-specific surface markers of human dental pulp cells (hDPCs) by performing decoy immunization. Among them, a monoclonal antibody against the cell surface antigen of the undifferentiated hDPCs (named UPSA-1) was purified and its heavy and light chain consensus regions were analyzed. The cell surface binding affinity of UPSA-1 mAb on the undifferentiated hDPCs was stronger than that on the differentiated cells. When tunicamycin was applied to hDPSCs during culture, the cell surface binding affinity of the antibody was dramatically decreased, and dentinogenic differentiation was reduced. The purified UPSA-1 antigen band resulting from immunoprecipitation disappeared or shifted down on the SDS-PAGE by deglycosylation. These data suggested that glycosylation on the cell surface might be a marker of an undifferentiated state, and that UPSA-1 mAb might be useful for identifying the carbohydrate moiety on the cell surface of undifferentiated pulp cells. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Monoclonal IgA Antibodies for Aflatoxin Immunoassays

PubMed Central

Ertekin, Özlem; Pirinçci, Şerife Şeyda; Öztürk, Selma

2016-01-01

Antibody based techniques are widely used for the detection of aflatoxins which are potent toxins with a high rate of occurrence in many crops. We developed a murine monoclonal antibody of immunoglobulin A (IgA) isotype with a strong binding affinity to aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2) and aflatoxin M1 (AFM1). The antibody was effectively used in immunoaffinity column (IAC) and ELISA kit development. The performance of the IACs was compatible with AOAC performance standards for affinity columns (Test Method: AOAC 991.31). The total binding capacity of the IACs containing our antibody was 111 ng, 70 ng, 114 ng and 73 ng for AFB1, AFB2, and AFG1 andAFG2, respectively. Furthermore, the recovery rates of 5 ng of each AF derivative loaded to the IACs were determined as 104.9%, 82.4%, 85.5% and 70.7% for AFB1, AFB2, AFG1 and AFG2, respectively. As for the ELISA kit developed using non-oriented, purified IgA antibody, we observed a detection range of 2–50 µg/L with 40 min total test time. The monoclonal antibody developed in this research is hitherto the first presentation of quadruple antigen binding IgA monoclonal antibodies in mycotoxin analysis and also the first study of their utilization in ELISA and IACs. IgA antibodies are valuable alternatives for immunoassay development, in terms of both sensitivity and ease of preparation, since they do not require any orientation effort. PMID:27187470

[Biotechnological advances in monoclonal antibody therapy: the RANK ligand inhibitor antibody].

PubMed

Kiss, Emese; Kuluncsics, Zénó; Kiss, Zoltán; Poór, Gyula

2010-12-26

Biological drugs have been used since the middle of the last century in medicine. Nowadays we are witnesses of the intensive development and wider administration of these drugs in clinical practice. Around 250 biological drugs are available and more than 350 million patients have been treated since their marketed authorization. Among the biologics there are protein based macromolecules, which mass production can be performed with the help of biotechnology. This term referring to the use of living organisms for production of molecules, was introduced by the Hungarian engineer, Károly Ereky. The present review focuses on the research, production and development of monoclonal antibodies manufactured by biotechnology. Some steps of this development have changed our immunological knowledge and the outcome of several diseases. The development of antibodies was highly recognized by two Nobel prizes. Authors detail the structure and functions of immunoglobulins, and their development, including fully human monoclonal antibodies. The RANKL inhibitor denosumab, a fully human IgG2 monoclonal antibody belongs to this latter group and it is available for treatment of osteoporosis. Authors also summarize the basic process of bone metabolism and the benefits of RANK ligand inhibition.

Monoclonal antibodies bind identically to both spores and hyphae of Aspergillus fumigatus.

PubMed

Reijula, K E; Kurup, V P; Kumar, A; Fink, J N

1992-05-01

Immunoelectron microscopy (IEM) was used to determine the binding of six monoclonal antibodies (MoAbs) produced against Aspergillus fumigatus antigens present on or within the conidia and hyphae of the fungus. Antigen-antibody complexes were demonstrated in EM using labelled colloidal gold particles (15 nm). Three out of 6 MoAbs (C9, F12 and H10) reacted only with the cytoplasmic components of A. fumigatus while the remaining three (B12, F6G5 and D6E6) showed reactivity to both cytoplasm and cell wall of the conidia and hyphae. The results indicate that IEM is of considerable value in determining and selecting monoclonal antibodies having specific reactivity with diverse antigenic components.

Scintigraphy of normal mouse ovaries with monoclonal antibodies to ZP-2, the major zona pellucida protein

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

East, I.J.; Keenan, A.M.; Larson, S.M.

1984-08-31

The zona pellucida is an extracellular glycocalyx, made of three sulfated glycoproteins, that surrounds mammalian oocytes. Parenterally administered monoclonal antibodies specific for ZP-2, the most abundant zona protein, localize in the zona pellucida. When labeled with iodine-125, these monoclonal antibodies demonstrate a remarkably high target-to-nontarget tissue ratio and provide clear external radioimaging of ovarian tissue.

Cloning and Characterization of a Hybridoma Secreting a 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Specific Monoclonal Antibody and Recombinant F(ab)

PubMed Central

Wanczyk, Heather; Barker, Tolga; Rood, Debra; Zapata, Daniel I.; Howell, Amy R.; Richardson, Stewart K.; Zinckgraf, John; Marusov, Gregory P.; Lynes, Michael A.; Silbart, Lawrence K.

2013-01-01

Smokeless tobacco products have been associated with increased risks of oro-pharyngeal cancers, due in part to the presence of tobacco-specific nitrosamines (TSNAs) such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These potent carcinogens are formed during tobacco curing and as a result of direct nitrosation reactions that occur in the oral cavity. In the current work we describe the isolation and characterization of a hybridoma secreting a high-affinity, NNK-specific monoclonal antibody. A structurally-related benzoyl derivative was synthesized to facilitate coupling to NNK-carrier proteins, which were characterized for the presence of the N-nitroso group using the Griess reaction, and used to immunize BALB/c mice. Splenocytes from mice bearing NNK-specific antibodies were used to create hybridomas. Out of four, one was selected for subcloning and characterization. Approximately 99% of the monoclonal antibodies from this clone were competitively displaced from plate-bound NNKB conjugates in the presence of free NNK. The affinity of the monoclonal antibody to the NNKB conjugates was Kd = 2.93 nM as determined by surface plasmon resonance. Free nicotine was a poor competitor for the NNKB binding site. The heavy and light chain antibody F(ab) fragments were cloned, sequenced and inserted in tandem into an expression vector, with an FMDV Furin 2A cleavage site between them. Expression in HEK 293 cells revealed a functional F(ab) with similar binding features to that of the parent hybridoma. This study lays the groundwork for synthesizing transgenic tobacco that expresses carcinogen-sequestration properties, thereby rendering it less harmful to consumers. PMID:23518474

A monoclonal antibody for distinction of invasive and noninvasive clinical isolates of Entamoeba histolytica.

PubMed Central

Gonzalez-Ruiz, A; Haque, R; Rehman, T; Aguirre, A; Jaramillo, C; Castañon, G; Hall, A; Guhl, F; Ruiz-Palacios, G; Warhurst, D C

1992-01-01

Approximately 10% of the world population is infected with Entamoeba histolytica, but only 10% of the carriers develop symptomatic amebiasis. This discrepancy could be explained by the genotypic differences between the morphologically indistinguishable invasive and noninvasive strains of E. histolytica currently identified by zymodeme analysis, a technique that is unsuitable for routine diagnostic laboratories. Here we report the production of a monoclonal antibody against E. histolytica and its use in an immunofluorescence assay to identify invasive isolates cultured from stool samples of infected patients in several regions where amebiasis is endemic: Bangladesh, Colombia, and Mexico. After testing a total of 88 E. histolytica isolates, the correlation between zymodeme characterization and the immunofluorescence assay with the invasive isolate-specific monoclonal antibody was 100%. The epitope detected by the invasive isolate-specific monoclonal antibody resides in a previously undescribed internal protein with molecular masses of 84 and 81 kDa in axenic and polyxenic E. histolytica strains, respectively. Images PMID:1452651

Identification of a Vesicular-Arbuscular Mycorrhizal Fungus by Using Monoclonal Antibodies in an Enzyme-Linked Immunosorbent Assay †

PubMed Central

Wright, Sara F.; Morton, Joseph B.; Sworobuk, Janis E.

1987-01-01

Spore morphology is currently used to identify species of vesicular-arbuscular mycorrhizal fungi. We report the first use of a highly specific immunological method for identification of a vesicular-arbuscular mycorrhizal fungus. Two monoclonal antibodies were produced against Glomus occultum. Monoclonal antibodies reacted strongly with both spores and hyphae in an indirect enzyme-linked immunosorbent assay. All other mycorrhizal (29 species) and nonmycorrhizal (5 species) fungi tested were nonreactive with the monoclonal antibodies. A single spore of G. occultum was detectable in the presence of high numbers of spores of other vesicular-arbuscular mycorrhizal fungi. Variation in the reaction of G. occultum isolates from West Virginia, Florida, and Colombia suggests that monoclonal antibodies may differentiate strains. PMID:16347441

Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A

NASA Astrophysics Data System (ADS)

Leserman, Lee D.; Barbet, Jacques; Kourilsky, François; Weinstein, John N.

1980-12-01

Many applications envisioned for liposomes in cell biology and chemotherapy require their direction to specific cellular targets1-3. The ability to use antibody as a means of conferring specificity to liposomes would markedly increase their usefulness. We report here a method for covalently coupling soluble proteins, including monoclonal antibody and Staphylococcus aureus protein A (ref. 4), to small sonicated liposomes, by using the heterobifunctional cross-linking reagent N-hydroxysuccinimidyl 3-(2-pyridyldithio)propionate (SPDP, Pharmacia). Liposomes bearing covalently coupled mouse monoclonal antibody against human β2-microglobulin [antibody B1.1G6 (IgG2a, κ) (B. Malissen et al., in preparation)] bound specifically to human, but not to mouse cells. Liposomes bearing protein A became bound to human cells previously incubated with the B1.1G6 antibody, but not to cells incubated without antibody. The coupling method results in efficient binding of protein to the liposomes without aggregation and without denaturation of the coupled ligand; at least 60% of liposomes bound functional protein. Further, liposomes did not leak encapsulated carboxyfluorescein (CF) as a consequence of the reaction.

Preparation of monoclonal antibody of anti-feline calicivirus and establishment of double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

PubMed

Yuan, B; Ai, C-X; Yuan, L; Gao, W; Hu, J-P; Chen, J; Ren, W-Z

2014-09-12

This study aimed to prepare monoclonal antibody of feline calicivirus (FCV) and identify its basic biological characteristics. Saturated ammonium sulfate precipitation, combined differential centrifugation, and cesium chloride density gradient centrifugation were used for the purification of FCV. The purified FCV was used as antigen to immunize BALB/c mice. The hybridoma lines of anti-FCV monoclonal antibodies were established using cell fusion and hybridoma screening techniques. The subtypes of the monoclonal antibody were identified. The results showed that 3 strains of hybridoma cell lines stably secreted anti-FCV monoclonal antibody; they were named as D8, E5, and H4. The D8 and E5 were IgM subtype antibodies, and H4 was IgG2b subtype antibody. The monoclonal antibody obtained shared no cross-reactivity with feline parvovirus, canine parvovirus, and canine distemper virus. According to the different recognition sites of 2 monoclonal antibodies E5 and H4 to the FCV, they were used to coat microtiter plates and prepare 2 enzyme-labeled secondary antibodies to establish double-antibody sandwich enzyme-linked immunosorbent assay detecting method.

High-Sensitivity Monoclonal Antibodies Specific for Homoserine Lactones Protect Mice from Lethal Pseudomonas aeruginosa Infections

PubMed Central

Downham, Christina; Broadbent, Ian; Charlton, Keith; Porter, Andrew J.

2014-01-01

A number of bacteria, including pathogens like Pseudomonas aeruginosa, utilize homoserine lactones (HSLs) as quorum sensing (QS) signaling compounds and engage in cell-to-cell communication to coordinate their behavior. Blocking this bacterial communication may be an attractive strategy for infection control as QS takes a central role in P. aeruginosa biology. In this study, immunomodulation of HSL molecules by monoclonal antibodies (MAbs) was used as a novel approach to prevent P. aeruginosa infections and as tools to detect HSLs in bodily fluids as a possible first clue to an undiagnosed Gram-negative infection. Using sheep immunization and recombinant antibody technology, a panel of sheep-mouse chimeric MAbs were generated which recognized HSL compounds with high sensitivity (nanomolar range) and cross-reactivity. These MAbs retained their nanomolar sensitivity in complex matrices and were able to recognize HSLs in P. aeruginosa cultures grown in the presence of urine. In a nematode slow-killing assay, HSL MAbs significantly increased the survival of worms fed on the antibiotic-resistant strain PA058. The therapeutic benefit of these MAbs was further studied using a mouse model of Pseudomonas infection in which groups of mice treated with HSL-2 and HSL-4 MAbs survived, 7 days after pathogen challenge, in significantly greater numbers (83 and 67%, respectively) compared with the control groups. This body of work has provided early proof-of-concept data to demonstrate the potential of HSL-specific, monoclonal antibodies as theranostic clinical leads suitable for the diagnosis, prevention, and treatment of life-threatening bacterial infections. PMID:24185854

The Case for Adjunctive Monoclonal Antibody Immunotherapy in Schizophrenia.

PubMed

Miller, Brian J; Buckley, Peter F

2016-06-01

This article presents the case in favor of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia. Evidence for prenatal and premorbid immune risk factors for the development of schizophrenia in the offspring is highlighted. Then key evidence for immune dysfunction in patients with schizophrenia is considered. Next, previous trials of adjunctive anti-inflammatory or other immunotherapy in schizophrenia are discussed. Then evidence for psychosis as a side effect of immunotherapy for other disorders is discussed. Also presented is preliminary evidence for adjunctive monoclonal antibody immunotherapy in psychiatric disorders. Finally, important considerations in the design and implementation of clinical trials of adjunctive monoclonal antibody immunotherapy in schizophrenia are discussed. Copyright © 2016 Elsevier Inc. All rights reserved.

Large Scale Generation and Characterization of Anti-Human IgA Monoclonal Antibody in Ascitic Fluid of Balb/c Mice

PubMed Central

Ezzatifar, Fatemeh; Majidi, Jafar; Baradaran, Behzad; Aghebati Maleki, Leili; Abdolalizadeh, Jalal; Yousefi, Mehdi

2015-01-01

Purpose: Monoclonal antibodies are potentially powerful tools used in biomedical research, diagnosis, and treatment of infectious diseases and cancers. The monoclonal antibody against Human IgA can be used as a diagnostic application to detect infectious diseases. The aim of this study was to improve an appropriate protocol for large-scale production of mAbs against IgA. Methods: For large-scale production of the monoclonal antibody, hybridoma cells that produce monoclonal antibodies against Human IgA were injected intraperitoneally into Balb/c mice that were previously primed with 0.5 ml Pristane. After ten days, ascitic fluid was harvested from the peritoneum of each mouse. The ELISA method was carried out for evaluation of the titration of produced mAbs. The ascitic fluid was investigated in terms of class and subclass by a mouse mAb isotyping kit. MAb was purified from the ascitic fluid by ion exchange chromatography. The purity of the monoclonal antibody was confirmed by SDS-PAGE, and the purified monoclonal antibody was conjugated with HRP. Results: Monoclonal antibodies with high specificity and sensitivity against Human IgA were prepared by hybridoma technology. The subclass of antibody was IgG1 and its light chain was the kappa type. Conclusion: This conjugated monoclonal antibody could have applications in designing ELISA kits in order to diagnose different infectious diseases such as toxoplasmosis and H. Pylori. PMID:25789225

Development of a highly specific HER2 monoclonal antibody for immunohistochemistry using protein microarray chips

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

Qi, Lili; Zhou, Lixin; Lu, Mingmin

HER2 is an orphan receptor tyrosine kinase of the EGFR families and is considered to be a key tumor driver gene [1]. Breast cancer and gastric cancer with HER2 amplification can be effectively treated by its neutralizing antibody, Herceptin. In clinic, Immunohistochemistry (IHC) was used as the primary screening method to diagnose HER2 amplification [2]. However, recent evidence suggested that the frequently used rabbit HER2 antibody 4B5 cross reacted with another family member HER4 [3]. IHC staining with 4B5 also indicated that there was strong non-specific cytoplasmic and nuclear signals in normal gastric mucosal cells and some gastric cancer samples.more » Using a protein lysate array which covers 85% of the human proteome, we have confirmed that the 4B5 bound to HER4 and a nuclear protein ZSCAN18 besides HER2. The non-specific binding accounts for the unexpected cytoplasmic and nuclear staining of 4B5 of normal gastric epithelium. Finally, we have developed a novel mouse HER2 monoclonal antibody UMAB36 with similar sensitivity to 4B5 but only reacted to HER2 across the 17,000 proteins on the protein chip. In 129 breast cancer and 158 gastric cancer samples, UMAB36 showed 100% sensitivity and specificity comparing to the HER2 FISH reference results with no unspecific staining in the gastric mucosa layer. Therefore, UMAB36 could provide as an alternative highly specific IHC reagent for testing HER2 amplification in gastric cancer populations. - Highlights: • HER2 antibody 4B5 cross-interacts with HER4 and ZSCAN18, which would interfere with the accuracy of IHC diagnosis of HER2. • A HER2 antibody UMAB36 has been developed with high sensitivity and specificity and can be utilized in HER2 diagnosis. • Protein lysate array is a novel strategy to screen for highly specific antibody.« less

A survey of manufacturing and handling practices for monoclonal antibodies by pharmacy, nursing and medical personnel.

PubMed

Alexander, M; King, J; Lingaratnam, S; Byrne, J; MacMillan, K; Mollo, A; Kirsa, S; Green, M

2016-04-01

There is a paucity of data available to assess the occupational health and safety risk associated with exposure to monoclonal antibodies. Industry standards and published guidelines are conflicting or outdated. Guidelines offer contrary recommendations based on an array of methodological approaches. This survey aimed to describe current practices, beliefs and attitudes relating to the handling of monoclonal antibodies by Australian medical, nursing and pharmacy clinicians. An electronic survey was distributed between June and September 2013. Respondents were surveyed on three focus areas: institutional guideline availability and content, current practices and attitudes. Demographic data relating to respondent and primary place of practice were also collected. A total of 222 clinicians completed the survey, with representation from all targeted professional groups and from a variety of geographic locations. 92% of respondents reported that their institution prepared or administered monoclonal antibodies, with 87% specifically handling anti-cancer monoclonal antibodies. Monoclonal antibodies were mostly prepared onsite (84-90%) and mostly within pharmacy clean-rooms (75%) and using cytotoxic cabinets (61%). 43% of respondents reported access to institutional monoclonal antibody handling guidelines with risk reduction strategies including training and education (71%), spill and waste management (71%), procedures for transportation (57%) and restricted handling (50%). Nurses had a stronger preference towards pharmacy manufacturing than both doctors and pharmacists for a range of clinical scenarios. 95% of all respondents identified that professional or regulatory body guidelines are an important resource when considering handling practices. Monoclonal antibodies are most commonly handled according to cytotoxic drug standards and often in the absence of formal guidelines. © The Author(s) 2014.

Generation and Characterization of Anti-CD34 Monoclonal Antibodies that React with Hematopoietic Stem Cells

PubMed Central

Aghebati Maleki, Leili; Majidi, Jafar; Baradaran, Behzad; Movassaghpour, Aliakbar; Abdolalizadeh, Jalal

2014-01-01

CD34 is a type I membrane protein with a molecular mass of approximately 110 kDa. This antigen is associated with human hematopoietic progenitor cells and is a differentiation stage-specific leukocyte antigen. In this study we have generated and characterized monoclonal antibodies (mAbs) directed against a CD34 marker. Mice were immunized with two keyhole lympet hemocyanin (KLH)-conjugated CD34 peptides. Fused cells were grown in hypoxanthine, aminopterine and thymidine (HAT) selective medium and cloned by the limiting dilution (L.D) method. Several monoclones were isolated by three rounds of limited dilutions. From these, we chose stable clones that presented sustained antibody production for subsequent characterization. Antibodies were tested for their reactivity and specificity to recognize the CD34 peptides and further screened by enzyme-linked immunosorbent assay (ELISA) and Western blotting analyses. One of the mAbs (3D5) was strongly reactive against the CD34 peptide and with native CD34 from human umbilical cord blood cells (UCB) in ELISA and Western blotting analyses. The results have shown that this antibody is highly specific and functional in biomedical applications such as ELISA and Western blot assays. This monoclonal antibodies (mAb) can be a useful tool for isolation and purification of human hematopoietic stem cells (HSCs). PMID:24611141

An Immunoglobulin G1 Monoclonal Antibody Highly Specific to the Wall of Cryptosporidium Oocysts

PubMed Central

Weir, C.; Vesey, G.; Slade, M.; Ferrari, B.; Veal, D. A.; Williams, K.

2000-01-01

The detection of Cryptosporidium oocysts in drinking water is critically dependent on the quality of immunofluorescent reagents. Experiments were performed to develop a method for producing highly specific antibodies to Cryptosporidium oocysts that can be used for water testing. BALB/c mice were immunized with six different antigen preparations and monitored for immunoglobulin G (IgG) and IgM responses to the surface of Cryptosporidium oocysts. One group of mice received purified oocyst walls, a second group received a soluble protein preparation extracted from the outside of the oocyst wall, and the third group received whole inactivated oocysts. Three additional groups were immunized with sequentially prepared oocyst extracts to provide for a comparison of the immune response. Mice injected with the soluble protein extract demonstrated an IgG response to oocysts surface that was not seen in the whole-oocyst group. Mice injected with whole oocysts showed an IgM response only, while mice injected with purified oocyst walls showed little increase in IgM or IgG levels. Of the additional reported preparations only one, BME (2-mercaptoethanol treated), produced a weak IgM response to the oocyst wall. A mouse from the soluble oocyst extract group yielding a high IgG response was utilized to produce a highly specific IgG1 monoclonal antibody (Cry104) specific to the oocyst surface. Comparative flow cytometric analysis indicated that Cry104 has a higher avidity and specificity to oocysts in water concentrates than other commercially available antibodies. PMID:10973448

Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis.

PubMed

Wootla, Bharath; Watzlawik, Jens O; Stavropoulos, Nikolaos; Wittenberg, Nathan J; Dasari, Harika; Abdelrahim, Murtada A; Henley, John R; Oh, Sang-Hyun; Warrington, Arthur E; Rodriguez, Moses

2016-06-01

Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside.

Monoclonal antibody to a cancer-specific and drug-responsive hydroquinone (NADH) oxidase from the sera of cancer patients

NASA Technical Reports Server (NTRS)

Cho, NaMi; Chueh, Pin-Ju; Kim, Chinpal; Caldwell, Sara; Morre, Dorothy M.; Morre, D. James

2002-01-01

Monoclonal antibodies were generated in mice to a 34-kDa circulating form of a drug-responsive hydroquinone (NADH) oxidase with a protein disulfide-thiol interchange activity specific to the surface of cancer cells and the sera of cancer patients. Screening used Western blots with purified 34-kDa tNOX from HeLa cells and the sera of cancer patients. Epitopes were sought that inhibited the drug-responsive oxidation of NADH with the sera of cancer patients, but which had no effect on NADH oxidation with the sera of healthy volunteers. Two such antisera were generated. One, designated monoclonal antibody (mAb) 12.1, was characterized extensively. The NADH oxidase activity inhibited by mAb 12.1 also was inhibited by the quinone site inhibitor capsaicin (8-methyl- N-vanillyl-6-noneamide). The inhibition was competitive for the drug-responsive protein disulfide-thiol interchange activity assayed either by restoration of activity to scrambled RNase or by cleavage of a dithiodipyridine substrate, and was uncompetitive for NADH oxidation. Both the mAb 12.1 and the postimmune antisera immunoprecipitated drug-responsive NOX activity and identified the same 34-kDa tNOX protein in the sera of cancer patients that was absent from sera of healthy volunteers, and was utilized as immunogen. Preimmune sera from the same mouse as the postimmune antisera was without effect. Both mouse ascites containing mAb 12.1 and postimmune sera (but not preimmune sera) slowed the growth of human cancer cell lines in culture, but did not affect the growth of non-cancerous cell lines. Immunocytochemical and histochemical findings showed that mAb 12.1 reacted with the surface membranes of human carcinoma cells and tissues.

Palladium-109 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

The invention consists of new monoclonal antibodies labelled with Palladium 109, a beta-emitting radionuclide, the method of preparing this material, and its use in the radiotherapy of melanoma. The antibodies are chelate-conjugated and demonstrate a high uptake in melanomas. (ACR)

Monoclonal Antibody Fragments for Targeting Therapeutics to Growth Plate Cartilage | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at The Eunice Kennedy Shriver National Institute on Child Health and Human Development (NICHD) have discovered monoclonal antibodies that bind to matrilin-3, a protein specifically expressed in cartilage tissue, that could be used for treating or inhibiting growth plate disorders, such as a skeletal dysplasia or short stature. The monoclonal antibodies can also be used to target therapeutic agents, such as anti-arthritis agents, to cartilage tissue. NICHD seeks statements of capability or interest from parties interested in collaborative research to co-develop, evaluate, or commercialize treatment of skeletal disorders using targeting antibodies.

[Preparation and application of monoclonal antibodies against DR region of Na+-K+-ATPase α1 subunit].

PubMed

Yan, Xiaofei; Wu, Litao; DU, Xiaojuan; Li, Jing; Zhang, Fujun; Han, Yan; Lyu, Shemin; Li, Dongmin

2016-12-01

Objective To prepare monoclonal antibodies against DR region (897DVEDSYGQQWTYEQR911) of Na + -K + -ATPase α1 subunit and identify their properties. Methods BALB/c mice were immunized with DR-keyholelimpet hemocyanin (KLH). Splenocytes from the immunized mice were collected and subsequently fused with SP2/0 mouse myeloma cells. Positive hybridoma clones were obtained after cell fusion and selection. ELISA was used to detect DR antibody titer in the cell supernatants. DR region-specific monoclonal antibodies were analyzed by dot blotting, Western blotting and immunofluorescence assay. Na + -K + -ATPase activity was detected by SensoLyte R FDP Protein Phosphatase Assay Kit and the protective effect of the monoclonal antibody against high glucose-induced cell injury was assessed in H9c2 cells. Results Three hybridoma cell lines which secreted stable DR monoclonal antibody were obtained. The strongest positive cell line, named DRm217, was selected to prepare ascites. Dot blotting, Western blotting and immunofluorescence assay showed that DRm217 recognized specially DR region of Na + -K + -ATPase and bound on H9c2 cell membranes. DRm217 stimulated Na + -K + -ATPase activity and alleviated high glucose-induced H9c2 cells injury. Conclusion The monoclonal antibodies against DR region of Na + -K + -ATPase α1 subunit is prepared.

[Construction of human phage antibody library and screening for human monoclonal antibodies of amylin].

PubMed

Gong, Qian; Li, Chang-ying; Chang, Ji-wu; Zhu, Tie-hong

2012-06-01

To screen monoclonal antibodies to amylin from a constructed human phage antibody library and identify their antigenic specificity and combining activities. The heavy chain Fd fragment and light chain of human immunoglobulin genes were amplified from peripheral blood lymphocytes of healthy donors using RT-PCR, and then inserted into phagemid pComb3XSS to generate a human phage antibody library. The insertion of light chain or heavy chain Fd genes were identified by PCR after the digestion of Sac I, Xba I, Xho Iand Spe I. One of positive clones was analyzed by DNA sequencing. The specific anti-amylin clones were screened from antibody library against human amylin antigens and then the positive clones were determined by Phage-ELISA analysis. A Fab phage antibody library with 0.8×10(8); members was constructed with the efficacy of about 70%. DNA sequence analysis indicated V(H); gene belonged to V(H);3 gene family and V(λ); gene belonged to the V(λ); gene family. Using human amylin as panning antigen, specific anti-amylin Fab antibodies were enriched by screening the library for three times. Phage-ELISA assay showed the positive clones had very good specificity to amylin antigen. The successful construction of a phage antibody library and the identification of anti-amylin Fab antibodies provide a basis for further study and preparation of human anti-amylin antibodies.

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model.

PubMed

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Rajendran, Madhusudan; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R; García-Sastre, Adolfo; Basler, Christopher F; Munoz-Fontela, Cesar; Krammer, Florian

2017-08-15

Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2 -/- mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro , suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies, including two

Novel Cross-Reactive Monoclonal Antibodies against Ebolavirus Glycoproteins Show Protection in a Murine Challenge Model

PubMed Central

Duehr, James; Wohlbold, Teddy John; Oestereich, Lisa; Chromikova, Veronika; Amanat, Fatima; Gomez-Medina, Sergio; Mena, Ignacio; tenOever, Benjamin R.; García-Sastre, Adolfo; Basler, Christopher F.

2017-01-01

ABSTRACT Out of an estimated 31,100 cases since their discovery in 1976, ebolaviruses have caused approximately 13,000 deaths. The vast majority (∼11,000) of these occurred during the 2013-2016 West African epidemic. Three out of five species in the genus are known to cause Ebola Virus Disease in humans. Several monoclonal antibodies against the ebolavirus glycoprotein are currently in development as therapeutics. However, there is still a paucity of monoclonal antibodies that can cross-react between the glycoproteins of different ebolavirus species, and the mechanism of these monoclonal antibody therapeutics is still not understood in detail. Here, we generated a panel of eight murine monoclonal antibodies (MAbs) utilizing a prime-boost vaccination regimen with a Zaire ebolavirus glycoprotein expression plasmid followed by infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. We tested the binding breadth of the resulting monoclonal antibodies using a set of recombinant surface glycoproteins from Reston, Taï Forest, Bundibugyo, Zaire, Sudan, and Marburg viruses and found two antibodies that showed pan-ebolavirus binding. An in vivo Stat2−/− mouse model was utilized to test the ability of these MAbs to protect from infection with a vesicular stomatitis virus expressing the Zaire ebolavirus glycoprotein. Several of our antibodies, including the broadly binding ones, protected mice from mortality despite lacking neutralization capability in vitro, suggesting their protection may be mediated by Fc-FcR interactions. Indeed, three antibodies displayed cellular phagocytosis and/or antibody-dependent cell-mediated cytotoxicity in vitro. Our antibodies, specifically the two identified cross-reactive monoclonal antibodies (KL-2E5 and KL-2H7), might add to the understanding of anti-ebolavirus humoral immunity. IMPORTANCE This study describes the generation of a panel of novel anti-ebolavirus glycoprotein monoclonal antibodies

Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq.

PubMed

Busby, Michele; Xue, Catherine; Li, Catherine; Farjoun, Yossi; Gienger, Elizabeth; Yofe, Ido; Gladden, Adrianne; Epstein, Charles B; Cornett, Evan M; Rothbart, Scott B; Nusbaum, Chad; Goren, Alon

2016-01-01

The robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: They are non-renewable, vary in performance between lots and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells. Overall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody. Altogether, we found that monoclonal antibodies as a class perform equivalently to polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

Mouse monoclonal antibodies against human c-Mpl and characterization for flow cytometry applications.

PubMed

Abbott, Christina; Huang, Guo; Ellison, Aaron R; Chen, Ching; Arora, Taruna; Szilvassy, Stephen J; Wei, Ping

2010-04-01

Mouse monoclonal antibodies (MAbs) against human c-Mpl, the cognate receptor for thrombopoietin (TPO), were generated using hybridoma technology and characterized by various assays to demonstrate their specificity and affinity. Two such MAbs, 1.6 and 1.75, were determined to be superior for flow cytometry studies and exhibited double-digit picomolar (pM) affinities to soluble human c-Mpl protein. Both MAbs specifically bound to cells engineered to overexpress human c-Mpl protein, immortalized human hematopoietic cell lines that express endogenous c-Mpl, primary human bone marrow and peripheral blood-derived CD34(+) cells, and purified human platelets. No binding was detected on cell lines that did not express c-Mpl. Receptor competition and siRNA knock-down studies further confirmed the specificity of antibodies 1.6 and 1.75 for human c-Mpl. In contrast to these newly generated MAbs, none of eight commercially available anti-c-Mpl antibodies tested were found to bind specifically to human c-Mpl and were thus shown to be unsuitable for flow cytometry studies. Monoclonal antibodies 1.6 and 1.75 will therefore be useful flow cytometry reagents to detect cell surface c-Mpl expression.

Improved monoclonal antibodies to halodeoxyuridine

DOEpatents

Vanderlaan, M.; Dolbeare, F.A.; Gray, J.W.; Thomas, C.B.

1983-10-18

The development, method of production, characterization and methods of use of two hybridomas, CIdU-1 (ATCC Accession No. HB-8321) and CIdU-2 (ATCC Accession No. HB-8320), are described. These secrete IgG/sub 1/(K) immunoglobulins that react with halodeoxyuridine (HdU or halodU) such as bromo, chloro, fluoro and iodo deoxyuridine (BrdU, CldU, FdU and IdU), whether these are free in solution or incorporated into single stranded DNA in whole cells. The antibodies do not react with naturally occurring free nucleic acids or with deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) polymers. These antibodies are suitable for use in enzyme immunoassays for free CldU, FdU, IdU and BrdU and for detecting cells with these nucleotides incorporated into them. The monoclonal antibodies are useful in the detection of the sensitivity of tumor cells to specific chemotherapeutic agents, in the measurement of the rate of cellular DNA synthesis, in the measurement of the rate of proliferation of normal and malignant cells and in the detection of HPRT deficiency in cells. 1 tab.

Altered antigenicity of human monoclonal antibodies derived from human-mouse heterohybridomas.

PubMed

Kan-Mitchell, J; Andrews, K L; Gallardo, D; Mitchell, M S

1987-04-01

We have generated milligram quantities of human monoclonal antibodies (Hu-MAbs) in the ascites of pristane-primed nude mice injected with human-mouse heterohybridomas. After contaminating mouse immunoglobulins were removed by affinity chromatography, an enzyme immunosorbent assay (EIA) was used to measure the concentrations of human immunoglobulins. Ten different partially purified preparations were tested. The titration curves with all 5 IgG Hu-MAbs were unusual, reaching a plateau at a very low apparent maximum concentration of antibody. In contrast, the EIA yielded more usual titration curves and thus apparently more reliable estimates of the concentrations of 4 IgM and 1 IgA monoclonal antibodies. An analogous EIA for the quantitation of mouse IgG monoclonal antibodies also gave accurate estimates. To understand the nature of the discrepancy with human IgG, 5 Hu-MAbs of the 3 classes (2 IgG, 2 pentameric IgM and 1 IgA) were purified to homogeneity for a more detailed analysis. The inability to quantitate the human IgG monoclonal antibodies by EIA was not due to defective molecules, as shown by SDS polyacrylamide gel electrophoresis. The human IgG monoclonal antibodies were found to consist of intact heavy and light chains, as were the IgM and IgA antibodies. The possibility that the human IgG monoclonal antibodies differed antigenically from polyclonal IgG was explored by comparing the concentrations by EIA with the protein concentrations determined by absorbance at 280 nm. This analysis permitted a comparison of the detectability of antigenic determinants on Hu-MAbs with those on polyclonal Ig with goat antibodies to Ig or Ig subclass. The IgG monoclonal antibodies differed from polyclonal IgG in both their heavy and light chains. Goat antiserum monospecific for the gamma chain in fact underestimated the concentration by as much as one hundred-fold. IgM and IgA monoclonal antibodies were less antigenically distinct from their polyclonal counterparts even

Bivalent monoclonal IgY antibody formats by conversion of recombinant antibody fragments.

PubMed

Greunke, Kerstin; Spillner, Edzard; Braren, Ingke; Seismann, Henning; Kainz, Sabine; Hahn, Ulrich; Grunwald, Thomas; Bredehorst, Reinhard

2006-07-13

Monoclonal IgY have the potential to become unique tools for diagnostic research and therapeutic purposes since avian antibodies provide several advantages due to their phylogenetic difference when compared to mammalian antibodies. The mechanism of avian immunoglobulin gene diversification renders chicken an excellent source for the generation of recombinant scFv as well as Fab antibody libraries of high diversity. One major limitation of these antibody fragments, however, is their monovalent format, impairing the functional affinity of the molecules and, thereby, their applicability in prevalent laboratory methods. In this study, we generated vectors for conversion of avian recombinant antibody fragments into different types of bivalent IgY antibody formats. To combine the properties of established mammalian monoclonal antibodies with those of IgY constant domains, we additionally generated bivalent murine/avian chimeric antibody constructs. When expressed in HEK-293 cells, all constructs yielded bivalent disulfide-linked antibodies, which exhibit a glycosylation pattern similar to that of native IgY as assessed by lectin blot analysis. After purification by one step procedures, the chimeric and the entire avian bivalent antibody formats were analyzed for antigen binding and interaction with secondary reagents. The data demonstrate that all antibody formats provide comparable antigen binding characteristics and the well established properties of avian constant domains.

Precipitation of a monoclonal antibody by soluble tungsten.

PubMed

Bee, Jared S; Nelson, Stephanie A; Freund, Erwin; Carpenter, John F; Randolph, Theodore W

2009-09-01

Tungsten microparticles may be introduced into some pre-filled syringes during the creation of the needle hole. In turn, these microcontaminants may interact with protein therapeutics to produce visible particles. We found that soluble tungsten polyanions formed in acidic buffer below pH 6.0 can precipitate a monoclonal antibody within seconds. Soluble tungsten in pH 5.0 buffer at about 3 ppm was enough to cause precipitation of a mAb formulated at 0.02 mg/mL. The secondary structure of the protein was near-native in the collected precipitate. Our observations are consistent with the coagulation of a monoclonal antibody by tungsten polyanions. Tungsten-induced precipitation should only be a concern for proteins formulated below about pH 6.0 since tungsten polyanions are not formed at higher pHs. We speculate that the heterogenous nature of particle contamination within the poorly mixed syringe tip volume could mean that a specification for tungsten contamination based on the entire syringe volume is not appropriate. The potential potency of tungsten metal contamination is highlighted by the small number of particles that would be required to generate soluble tungsten levels needed to coagulate this antibody at pH 5.0.

Precipitation of a Monoclonal Antibody by Soluble Tungsten

PubMed Central

Bee, Jared S.; Nelson, Stephanie A.; Freund, Erwin; Carpenter, John F.; Randolph, Theodore W.

2009-01-01

Tungsten microparticles may be introduced into some pre-filled syringes during the creation of the needle hole. In turn, these microcontaminants may interact with protein therapeutics to produce visible particles. We found that soluble tungsten polyanions formed in acidic buffer below pH 6.0 can precipitate a monoclonal antibody within seconds. Soluble tungsten in pH 5.0 buffer at about 3 ppm was enough to cause precipitation of a mAb formulated at 0.02 mg/mL. The secondary structure of the protein was near-native in the collected precipitate. Our observations are consistent with the coagulation of a monoclonal antibody by tungsten polyanions. Tungsten-induced precipitation should only be a concern for proteins formulated below about pH 6.0 since tungsten polyanions are not formed at higher pHs. We speculate that the heterogenous nature of particle contamination within the poorly mixed syringe tip volume could mean that a specification for tungsten contamination based on the entire syringe volume is not appropriate. The potential potency of tungsten metal contamination is highlighted by the small number of particles that would be required to generate soluble tungsten levels needed to coagulate this antibody at pH 5.0. PMID:19230018

Pathogenic and Nonpathogenic Strains of Entamoeba histolytica can be Differentiated by Monoclonal Antibodies to the Galactose-Specific Adherence Lectin

DTIC Science & Technology

1991-04-01

AD- A235 913 DEVELOPMENT Ei ENGINEERING CENTER CRDEC-TR-268 PATHOGENIC AND NONPATHOGENIC STRAINS OF ENTAMOEBA HISTOLYTICA CAN BE DIFFERENTIATED BY...Pathogenic and Nonpathogenic Strains of Entamoeba Histolytica can be Differentiated by Monoclonal PR-IFJlX2XXRPEW Antibodies to the Galactose-Specific...galactose lectin produced by Entamoeba histolytica provide the basis for development of a model system for the environmental detection of adherence and

Using Monoclonal Antibodies to Prevent Mucosal Transmission of Epidemic Infectious Diseases

PubMed Central

Zeitlin, Larry; Cone, Richard A.

1999-01-01

Passive immunization with antibodies has been shown to prevent a wide variety of diseases. Recent advances in monoclonal antibody technology are enabling the development of new methods for passive immunization of mucosal surfaces. Human monoclonal antibodies, produced rapidly, inexpensively, and in large quantities, may help prevent respiratory, diarrheal, and sexually transmitted diseases on a public health scale. PMID:10081672

Recent Advances in Monoclonal Antibody Therapies for Multiple Sclerosis

PubMed Central

Stavropoulos, Nikolaos; Wittenberg, Nathan J.; Dasari, Harika; Abdelrahim, Murtada A.; Henley, John R.; Oh, Sang-Hyun; Warrington, Arthur E.; Rodriguez, Moses

2016-01-01

Introduction Multiple sclerosis (MS) is the most common chronic inflammatory, demyelinating disease of the CNS and results in neurological disability. Existing immunomodulatory and immunosuppressive approaches lower the number of relapses but do not cure or reverse existing deficits nor improve long-term disability in MS patients. Areas Covered Monogenic antibodies were described as treatment options for MS, however the immunogenicity of mouse antibodies hampered the efficacy of potential therapeutics in humans. Availability of improved antibody production technologies resulted in a paradigm shift in MS treatment strategies. In this review, an overview of immunotherapies for MS that use conventional monoclonal antibodies reactive to immune system and their properties and mechanisms of action will be discussed, including recent advances in MS therapeutics and highlight natural autoantibodies (NAbs) that directly target CNS cells. Expert Opinion Recent challenges for MS therapy are the identification of relevant molecular and cellular targets, time frame of treatment, and antibody toxicity profiles to identify safe treatment options for MS patients. The application of monoclonal antibody therapies with better biological efficacy associated with minimum side effects possesses huge clinical potential. Advances in monoclonal antibody technologies that directly target cells of nervous system may promote the CNS regeneration field from bench to bedside. PMID:26914737

Influence of pH on heat-induced aggregation and degradation of therapeutic monoclonal antibodies.

PubMed

Ishikawa, Tomoyoshi; Ito, Takahiko; Endo, Ryosuke; Nakagawa, Keiko; Sawa, Eiji; Wakamatsu, Kaori

2010-01-01

Monoclonal antibodies are widely used for the treatment of various diseases, and because therapeutic monoclonal antibodies are stored in an aqueous solution or in a lyophilized state, the preparation of a stabilizing formulation that prevents their deterioration (degradation and aggregation) is crucial. Given the structural similarities of the immunoglobulin G (IgG) framework regions and a diversity of only four subclasses, we aimed to find common conditions that stabilize many different antibodies. In this study, we analyzed the effect of pH (the most critical factor in establishing a stable formulation) on human monoclonal antibodies from subclasses IgG1, IgG2, and IgG4, all of which have been utilized in antibody therapeutics. We found that human IgGs are stable with minimal heat-induced degradation and aggregation at pH 5.0-5.5 irrespective of their subclass. We also found that IgG1 is more susceptible to fragmentation, whereas IgG4 is more susceptible to aggregation. This basic information emphasizing the influence of pH on IgG stability should facilitate the optimization of formulation conditions tailored to individual antibodies for specific uses.

PMab-52: Specific and Sensitive Monoclonal Antibody Against Cat Podoplanin for Immunohistochemistry.

PubMed

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Harada, Hiroyuki; Kagawa, Yumiko; Ichii, Osamu; Konnai, Satoru; Kaneko, Mika K; Kato, Yukinari

2017-10-01

Podoplanin (PDPN) is expressed in several normal tissues, such as lymphatic endothelial cells, podocytes of renal glomerulus, and type I alveolar cells of lung. PDPN activates platelet aggregation by binding to C-type lectin-like receptor-2 (CLEC-2) on platelet. Although monoclonal antibodies (mAbs) against human PDPN, mouse PDPN, rat PDPN, rabbit PDPN, dog PDPN, and bovine PDPN have been established, anticat PDPN (cPDPN) mAbs have not been developed. In this study, we immunized mice with Chinese hamster ovary (CHO)-K1 cell lines expressing cPDPN, and developed anti-cPDPN mAbs. One of the clones, PMab-52 (IgM, kappa), detected cPDPN specifically in flow cytometry and Western blot analysis. PMab-52 is also useful for detecting feline squamous cell carcinoma cells in immunohistochemical analysis. PMab-52 is expected to be useful for investigating the function of cPDPN in feline carcinomas.

Immunochemical identification of insect hemocyte populations: monoclonal antibodies distinguish four major hemocyte types in manduca sexta.

PubMed

Willott, E; Trenczek, T; Thrower, L W; Kanost, M R

1994-12-01

We have made 140 monoclonal antibodies to hemocytes (insect blood cells) from Manduca sexta. Four of these antibodies, when used in immunofluorescent microscopy of fixed hemocytes, distinguish the four main morphologically distinct hemocyte types. Plasmatocytes, granular cells, and oenocytoids are each recognized by a unique antibody specific to that type; spherulocytes are recognized by an antibody that also binds to plasmatocytes. When used in flow cytometry with nonfixed hemocytes, three of the four antibodies bind their respective cells; the oenocytoid marker failed to bind to any hemocytes. This set of four monoclonal antibodies may be useful for labeling individual cell types and for separating the different hemocyte types for further study of hemocyte functions.

The detection of hemorrhagic proteins in snake venoms using monoclonal antibodies against Virginia opossum (Didelphis virginiana) serum.

PubMed

Sánchez, E E; García, C; Pérez, J C; De La Zerda, S J

1998-10-01

Most snakes and a few warm-blooded animals have a resistance to snake venoms because of naturally occurring antihemorrhagins found in their sera. The antihemorrhagins in serum of Virginia opossum (Didelphis virginiana) neutralize hemorrhagic activity by binding to hemorrhagins in snake venoms. The binding characteristic of antihemorrhagins in D. virginiana serum was used to develop a five-step western blot. The detection of hemorrhagic proteins were measured indirectly with antihemorrhagins in Virginia opossum serum and with DV-2LD#2, a monoclonal antibody specific for Virginia opossum antihemorrhagins. Snake venoms were separated by native-PAGE, transferred to a Millipore Immobilon-P membrane and then incubated with crude Virginia opossum serum. The hemorrhagins in snake venom bind to antihemorrhagins in Virginia opossum serum which react with DV-2LD#2 a monoclonal antibody that is specific for Virginia opossum antihemorrhagins. DV-2LD#2 monoclonal antibody inhibits antihemorrhagic activity in Virginia opossum serum when mixed in equal amounts. The inhibition of antihemorrhagins by DV-2LD#2 monoclonal antibody suggests specificity. DV-2LD#2 monoclonal antibody does not recognize antihemorrhagins in gray woodrat (Neotoma micropus) serum. The five-step western blot reveals two well-defined bands which represent hemorrhagins found in Western diamondback rattlesnake (Crotalus atrox) venom. Venoms from 15 different snake species were examined to determine the usefulness of the five-step western blot. Other hemorrhagic venoms (Great Basin rattlesnake (C. viridis lutosus), Prairie rattlesnake (C. viridis viridis), Tancitaran dusky rattlesnake (C. pusillus), Northern Mojave rattlesnake (C. scutulatus scutulatus type B) and Northern Pacific rattlesnake (C. v. oreganus)) had one single band in the five-step western blot. DV-2LD#2 did not bind to the non-hemorrhagic venoms and reacted with 50% of the hemorrhagic venoms used in this study. The monoclonal antibody, CAH

Monoclonal Antibody Analysis of Keratin Expression in the Central Nervous System

NASA Astrophysics Data System (ADS)

Franko, Maryellen C.; Gibbs, Clarence J.; Rhoades, Dorothy A.; Carleton Gajdusek, D.

1987-05-01

A monoclonal antibody directed against a 65-kDa brain protein demonstrates an epitope found in keratin from human epidermis. By indirect immunofluorescence, the antibody decorates intracytoplasmic filaments in a subclass of astrocytes and Purkinje cells of adult hamster brain. Double-label immunofluorescence study using antibody to glial fibrillary acidic protein and this antibody reveals the 65-kDa protein to be closely associated with glial filaments in astrocytes of fetal mouse brain cultures. Immunoblot analysis of purified human epidermal keratin and hamster brain homogenate confirms the reactivity of this antibody to epidermal keratin polypeptides. All the major epidermal keratins were recognized by this antibody. It did not bind to the remaining major intermediate filament proteins. These findings suggest that monoclonal antibody 34C9 recognizes a cytoskeletal structure connected with intermediate filaments. In addition, the monoclonal antibody demonstrates that epidermal keratins share an epitope not only among themselves but also with a ``neural keratin.''

Next generation and biosimilar monoclonal antibodies

PubMed Central

2011-01-01

The Next Generation and Biosimilar Monoclonal Antibodies: Essential Considerations Towards Regulatory Acceptance in Europe workshop, organized by the European Centre of Regulatory Affairs Freiburg (EUCRAF), was held February 3–4, 2011 in Freiburg, Germany. The workshop attracted over 100 attendees from 15 countries, including regulators from 11 agencies, who interacted over the course of two days. The speakers presented their authoritative views on monoclonal antibodies (mAbs) as attractive targets for development, the experience to date with the regulatory process for biosimilar medicinal products, the European Medicines Agency draft guideline on biosimilar mAbs, as well as key elements in the development of mAbs. Participants engaged in many lively discussions, and much speculation on the nature of the quality, non-clinical and clinical requirements for authorization of biosimilar mAbs. PMID:21487235

Imaging of myocardial infarction in dogs and humans using monoclonal antibodies specific for human myosin heavy chains

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

Leger, J.; Chevalier, J.; Larue, C.

1991-08-01

The use of three different monoclonal antibodies specific for human ventricular myosin heavy chains in the visualization of the location and extent of necrosis in dogs with experimental acute myocardial infarction and in humans is described. Using a classic immunohistochemical method or ex vivo analysis of heart slices in dogs with acute myocardial infarction subjected to intravenous injection of unlabeled antimyosin antibodies or antimyosin antibodies labeled with indium-111, it was observed that all antibody fragments specifically reached the targeted necrotic zone less than 2 h after antibody injection and remained bound for up to 24 h. In a limited butmore » significant number of cases (5 of the 12 humans and 11 of 43 dogs), it was possible to image the necrotic zone in vivo as early as 2 to 4 h after antibody injection. In other cases, individual blood clearance variations retarded or even prevented in vivo necrosis detection. Higher antimyosin fixation values were obtained in the necrotic zones in dogs with a rapid blood clearance relative to that of the other dogs. It is concluded that antimyosin antibodies always reached necrotic areas within 2 h. If blood clearance was rapid, in vivo imaging of the necrotic area was possible 2 to 6 h after necrosis, even in humans. In some cases, however, uncontrolled individual variations in the timing required for sufficient blood clearance hampered this rapid in vivo detection of myocardial necrosis.« less

The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line

NASA Technical Reports Server (NTRS)

Smiley, S. A.; Gillock, E. T.; Black, M. C.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1997-01-01

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Novel monoclonal antibodies to study tissue regeneration in planarians.

PubMed

Ross, Kelly G; Omuro, Kerilyn C; Taylor, Matthew R; Munday, Roma K; Hubert, Amy; King, Ryan S; Zayas, Ricardo M

2015-01-21

Planarians are an attractive model organism for studying stem cell-based regeneration due to their ability to replace all of their tissues from a population of adult stem cells. The molecular toolkit for planarian studies currently includes the ability to study gene function using RNA interference (RNAi) and observe gene expression via in situ hybridizations. However, there are few antibodies available to visualize protein expression, which would greatly enhance analysis of RNAi experiments as well as allow further characterization of planarian cell populations using immunocytochemistry and other immunological techniques. Thus, additional, easy-to-use, and widely available monoclonal antibodies would be advantageous to study regeneration in planarians. We have created seven monoclonal antibodies by inoculating mice with formaldehyde-fixed cells isolated from dissociated 3-day regeneration blastemas. These monoclonal antibodies can be used to label muscle fibers, axonal projections in the central and peripheral nervous systems, two populations of intestinal cells, ciliated cells, a subset of neoblast progeny, and discrete cells within the central nervous system as well as the regeneration blastema. We have tested these antibodies using eight variations of a formaldehyde-based fixation protocol and determined reliable protocols for immunolabeling whole planarians with each antibody. We found that labeling efficiency for each antibody varies greatly depending on the addition or removal of tissue processing steps that are used for in situ hybridization or immunolabeling techniques. Our experiments show that a subset of the antibodies can be used alongside markers commonly used in planarian research, including anti-SYNAPSIN and anti-SMEDWI, or following whole-mount in situ hybridization experiments. The monoclonal antibodies described in this paper will be a valuable resource for planarian research. These antibodies have the potential to be used to better understand

A monoclonal antibody IMab-1 specifically recognizes IDH1R132H, the most common glioma-derived mutation.

PubMed

Kato, Yukinari; Jin, Genglin; Kuan, Chien-Tsun; McLendon, Roger E; Yan, Hai; Bigner, Darell D

2009-12-18

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1(R132H)) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1(R132H), were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1(R132H) peptide, but not with wild type IDH1 (IDH1(wt)) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1(R132H) protein, not IDH1(wt) protein or the other IDH1 mutants, indicating that IMab-1 is IDH1(R132H)-specific. Furthermore, IMab-1 specifically stained the IDH1(R132H)-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1(R132H)-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1(R132H)-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.

ERBB oncogene proteins as targets for monoclonal antibodies.

PubMed

Polanovski, O L; Lebedenko, E N; Deyev, S M

2012-03-01

General properties of the family of tyrosine kinase ERBB receptors are considered in connection with their role in the generation of cascades of signal transduction in normal and tumor cells. Causes of acquisition of oncogene features by genes encoding these receptors and their role in tumorigenesis are analyzed. Anti-ERBB monoclonal antibodies approved for therapy are described in detail, and mechanisms of their antitumor activity and development of resistance to them are reviewed. The existing and the most promising strategies for creating and using monoclonal antibodies and their derivatives for therapy of cancer are discussed.

Comparison of humanized IgG and FvFc anti-CD3 monoclonal antibodies expressed in CHO cells.

PubMed

Serpieri, Flavia; Inocencio, Andre; de Oliveira, Jose Marcelino; Pimenta, Alécio A; Garbuio, Angélica; Kalil, Jorge; Brigido, Marcelo M; Moro, Ana Maria

2010-07-01

Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Tracking the emerging human pathogen Pseudallescheria boydii by using highly specific monoclonal antibodies.

PubMed

Thornton, Christopher R

2009-05-01

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 kappa-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.

Monoclonal antibody proteomics: use of antibody mimotope displaying phages and the relevant synthetic peptides for mAb scouting.

PubMed

Hajdú, István; Flachner, Beáta; Bognár, Melinda; Végh, Barbara M; Dobi, Krisztina; Lőrincz, Zsolt; Lázár, József; Cseh, Sándor; Takács, László; Kurucz, István

2014-08-01

Monoclonal antibody proteomics uses nascent libraries or cloned (Plasmascan™, QuantiPlasma™) libraries of mAbs that react with individual epitopes of proteins in the human plasma. At the initial phase of library creation, cognate protein antigen and the epitope interacting with the antibodies are not known. Scouting for monoclonal antibodies (mAbs) with the best binding characteristics is of high importance for mAb based biomarker assay development. However, in the absence of the identity of the cognate antigen the task represents a challenge. We combined phage display, and surface plasmon resonance (Biacore) experiments to test whether specific phages and the respective mimotope peptides obtained from large scale studies are applicable to determine key features of antibodies for scouting. We show here that mAb captured phage-mimotope heterogeneity that is the diversity of the selected peptide sequences, is inversely correlated with an important binding descriptor; the off-rate of the antibodies and that represents clues for driving the selection of useful mAbs for biomarker assay development. Carefully chosen synthetic mimotope peptides are suitable for specificity testing in competitive assays using the target proteome, in our case the human plasma. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparation and characterization of monoclonal antibodies recognizing unique epitopes on sexually differentiated rat liver cytochrome P-450 isozymes.

PubMed

Morgan, E T; Rönnholm, M; Gustafsson, J A

1987-07-14

Cytochrome P-450 isozymes P-450(16 alpha), P-450(15 beta), and P-450DEa are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to P-450(15 beta) and one antibody to P-450(16 alpha) that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among P-450(16 alpha), P-450(15 beta), and P-450DEa. All of the P-450(15 beta-specific antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay. Western immunoblotting, and antibody-Sepharose immunoadsorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for P-450(15 beta) by these criteria. However, the apparent specificities of antibodies F3 and F20 for P-450(15 beta), and of M16 for P-450(16 alpha), were highly dependent on the analytical technique used. The five anti-P-450(15 beta) antibodies all inhibited the catalytic activity of microsomal P-450(15 beta), by a maximum of 70%. However, they also produced a similar inhibition of microsomal P-450(16 alpha-specific antibody M16 and F23 have a low-affinity interaction with an epitope on P-450(16 alpha). The P-450(16 alpha)-specific antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)

[A New Simple Technique for Producing Labeled Monoclonal Antibodies for Antibody Pair Screening in Sandwich-ELISA].

PubMed

Zaripov, M M; Afanasieva, G V; Glukhova, X A; Trizna, Y A; Glukhov, A S; Beletsky, I P; Prusakova, O V

2015-01-01

A simple and fast method for obtaining biotin-labeled monoclonal antibodies was developed usingcontent of hybridoma culture supernatant sufficient to select antibody pairs in sandwich ELISA. The method consists in chemical biotinylation of antigen-bound antibodies in a well of ELISA plate. Using as an example target Vaccinia virus A27L protein it was shown that the yield of biotinylated reactant is enough to set comprehensive sandwich ELISA for a moderate size panel of up to 25 monoclonal antibodies with an aim to determine candidate pairs. The technique is a cheap and effective solution since it avoids obtaining preparative amounts of antibodies.

Serological diversity demonstrable by a set of monoclonal antibodies to eight serotypes of the mutans streptococci.

PubMed

Ota, F; Ota, M; Mahmud, Z H; Mohammad, A; Yamato, M; Kassu, A; Kato, Y; Tomotake, H; Batoni, G; Campa, M

2006-01-01

A set of monoclonal antibodies were prepared by the conventional cell fusion of myeloma cells (SP2/0-Ag14) with spleen cells from BALB/c mice immunised with whole cells of a strain of mutans streptococci. Their specificities were examined against 35 reference strains of mutans streptococci, 34 reference strains of other oral streptococci and 8 reference strains of other microorganisms often inhabiting the oral cavity. Specificity was examined by enzyme immunoassay using whole cells. A total of 52 strains, consisting of 19 strains isolated in Japan, 19 strains isolated in Italy and 14 strains isolated in England, were characterised by conventional physiological and biochemical tests and then serotyped by the use of 8 monoclonal antibodies with different specificities. They were also confirmed by guanine-plus-cytosine contents of their nucleic acid and DNA-DNA hybridisation test. The results indicated that all monoclonal antibodies are useful for identification of 8 serotypes of the mutans streptococci responsible for dental caries. They also suggest the existence of more serological varieties among mutans species.

Development of monoclonal antibodies against axenic amastigotes of Leishmania infantum strain in Iran: implication for diagnosis of Kala-azar

PubMed Central

Nourizadeh, Ezzat; Zargar, Seyed Jalal; Alimohammadian, Mohammad Hossein; Ajdary, Soheila; Mahdavi, Mahdi

2018-01-01

Objective(s): Leishmaniasis is endemic in 88 countries. Amastigote forms of Leishmania are experts at exploiting host cell processes to establish infection. Monoclonal antibodies are key reagents used in the diagnosis of infectious and non-infectious diseases. The aim of this study was to produce monoclonal antibodies against axenic amastigotes of the Leishmania infantum strain in Iran. Materials and Methods: First, standard strains were cultured and axenic amastigote antigens of L. infantum were obtained. Since then, BALB/c smice were immunized and antibody titers were determined. For hybridoma cell formation, lymphocytes isolated from spleen of immunized mice and myeloma cells were fused at a ratio of 10 to 1 in the presence of polyethylene glycol, followed by limiting dilution for the isolation of monoclones. Subsequently, antibody isotypes were determined by using the isotyping kit. The best clone was injected intraperitoneally to pristane-primed mice for large scale production of monoclonal antibodies. The specificity of antibody was determined with Western blotting. Results: Approximately 25 positive monoclones were obtained, of which four hybrids producing anti-amastigotes L. infantum monoclonal antibodies with high optical density (OD), selected and designated as 8D2 FVI6, 8D2 FVI3, 6G2 FV4 and 6G2 FV3. Results from isotype determination showed the IgG2b sub-class in 6G2FV2 and 8D2FVI6 monoclones. Conclusion: This study produced monoclonal antibody against amastigotes of Iranian strain of L. infantum for the first time. These antibodies have reactivity against Iranian strain of L. infantum and can be used in the diagnosis of Kala-azar.

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.

Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

PubMed

Rahbarizadeh, F; Rasaee, M J; Madani, R; Rahbarizadeh, M H; Omidfar, K

2000-10-01

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.

Production of a broad-specificity monoclonal antibody and application as a receptor to detection amatoxins in mushroom.

PubMed

He, Kuo; Mao, Qingwen; Zang, Xiuyuan; Zhang, Yanyu; Li, Hui; Zhang, Donghao

2017-09-01

In this study, we report the production of a monoclonal broad-specificity monoclonal antibody (mAb) specific for amatoxins and development of an indirect competitive immunoassay for detection of amatoxins in mushroom samples. In the assay, the complete antigen (α-amanitin-OVA) was used as coating antigen, and amatoxins as competitor competes with coating antigen to bind with mAb. Using this approach, The half-maximum inhibition concentrations (IC 50 ) of α-amanitin, β-amanitin and γ-amanitin, and limits of detection (LODs, IC 15 ) were 66.3, 97.4, 163.1 ng/mL and 0.91, 0.98, 0.89 ng/mL, respectively. The LODs for α-amanitin, β-amanitin and γ-amanitin in mushroom samples were 4.55, 4.9, and 4.45 ng/mL. The spiked results were also confirmed by HPLC, which showed a good correlation (R 2  = 0.996) between the two methods. The results indicated that the developed assay was reliable and suitable for the detection of amatoxins in mushroom samples. Copyright © 2017. Published by Elsevier Ltd.

Growth inhibition of tumor cells in vitro by using monoclonal antibodies against gonadotropin-releasing hormone receptor.

PubMed

Lee, Gregory; Ge, Bixia

2010-07-01

As the continuation of a previous study, synthetic peptides corresponding to the extracellular domains of human gonadotropin-releasing hormone (GnRH) receptor were used to generate additional monoclonal antibodies which were further characterized biochemically and immunologically. Among those identified to recognize GnRH receptor, monoclonal antibodies designated as GHR-103, GHR-106 and GHR-114 were found to exhibit high affinity (Kd < or = 1 x 10(-8) M) and specificity to GnRH receptor as judged by the whole cell binding immunoassay and Western blot assay. Both anti-GnRH receptor monoclonal antibodies and GnRH were shown to compete for the same binding site of GnRH receptor on the surface of cultured cancer cells. Growth inhibitions of cancer cells cultured in vitro were demonstrated by cellular apoptosis experiments (TUNEL and MTT assays) under different conditions of treatment with GHR-106 monoclonal antibody or GnRH analogs. It was generally observed that both GnRH I and GHR-106 effectively induce the apoptosis of cultured cancer cells as determined by TUNEL and MTT assays. Consistently, suppressions of gene expressions at mRNA levels were demonstrated with several ribosomal proteins (P0, P1, P2 and L37), when cancer cells were incubated with GnRH or GHR-106. The widespread expressions of GnRH receptor in almost all of the studied human cancer cell lines were also demonstrated by RT-PCR and Western blot assay, as well as indirect immunofluorescence assay with either of these monoclonal antibodies as the primary antibody. In view of the longer half life of antibodies as compared to that of GnRH or its analogs, anti-GnRH receptor monoclonal antibodies in humanized forms could function as GnRH analogs and serve as an ideal candidate of anti-cancer drugs for therapeutic treatments of various cancers in humans as well as for fertility regulations.

Challenges and opportunities for monoclonal antibody therapy in veterinary oncology.

PubMed

Beirão, Breno C B; Raposo, Teresa; Jain, Saurabh; Hupp, Ted; Argyle, David J

2016-12-01

Monoclonal antibodies (mAbs) have come to dominate the biologics market in human cancer therapy. Nevertheless, in veterinary medicine, very few clinical trials have been initiated using this form of therapy. Some of the advantages of mAb therapeutics over conventional drugs are high specificity, precise mode of action and long half-life, which favour infrequent dosing of the antibody. Further advancement in the field of biomedical sciences has led to the production of different forms of antibodies, such as single chain antibody fragment, Fab, bi-specific antibodies and drug conjugates for use in diagnostic and therapeutic purposes. This review describes the potential for mAbs in veterinary oncology in supporting both diagnosis and therapy of cancer. The technical and financial hurdles to facilitate clinical acceptance of mAbs are explored and insights into novel technologies and targets that could support more rapid clinical development are offered. Copyright © 2016 Elsevier Ltd. All rights reserved.

Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

NASA Astrophysics Data System (ADS)

Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

1993-05-01

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Microbials for the production of monoclonal antibodies and antibody fragments.

PubMed

Spadiut, Oliver; Capone, Simona; Krainer, Florian; Glieder, Anton; Herwig, Christoph

2014-01-01

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Copyright © 2013 Elsevier Ltd. All rights reserved.

Development of an Antigen Capture Enzyme-Linked Immunosorbent Assay for Virus Detection Based on Porcine Epidemic Diarrhea Virus Monoclonal Antibodies

PubMed Central

Wang, Zanyu; Jiyuan, Yin; Su, Chen; Xinyuan, Qiao

2015-01-01

Abstract Porcine epidemic diarrhea virus (PEDV), a coronavirus, can cause acute diarrhea and dehydration in pigs. In the current study, two positive monoclonal cell lines (5D7 and 3H4) specific for PEDV were established, and the immunoreactivity of the monoclonal antibodies was confirmed by immunofluorescence and dot-immunobinding assays. A method, termed antigen capture enzyme-linked immunosorbent assay (AC-ELISA), which used the monoclonal antibody 5D7 as the detecting antibody and rabbit antiserum of PEDV protein S as the capture antibody, was developed. Compared with the reverse transcription polymerase chain reaction method of detecting PEDV in fecal samples, AC-ELISA showed similar sensitivity and specificity. These results suggested that AC-ELISA would be useful for the diagnosis and epidemiological studies of PEDV. PMID:25658793

Drug-protein conjugates: haptenation of 1-methyl-10 alpha-methoxydihydrolysergol and 5-bromonicotinic acid to albumin for the production of epitope-specific monoclonal antibodies against nicergoline.

PubMed

Gabor, F; Hamilton, G; Pittner, F

1995-09-01

Two types of monoclonal antibodies were used for the determination of nicergoline in biological matrices. The antibodies were prepared with the hydrolysis products 5-bromonicotinic acid and 1-methyl-10 alpha-methoxydihydrolysergol after hemisuccinoylation to haptens. The current amide bond-generating methods (mixed anhydride-, carbodiimide-, carbodiimide/sulfo-N-hydroxysuccinimide-, and dicyclohexylcarbodiimide/N-hydroxysuccinimide methods) were used in bovine serum albumin (BSA)-coupling techniques and yielded conjugates that were haptenated to varying extents. The conjugates exhibiting 23 mol of 1-methyl-10 alpha-methoxydihydrolysergol (MMD) or 41 mol of 5-bromonicotinic acid (BNA) per mole of BSA were used for both immunization of mice and for coating the wells of the microtiter plates to select hybridomas and investigate specificity of the obtained antibodies. The results of hapten-inhibition ELISA using antigen-coated wells indicate that the supernatant of MMD-specific hybridoma exhibited 50% inhibition of antibody binding at 17 +/- 2 micrograms of MMD and at 24.5 +/- 2 micrograms of nicergoline, and the BNA-specific hybridoma exhibited similar inhibition at 147 +/- 6 micrograms of BNA and 500 +/- 30 micrograms of nicergoline. A main requirement for analytical purposes is that two different types of monoclonal antibodies recognize two different epitopes on nicergoline and its main metabolite, as shown by hapten-inhibition ELISA.

Investigation of a panel of monoclonal antibodies and polyclonal sera against anthrax toxins resulted in identification of an anti-lethal factor antibody with disease-enhancing characteristics.

PubMed

Kulshreshtha, Parul; Tiwari, Ashutosh; Priyanka; Joon, Shikha; Sinha, Subrata; Bhatnagar, Rakesh

2015-12-01

Hybridomas were created using spleen of mice that were actively immunized with rLFn (recombinant N-terminal domain of lethal factor). Later on, separate group of mice were immunized with rLFn to obtain a polyclonal control for passive immunization studies of monoclonal antibodies. This led to the identification of one cohort of rLFn-immnized mice that harboured disease-enhancing polyclonal antibodies. At the same time, the monoclonal antibodies secreted by all the hybridomas were being tested. Two hybridomas secreted monoclonal antibodies (H10 and H8) that were cross-reactive with EF (edema factor) and LF (lethal factor), while the other two hybridomas secreted LF-specific antibodies (H7 and H11). Single chain variable fragment (LETscFv) was derived from H10 hybridoma. H11 was found to have disease-enhancing property. Combination of H11 with protective monoclonal antibodies (H8 and H10) reduced its disease enhancing nature. This in vitro abrogation of disease-enhancement provides the proof of concept that in polyclonal sera the disease enhancing character of a fraction of antibodies is overshadowed by the protective nature of the rest of the antibodies generated on active immunization. Copyright © 2015. Published by Elsevier Ltd.

Specific ion-protein interactions dictate solubility behavior of a monoclonal antibody at low salt concentrations.

PubMed

Zhang, Le; Zhang, Jifeng

2012-09-04

The perturbation of salt ions on the solubility of a monoclonal antibody was systematically studied at various pHs in Na(2)SO(4), NaNO(3), NaCl, NaF, MgSO(4), Mg(NO(3))(2) and MgCl(2) solutions below 350 mM. At pH 7.1, close to the pI, all of the salts increased the solubility of the antibody, following the order of SO(4)(2-) > NO(3)(-) > Cl(-) > F(-) for anions and Mg(2+) > Na(+) for cations. At pH 5.3 where the antibody had a net positive charge, the anions initially followed the order of SO(4)(2-) > NO(3)(-) > Cl(-) > F(-) for effectiveness in reducing the solubility and then switched to increasing the solubility retaining the same order. Furthermore, the antibody was more soluble in the Mg(2+) salt solutions than in the corresponding Na(+) salt solutions with the same anion. At pH 9.0 where the antibody had a net negative charge, an initial decrease in the protein solubility was observed in the solutions of the Mg(2+) salts and NaF, but not in the rest of the Na(+) salt solutions. Then, the solubility of the antibody was increased by the anions in the order of SO(4)(2-) > NO(3)(-) > Cl(-) > F(-). The above complex behavior is explained based on the ability of both cation and anion from a salt to modulate protein-protein interactions through their specific binding to the protein surface.

Coarse grained modeling of transport properties in monoclonal antibody solution

NASA Astrophysics Data System (ADS)

Swan, James; Wang, Gang

Monoclonal antibodies and their derivatives represent the fastest growing segment of the bio pharmaceutical industry. For many applications such as novel cancer therapies, high concentration, sub-cutaneous injections of these protein solutions are desired. However, depending on the peptide sequence within the antibody, such high concentration formulations can be too viscous to inject via human derived force alone. Understanding how heterogenous charge distribution and hydrophobicity within the antibodies leads to high viscosities is crucial to their future application. In this talk, we explore a coarse grained computational model of therapeutically relevant monoclonal antibodies that accounts for electrostatic, dispersion and hydrodynamic interactions between suspended antibodies to predict assembly and transport properties in concentrated antibody solutions. We explain the high viscosities observed in many experimental studies of the same biologics.

Development of monoclonal antibodies that recognize Treponema pallidum.

PubMed Central

Saunders, J M; Folds, J D

1983-01-01

We developed a panel of monoclonal antibodies to Treponema pallidum (Nichols) antigens, some of which recognize treponemal antigens on T. pallidum (Nichols), T. pallidum strain 14, and Treponema phagedenis biotype Reiter. The antibodies were detected by either an enzyme-linked immunosorbent assay or a radioimmunoassay. PMID:6347899

Preparation and characterization of monoclonal antibody against melatonin.

PubMed

Soukhtanloo, Mohammad; Ansari, Mohammad; Paknejad, Maliheh; Parizadeh, Mohammad Reza; Rasaee, Mohammad Javad

2008-06-01

Anti-melatonin monoclonal antibodies (MAb) were prepared following coupling melatonin to bovine serum albumin (BSA) by Mannich reaction. Balb/c mice were immunized via injection of the melatonin-BSA intraperitonally. The spleen cells producing high titer of antibody were fused with myeloma cells of SP2/0 origin. After two limiting dilutions, two stable clones (AS-H10 and AS-D26) exhibiting best properties were selected for further studies. The class and subclass of two MAbs were found to be IgG(1) and IgG(2a) with lambda and kappa light chains, respectively. Antibodies secreted by these two clones showed high affinity of about 10(9)M(1). Study of the specificity criteria showed that these clones had no cross reactivity with indolic, aromatic, and imidazole ring-containing compounds, and had high specificity towards melatonin. The calibration curve was constructed with a sensitivity range of 10 ng/mL to 10 microg/mL. In conclusion, these MAbs may be useful for immunoassay of melatonin.

A Spectrum of Monoclonal Antibodies Reactive with Human Mammary Tumor Cells

NASA Astrophysics Data System (ADS)

Colcher, D.; Horan Hand, P.; Nuti, M.; Schlom, J.

1981-05-01

Splenic lymphocytes of mice, immunized with membrane-enriched fractions of metastatic human mammary carcinoma tissues, were fused with the NS-1 non-immunoglobulin-secreting murine myeloma cell line. This resulted in the generation of hybridoma cultures secreting immunoglobulins reactive in solid-phase radioimmunoassays with extracts of metastatic mammary carcinoma cells from involved livers, but not with extracts of apparently normal human liver. As a result of further screening of immunoglobulin reactivities and double cloning of cultures, 11 monoclonal antibodies were chosen that demonstrated reactivities with human mammary tumor cells and not with apparently normal human tissues. These monoclonal antibodies could be placed into at least five major groups on the basis of their differential binding to the surface of various live human mammary tumor cells in culture, to extracts of mammary tumor tissues, or to tissue sections of mammary tumor cells studied by the immunoperoxidase technique. Whereas a spectrum of reactivities to mammary tumors was observed with the 11 monoclonal antibodies, no reactivity was observed to apparently normal cells of the following human tissues: breast, lymph node, lung, skin, testis, kidney, thymus, bone marrow, spleen, uterus, thyroid, intestine, liver, bladder, tonsils, stomach, prostate, and salivary gland. Several of the antibodies also demonstrated a ``pancarcinoma'' reactivity, showing binding to selected non-breast carcinomas. None of the monoclonal antibodies showed binding to purified ferritin or carcinoembryonic antigen. Monoclonal antibodies of all five major groups, however, demonstrated binding to human metastatic mammary carcinoma cells both in axillary lymph nodes and at distal sites.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, Larry H.; Vanderlaan, Martin; Watkins, Bruce E.; Van Emon, Jeanette M.; Bigbee, Carolyn L.

1992-01-01

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples.

[Identification of Env-specific monoclonal antibodies from Chinese HIV-1 infected person by magnetic beads separating B cells and single cell RT-PCR cloning].

PubMed

Huang, Xiang-Ying; Yu, Shuang-Qing; Cheng, Zhan; Ye, Jing-Rong; Xu, Ke; Feng, Xia; Zeng, Yi

2013-04-01

To establish a simple and practical method for screening of Env-specific monoclonal antibodies from HIV-1 infected individuals. Human B cells were purified by negative sorting from PBMCs and memory B cells were further enriched using anti-CD27 microbeads. Gp120 antigen labbled with biotin was incubated with memory B cells to specifically bind IgG on cells membrane. The memory B cells expressing the Env-specific antibody were harvested by magnetic beads separating, counted and diluted to the level of single cell in each PCR well that loading with catch buffer containing RNase inhibitor to get RNAs. The antibody genes were amplified by single cell RT-PCR and nested PCR, cloned into eukaryotic expression vectors and transfected into 293T cells. The binding activity of recombinant antibodies to Env were tested by ELISA. Three monocolonal Env-specific antibodies were isolated from one HIV-1 infected individual. We can obtain Env-specific antibody by biotin labbled antigen, magnetic beads separating technique coupled with single cell RT-PCR and expression cloning.

Production of monoclonal antibody to acaricide dicofol and its derivatives.

PubMed

Hongsibsong, Surat; Prapamontol, Tippawan; Suphavilai, Chaisuree; Wipasa, Jiraprapa; Pattarawarapan, Mookda; Kasinrerk, Watchara

2010-12-01

In Thailand detection of acaricide dicofol residues has been sporadically performed due to the limitation of analytical techniques. Conventional analytical methods for detecting dicofol residues most often use chromatographic-based techniques. Our ultimate aim is to develop an alternative method for rapidly analyzing dicofol residues in vegetables and fruit samples. Here we report the production of monoclonal antibodies specific to dicofol and its derivatives. Hapten-protein carriers were prepared by linking succinic anhydride to dichlorobenzhydrol (DCBH), which was then conjugated to bovine serum albumin (BSA) and oval albumin (OVA). DCBH-BSA conjugate was used as immunogen while DCBH-OVA conjugate was used as capture antigen for competitive inhibition assay. Female BALB/c mice were immunized with DCBH-BSA conjugate subcutaneously, and antibody (Ab) level was determined 2 weeks after the last immunization. Spleen cells producing high titer antibody were isolated and fused with myeloma cells of P3.X6.Ag8.653. After limiting dilutions, antibody produced by one clone had high affinity, which was found to be of IgG1 with κ light chain. Specificity and inhibition concentrations of the monoclonal antibody (MAb) were determined by competitive indirect ELISA with dicofol, and its 50% (IC(50)) was 0.28 μg/mL. Working ranges of the developed immunoassay were from 0.07 to 25 μg/mL. Hence, the prepared MAb will be able to be applied for immunoassay development for detecting dicofol residue in vegetables and fruits far below the maximum residue limit such that 5 g of fruits and berries can be detected below 0.1 mg/kg.

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.

Conserved and Variant Epitopes of Plasmodium vivax Duffy Binding Protein as Targets of Inhibitory Monoclonal Antibodies

PubMed Central

Ntumngia, Francis B.; Schloegel, Jesse; Barnes, Samantha J.; McHenry, Amy M.; Singh, Sanjay; King, Christopher L.

2012-01-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains. PMID:22215740

Conserved and variant epitopes of Plasmodium vivax Duffy binding protein as targets of inhibitory monoclonal antibodies.

PubMed

Ntumngia, Francis B; Schloegel, Jesse; Barnes, Samantha J; McHenry, Amy M; Singh, Sanjay; King, Christopher L; Adams, John H

2012-03-01

The Duffy binding protein (DBP) is a vital ligand for Plasmodium vivax blood-stage merozoite invasion, making the molecule an attractive vaccine candidate against vivax malaria. Similar to other blood-stage vaccine candidates, DBP allelic variation eliciting a strain-specific immunity may be a major challenge for development of a broadly effective vaccine against vivax malaria. To understand whether conserved epitopes can be the target of neutralizing anti-DBP inhibition, we generated a set of monoclonal antibodies to DBP and functionally analyzed their reactivity to a panel of allelic variants. Quantitative analysis by enzyme-linked immunosorbent assay (ELISA) determined that some monoclonal antibodies reacted strongly with epitopes conserved on all DBP variants tested, while reactivity of others was allele specific. Qualitative analysis characterized by anti-DBP functional inhibition using an in vitro erythrocyte binding inhibition assay indicated that there was no consistent correlation between the endpoint titers and functional inhibition. Some monoclonal antibodies were broadly inhibitory while inhibition of others varied significantly by target allele. These data demonstrate a potential for vaccine-elicited immunization to target conserved epitopes but optimization of DBP epitope target specificity and immunogenicity may be necessary for protection against diverse P. vivax strains.

Gaining insights into the consequences of target-mediated drug disposition of monoclonal antibodies using quasi-steady-state approximations.

PubMed

Grimm, Hans Peter

2009-10-01

Target-mediated drug disposition (TMDD) is frequently reported for therapeutic monoclonal antibodies and is linked to the high affinity and high specificity of antibody molecules for their target. Understanding TMDD of a monoclonal antibody should go beyond the empirical description of its non-linear PK since valuable insights on the antibody-target interaction itself can be gained. This makes its mechanistic understanding precious for the drug development process, in particular for the optimization of new antibody molecules, for the design and interpretation of pharmacokinetic studies, and possibly even for the evaluation of efficacy and dose selection of drug candidates. Using the observation that the molecular (microscopic) processes are usually much more rapid than the pharmacokinetic (macroscopic) processes, a series of quasi-steady-state conditions on the microscopic level is proposed to bridge the gap between simple empirical and complex mechanistic descriptions of TMDD. These considerations show the impact of parameters such as target turnover, target expression, and target accessibility on the pharmacokinetics and pharmacodynamics of monoclonal antibodies.

Production and Characterization of Monoclonal Antibody against Recombinant Virus Coat Protein CP42.

PubMed

Shibaei, Naeimeh; Majidi, Jafar; Razavi, Khadijeh; Karkhane, Ali Asghar; Sokhandan-Bashir, Nemat; Aghebati-Maleki, Leili

2017-02-01

There are many studies related to the production of a ELISA kit for diagnosing virus infections. However, production of most kits depends on purification of whole virus particles, which involves the use of costly equipment and reagents. The purpose of this study was to check out if the anti-CP42 antibodies could be used as a diagnostic assay for detection of Grapevine fanleaf Virus (GFLV). In this study, recombinant GFLV coat protein gene related to selected antigenic determinants was inserted into pET-28a bacterial expression vector and the construct (pET-28a CP42) was cloned into E. coli strain (DE3). Expressed protein was verified with western blotting assay by the use of commercially available anti-GFLV antibody. The recombinant protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) resin. Balb/c mice were immunized with purified protein and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by ELISA using CP42 as coating antigen. The results showed that monoclonal antibody (MAb) specific to CP42 has been successfully generated. Efficiency of produced antibody was analyzed by ELISA and western blotting assay using some confirmed grapevine samples. The infection was confirmed previously based on morphological features and ELISA assay, performed using commercial anti-GFLV antibody. The monoclonal antibody reacted with antigen in ELISA and immunoblot method. Our results demonstrated that anti recombinant CP42 monoclonal antibodies are able to diagnose whole virus in infected grapevine sample using ELISA test.

Monoclonal Antibodies Radiolabeling with Rhenium-188 for Radioimmunotherapy

PubMed Central

Martini, Petra; Pasquali, Micol

2017-01-01

Rhenium-188, obtained from an alumina-based tungsten-188/rhenium-188 generator, is actually considered a useful candidate for labeling biomolecules such as antibodies, antibody fragments, peptides, and DNAs for radiotherapy. There is a widespread interest in the availability of labeling procedures that allow obtaining 188Re-labeled radiopharmaceuticals for various therapeutic applications, in particular for the rhenium attachment to tumor-specific monoclonal antibodies (Mo)Abs for immunotherapy. Different approaches have been developed in order to obtain 188Re-radioimmunoconjugates in high radiochemical purity starting from the generator eluted [188Re]ReO4−. The aim of this paper is to provide a short overview on 188Re-labeled (Mo)Abs, focusing in particular on the radiolabeling methods, quality control of radioimmunoconjugates, and their in vitro stability for radioimmunotherapy (RIT), with particular reference to the most important contributions published in literature in this topic. PMID:28951872

Monoclonal Antibodies Radiolabeling with Rhenium-188 for Radioimmunotherapy.

PubMed

Uccelli, Licia; Martini, Petra; Pasquali, Micol; Boschi, Alessandra

2017-01-01

Rhenium-188, obtained from an alumina-based tungsten-188/rhenium-188 generator, is actually considered a useful candidate for labeling biomolecules such as antibodies, antibody fragments, peptides, and DNAs for radiotherapy. There is a widespread interest in the availability of labeling procedures that allow obtaining 188 Re-labeled radiopharmaceuticals for various therapeutic applications, in particular for the rhenium attachment to tumor-specific monoclonal antibodies (Mo)Abs for immunotherapy. Different approaches have been developed in order to obtain 188 Re-radioimmunoconjugates in high radiochemical purity starting from the generator eluted [ 188 Re]ReO 4 - . The aim of this paper is to provide a short overview on 188 Re-labeled (Mo)Abs, focusing in particular on the radiolabeling methods, quality control of radioimmunoconjugates, and their in vitro stability for radioimmunotherapy (RIT), with particular reference to the most important contributions published in literature in this topic.

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques.

PubMed

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A; Veazey, Ronald S

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross-react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define "memory" T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in approximately 44% of rhesus macaques (Macaca mulatta) of Indian but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques.

Differential cross-reactivity of monoclonal antibody OPD4 (anti-CD45RO) in macaques

PubMed Central

Wang, Xiaolei; Pahar, Bapi; Rasmussen, Terri; Alvarez, Xavier; Dufour, Jason; Rasmussen, Kelsi; Lackner, Andrew A.; Veazey, Ronald S.

2008-01-01

Immunologic research in nonhuman primates is occasionally limited by the availability of reagents that cross react in nonhuman primates. One major limitation has been the lack of a monoclonal antibody to CD45RO. Although the monoclonal antibody UCHL-1 is used to detect CD45RO isoforms in humans, it does not react with nonhuman primates, mandating the use of alternative strategies to define “memory” T cell responses in nonhuman primates. The current study examined the reactivity and specificity of another antibody against CD45RO, clone OPD4, in macaques. Here we demonstrate that OPD4 specifically labels memory CD4+ T cells in ~44% of rhesus macaques (Macaca mulatta) of Indian, but not Chinese origin. In contrast, tissues from pigtail macaques (Macaca nemestrina) react with this clone, indicating that OPD4 may be useful for examining memory CD4+ T cells in certain macaques, but its utility may be limited in other species or even among individual macaques. PMID:18304631

Emerging monoclonal antibodies for the treatment of renal cell carcinoma (RCC).

PubMed

Atkins, Michael B; Philips, George K

2016-09-01

Advanced renal cell carcinoma (RCC) was considered refractory to most cancer therapies until the 1980s, after which immune modulating agents and targeted agents were developed. Recently the rapid development of therapeutic monoclonal antibodies targeting immune checkpoint pathways has provided significant clinical benefit in patients with many distinct cancer types. Nivolumab, an anti-PD1 monoclonal antibody showed improvement in response rate and overall survival in patients with previously treated RCC and received US FDA approval in late 2015. Current efforts with anti-PD1-based therapy include combinations with ipilimumab and with VEGF pathway blockers in the hopes on building on the activity of single agent therapy. We describe our current understanding of tumor immunology including the basis of the tumor-specific immune response and the adaptive mechanisms used by the tumor for immune escape. We describe the mechanisms of action as well as the therapeutic application of the antibodies, ipilimumab, nivolumab and atezolizumab in patients with RCC. We identify key areas of active research in biomarker development and combination therapies. Clinical trials and the field of RCC therapeutics are expected to move in the direction of combination therapies using immune checkpoint inhibitors, extending overall survival as a benchmark for new drug approvals, and biomarker validation for improved selection of patients for specific therapies.

Selectivity verification of cardiac troponin monoclonal antibodies for cardiac troponin detection by using conventional ELISA

NASA Astrophysics Data System (ADS)

Fathil, M. F. M.; Arshad, M. K. Md; Gopinath, Subash C. B.; Adzhri, R.; Ruslinda, A. R.; Hashim, U.

2017-03-01

This paper presents preparation and characterization of conventional enzyme-linked immunosorbent assay (ELISA) for cardiac troponin detection to determine the selectivity of the cardiac troponin monoclonal antibodies. Monoclonal antibodies, used to capture and bind the targets in this experiment, are cTnI monoclonal antibody (MAb-cTnI) and cTnT monoclonal antibody (MAb-cTnT), while both cardiac troponin I (cTnI) and T (cTnT) are used as targets. ELISA is performed inside two microtiter plates for MAb-cTnI and MAb-cTnT. For each plate, monoclonal antibodies are tested by various concentrations of cTnI and cTnT ranging from 0-6400 µg/l. The binding selectivity and level of detection between monoclonal antibodies and antigen are determined through visual observation based on the color change inside each well on the plate. ELISA reader is further used to quantitatively measured the optical density of the color changes, thus produced more accurate reading. The results from this experiment are utilized to justify the use of these monoclonal antibodies as bio-receptors for cardiac troponin detection by using field-effect transistor (FET)-based biosensors coupled with substrate-gate in the future.

Monoclonal antibodies to synthetic pyrethroids and method for detecting the same

DOEpatents

Stanker, L.H.; Vanderlaan, M.; Watkins, B.E.; Van Emon, J.M.; Bigbee, C.L.

1992-04-28

Methods are described for making specific monoclonal antibodies which may be used in a sensitive immunoassay for detection of synthetic pyrethroids in foods and environmental samples. Appropriate sample preparation and enzyme amplification of the immunoassay for this widely-used class of pesticides permits detection at low levels in laboratory and field tested samples. 6 figs.

[Preparation and characterization of monoclonal antibodies against Micrococcus luteus Rpf domain].

PubMed

Fan, Ai-lin; Shi, Chang-hong; Su, Ming-quan; Ma, Jing; Bai, Yin-lan; Cheng, Xiao-dong; Xu, Zhi-kai; Hao, Xiao-ke

2008-05-01

To express Micrococcus luteus Rpf domain in prokaryotic cells and prepare monoclonal antibodies against Rpf domain. The gene encoding Micrococcus luteus Rpf domain was amplified from genome of Micrococcus luteus by polymerase chain reaction(PCR), and inserted into cloning vector pUC-19. After sequenced, Micrococcus luteus Rpf domain gene was subcloned into the expression vector pPro-EXHT and transfected into E.coli DH5alpha. After induced by IPTG, the bacteria controlled by T7 promoter expressed the fused Micrococcus luteus Rpf domain protein with a hexahistidine tail at its N-terminal and the target protein was purified under denaturing conditions. Using this protein as antigen to immunize the BALB/c mice and prepare monoclonal antibodies against Micrococcus luteus Rpf domain. Then specifities and relative affinities of mAbs were identified by ELISA. The fusion protein was purified by metal chelate affinity chromatography under denaturing condition. Three cloned mAbs were prepared from the mice immunized by Rpf domain. All of them could recognize Rpf domain. specifically. The prepared mAbs against Rpf domain have strong specificity with high titers, which provides useful tools for further study of the function of Rpf domain in TB prevention.

Production of polyclonal and monoclonal antibodies against group A, B, and C capsular polysaccharides of Neisseria meningitidis and preparation of latex reagents.

PubMed Central

Nato, F; Mazie, J C; Fournier, J M; Slizewicz, B; Sagot, N; Guibourdenche, M; Postic, D; Riou, J Y

1991-01-01

Polyclonal and monoclonal antibodies against capsular polysaccharides of Neisseria meningitidis serogroups A, B, and C were produced in order to develop immunological reagents allowing both the detection of soluble antigens during meningococcal meningitis and antigenic serogrouping of N. meningitidis cultures. The performance characteristics of monoclonal and polyclonal antibody latex reagents were compared. For the detection of soluble polysaccharide antigen, polyclonal antibody latex reagent was selected for N. meningitidis A and C. The latex reagent prepared with polyclonal antibodies against N. meningitidis B could not detect capsular polysaccharide even at 1 mg/ml. The monoclonal antibody B latex reagent which detected 100 ng of polysaccharide per ml was therefore chosen. For the serogroup identification of N. meningitidis, the use of a confirmatory test results in an overall specificity of 100% with polyclonal or monoclonal antibody latex reagents. PMID:1909346

Epitope characterization of pre-existing and developing antibodies to an aglycosylated monoclonal antibody therapeutic of G1m17,1 allotype.

PubMed

Tatarewicz, Suzanna M; Juan, Gloria; Swanson, Steven J; Moxness, Michael S

2012-08-31

Allotypes of IgG1 molecules can influence the immunogenicity of therapeutic monoclonal antibodies and may account for the presence of some pre-existing antibodies. An electrochemiluminescent (ECL) bridging immunoassay was used to characterize the binding epitopes of anti-therapeutic antibodies (ATAs) in a Phase 1 single ascending dose clinical trial of a therapeutic aglycosylated IgG1monoclonal antibody (mAb). There was no evidence for ATAs specific for a possible neo-epitope created due to the lack of glycosylation. ATAs that developed post-treatment were specific for the F(ab')2, whereas, pre-existing ATAs were specific to the Fc region. Further characterization of the pre-existing ATAs identified the specific epitope to be the G1m1 allotype determinant in the Fc of the therapeutic. A novel competitive bridging assay was developed to verify that serum IgG1 from subjects with pre-existing anti-G1m1 antibodies was homozygous for the antithetical allotype (G1m3). The endogenous G1m allotype of all subjects was assessed and correlation to ATA incidence and adverse events was evaluated. Interestingly, the pre-existing anti-allotype antibody in subjects persisted but was not augmented after dosing, indicating the lack of a secondary immune response to this epitope. These studies indicate the relationship of the therapeutic allotype and the corresponding allotype of subjects is an important component to further understand the impact of immunogenicity on the safety and efficacy of therapeutic antibodies. Copyright © 2012 Elsevier B.V. All rights reserved.

Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies.

PubMed

Perruchini, Claire; Pecorari, Frederic; Bourgeois, Jean-Pierre; Duyckaerts, Charles; Rougeon, François; Lafaye, Pierre

2009-11-01

Camelids produce antibodies made of homodimeric heavy chains, and the antigen-binding region being composed of a single domain called VHH. These VHHs are much smaller than complete IgG. They are also more thermostable and more soluble in water; they should, therefore, diffuse more readily in the tissues. VHHs, expressed in bacteria, are easier to produce than conventional monoclonal antibodies. Because of these special characteristics, these antibody fragments could have interesting developments in immunohistochemistry and in the development of biomarkers. To test the possibility of their use in immunohistochemistry (IHC), we selected the glial fibrillary acidic protein (GFAP), a well-known marker of astrocytes. One alpaca (Lama pacos) was immunized against GFAP. Lymphocytes were isolated; the DNA was extracted; the VHH-coding sequences were selectively amplified. Three VHHs with a high affinity for GFAP and their corresponding mRNA were selected by ribosome display. Large quantities of the recombinant VHHs coupled with different tags were harvested from transfected bacteria. One of them was shown to immunolabel strongly and specifically to GFAP of human astrocytes in tissue sections. The quality of the IHC was comparable or, in some aspects, superior to the quality obtained with conventional IgG. The VHH was shown to diffuse on a longer distance than conventional monoclonal antibodies in fixed cortical tissue: a property that may be useful in immunolabeling of thick sections.

Topography of the casein micelle surface by surface plasmon resonance (SPR) using a selection of specific monoclonal antibodies.

PubMed

Dupont, Didier; Johansson, Annette; Marchin, Stephane; Rolet-Repecaud, Odile; Marchesseau, Sylvie; Leonil, Joelle

2011-08-10

Several theoretical models of the casein micelle structure have been proposed in the past, but the exact organization of the four individual caseins (α(s1), α(s2), β, and κ) within this supramolecular structure remains unknown. The present study aims at determining the topography of the casein micelle surface by following the interaction between 44 monoclonal antibodies specific for different epitopes of α(s1)-, α(s2)-, β-, and κ-casein and the casein micelle in real time and no labeling using a surface plasmon resonance (SPR)-based biosensor. Although the four individual caseins were found to be accessible for antibody binding, data confirmed that the C-terminal extremity of κ-casein was highly accessible and located at the periphery of the structure. When casein micelles were submitted to proteolysis, the C-terminal extremity of κ-casein was rapidly hydrolyzed. Disintegration of the micellar structure resulted in an increased access for antibodies to hydrophobic areas of α(s1)- and α(s2)-casein.

Production of anti-CD14 monoclonal antibody using synthetic peptide of human CD14 as immunizing antigen.

PubMed

Maleki, Leili Aghebati; Shanehbandi, Dariush; Majidi, Jafar; Yusefi, Mehdi; Abdolalizadeh, Jalal; Orangi, Mona; Baradaran, Behzad

2013-01-01

CD14 is a myeloid differentiation antigen expressed primarily on peripheral blood monocytes, dendritic cells and macrophages. It is a key regulator of inflammatory responses to gram-negative bacteria, oxidative burst and septic shock. The aim of this study was to produce and characterize monoclonal antibody against CD14 for use in detection and diagnosis of monocytes. To produce MAb against CD14 protein, mice were immunized with two KLH-conjugated CD14 peptides. The spleen cells of the immunized mice were then fused with SP2/0 by hybridoma technique. Fused cells were grown in selective medium and cloned by limiting dilution method. The desired clones were selected and supernatants of hybridoma cells were screened by ELISA for antibody. Monoclonal antibody was purified by chromatography and confirmed by SDS-PAGE. Finally, immunoblotting and flowcytometry were recruited to explore the specificity of the MAb. Our results showed successful production and characterization of anti CD14 monoclonal antibody. The MAb was IgG2a with Kappa light chain and immunobloting and flowcytometry results demonstrated specific reactivity of this MAb with CD14. The results show that, the produced anti- CD14 MAb is highly specific and functional in biomedical applications such as flow cytometry and western blotting and could be utilized for identification of monocytes.

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-8

USDA-ARS?s Scientific Manuscript database

Interleukin-8 (IL-8) is a key mediator in neutrophil-mediated acute proinflammatory response and activates multiple signaling pathways downstream of two receptors (CXCR1 and CXCR2). However, there have not been any monoclonal antibodies which specifically detect chicken IL8 (chIL8) and define its b...

Immunoactive two-dimensional self-assembly of monoclonal antibodies in aqueous solution revealed by atomic force microscopy

NASA Astrophysics Data System (ADS)

Ido, Shinichiro; Kimiya, Hirokazu; Kobayashi, Kei; Kominami, Hiroaki; Matsushige, Kazumi; Yamada, Hirofumi

2014-03-01

The conformational flexibility of antibodies in solution directly affects their immune function. Namely, the flexible hinge regions of immunoglobulin G (IgG) antibodies are essential in epitope-specific antigen recognition and biological effector function. The antibody structure, which is strongly related to its functions, has been partially revealed by electron microscopy and X-ray crystallography, but only under non-physiological conditions. Here we observed monoclonal IgG antibodies in aqueous solution by high-resolution frequency modulation atomic force microscopy (FM-AFM). We found that monoclonal antibodies self-assemble into hexamers, which form two-dimensional crystals in aqueous solution. Furthermore, by directly observing antibody-antigen interactions using FM-AFM, we revealed that IgG molecules in the crystal retain immunoactivity. As the self-assembled monolayer crystal of antibodies retains immunoactivity at a neutral pH and is functionally stable at a wide range of pH and temperature, the antibody crystal is applicable to new biotechnological platforms for biosensors or bioassays.

Development and characterization of a monoclonal antibody to human embryonal carcinoma

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

Khazaeli, M.B.; Beierwaltes, W.H.; Pitt, G.S.

1987-06-01

A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian,more » 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with /sup 131/I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21.« less

A monoclonal antibody IMab-1 specifically recognizes IDH1{sup R132H}, the most common glioma-derived mutation

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

Kato, Yukinari, E-mail: yukinari-k@bea.hi-ho.ne.jp; The Oncology Research Center, Research Institute for Advanced Molecular Epidemiology, Yamagata University, 2-2-2 Iida-nishi, Yamagata 990-9585; Jin, Genglin

2009-12-18

IDH1 (isocitrate dehydrogenase 1) mutations have been identified as early and frequent genetic alterations in astrocytomas, oligodendrogliomas, and oligoastrocytomas as well as secondary glioblastomas. In contrast, primary glioblastomas very rarely contain IDH1 mutations, although primary and secondary glioblastomas are histologically indistinguishable. The IDH1 mutations are remarkably specific to a single codon in the conserved and functionally important Arg132 in IDH1. In gliomas, the most frequent IDH1 mutations (>90%) were G395A (R132H). In this study, we immunized mice with R132H-containing IDH1 (IDH1{sup R132H}) peptide. After cell fusion using Sendai virus envelope, the monoclonal antibodies (mAbs), which specifically reacted with IDH1{sup R132H},more » were screened in ELISA. One of the mAbs, IMab-1 reacted with the IDH1{sup R132H} peptide, but not with wild type IDH1 (IDH1{sup wt}) peptide in ELISA. In Western-blot analysis, IMab-1 reacted with only the IDH1{sup R132H} protein, not IDH1{sup wt} protein or the other IDH1 mutants, indicating that IMab-1 is IDH1{sup R132H}-specific. Furthermore, IMab-1 specifically stained the IDH1{sup R132H}-expressing cells in astrocytomas in immunohistochemistry, whereas it did not react with IDH1{sup R132H}-negative primary glioblastoma sections. In conclusion, we established an anti-IDH1{sup R132H}-specific monoclonal antibody IMab-1, which should be significantly useful for diagnosis and biological evaluation of mutation-bearing gliomas.« less

Analysis of Defined Combinations of Monoclonal Antibodies in Anthrax Toxin Neutralization Assays and Their Synergistic Action

PubMed Central

Ngundi, Miriam M.; Meade, Bruce D.; Little, Stephen F.; Quinn, Conrad P.; Corbett, Cindi R.; Brady, Rebecca A.

2012-01-01

Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

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

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to exploremore » the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.« less

Generation of Recombinant Porcine Parvovirus Virus-Like Particles in Saccharomyces cerevisiae and Development of Virus-Specific Monoclonal Antibodies

PubMed Central

Tamošiūnas, Paulius Lukas; Petraitytė-Burneikienė, Rasa; Lasickienė, Rita; Sereika, Vilimas; Lelešius, Raimundas; Žvirblienė, Aurelija; Sasnauskas, Kęstutis

2014-01-01

Porcine parvovirus (PPV) is a widespread infectious virus that causes serious reproductive diseases of swine and death of piglets. The gene coding for the major capsid protein VP2 of PPV was amplified using viral nucleic acid extract from swine serum and inserted into yeast Saccharomyces cerevisiae expression plasmid. Recombinant PPV VP2 protein was efficiently expressed in yeast and purified using density gradient centrifugation. Electron microscopy analysis of purified PPV VP2 protein revealed the self-assembly of virus-like particles (VLPs). Nine monoclonal antibodies (MAbs) against the recombinant PPV VP2 protein were generated. The specificity of the newly generated MAbs was proven by immunofluorescence analysis of PPV-infected cells. Indirect IgG ELISA based on the recombinant VLPs for detection of PPV-specific antibodies in swine sera was developed and evaluated. The sensitivity and specificity of the new assay were found to be 93.4% and 97.4%, respectively. In conclusion, yeast S. cerevisiae represents a promising expression system for generating recombinant PPV VP2 protein VLPs of diagnostic relevance. PMID:25045718

The Use of Monoclonal Antibodies in Human Prion Disease

NASA Astrophysics Data System (ADS)

Bodemer, Walter

Detection of PrP and its pathological isoform(s) is the key to understanding the etiology and pathogenesis of transmissible spongiform encephalopathy. There is ample evidence that PrP isoforms constitute a major component of an unknown and perhaps unconventional infectious agent. An etiological relationship between human and zoonotic transmissible spongiform encephalopathies may be revealed with monoclonal antibodies. Knowledge of the conformational transition rendering a nonpathogenic, almost ubiquitous cellular protein into a pathogenic one is crucial to defining pathomechanisms. The stepwise or even continuous formation of pathogenic molecules can be monitored. Any improvement in the early diagnosis could help to conceive new therapeutic measures which are not currently available. Determination of PrP isoforms in tissue, cells, or body fluids may be of prognostic value. Many experimental approaches in molecular medicine and molecular biology of the prion protein already rely on monoclonal antibodies. Recombinant antibodies such as the single-chain Fv may soon replace traditional hybridoma techniques. Binding affinity can easily be manipulated by a number of techniques, including in vitro mutagenesis - a step which could never be carried out using the traditional hybridoma technology. Monoclonal antibodies are and will remain an essential support for ongoing research on the prion protein in general and on the unconventional infectious prions.

Isolation of HIV-1-Neutralizing Mucosal Monoclonal Antibodies from Human Colostrum

PubMed Central

Friedman, James; Alam, S. Munir; Shen, Xiaoying; Xia, Shi-Mao; Stewart, Shelley; Anasti, Kara; Pollara, Justin; Fouda, Genevieve G.; Yang, Guang; Kelsoe, Garnett; Ferrari, Guido; Tomaras, Georgia D.; Haynes, Barton F.; Liao, Hua-Xin

2012-01-01

Background Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. Methods We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). Results The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. Conclusions These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces. PMID:22624058

Evaluation of Ion Mobility-Mass Spectrometry for Comparative Analysis of Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Ferguson, Carly N.; Gucinski-Ruth, Ashley C.

2016-05-01

Analytical techniques capable of detecting changes in structure are necessary to monitor the quality of monoclonal antibody drug products. Ion mobility mass spectrometry offers an advanced mode of characterization of protein higher order structure. In this work, we evaluated the reproducibility of ion mobility mass spectrometry measurements and mobiligrams, as well as the suitability of this approach to differentiate between and/or characterize different monoclonal antibody drug products. Four mobiligram-derived metrics were identified to be reproducible across a multi-day window of analysis. These metrics were further applied to comparative studies of monoclonal antibody drug products representing different IgG subclasses, manufacturers, and lots. These comparisons resulted in some differences, based on the four metrics derived from ion mobility mass spectrometry mobiligrams. The use of collision-induced unfolding resulted in more observed differences. Use of summed charge state datasets and the analysis of metrics beyond drift time allowed for a more comprehensive comparative study between different monoclonal antibody drug products. Ion mobility mass spectrometry enabled detection of differences between monoclonal antibodies with the same target protein but different production techniques, as well as products with different targets. These differences were not always detectable by traditional collision cross section studies. Ion mobility mass spectrometry, and the added separation capability of collision-induced unfolding, was highly reproducible and remains a promising technique for advanced analytical characterization of protein therapeutics.

Determination of influenza B identity and potency in quadrivalent inactivated influenza vaccines using lineage-specific monoclonal antibodies

PubMed Central

Verma, Swati; Soto, Jackeline; Vasudevan, Anupama; Schmeisser, Falko; Alvarado-Facundo, Esmeralda; Wang, Wei; Weiss, Carol D.

2017-01-01

Co-circulation of two antigenically and genetically distinct lineages of influenza B virus, represented by prototype viruses B/Victoria/2/1987 and B/Yamagata/16/1988, has led to the development of quadrivalent influenza vaccines that contain two influenza B antigens. The inclusion of two influenza B antigens presents challenges for the production and regulation of inactivated quadrivalent vaccines, including the potential for cross-reactivity of the reagents used in identity and potency assays because of the relative close relatedness of the hemagglutinin (HA) from the two virus lineages. Monoclonal antibodies (mAbs) specific for the two lineages of influenza B HA were generated and characterized and used to set-up simple identity tests that distinguish the influenza B antigens in inactivated trivalent and quadrivalent vaccines. The lineage-specific mAbs bound well to the HA of influenza B strains included in influenza vaccines over a period of more than 10 years, suggesting that identity tests using such lineage-specific mAbs would not necessarily have to be updated with every influenza B vaccine strain change. These lineage-specific mAbs were also used in an antibody capture ELISA format to quantify HA in vaccine samples, including monovalent, trivalent, and quadrivalent vaccine samples from various manufacturers. The results demonstrated correlation with HA values determined by the traditional single radial immunodiffusion (SRID) assay. Further, the antibody-capture ELISA was able to distinguish heat-stressed vaccine from unstressed vaccine, and was similar to the SRID in quantifying the resultant loss of potency. These mAb reagents should be useful for further development of antibody-based alternative influenza B identity and potency assays. PMID:28423025

Determination of influenza B identity and potency in quadrivalent inactivated influenza vaccines using lineage-specific monoclonal antibodies.

PubMed

Verma, Swati; Soto, Jackeline; Vasudevan, Anupama; Schmeisser, Falko; Alvarado-Facundo, Esmeralda; Wang, Wei; Weiss, Carol D; Weir, Jerry P

2017-01-01

Co-circulation of two antigenically and genetically distinct lineages of influenza B virus, represented by prototype viruses B/Victoria/2/1987 and B/Yamagata/16/1988, has led to the development of quadrivalent influenza vaccines that contain two influenza B antigens. The inclusion of two influenza B antigens presents challenges for the production and regulation of inactivated quadrivalent vaccines, including the potential for cross-reactivity of the reagents used in identity and potency assays because of the relative close relatedness of the hemagglutinin (HA) from the two virus lineages. Monoclonal antibodies (mAbs) specific for the two lineages of influenza B HA were generated and characterized and used to set-up simple identity tests that distinguish the influenza B antigens in inactivated trivalent and quadrivalent vaccines. The lineage-specific mAbs bound well to the HA of influenza B strains included in influenza vaccines over a period of more than 10 years, suggesting that identity tests using such lineage-specific mAbs would not necessarily have to be updated with every influenza B vaccine strain change. These lineage-specific mAbs were also used in an antibody capture ELISA format to quantify HA in vaccine samples, including monovalent, trivalent, and quadrivalent vaccine samples from various manufacturers. The results demonstrated correlation with HA values determined by the traditional single radial immunodiffusion (SRID) assay. Further, the antibody-capture ELISA was able to distinguish heat-stressed vaccine from unstressed vaccine, and was similar to the SRID in quantifying the resultant loss of potency. These mAb reagents should be useful for further development of antibody-based alternative influenza B identity and potency assays.

Mining Naïve Rabbit Antibody Repertoires by Phage Display for Monoclonal Antibodies of Therapeutic Utility.

PubMed

Peng, Haiyong; Nerreter, Thomas; Chang, Jing; Qi, Junpeng; Li, Xiuling; Karunadharma, Pabalu; Martinez, Gustavo J; Fallahi, Mohammad; Soden, Jo; Freeth, Jim; Beerli, Roger R; Grawunder, Ulf; Hudecek, Michael; Rader, Christoph

2017-09-15

Owing to their high affinities and specificities, rabbit monoclonal antibodies (mAbs) have demonstrated value and potential primarily as basic research and diagnostic reagents, but, in some cases, also as therapeutics. To accelerate access to rabbit mAbs bypassing immunization, we generated a large naïve rabbit antibody repertoire represented by a phage display library encompassing >10 billion independent antibodies in chimeric rabbit/human Fab format and validated it by next-generation sequencing. Panels of rabbit mAbs selected from this library against two emerging cancer targets, ROR1 and ROR2, revealed high diversity, affinity, and specificity. Moreover, ROR1- and ROR2-targeting rabbit mAbs demonstrated therapeutic utility as components of chimeric antigen receptor-engineered T cells, further corroborating the value of the naïve rabbit antibody library as a rich and virtually unlimited source of rabbit mAbs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, Martin; Watkins, Bruce E.; Stanker, Larry H.

1991-01-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described.

Structure of solid tumors and their vasculature: Implications for therapy with monoclonal antibodies

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

Dvorak, H.F.; Nagy, J.A.; Dvorak, A.M.

Delivery of monoclonal antibodies to solid tumors is a vexing problem that must be solved if these antibodies are to realize their promise in therapy. Such success as has been achieved with monoclonal antibodies is attributable to the local hyperpermeability of the tumor vasculature, a property that favors antibody extravasation at tumor sites and that is mediated by a tumor-secreted vascular permeability factor. However, leaky tumor blood vessels are generally some distance removed from target tumor cells, separated by stroma and by other tumor cells that together represent significant barriers to penetration by extravasated monoclonal antibodies. For this reason, alternativemore » approaches may be attractive. These include the use of antibody-linked cytotoxins, which are able to kill tumor cells without immediate contact, and direction of antibodies against nontumor cell targets, for example, antigens unique to the tumor vascular endothelium or to tumor stroma. 50 refs.« less

Purification and characterization of monoclonal antibodies to alpha-linolenic acid.

PubMed

Buffière, F; Cook-Moreau, J; Gualde, N; Rigaud, M

1989-01-01

The covalently linked antigenic complex, bovine serum albumin-alpha-linolenic acid, was used to immunize Balb/c mice against the hapten. Hybridization between splenocytes and the myeloma cell line, P 3 X63 Ag 8,651, resulted in stable clones synthesizing monoclonal antibodies (Mab) that were subsequently purified and characterized. Four Mab (A, B, C, D) were retained and their specificities studied by ELISA. Antibody D only recognized 18-carbon fatty acids having a cis,cis,-cis-1,4,7 unsaturated system in the omega-3 position: it was specific for alpha-linolenic acid. B recognized all fatty acids containing the structure cis,cis,cis-1,4,7-octatriene. A and C recognized polyunsaturated fatty acids with a degree of unsaturation superior to two double bonds.

Monoclonal antibody binding to the macrophage-specific receptor sialoadhesin alters the phagocytic properties of human and mouse macrophages.

PubMed

De Schryver, Marjorie; Cappoen, Davie; Elewaut, Dirk; Nauwynck, Hans J; Maes, Louis; Caljon, Guy; Cos, Paul; Delputte, Peter L

2017-02-01

Sialoadhesin (Sn) is a surface receptor expressed on macrophages in steady state conditions, but during inflammation, Sn can be upregulated both on macrophages and on circulating monocytes. It was shown for different species that Sn becomes internalized after binding with monoclonal antibodies. These features suggest that Sn is a potential target for immunotherapies. In this study, human and mouse macrophages were treated with anti-Sn monoclonal antibodies or F(ab') 2 fragments and the effect of their binding to Sn on phagocytosis was analyzed. Binding of antibodies to Sn resulted in delayed and reduced phagocytosis of fluorescent beads. No effect was observed on Fc-mediated phagocytosis or phagocytosis of bacteria by human macrophages. In contrast, an enhanced phagocytosis of bacteria by mouse macrophages was detected. These results showed that stimulation of Sn could have different effects on macrophage phagocytosis, depending both on the type of phagocytosis and cellular background. Copyright © 2016 Elsevier Inc. All rights reserved.

Human Monoclonal Antibody-Based Therapy in the Treatment of Invasive Candidiasis

PubMed Central

Bugli, Francesca; Cacaci, Margherita; Martini, Cecilia; Torelli, Riccardo; Posteraro, Brunella; Sanguinetti, Maurizio; Paroni Sterbini, Francesco

2013-01-01

Invasive candidiasis (IC) represents the leading fungal infection of humans causing life-threatening disease in immunosuppressed and neutropenic individuals including also the intensive care unit patients. Despite progress in recent years in drugs development for the treatment of IC, morbidity and mortality rates still remain very high. Historically, cell-mediated immunity and innate immunity are considered to be the most important lines of defense against candidiasis. Nevertheless recent evidence demonstrates that antibodies with defined specificities could act with different degrees showing protection against systemic and mucosal candidiasis. Mycograb is a human recombinant monoclonal antibody against heat shock protein 90 (Hsp90) that was revealed to have synergy when combined with fluconazole, caspofungin, and amphotericin B against a broad spectrum of Candida species. Furthermore, recent studies have established an important role for Hsp90 in mediating Candida resistance to echinocandins, giving to this antibody molecule even more attractive biological properties. In response to the failure of marketing authorization by the CHMP (Committee for Medicinal Products for Human Use) a new formulation of Mycograb, named Mycograb C28Y variant, with an amino acid substitution was developed in recent years. First data on Mycograb C28Y variant indicate that this monoclonal antibody lacked efficacy in a murine candidiasis model. PMID:23878583

Discovery of a Chemical Modification by Citric Acid in a Recombinant Monoclonal Antibody

PubMed Central

2015-01-01

Recombinant therapeutic monoclonal antibodies exhibit a high degree of heterogeneity that can arise from various post-translational modifications. The formulation for a protein product is to maintain a specific pH and to minimize further modifications. Generally Recognized as Safe (GRAS), citric acid is commonly used for formulation to maintain a pH at a range between 3 and 6 and is generally considered chemically inert. However, as we reported herein, citric acid covalently modified a recombinant monoclonal antibody (IgG1) in a phosphate/citrate-buffered formulation at pH 5.2 and led to the formation of so-called “acidic species” that showed mass increases of 174 and 156 Da, respectively. Peptide mapping revealed that the modification occurred at the N-terminus of the light chain. Three additional antibodies also showed the same modification but displayed different susceptibilities of the N-termini of the light chain, heavy chain, or both. Thus, ostensibly unreactive excipients under certain conditions may increase heterogeneity and acidic species in formulated recombinant monoclonal antibodies. By analogy, other molecules (e.g., succinic acid) with two or more carboxylic acid groups and capable of forming an anhydride may exhibit similar reactivities. Altogether, our findings again reminded us that it is prudent to consider formulations as a potential source for chemical modifications and product heterogeneity. PMID:25136741

Monoclonal Antibodies Against Fusicoccin with Binding Characteristics Similar to the Putative Fusicoccin Receptor of Higher Plants 1

PubMed Central

Feyerabend, Martin; Weiler, Elmar W.

1987-01-01

Monoclonal antibodies were raised against fusicoccin. The toxin, linked to bovine serum albumin through its t-pentenyl moiety, served as immunogen. Hybridomas secreting anti-fusicoccin antibodies were screened by radioimmunoassay employing a novel radioactive derivative, [3H]-nor-fusicoccin-alcohol of high specific activity (1.5 × 1014Bq/mole). The two monoclonal antibodies reported here are of high apparent affinity for fusicoccin (0.71 × 10−9 molar and 1.85 × 10−9 molar). This is comparable to the apparent affinity of rabbit antiserum raised against the same type of conjugate (9.3 × 10−9 molar). A method for the single step purification of the monoclonal antibodies from ascites fluid is reported. A solid-phase immunoassay, using alkaline phosphatase as enzyme, exhibits a measuring range from 0.1 to 1.5 picomoles (about 70 picograms to 1 nanogram) of fusicoccin. The displacement of [3H]-nor-fusicoccin-alcohol from the antibodies by compounds structurally related to fusicoccin exhibits similar selectivity as a microsomal binding assay with the same tracer as radiolabeled probe. Images Fig. 2 PMID:16665786

Use of polyclonal and monoclonal antibodies to study hCG-receptor interactions

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

Milius, R.P.

1985-01-01

Although the glycoprotein hormones lutropin (LH), follitropin (FSH), and thyrotropin (TSH) bind to different receptors, each contains an identical alpha subunit. Specificity is somehow endowed by theta subunits which are distinct for each hormone. Human choriogonadotropin (hCG) is a natural LH analog that contains a beta subunit nearly identical to that of LH. The roles of these subunits in the recognition and high affinity binding of hCG to receptor was examined. Polyclonal and monoclonal antibodies specific for the individual subunits of hCG were used to probe the hormone-receptor interaction. Conformation-specific and sequence-specific antibodies were examined for their abilities to bindmore » Triton X-100-solubilized /sup 125/I-hCG-receptor complex and to inhibit hormone binding to crude rat ovarian membranes containing receptor. Even though the immunoreactive sites are not located on the receptor binding surface of the beta subunit, most, but not all, of these polyclonal and monoclonal antibodies were able to inhibit /sup 125/I-hCG binding to receptor. Although the inhibition of binding may be due to steric interference due to the size of the antibody molecules, a two-step model for hCG binding to receptor is presented that also explains these results. In this model, the beta subunit initially binds with the receptor with a highly specific but low affinity interaction. This activates a site for the high affinity binding of the alpha subunit and stabilization of the complex. This is an attractive model as it may be applied to other glycoprotein hormones sharing an alpha subunit.« less

Indium-111 labeled anti-melanoma monoclonal antibodies

DOEpatents

Srivastava, S.C.; Fawwaz, R.A.; Ferrone, S.

1984-04-30

A monoclonal antibody to a high molecular weight melanoma-associated antigen was chelated and radiolabeled with indium-111. This material shows high affinity for melanoma and thus can be used in the detection, localization and imaging of melanoma. 1 figure.

Epidermal growth factor receptor mutations in adenocarcinoma in situ and minimally invasive adenocarcinoma detected using mutation-specific monoclonal antibodies.

PubMed

Nakamura, Haruhiko; Koizumi, Hirotaka; Kimura, Hiroyuki; Marushima, Hideki; Saji, Hisashi; Takagi, Masayuki

2016-09-01

Epidermal growth factor receptor (EGFR) mutation rates in adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) were studied using both DNA analysis and mutation-specific immunohistochemistry. The peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp method was used to detect mutations in exons 18, 19, 20, and 21 of the EGFR gene in DNA samples extracted from paraffin-embedded tissue sections. Simultaneously, immunohistochemical analysis with two EGFR mutation-specific monoclonal antibodies was used to identify proteins resulting from an in-frame deletion in exon 19 (E746_A750del) and a point mutation replacing leucine with arginine at codon 858 of exon 21 (L858R). Forty-three tumors (22 AIS and 21 MIA) were examined. The EGFR mutation rate in AIS detected by DNA analysis was 27.3% (L858R, 5/22; exon 19 deletion,1/22), whereas that detected in MIA was 42.9% (L858R,4/21; exon 19 deletion,5/21). Mutations detected by immunohistochemical analysis included 22.7% (L858R, 4/22; exon 19 deletion, 1/22) in AIS and 42.9% (L858R, 4/21; exon 19 deletion, 5/21) in MIA. Although some results were contradictory, concordant results were obtained using both assays in 38 of 43 cases (88.4%). DNA and immunohistochemical analyses revealed similar EGFR mutation rates in both MIA and AIS, suggesting that mutation-specific monoclonal antibodies are useful to confirm DNA assay results. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Development of broad-spectrum human monoclonal antibodies for rabies post-exposure prophylaxis.

PubMed

De Benedictis, Paola; Minola, Andrea; Rota Nodari, Elena; Aiello, Roberta; Zecchin, Barbara; Salomoni, Angela; Foglierini, Mathilde; Agatic, Gloria; Vanzetta, Fabrizia; Lavenir, Rachel; Lepelletier, Anthony; Bentley, Emma; Weiss, Robin; Cattoli, Giovanni; Capua, Ilaria; Sallusto, Federica; Wright, Edward; Lanzavecchia, Antonio; Bourhy, Hervé; Corti, Davide

2016-04-01

Currently available rabies post-exposure prophylaxis (PEP) for use in humans includes equine or human rabies immunoglobulins (RIG). The replacement of RIG with an equally or more potent and safer product is strongly encouraged due to the high costs and limited availability of existing RIG. In this study, we identified two broadly neutralizing human monoclonal antibodies that represent a valid and affordable alternative to RIG in rabies PEP. Memory B cells from four selected vaccinated donors were immortalized and monoclonal antibodies were tested for neutralizing activity and epitope specificity. Two antibodies, identified as RVC20 and RVC58 (binding to antigenic site I and III, respectively), were selected for their potency and broad-spectrum reactivity. In vitro, RVC20 and RVC58 were able to neutralize all 35 rabies virus (RABV) and 25 non-RABV lyssaviruses. They showed higher potency and breath compared to antibodies under clinical development (namely CR57, CR4098, and RAB1) and commercially available human RIG. In vivo, the RVC20-RVC58 cocktail protected Syrian hamsters from a lethal RABV challenge and did not affect the endogenous hamster post-vaccination antibody response. © 2016 Humabs BioMed SA Published under the terms of the CC BY 4.0 license.

[Production of the monoclonal antibodies to the rabies virus nucleoprotein].

PubMed

Gribencha, S V; Kozlov, A Iu; Kostina, L V; Elakov, A L; Losich, M A; Tsibezov, V V; Zaberezhnyĭ, A D; Aliper, T I

2013-01-01

Five hybridomas secreting monoclonal antibodies (MAbs) for the nucleocapsid protein of the rabies virus were obtained through the fusion of the SP2/0 murine myeloma cells with splenocytes of BALB/c mice immunized with fixed rabies virus (CVS strain). All hybridomas secret MAbs of the IgG class that display different specificity to the nucleocapsids of rabies and rabies-related viruses. MAbs 2ell showed the specificity for the prevalent in Russia rabies viruses that are similar to commercially available anti-rabies conjugate.

Immunohistochemical and ultrastructural detection of advanced glycation end products in atherosclerotic lesions of human aorta with a novel specific monoclonal antibody.

PubMed Central

Kume, S.; Takeya, M.; Mori, T.; Araki, N.; Suzuki, H.; Horiuchi, S.; Kodama, T.; Miyauchi, Y.; Takahashi, K.

1995-01-01

To elucidate the deposition of advanced glycation end products (AGEs) in aortic atherosclerosis, aortic walls were obtained from 25 autopsy cases and examined immunohistochemically and immunoelectron microscopically with a monoclonal antibody specific for AGEs, 6D12. Among the autopsy cases, atherosclerotic lesions were found in the aortas of 22 cases and were composed of diffuse intimal thickening, fatty streaks, atherosclerotic plaques, and/or complicated lesions. In these cases, intracellular AGE accumulation was demonstrated in the intimal lesions of aortic atherosclerosis in 12 cases. Compared with the diffuse intimal thickening, intracellular AGE accumulation was marked in the fatty streaks and atherosclerotic plaques. Immunohistochemical double staining with 6D12 and monoclonal antibodies for macrophages or muscle actin or a polyclonal antibody for scavenger receptors demonstrated that the AGE accumulation in macrophages or their related foam cells was marked in the diffuse intimal thickening and fatty streak lesions and that almost all macrophages and macrophage-derived foam cells possessed scavenger receptors. Immunoelectron microscopic observation revealed the localization of 6D12-positive reaction in lysosomal lipid vacuoles or electron-dense granules of the foam cells. These results indicate that AGE accumulation occurs in macrophages, smooth muscle cells, and their related foam cells. Images Figure 2 Figure 3 Figure 6 PMID:7545874

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

PubMed

Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

2002-10-01

We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed

Heyworth, M F; Ho, K E; Pappo, J

1989-11-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice.

Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

PubMed Central

Wang, Joshua W.; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P.; Macgregor-Das, Anne; Fogel, Jessica M.; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L.; Gravitt, Patti E.; Trimble, Cornelia L.

2015-01-01

Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies. Therefore, we screened a total of 1,078 serum samples for HPV16 L2 reactivity, and these were obtained from four prior clinical studies: a population-based (n = 880) surveillance study with a high-risk HPV DNA prevalence of 10.8%, a cohort study of women (n = 160) with high-grade cervical intraepithelial neoplasia (CIN), and two phase II trials in women with high-grade vulvar intraepithelial neoplasia (VIN) receiving imiquimod therapy combined with either photodynamic therapy (PDT) (n = 19) or vaccination with a fusion protein comprising HPV16 L2, E7, and E6 (TA-CIN) (n = 19). Sera were screened sequentially by HPV16 L2 enzyme-linked immunosorbent assay (ELISA) and then Western blot. Seven of the 1,078 serum samples tested had L2-specific antibodies, but none were detectably neutralizing for HPV16. To develop a standard, we substituted human IgG1 sequences into conserved regions of two rodent monoclonal antibodies (MAbs) specific for neutralizing epitopes at HPV16 L2 residues 17 to 36 and 58 to 64, creating JWW-1 and JWW-2, respectively. These chimeric MAbs retained neutralizing activity and together reacted with 33/34 clinically relevant HPV types tested. In conclusion, our inability to identify an HPV16 L2-specific neutralizing antibody response even in the sera of patients with active genital HPV disease suggests the subdominance of L2 protective epitopes and the value of the chimeric MAbs JWW-1 and JWW-2 as standards for immunoassays to measure L2-specific human antibodies. PMID:25972404

Phase Separation in Solutions of Monoclonal Antibodies

NASA Astrophysics Data System (ADS)

Benedek, George; Wang, Ying; Lomakin, Aleksey; Latypov, Ramil

2012-02-01

We report the observation of liquid-liquid phase separation (LLPS) in a solution of humanized monoclonal antibodies, IgG2, and the effects of human serum albumin, a major blood protein, on this phase separation. We find a significant reduction of phase separation temperature in the presence of albumin, and a preferential partitioning of the albumin into the antibody-rich phase. We provide a general thermodynamic analysis of the antibody-albumin mixture phase diagram and relate its features to the magnitude of the effective inter-protein interactions. Our analysis suggests that additives (HSA in this report), which have moderate attraction with antibody molecules, may be used to forestall undesirable protein condensation in antibody solutions. Our findings are relevant to understanding the stability of pharmaceutical solutions of antibodies and the mechanisms of cryoglobulinemia.

Vaccine and Monoclonal Antibody That Enhance Mouse Resistance to Candidiasis ▿

PubMed Central

Xin, Hong; Cutler, Jim E.

2011-01-01

Previously we showed that antibodies specific for the glycan β-1,2-mannotriose [β-(Man)3] on the cell surface of Candida albicans protect mice against disseminated candidiasis (H. Xin, S. Dziadek, D. R. Bundle, and J. E. Cutler, Proc. Natl. Acad. Sci. U. S. A. 105:13526–13531, 2008). Furthermore, six 14-mer peptides that are within the N-terminal portion of C. albicans wall proteins were conjugated to the glycan in an attempt to create immunogenic glycopeptide conjugates. By a dendritic cell (DC)-based immunization approach, all were immunogenic and three of the six conjugates induced a high degree of protection in mice. Interestingly, whereas all six peptides induced antibody responses when used alone to pulse DCs for subsequent immunizations, three peptides induced protection, and one in particular, peptide Fba (derived from fructose-bisphosphate aldolase), induced robust protective responses and is the focus of the current work. Fba peptide is not restricted by the major histocompatibility complex class II (MHC-II), as it induced anti-Fba antibodies in mice of different H-2 haplotypes and in rabbits. Furthermore, the peptide induced protection against disease caused by different C. albicans strains. Partial protection was achieved when alum was used in place of DCs for Fba immunizations. The passive transfer of immune sera from Fba-vaccinated mice, but not immune serum preabsorbed with fungal cells, conferred protection in naïve mice. This result, along with our finding that a monoclonal antibody specific for the peptide, E2-9 (IgM), protected mice against candidiasis, provide strong evidence that antibodies contribute to protection. Our work demonstrates the utility of cell wall peptides alone or as glycopeptides in vaccines designed for the induction of immunity against candidiasis and monoclonal antibodies as a rapid immunoprotective approach against the disease. PMID:21832099

Monoclonal antibodies for the separate detection of halodeoxyuridines and method for their use

DOEpatents

Vanderlaan, M.; Watkins, B.E.; Stanker, L.H.

1991-10-01

Monoclonal antibodies are described which have specific affinities for halogenated nucleoside analogs and are preferentially selective for one particular halogen. Such antibodies, when incorporated into immunochemical reagents, may be used to identify and independently quantify the cell division character of more than one population or subpopulation in flow cytometric measurements. Independent assessment of division activity in cell sub-populations facilitates selection of appropriate time and dose for administration of anti-proliferative agents. The hybridomas which secrete halogen selective antibodies and the method of making them are described. 14 figures.

An antagonistic monoclonal antibody (B-N6) specific for the human neurotensin receptor-1.

PubMed

Ovigne, J M; Vermot-Desroches, C; Lecron, J C; Portier, M; Lupker, J; Pecceu, F; Wijdenes, J

1998-06-01

The neuropeptide neurotensin (NT) interacts with two types of human receptors (hNTR) termed hNTR-1 and hNTR-2. This study describes a monoclonal antibody (MAb) specific for hNTR-1, B-N6. This MAb binds specifically to hNTR-1, but not to hNTR-2 transfected CHO cells. B-N6 and NT display a reciprocal competition and react in a similar way to trypsin, suggesting that the B-N6 epitope is at or close to the NT binding site on the third extracellular loop. Unlike B-N6, NT induces hNTR-1 internalization. Although neither NT-FITC nor B-N6 binding was detected by flow cytometry on different human cells, specific mRNA expression for hNTR-1 was detected in these cells. In CHO cells expressing hNTR-1 and a luciferase gene coupled to the krox24 reporter, B-N6 and the antagonist SR 48692 inhibited NT-induced intracellular activation of krox24 in a dose-dependent manner. From these results it is concluded that B-N6 is an antagonistic anti-hNTR-1 MAb.

Prevention of Herpes Simplex Virus Induced Stromal Keratitis by a Glycoprotein B-Specific Monoclonal Antibody

PubMed Central

Krawczyk, Adalbert; Dirks, Miriam; Kasper, Maren; Buch, Anna; Dittmer, Ulf; Giebel, Bernd; Wildschütz, Lena; Busch, Martin; Goergens, Andre; Schneweis, Karl E.; Eis-Hübinger, Anna M.; Sodeik, Beate; Heiligenhaus, Arnd; Roggendorf, Michael; Bauer, Dirk

2015-01-01

The increasing incidence of acyclovir (ACV) and multidrug-resistant strains in patients with corneal HSV-1 infections leading to Herpetic Stromal Keratitis (HSK) is a major health problem in industrialized countries and often results in blindness. To overcome this obstacle, we have previously developed an HSV-gB-specific monoclonal antibody (mAb 2c) that proved to be highly protective in immunodeficient NOD/SCID-mice towards genital infections. In the present study, we examined the effectivity of mAb 2c in preventing the immunopathological disease HSK in the HSK BALB/c mouse model. Therefore, mice were inoculated with HSV-1 strain KOS on the scarified cornea to induce HSK and subsequently either systemically or topically treated with mAb 2c. Systemic treatment was performed by intravenous administration of mAb 2c 24 h prior to infection (pre-exposure prophylaxis) or 24, 40, and 56 hours after infection (post-exposure immunotherapy). Topical treatment was performed by periodical inoculations (5 times per day) of antibody-containing eye drops as control, starting at 24 h post infection. Systemic antibody treatment markedly reduced viral loads at the site of infection and completely protected mice from developing HSK. The administration of the antiviral antibody prior or post infection was equally effective. Topical treatment had no improving effect on the severity of HSK. In conclusion, our data demonstrate that mAb 2c proved to be an excellent drug for the treatment of corneal HSV-infections and for prevention of HSK and blindness. Moreover, the humanized counterpart (mAb hu2c) was equally effective in protecting mice from HSV-induced HSK when compared to the parental mouse antibody. These results warrant the future development of this antibody as a novel approach for the treatment of corneal HSV-infections in humans. PMID:25587898

Radiolabeled monoclonal antibodies (for the radiology administrator).

PubMed

Wahl, R

1992-11-01

Cheaper, faster, safer, these are not the attributes of 1993 automobiles, but criteria for new diagnostic tests in medicine. To achieve these characteristics, medicine is increasingly looking to biotechnology for answers. And the mother of all biotechnology is monoclonal antibody research. In past issues, Administrative Radiology published articles discussing the role of biotechnology in the development of radiopharmaceuticals used in nuclear medicine. In this issue, Richard Wahl, M.D., reviews, in plain talk, the current status and prospects for diagnostic imaging with radiolabeled monoclonal antibodies. Are there any such procedures of value today? Are there any that are FDA approved? Will there ever be such agents that are either useful or approved? If so, will any insurance carrier pay for them? For the answers to these and other "hot" questions, the reader is encouraged to continue on and read this month's Technology Review section.

Monoclonal antibodies to Mojave toxin and use for isolation of cross-reacting proteins in Crotalus venoms.

PubMed

Rael, E D; Salo, R J; Zepeda, H

1986-01-01

Hybridomas secreting monoclonal antibodies against Mojave toxin were established. The antibodies were used for identifying cross-reacting proteins in individual C. s. scutulatus and other Crotalus venoms and to isolate Mojave toxin. The antibodies recognized five bands with a pI range from 5.1 to 6.1 in immunoblots of electrofocused crude venom and Mojave toxin purified by immunoaffinity chromatography. The specificity of the antibodies was for the basic subunit of the toxin, which resolved into four bands of pI between 9.3 and 9.6. Individual C. s. scutulatus venoms of snakes from Texas and southern Arizona had multiple bands with pI's ranging from 4.9 to 6.3. Cross-reacting proteins were also recognized by the antibodies in the electrophoresed venoms of C. basiliscus, C. d. durissus, C. d. terrificus, C. h. horridus and C. v. concolor, and may be isolated by immunoaffinity chromatography with the monoclonal antibodies.

Identification of secreted and membrane-bound bat immunoglobulin using a Microchiropteran-specific mouse monoclonal antibody.

PubMed

Lee, William T; Jones, Derek D; Yates, Jennifer L; Winslow, Gary M; Davis, April D; Rudd, Robert J; Barron, Christopher T; Cowan, Cailyn

2016-12-01

Bat immunity has received increasing attention because some bat species are being decimated by the fungal disease, White Nose Syndrome, while other species are potential reservoirs of zoonotic viruses. Identifying specific immune processes requires new specific tools and reagents. In this study, we describe a new mouse monoclonal antibody (mAb) reactive with Eptesicus fuscus immunoglobulins. The epitope recognized by mAb BT1-4F10 was localized to immunoglobulin light (lambda) chains; hence, the mAb recognized serum immunoglobulins and B lymphocytes. The BT1-4F10 epitope appeared to be restricted to Microchiropteran immunoglobulins and absent from Megachiropteran immunoglobulins. Analyses of sera and other E. fuscus fluids showed that most, if not all, secreted immunoglobulins utilized lambda light chains. Finally, mAb BT1-4F10 permitted the identification of B cell follicles in splenic white pulp. This Microchiropteran-specific mAb has potential utility in seroassays; hence, this reagent may have both basic and practical applications for studying immune process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mice are actively immunized after passive monoclonal antibody prophylaxis and ricin toxin challenge. (Reannouncement with new availability information)

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

Lemley, P.V.; Wright, D.C.

1992-12-31

Mice passively immunized by a protective, anti-ricin A-chain monoclonal antibody, then challenged intravenously with ricin, were protected from a subsequent ricin challenge, and were actively immunized. Two significant advantages accrued from this experiment: the monoclonal antibody neutralized the toxicity of the ricin immunogen, and active immunization was achieved with very low antigen load (approx. 0.5 micrograms/mouse). We ruled out the possibility that residual monoclonal antibody provided the protection by using three independent criteria. There was significant (four orders of magnitude) enhancement of the immune response in the presence of the monoclonal antibody; control immunizations of mice with ricin A-chain, ricinmore » B-chain or either chain with the monoclonal antibody did not induce active immunity; and the active immunization could not be replicated when protective goat polyclonal antibody was substituted for the monoclonal antibody. Because high titers were achieved rapidly without any adjuvant, we are currently investigating haptenized ricin to determine if anti-hapten monoclonal antibodies can be produced by this refined procedure.« less

Emerging monoclonal antibodies against Clostridium difficile infection.

PubMed

Péchiné, Séverine; Janoir, Claire; Collignon, Anne

2017-04-01

Clostridium difficile infections are characterized by a high recurrence rate despite antibiotic treatments and there is an urgent need to develop new treatments such as fecal transplantation and immonotherapy. Besides active immunotherapy with vaccines, passive immunotherapy has shown promise, especially with monoclonal antibodies. Areas covered: Herein, the authors review the different assays performed with monoclonal antibodies against C. difficile toxins and surface proteins to treat or prevent primary or recurrent episodes of C. difficile infection in animal models and in clinical trials as well. Notably, the authors lay emphasis on the phase III clinical trial (MODIFY II), which allowed bezlotoxumab to be approved by the Food and Drug Administration and the European Medicines Agency. They also review new strategies for producing single domain antibodies and nanobodies against C. difficile and new approaches to deliver them in the digestive tract. Expert opinion: Only two human Mabs against TcdA and TcdB have been tested alone or in combination in clinical trials. However, many animal model studies have provided rationale for the use of Mabs and nanobodies in C. difficile infection and pave the way for further clinical investigation.

Intermolecular Interactions and the Viscosity of Highly Concentrated Monoclonal Antibody Solutions.

PubMed

Binabaji, Elaheh; Ma, Junfen; Zydney, Andrew L

2015-09-01

The large increase in viscosity of highly concentrated monoclonal antibody solutions can be challenging for downstream processing, drug formulation, and delivery steps. The objective of this work was to examine the viscosity of highly concentrated solutions of a high purity IgG1 monoclonal antibody over a wide range of protein concentrations, solution pH, ionic strength, and in the presence / absence of different excipients. Experiments were performed with an IgG1 monoclonal antibody provided by Amgen. The steady-state viscosity was evaluated using a Rheometrics strain-controlled rotational rheometer with a concentric cylinder geometry. The viscosity data were well-described by the Mooney equation. The data were analyzed in terms of the antibody virial coefficients obtained from osmotic pressure data evaluated under the same conditions. The viscosity coefficient in the absence of excipients was well correlated with the third osmotic virial coefficient, which has a negative value (corresponding to short range attractive interactions) at the pH and ionic strength examined in this work. These results provide important insights into the effects of intermolecular protein-protein interactions on the behavior of highly concentrated antibody solutions.

Monoclonal Antibodies Passively Protect BALB/c Mice Against Burkholderia mallei Aerosol Challenge

DTIC Science & Technology

2006-03-01

November 2005 Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei , the... Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei , the etiologic... Burkholderia mallei auxotroph protects against aerosol-initiated glanders in mice. Vaccine 23:1986–1992. 17. Vyshelesskii, S. N. 1974. Glanders (Equinia). Tr

Monoclonal antibodies and toxins--a perspective on function and isotype.

PubMed

Chow, Siu-Kei; Casadevall, Arturo

2012-06-01

Antibody therapy remains the only effective treatment for toxin-mediated diseases. The development of hybridoma technology has allowed the isolation of monoclonal antibodies (mAbs) with high specificity and defined properties, and numerous mAbs have been purified and characterized for their protective efficacy against different toxins. This review summarizes the mAb studies for 6 toxins--Shiga toxin, pertussis toxin, anthrax toxin, ricin toxin, botulinum toxin, and Staphylococcal enterotoxin B (SEB)--and analyzes the prevalence of mAb functions and their isotypes. Here we show that most toxin-binding mAbs resulted from immunization are non-protective and that mAbs with potential therapeutic use are preferably characterized. Various common practices and caveats of protection studies are discussed, with the goal of providing insights for the design of future research on antibody-toxin interactions.

Development and characterization of mouse monoclonal antibodies reactive with chicken IL1 Beta

USDA-ARS?s Scientific Manuscript database

Two mouse monoclonal antibodies (mAbs) specific for chicken interleukin-1 Beta (chIL-1 Beta) were produced and characterized. Both mAbs identified a 66.0 kDa recombinant protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant...

Detection and Quantitation of Afucosylated N-Linked Oligosaccharides in Recombinant Monoclonal Antibodies Using Enzymatic Digestion and LC-MS

NASA Astrophysics Data System (ADS)

Du, Yi; May, Kimberly; Xu, Wei; Liu, Hongcheng

2012-07-01

The presence of N-linked oligosaccharides in the CH2 domain has a significant impact on the structure, stability, and biological functions of recombinant monoclonal antibodies. The impact is also highly dependent on the specific oligosaccharide structures. The absence of core-fucose has been demonstrated to result in increased binding affinity to Fcγ receptors and, thus, enhanced antibody-dependent cellular cytotoxicity (ADCC). Therefore, a method that can specifically determine the level of oligosaccharides without the core-fucose (afucosylation) is highly desired. In the current study, recombinant monoclonal antibodies and tryptic peptides from the antibodies were digested using endoglycosidases F2 and H, which cleaves the glycosidic bond between the two primary GlcNAc residues. As a result, various oligosaccharides of either complex type or high mannose type that are commonly observed for recombinant monoclonal antibodies are converted to either GlcNAc residue only or GlcNAc with the core-fucose. The level of GlcNAc represents the sum of all afucosylated oligosaccharides, whereas the level of GlcNAc with the core-fucose represents the sum of all fucosylated oligosaccharides. LC-MS analysis of the enzymatically digested antibodies after reduction provided a quick estimate of the levels of afucosylation. An accurate determination of the level of afucosylation was obtained by LC-MS analysis of glycopeptides after trypsin digestion.

Effect of kinase inhibitors on the therapeutic properties of monoclonal antibodies

PubMed Central

Duong, Minh Ngoc; Matera, Eva-Laure; Mathé, Doriane; Evesque, Anne; Valsesia-Wittmann, Sandrine; Clémenceau, Béatrice; Dumontet, Charles

2015-01-01

Targeted therapies of malignancies currently consist of therapeutic monoclonal antibodies and small molecule kinase inhibitors. The combination of these novel agents raises the issue of potential antagonisms. We evaluated the potential effect of 4 kinase inhibitors, including the Bruton tyrosine kinase inhibitor ibrutinib, and 3 PI3K inhibitors idelalisib, NVP-BEZ235 and LY294002, on the effects of the 3 monoclonal antibodies, rituximab and obinutuzumab (directed against CD20) and trastuzumab (directed against HER2). We found that ibrutinib potently inhibits antibody-dependent cell-mediated cytotoxicity exerted by all antibodies, with a 50% inhibitory concentration of 0.2 microM for trastuzumab, 0.5 microM for rituximab and 2 microM for obinutuzumab, suggesting a lesser effect in combination with obinutuzumab than with rituximab. The 4 kinase inhibitors were found to inhibit phagocytosis by fresh human neutrophils, as well as antibody-dependent cellular phagocytosis induced by the 3 antibodies. Conversely co-administration of ibrutinib with rituximab, obinutuzumab or trastuzumab did not demonstrate any inhibitory effect of ibrutinib in vivo in murine xenograft models. In conclusion, some kinase inhibitors, in particular, ibrutinib, are likely to exert inhibitory effects on innate immune cells. However, these effects do not compromise the antitumor activity of monoclonal antibodies in vivo in the models that were evaluated. PMID:25523586

Customizing monoclonal antibodies for the treatment of methamphetamine abuse: current and future applications.

PubMed

Peterson, Eric C; Gentry, W Brooks; Owens, S Michael

2014-01-01

Monoclonal antibody-based medications designed to bind (+)-methamphetamine (METH) with high affinity are among the newest approaches to the treatment of METH abuse and the associated medical complications. The potential clinical indications for these medications include treatment of overdose, reduction of drug dependence, and protection of vulnerable populations from METH-related complications. Research designed to discover and conduct preclinical and clinical testing of these antibodies suggests a scientific vision for how intact monoclonal antibody (mAb) (singular and plural) or small antigen-binding fragments of mAb could be engineered to optimize the proteins for specific therapeutic applications. In this review, we discuss keys to success in this development process including choosing predictors of specificity, efficacy, duration of action, and safety of the medications in disease models of acute and chronic drug abuse. We consider important aspects of METH-like hapten design and how hapten structural features influence specificity and affinity, with an example of a high-resolution X-ray crystal structure of a high-affinity antibody to demonstrate this structural relationship. Additionally, several prototype anti-METH mAb forms such as antigen-binding fragments and single-chain variable fragments are under development. Unique, customizable aspects of these fragments are presented with specific possible clinical indications. Finally, we discuss clinical trial progress of the first in kind anti-METH mAb, for which METH is the disease target instead of vulnerable central nervous system networks of receptors, binding sites, and neuronal connections. © 2014 Elsevier Inc. All rights reserved.

Determination of the specificity of monoclonal antibodies against Schistosoma mansoni CAA glycoprotein antigen using neoglycoconjugate variants.

PubMed

Carvalho de Souza, Adriana; van Remoortere, Alexandra; Hokke, Cornelis H; Deelder, André M; Vliegenthart, Johannes F G; Kamerling, Johannis P

2005-09-01

The immunogenic O-glycan of circulating anodic antigen (CAA) is a high-molecular-mass polysaccharide with the unique -->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GalpNAc-(1--> repeating unit. To obtain information at the molecular level about the specificity of monoclonal antibodies against CAA, the immunoreactivity of two series of bovine serum albumin-coupled synthetic oligosaccharides related to the CAA O-glycan was monitored using ELISA and surface plasmon resonance spectroscopy. The importance of the axial hydroxyl group of beta-D-GalpNAc for antibody binding was investigated using the following series of analogues: beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->O); beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O); and beta-D-GlcpA-(1-->3)-beta-D-GlcpNAc-(1-->6)-[beta-D-GlcpA-(1-->3)]-beta-D-GlcpNAc-(1-->O). In the second series of analogues, beta-D-Glcp6S-(1-->3)-beta-D-GalpNAc-(1-->O), beta-D-GalpNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), and beta-D-Glcp6S-(1-->3)-beta-D-Gal-pNAc-(1-->6)-[beta-D-Glcp6S-(1-->3)]-beta-D-GalpNAc-(1-->O), the native beta-D-GlcpA moiety was replaced by beta-D-Glcp6S to evaluate the influence of the nature of the charge on antibody recognition. Comparison of the immunoreactivity of these series with that measured for conjugates containing corresponding synthetic CAA fragments showed that the antibody binding levels can be correlated to the antibody specificity to CAA fragments. For the most reactive antibodies, the structural changes chosen (beta-D-GalpNAc replaced by beta-D-GlcpNAc, and beta-D-GlcpA replaced by beta-D-Glcp6S) completely eradicated the binding.

A Monoclonal Antibody Toolkit for C. elegans

PubMed Central

Hadwiger, Gayla; Dour, Scott; Arur, Swathi; Fox, Paul; Nonet, Michael L.

2010-01-01

Background Antibodies are critical tools in many avenues of biological research. Though antibodies can be produced in the research laboratory setting, most research labs working with vertebrates avail themselves of the wide array of commercially available reagents. By contrast, few such reagents are available for work with model organisms. Methodology/Principal Findings We report the production of monoclonal antibodies directed against a wide range of proteins that label specific subcellular and cellular components, and macromolecular complexes. Antibodies were made to synaptobrevin (SNB-1), a component of synaptic vesicles; to Rim (UNC-10), a protein localized to synaptic active zones; to transforming acidic coiled-coil protein (TAC-1), a component of centrosomes; to CENP-C (HCP-4), which in worms labels the entire length of their holocentric chromosomes; to ORC2 (ORC-2), a subunit of the DNA origin replication complex; to the nucleolar phosphoprotein NOPP140 (DAO-5); to the nuclear envelope protein lamin (LMN-1); to EHD1 (RME-1) a marker for recycling endosomes; to caveolin (CAV-1), a marker for caveolae; to the cytochrome P450 (CYP-33E1), a resident of the endoplasmic reticulum; to β-1,3-glucuronyltransferase (SQV-8) that labels the Golgi; to a chaperonin (HSP-60) targeted to mitochondria; to LAMP (LMP-1), a resident protein of lysosomes; to the alpha subunit of the 20S subcomplex (PAS-7) of the 26S proteasome; to dynamin (DYN-1) and to the α-subunit of the adaptor complex 2 (APA-2) as markers for sites of clathrin-mediated endocytosis; to the MAGUK, protein disks large (DLG-1) and cadherin (HMR-1), both of which label adherens junctions; to a cytoskeletal linker of the ezrin-radixin-moesin family (ERM-1), which localized to apical membranes; to an ERBIN family protein (LET-413) which localizes to the basolateral membrane of epithelial cells and to an adhesion molecule (SAX-7) which localizes to the plasma membrane at cell-cell contacts. In addition to working

Development and characterization of mouse monoclonal antibodies reactive with chicken IL-1ß

USDA-ARS?s Scientific Manuscript database

Two mouse monoclonal antibodies (mAbs) specific for chicken interleukin-1ß (chIL-1ß) were produced and characterized. Both mAbs identified a 66.0 kDa recombinant protein expressed in Escherichia coli by Western blot analysis that corresponded to the expected molecular weight of a recombinant fusion ...

Method of rapid production of hybridomas expressing monoclonal antibodies on the cell surface

DOEpatents

Meagher, Richard B.; Laterza, Vince

2006-12-12

The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

A Protein-Conjugate Approach to Develop a Monoclonal Antibody-Based Antigen Detection Test for the Diagnosis of Human Brucellosis

PubMed Central

Patra, Kailash P.; Saito, Mayuko; Atluri, Vidya L.; Rolán, Hortensia G.; Young, Briana; Kerrinnes, Tobias; Smits, Henk; Ricaldi, Jessica N.; Gotuzzo, Eduardo; Gilman, Robert H.; Tsolis, Renee M.; Vinetz, Joseph M.

2014-01-01

Human brucellosis is most commonly diagnosed by serology based on agglutination of fixed Brucella abortus as antigen. Nucleic acid amplification techniques have not proven capable of reproducibly and sensitively demonstrating the presence of Brucella DNA in clinical specimens. We sought to optimize a monoclonal antibody-based assay to detect Brucella melitensis lipopolysaccharide in blood by conjugating B. melitensis LPS to keyhole limpet hemocyanin, an immunogenic protein carrier to maximize IgG affinity of monoclonal antibodies. A panel of specific of monoclonal antibodies was obtained that recognized both B. melitensis and B. abortus lipopolysaccharide epitopes. An antigen capture assay was developed that detected B. melitensis in the blood of experimentally infected mice and, in a pilot study, in naturally infected Peruvian subjects. As a proof of principle, a majority (7/10) of the patients with positive blood cultures had B. melitensis lipopolysaccharide detected in the initial blood specimen obtained. One of 10 patients with relapsed brucellosis and negative blood culture had a positive serum antigen test. No seronegative/blood culture negative patients had a positive serum antigen test. Analysis of the pair of monoclonal antibodies (2D1, 2E8) used in the capture ELISA for potential cross-reactivity in the detection of lipopolysaccharides of E. coli O157:H7 and Yersinia enterocolitica O9 showed specificity for Brucella lipopolysaccharide. This new approach to develop antigen-detection monoclonal antibodies against a T cell-independent polysaccharide antigen based on immunogenic protein conjugation may lead to the production of improved rapid point-of-care-deployable assays for the diagnosis of brucellosis and other infectious diseases. PMID:24901521

Identification of whole pathogenic cells by monoclonal antibodies generated against a specific peptide from an immunogenic cell wall protein.

PubMed

Kaba, Hani E J; Maier, Natalia; Schliebe-Ohler, Nicole; Mayer, Yvonne; Müller, Peter P; van den Heuvel, Joop; Schuchhardt, Johannes; Hanack, Katja; Bilitewski, Ursula

2015-01-01

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Preparation and characterization of monoclonal antibody against digoxin.

PubMed

Kashanian, S; Rasaee, M J; Paknejad, M; Omidfar, K; Pour-Amir, M; Rajabi, Bazl M

2002-10-01

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

Generation and characterization of monoclonal antibodies against Giardia muris trophozoites.

PubMed Central

Heyworth, M F; Ho, K E; Pappo, J

1989-01-01

Mouse monoclonal antibodies (mAb) were produced against Giardia muris trophozoite surface antigens. To generate B-cell hybridomas, P3/NS1/1-Ag4-1 myeloma cells were fused with splenic lymphocytes from BALB/c mice that had been immunized parenterally with G. muris trophozoites. Hybridoma culture supernatants were screened for mAb by flow cytometry of G. muris trophozoites incubated with culture supernatant followed by fluorescein-conjugated anti-mouse IgG and IgM. Flow cytometry showed three types of trophozoite staining by mAb: (i) bright staining of greater than 90% of trophozoites, with aggregation of the organisms; (ii) bright staining of approximately 90% of trophozoites, with little or no aggregation; (iii) dull staining of approximately 20% of trophozoites, without aggregation. Western blotting of mAb on G. muris trophozoite antigens separated by polyacrylamide gel electrophoresis showed that a mAb exhibiting the third of these flow cytometry staining patterns recognized trophozoite antigens of MW approximately 31,000 and 35,000. Immunoprecipitation studies indicated that the same mAb specifically precipitated two 125I-labelled trophozoite surface antigens of MW approximately 30,000. Monoclonal antibodies generated in this study may facilitate the purification and biochemical characterization of trophozoite antigens that are targets for protective intestinal antibody in G. muris-infected mice. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:2592009

Prediction and Reduction of the Aggregation of Monoclonal Antibodies.

PubMed

van der Kant, Rob; Karow-Zwick, Anne R; Van Durme, Joost; Blech, Michaela; Gallardo, Rodrigo; Seeliger, Daniel; Aßfalg, Kerstin; Baatsen, Pieter; Compernolle, Griet; Gils, Ann; Studts, Joey M; Schulz, Patrick; Garidel, Patrick; Schymkowitz, Joost; Rousseau, Frederic

2017-04-21

Protein aggregation remains a major area of focus in the production of monoclonal antibodies. Improving the intrinsic properties of antibodies can improve manufacturability, attrition rates, safety, formulation, titers, immunogenicity, and solubility. Here, we explore the potential of predicting and reducing the aggregation propensity of monoclonal antibodies, based on the identification of aggregation-prone regions and their contribution to the thermodynamic stability of the protein. Although aggregation-prone regions are thought to occur in the antigen binding region to drive hydrophobic binding with antigen, we were able to rationally design variants that display a marked decrease in aggregation propensity while retaining antigen binding through the introduction of artificial aggregation gatekeeper residues. The reduction in aggregation propensity was accompanied by an increase in expression titer, showing that reducing protein aggregation is beneficial throughout the development process. The data presented show that this approach can significantly reduce liabilities in novel therapeutic antibodies and proteins, leading to a more efficient path to clinical studies. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

PubMed

Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

2017-12-01

Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

Lipopolysaccharide-Specific but Not Anti-Flagellar Immunoglobulin A Monoclonal Antibodies Prevent Salmonella enterica Serotype Enteritidis Invasion and Replication within HEp-2 Cell Monolayers

PubMed Central

Iankov, Ianko D.; Petrov, Dragomir P.; Mladenov, Ivan V.; Haralambieva, Iana H.; Mitov, Ivan G.

2002-01-01

The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens of Salmonella enterica serotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope of Salmonella lipopolysaccharide antigen alone prevented S. enterica serotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms. PMID:11854252

Monoclonal Antibodies and Toxins—A Perspective on Function and Isotype

PubMed Central

Chow, Siu-Kei; Casadevall, Arturo

2012-01-01

Antibody therapy remains the only effective treatment for toxin-mediated diseases. The development of hybridoma technology has allowed the isolation of monoclonal antibodies (mAbs) with high specificity and defined properties, and numerous mAbs have been purified and characterized for their protective efficacy against different toxins. This review summarizes the mAb studies for 6 toxins—Shiga toxin, pertussis toxin, anthrax toxin, ricin toxin, botulinum toxin, and Staphylococcal enterotoxin B (SEB)—and analyzes the prevalence of mAb functions and their isotypes. Here we show that most toxin-binding mAbs resulted from immunization are non-protective and that mAbs with potential therapeutic use are preferably characterized. Various common practices and caveats of protection studies are discussed, with the goal of providing insights for the design of future research on antibody-toxin interactions. PMID:22822456

Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains

PubMed Central

1988-01-01

We have prepared and characterized seven mouse monoclonal antibodies (SUK 1-7) to the 130-kD heavy chain of sea urchin egg kinesin. On immunoblots, SUK 3 and SUK 4 cross-reacted with Drosophila embryo 116- kD heavy chains, and SUK 4, SUK 5, SUK 6, and SUK 7 bound to the 120-kD heavy chains of bovine brain kinesin. Three out of seven monoclonal antikinesins (SUK 4, SUK 6, and SUK 7) caused a dose-dependent inhibition of sea urchin egg kinesin-induced microtubule translocation, whereas the other four monoclonal antibodies had no detectable effect on this motility. The inhibitory monoclonal antibodies (SUK 4, SUK 6, and SUK 7) appear to bind to spatially related sites on an ATP- sensitive microtubule binding 45-kD chymotryptic fragment of the 130-kD heavy chain, whereas SUK 2 binds to a spatially distinct site. None of the monoclonal antikinesins inhibited the microtubule activated MgATPase activity of kinesin, suggesting that SUK 4, SUK 6, and SUK 7 uncouple this MgATPase activity from motility. PMID:2974459

A murine monoclonal antibody based enzyme-linked immunosorbent assay for almond (Prunus dulcis L.) detection.

PubMed

Su, Mengna; Venkatachalam, Mahesh; Liu, Changqi; Zhang, Ying; Roux, Kenneth H; Sathe, Shridhar K

2013-11-13

A sandwich enzyme-linked immunosorbent assay (ELISA) using anti-almond soluble protein rabbit polyclonal antibodies as capture antibodies and murine monoclonal antibody 4C10 as the detection antibodies was developed. The assay is specific and sensitive (3-200 ng almond protein/mL) for almond detection. The standardized assay is accurate (<15% CV) and reproducible (intra- and inter assay variability <15% CV). The assay did not register any cross-reactivity with the tested food matrices, suggesting the assay to be almond amandin specific. The assay could detect the presence of declared almond in the tested matched commercial samples. Further, the assay reliably detected the presence of almonds in the laboratory prepared food samples spiked with almond flour.

Cell cycle phase dependent emergence of thymidylate synthase studied by monoclonal antibody (M-TS-4).

PubMed

Shibui, S; Hoshino, T; Iwasaki, K; Nomura, K; Jastreboff, M M

1989-05-01

A method of identifying thymidylate synthase (TS) at the cellular level was developed using anti-TS monoclonal antibody (M-TS-4), a monoclonal antibody created against purified TS from a HeLa cell line. In HeLa cells and four human glioma cell lines (U-251, U-87, 343-MGA, and SF-188), TS was identified primarily in the cytoplasm. Autoradiographic and flow cytometric studies showed that TS appeared mainly in the G1 phase and subsided early in the S phase; thus, the G1 phase can be divided into TS-positive and -negative fractions. Nuclear TS was not demonstrated unequivocally with M-TS-4, and the relationship between nuclear TS and DNA synthesis could not be determined. Although the percentage of TS-positive cells was larger than the S-phase fraction measured by autoradiography after a pulse of tritiated thymidine or by the immunoperoxidase method using BUdR, the ratios were within a similar range (1.2-1.4) in all cell lines studied. Therefore, the S-phase fraction can be estimated indirectly from the percentage of TS-positive cells measured by M-TS-4. Because the emergence of TS detected by our method is cell cycle dependent, M-TS-4 may be useful for biochemical studies of TS and for cytokinetic analysis.

Characterization of fimbriae of Actinomyces naeslundii N16 using monoclonal and polyclonal antibodies

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

Bragg, S.L.

1988-01-01

Two populations of fimbriae, which differ both in antigenicity and biological activity, have been identified on Actinomyces viscosus T14V cells. Although A. naeslundii serotype 1 isolates possess only one of these fimbrial populations (type 2 fimbriae), there was functional evidence to suggest that A. naeslundii serotype 3 strain N16 had both types of fimbriae. The purpose of this study was to characterize the fimbriae of A. naeslundii N16 immunologically by using both monoclonal and polyclonal antibodies. Three monoclonal antibodies (MAbs) to N16 were produced; all three bound to N16 fimbriae as determined by immunoelectron microscopy. In a solid-phase radioimmunoassay MAbmore » 3B5.A1 reacted with 100% of the A. naeslundii serotype 3 isolates tested, but it did not react with any heterologous isolates. Type 1 and type 2 fimbriae were detected in Lancefield extracts of N16 cells by crossed immunoelectrophoresis (XIEP) using rabbit antiserum against N16 whole cells. When {sup 125}I-MAb 3B5.A1 was also incorporated into the gel, autoradiography indicated that MAb 3B5.A1 was specific for type 2 fimbriae. The N16 type 2 fimbriae were purified by gel filtration and immunoaffinity chromatography on a MAb 3B5.A1 column. Fimbriae-specific polyclonal and monoclonal antibodies were used in various immunological assays to determine that (a) N16 type 1 fimbriae are not related antigenically to type 2 fimbriae, (b) each type of fimbriae has epitopes that are present on the corresponding fimbriae of certain heterologous strains, and (c) MAb 3B5.A1 recognizes a serotype-specific epitope residing on the type 2 fimbriae of A. naeslundii serotype 3 strains.« less

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.

Strategic deployment of CHO expression platforms to deliver Pfizer's Monoclonal Antibody Portfolio.

PubMed

Scarcelli, John J; Shang, Tanya Q; Iskra, Tim; Allen, Martin J; Zhang, Lin

2017-11-01

Development of stable cell lines for expression of large-molecule therapeutics represents a significant portion of the time and effort required to advance a molecule to enabling regulatory toxicology studies and clinical evaluation. Our development strategy employs two different approaches for cell line development based on the needs of a particular project: a random integration approach for projects where high-level expression is critical, and a site-specific integration approach for projects in which speed and reduced employee time spend is a necessity. Here we describe both our random integration and site-specific integration platforms and their applications in support of monoclonal antibody development and production. We also compare product quality attributes of monoclonal antibodies produced with a nonclonal cell pool or clonal cell lines derived from the two platforms. Our data suggests that material source (pools vs. clones) does not significantly alter the examined product quality attributes. Our current practice is to leverage this observation with our site-specific integration platform, where material generated from cell pools is used for an early molecular assessment of a given candidate to make informed decisions around development strategy. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1463-1467, 2017. © 2017 American Institute of Chemical Engineers.

Monoclonal antibodies against acetylcholinesterase from electric organs of Electrophorus and Torpedo.

PubMed

Musset, F; Frobert, Y; Grassi, J; Vigny, M; Boulla, G; Bon, S; Massoulié, J

1987-02-01

We studied the reactivity of monoclonal antibodies (mAbs) raised against acetylcholinesterase (AChE) purified from Electrophorus and Torpedo electric organs. We obtained IgG antibodies (Elec-21, Elec-106, Tor-3E5, Tor-ME8, Tor-1A5), all of them directed against the catalytic subunit of the corresponding species, with no significant cross-reactivity. These antibodies do not inhibit the enzyme and recognize all molecular forms, globular (G) and asymmetric (A). Tor-ME8 reacts specifically with the denatured A and G subunits of Torpedo AChE, in immunoblots. Several hybridomas raised against Electrophorus AChE produced IgM antibodies (Elec-39, Elec-118, Elec-121). These antibodies react with the A forms of Electrophorus electric organs and also with a subset of dimers (G2) from Torpedo electric organ. In addition, they react with a number of non-AChE components, in immunoblots. In contrast, they do not recognize AChE from other Electrophorus tissues or A forms from Torpedo electric organs.

Discovery and characterization of antibody variants using mass spectrometry-based comparative analysis for biosimilar candidates of monoclonal antibody drugs.

PubMed

Li, Wenhua; Yang, Bin; Zhou, Dongmei; Xu, Jun; Ke, Zhi; Suen, Wen-Chen

2016-07-01

Liquid chromatography mass spectrometry (LC-MS) is the most commonly used technique for the characterization of antibody variants. MAb-X and mAb-Y are two approved IgG1 subtype monoclonal antibody drugs recombinantly produced in Chinese hamster ovary (CHO) cells. We report here that two unexpected and rare antibody variants have been discovered during cell culture process development of biosimilars for these two approved drugs through intact mass analysis. We then used comprehensive mass spectrometry-based comparative analysis including reduced light, heavy chains, and domain-specific mass as well as peptide mapping analysis to fully characterize the observed antibody variants. The "middle-up" mass comparative analysis demonstrated that the antibody variant from mAb-X biosimilar candidate was caused by mass variation of antibody crystalline fragment (Fc), whereas a different variant with mass variation in antibody antigen-binding fragment (Fab) from mAb-Y biosimilar candidate was identified. Endoproteinase Lys-C digested peptide mapping and tandem mass spectrometry analysis further revealed that a leucine to glutamine change in N-terminal 402 site of heavy chain was responsible for the generation of mAb-X antibody variant. Lys-C and trypsin coupled non-reduced and reduced peptide mapping comparative analysis showed that the formation of the light-heavy interchain trisulfide bond resulted in the mAb-Y antibody variant. These two cases confirmed that mass spectrometry-based comparative analysis plays a critical role for the characterization of monoclonal antibody variants, and biosimilar developers should start with a comprehensive structural assessment and comparative analysis to decrease the risk of the process development for biosimilars. Copyright © 2016 Elsevier B.V. All rights reserved.

Development of an analytical method to assess the occupational health risk of therapeutic monoclonal antibodies using LC-HRMS.

PubMed

Reinders, Lars M H; Klassen, Martin D; Jaeger, Martin; Teutenberg, Thorsten; Tuerk, Jochen

2018-04-01

Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1 μg per sample for daratumumab and 25 μg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.

Monoclonal antibodies as cancer therapeutics.

PubMed

Elloumi, Jihene; Jellali, Karim; Jemel, Ikram; Aifa, Sami

2012-04-01

Three main targets were subjected for the most approved monoclonal antibodies (mAbs) in cancer therapy: EGFR in solid cancer, the clusters of differentiation in blood cancer and VEGF in angiogenesis. Meanwhile side effects, the elevated costs and resistance problems are limiting the efficiency of mAbs as targeted therapy. The combinatory therapy with chemo or radiotherapy has improved the efficiency of mAbs. The present review aims to shed more light on the immunotherapy and the related patents that were developed for cancer treatment.

Therapeutic monoclonal antibodies in human breast milk: a case study.

PubMed

Ross, Elle; Robinson, Steven E; Amato, Carol; McMillan, Colette; Westcott, Jay; Wolf, Tiffany; Robinson, William A

2014-04-01

Recently, therapeutic monoclonal antibodies have been introduced for the treatment of advanced melanoma and other diseases. It remains unclear whether these drugs can be safely administered to women who are breast feeding because of the potential hazardous side effects for nursing infants. One such therapy for metastatic melanoma is ipilimumab, a human monoclonal antibody that blocks cytotoxic T-lymphocyte-antigen-4, and is the preferred treatment for patients with metastatic melanoma when other molecular therapies are not viable. This study measured ipilimumab levels in the breast milk of a patient undergoing treatment that were enough to raise concerns for a nursing infant exposed to ipilimumab.

Fc Receptor-Mediated Activities of Env-Specific Human Monoclonal Antibodies Generated from Volunteers Receiving the DNA Prime-Protein Boost HIV Vaccine DP6-001.

PubMed

Costa, Matthew R; Pollara, Justin; Edwards, Regina Whitney; Seaman, Michael S; Gorny, Miroslaw K; Montefiori, David C; Liao, Hua-Xin; Ferrari, Guido; Lu, Shan; Wang, Shixia

2016-11-15

HIV-1 is able to elicit broadly potent neutralizing antibodies in a very small subset of individuals only after several years of infection, and therefore, vaccines that elicit these types of antibodies have been difficult to design. The RV144 trial showed that moderate protection is possible and that this protection may correlate with antibody-dependent cellular cytotoxicity (ADCC) activity. Our previous studies demonstrated that in an HIV vaccine phase I trial, the DP6-001 trial, a polyvalent Env DNA prime-protein boost formulation could elicit potent and broadly reactive, gp120-specific antibodies with positive neutralization activities. Here we report on the production and analysis of HIV-1 Env-specific human monoclonal antibodies (hMAbs) isolated from vaccinees in the DP6-001 trial. For this initial report, 13 hMAbs from four vaccinees in the DP6-001 trial showed broad binding to gp120 proteins of diverse subtypes both autologous and heterologous to vaccine immunogens. Equally cross-reactive Fc receptor-mediated functional activities, including ADCC and antibody-dependent cellular phagocytosis (ADCP) activities, were present with both immune sera and isolated MAbs, confirming the induction of nonneutralizing functional hMAbs by the DNA prime-protein boost vaccination. Elicitation of broadly reactive hMAbs by vaccination in healthy human volunteers confirms the value of the polyvalent formulation in this HIV vaccine design. The roles of Fc receptor-mediated protective antibody responses are gaining more attention due to their potential contribution to the low-level protection against HIV-1 infection that they provided in the RV144 trial. At the same time, information about hMabs from other human HIV vaccine studies is very limited. In the current study, both immune sera and monoclonal antibodies from vaccinated humans showed not only high-level ADCC and ADCP activities but also cross-subtype ADCC and ADCP activities when a polyvalent DNA prime-protein boost

Development and evaluation of monoclonal antibodies for paxilline

USDA-ARS?s Scientific Manuscript database

Paxilline (PAX) is a tremorgenic mycotoxin that has been found in perennial ryegrass infected with Acremonium lolii. To facilitate screening for this toxin, four murine monoclonal antibodies (mAbs) were developed. In competitive indirect enzyme-linked immunosorbent assays (CI-ELISAs) the concentrati...

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.

Development of a Specific Monoclonal Antibody for the Quantification of Artemisinin in Artemisia annua and Rat Serum.

PubMed

Guo, Suqin; Cui, Yongliang; Wang, Kunbi; Zhang, Wei; Tan, Guiyu; Wang, Baomin; Cui, Liwang

2016-03-01

Artemisinin, extracted from Artemisia annua, and its derivatives are important frontline antimalarials. To produce specific antibodies for the detection and quantification of artemisinin, artemisinin was transformed to 9-hydroxyartemisinin by microbial fermentation, which was used to prepare a 9-succinate artemisinin hapten for conjugation with ovalbumin. A monoclonal antibody (mAb), designated as 3H7A10, was selected from hybridoma cell lines which showed high specificity to artemisinin. No competitive inhibition was observed with artesunate, dihydroartemisinin, and artemether for up to 20,000 ng mL(-1). An indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed, which showed a concentration causing 50% of inhibition (IC50) for artemisinin as 2.6 ng mL(-1) and a working range of 0.6-11.5 ng mL(-1). The icELISA was applied for the quantification of artemisinin in crude extracts of wild A. annua and the study of pharmacokinetics of artemisinin in rat serum after intraperitoneal injection. The results were highly correlated with those determined by HPLC-UV analysis (R(2) = 0.9919). In comparison with reported antiartemisinin mAbs which have broad cross-reactivity with other artemisinin derivatives, the high specificity of 3H7A10 for artemisinin will enable development of methods for quantification of artemisinin in Artemisia plants and antimalarial drugs such as Arco and for pharmacokinetic studies.

Detection of Penicillinase in Milk by Sandwich ELISA Based Polyclonal and Monoclonal Antibody.

PubMed

Zhao, Yinli; Li, Guoxi

2016-01-01

A sandwich ELISA has been developed using polyclonal and monoclonal antibody for the determination of penicillinase in milk. For this purpose, specific polyclonal and monoclonal antibodies against penicillinase were generated and characterized. Using penicillinase standards prepared from 1-128 ng/mL, the method indicated that the detection limit of the sandwich ELISA, as measured in an ELISA plate reader, was as low as 0.86 ng/mL of penicillinase. For determine the accuracy, raw milk containing 2, 8, 32, and 64 ng/mL of penicillinase were tested by sandwich ELISA. Recoveries were from 93-97.5%, and the coefficient of variation [CV (%)] were from 5.55-8.38%. For interassay reproducibility, recoveries were from 89.5-95.1%, the coefficient of variation [CV (%)] were from 5.26-9.58%. This sandwich ELISA provides a useful screening method for quantitative detection of penicillinase in milk.

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.

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.

PubMed

Malakaneh, M; Rasaee, M J; Rahbarizadeh, F; Madani, R; Forozandeh, M M; Khabiri, K; Alimohammadian, M H

2001-04-01

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.

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.

Monoclonal antibodies specific to sailfish serum albumin: development of an assay for the identification of fish species in the field.

PubMed

Rossi, E A; Shepard, S R; Poyer, J C; Hartmann, J X

1992-06-01

Balb/c mice were immunized with albumin purified from sailfish (Istiophorus albicans) serum. Hybridomas were produced and screened by ELISA for reactivity with the purified albumins of sailfish, blue marlin (Makaira nigricans) and white marlin (Tetrapturus albidus). Monoclonal antibodies (MAbs) from 16 different clones exhibited activity against sailfish albumin. Thirteen of the MAbs showed cross-reactivity with the marlin species. Three MAbs exhibited distinct specificity for sailfish albumin. One of these species specific MAbs (M2D1) was conjugated to horseradish peroxidase (HRP) in order to construct an ELISA for identification of sailfish from serum. The ELISA for sailfish correctly identified eight sailfish from 26 billfish serum samples. The MAb-peroxidase conjugate was highly specific toward sailfish in that no reaction against heterologous species was detected.

Improving monoclonal antibody selection and engineering using measurements of colloidal protein interactions

PubMed Central

Geng, Steven B.; Cheung, Jason K.; Narasimhan, Chakravarthy; Shameem, Mohammed; Tessier, Peter M.

2014-01-01

A limitation of using monoclonal antibodies as therapeutic molecules is their propensity to associate with themselves and/or with other molecules via non-affinity (colloidal) interactions. This can lead to a variety of problems ranging from low solubility and high viscosity to off-target binding and fast antibody clearance. Measuring such colloidal interactions is challenging given that they are weak and potentially involve diverse target molecules. Nevertheless, assessing these weak interactions – especially during early antibody discovery and lead candidate optimization – is critical to preventing problems that can arise later in the development process. Here we review advances in developing and implementing sensitive methods for measuring antibody colloidal interactions as well as using these measurements for guiding antibody selection and engineering. These systematic efforts to minimize non-affinity interactions are expected to yield more effective and stable monoclonal antibodies for diverse therapeutic applications. PMID:25209466

Establishment and characterization of monoclonal and polyclonal antibodies against human intestinal fatty acid-binding protein (I-FABP) using synthetic regional peptides and recombinant I-FABP.

PubMed

Kajiura, Satoshi; Yashiki, Tetsuya; Funaoka, Hiroyuki; Ohkaru, Yasuhiko; Nishikura, Ken; Kanda, Tatsuo; Ajioka, Yoichi; Igarashi, Michihiro; Hatakeyama, Katsuyoshi; Fujii, Hiroshi

2008-01-01

We have succeeded in raising highly specific anti-human intestinal fatty acid-binding protein (I-FABP) monoclonal antibodies by immunizing animals with three synthetic regional peptides, i.e., the amino terminal (RP-1: N-acetylated 1-19-cysteine), middle portion (RP-2: cysteinyl-91-107) and carboxylic terminal (RP-3: cysteinyl-121-131) regions of human I-FABP, and the whole I-FABP molecule as antigens. We also raised a polyclonal antibody by immunizing with a recombinant (r) I-FABP. To ascertain the specificity of these antibodies for human I-FABP, the immunological reactivity of each was examined by a binding assay using rI-FABP, partially purified native I-FABP and related proteins such as liver-type (L)-FABP, heart-type (H)-FABP, as well as the regional peptides as reactants, and by Western blot analysis. In addition, the expression and distribution of I-FABP in the human gastrointestinal tract were investigated by an immunohistochemical technique using a carboxylic terminal region-specific monoclonal antibody, 8F9, and a polyclonal antibody, DN-R2. Our results indicated that both the monoclonal and polyclonal antibodies established in this study were highly specific for I-FABP, but not for L-FABP and H-FABP. Especially, the monoclonal antibodies raised against the regional peptides, showed regional specificity for the I-FABP molecule. Immunoreactivity of I-FABP was demonstrated in the mucosal epithelium of the jejunum and ileum by immunohistochemical staining, and the immunoreactivity was based on the presence of the whole I-FABP molecule but not the presence of any precursors or degradation products containing a carboxylic terminal fragment. It is concluded that some of these monoclonal and polyclonal antibodies, such as 8F9, 4205, and DN-R2, will be suitable for use in research on the immunochemistry and clinical chemistry of I-FABP because those antibodies can recognize both types of native and denatured I-FABP. In order to detect I-FABP in blood samples, it

Rapid preparative separation of monoclonal antibody charge variants using laterally-fed membrane chromatography.

PubMed

Sadavarte, Rahul; Madadkar, Pedram; Filipe, Carlos Dm; Ghosh, Raja

2018-01-15

Monoclonal antibodies undergo various forms of chemical transformation which have been shown to cause loss in efficacy and alteration in pharmacokinetic properties of these molecules. Such modified antibody molecules are known as variants. They also display physical properties such as charge that are different from intact antibody molecules. However, the difference in charge is very subtle and separation based on it is quite challenging. Charge variants are usually separated using ion-exchange column chromatography or isoelectric focusing. In this paper, we report a rapid and scalable method for fractionating monoclonal antibody charge variants, based on the use of cation exchange laterally-fed membrane chromatography (LFMC). Starting with a sample of monoclonal antibody hIgG1-CD4, three well-resolved fractions were obtained using either pH or salt gradient. These fractions were identified as acidic, neutral and basic variants. Each of these fractions contained intact heavy and light chains and so antibody fragmentation had no role in variant generation. The separation was comparable to that using column chromatography but was an order of magnitude faster. Copyright © 2017 Elsevier B.V. All rights reserved.

Frontiers of monoclonal antibodies: Applications in medical practices.

PubMed

Ghagane, Shridhar C; Puranik, Sridevi I; Gan, Siew Hua; Hiremath, Murigendra B; Nerli, R B; Ravishankar, M V

2017-01-01

With the flourishing of innovation in drug discovery into a new era of personalized therapy, the use of monoclonal antibodies (mAbs) in the treatment of various ailments lies at the forefront. Major improvements in genetic sequencing and biomedical techniques as well as research into mAbs emphasize on determining new targets for advanced therapy while maximizing efficacy for clinical application. However, a balance has to be achieved concerning developing a target with low toxicity combined with high specificity and versatility, to allow a specific antibody to facilitate several biotic effects, ranging from neutralization of virus mechanisms to modulation of immune response and maintaining low global economic cost. Presently, there are approximately 30 mAbs' permitted for therapeutic use with many more being tested in clinical trials. Nevertheless, the heavy cost of mAbs' production, stowage and management as well as the subsequent hindrances to their development are outweighed by mAbs' clinical advantages. Compared to conventional drugs, since mAbs use as pharmacologic iotas have specific physical features and modes of action, they should be considered as a discrete therapeutic category. In this review, the history of mAb generation and the innovative technological applications of mAbs that has advanced in clinical practices is reviewed.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules.

PubMed

Hilton, H G; Parham, P

2013-04-01

Monoclonal antibodies with specificity for human leukocyte antigen (HLA) class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing three to six HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with 1 of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 not only exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1 µg/ml), PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50 µg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23 and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. © 2013 John Wiley & Sons A/S.

Direct binding to antigen-coated beads refines the specificity and cross-reactivity of four monoclonal antibodies that recognize polymorphic epitopes of HLA class I molecules

PubMed Central

Hilton, Hugo G; Parham, Peter

2013-01-01

Monoclonal antibodies with specificity for HLA class I determinants of HLA were originally characterized using serological assays in which the targets were cells expressing 3-6 HLA class I variants. Because of this complexity, the specificities of the antibodies were defined indirectly by correlation. Here we use a direct binding assay, in which the targets are synthetic beads coated with one of 111 HLA class I variants, representing the full range of HLA-A, -B and -C variation. We studied one monoclonal antibody with monomorphic specificity (W6/32) and four with polymorphic specificity (MA2.1, PA2.1, BB7.2 and BB7.1) and compared the results with those obtained previously. W6/32 reacted with all HLA class I variants. MA2.1 exhibits high specificity for HLA-A*02, -B*57 and -B*58, but also exhibited cross-reactivity with HLA-A*11 and -B*15:16. At low concentration (1μg/ml) PA2.1 and BB7.2 were both specific for HLA-A*02 and -A*69, and at high concentration (50μg/ml) exhibited significant cross-reactions with HLA-A*68, -A*23, and -A*24. BB7.1 exhibits specificity for HLA-B*07 and -B*42, as previously described, but reacts equally well with HLA-B*81, a rare allotype defined some 16 years after the description of BB7.1. The results obtained with cell-based and bead-based assays are consistent and, in combination with amino acid sequence comparison, increase understanding of the polymorphic epitopes recognized by the MA2.1, PA2.1, BB7.2 and BB7.1 antibodies. Comparison of two overlapping but distinctive bead sets from two sources gave similar results, but the overall levels of binding were significantly different. Several weaker reactions were observed with only one of the bead sets. PMID:23510417

Magnetic nanoparticle based purification and enzyme-linked immunosorbent assay using monoclonal antibody against enrofloxacin

PubMed Central

Kim, Nam-Gun; Kim, Myeong-Ae; Park, Young-Il; Jung, Tae-Sung; Son, Seong-Wan; So, ByungJae

2015-01-01

Monoclonal anti-enrofloxacin antibody was prepared for a direct competitive enzyme-linked immunosorbent assay (ELISA) and purification system using monoclonal antibody (mAb) coupled magnetic nanoparticles (MNPs). The IC50 values of the developed mAb for enrofloxacin (ENR), ciprofloxacin, difloxacin, sarafloxacin, pefloxacin, and norfloxacin were 5.0, 8.3, 9.7, 21.7, 36.0, and 63.7 ng/mL, respectively. The lowest detectable level of ENR was 0.7 ng/mL in the prepared ELISA system. To validate the developed ELISA in the food matrix, known amounts of ENR were spiked in meat and egg samples at 10, 20 and 30 ng/mL. Recoveries for ENR ranged from 72.9 to 113.16% with a coefficient of variation (CV) of 2.42 to 10.11%. The applicability of the mAb-MNP system was verified by testing the recoveries for ENR residue in three different matrices. Recoveries for ENR ranged from 75.16 to 86.36%, while the CV ranged from 5.08 to 11.53%. Overall, ENR-specific monoclonal antibody was prepared and developed for use in competitive to ELISAs for the detection of ENR in animal meat samples. Furthermore, we suggest that a purification system for ENR using mAb-coupled MNPs could be useful for determination of ENR residue in food. PMID:26040610

Antibody-Dependent Cell-Mediated Cytotoxicity Effector-Enhanced EphA2 Agonist Monoclonal Antibody Demonstrates Potent Activity against Human Tumors1

PubMed Central

Bruckheimer, Elizabeth M; Fazenbaker, Christine A; Gallagher, Sandra; Mulgrew, Kathy; Fuhrmann, Stacy; Coffman, Karen T; Walsh, William; Ready, Shannon; Cook, Kim; Damschroder, Melissa; Kinch, Michael; Kiener, Peter A; Woods, Rob; Gao, Changshou; Dall'Acqua, William; Wu, Herren; Coats, Steven

2009-01-01

EphA2 is a receptor tyrosine kinase that has been shown to be overexpressed in a variety of human tumor types. Previous studies demonstrated that agonist monoclonal antibodies targeting EphA2 induced the internalization and degradation of the receptor, thereby abolishing its oncogenic effects. In this study, the in vitro and in vivo antibody-dependent cell-mediated cytotoxicity (ADCC) activity of EphA2 effector-enhanced agonist monoclonal antibodies was evaluated. With tumor cell lines and healthy human peripheral blood monocytes, the EphA2 antibodies demonstrated ∼80% tumor cell killing. In a dose-dependent manner, natural killer (NK) cells were required for the in vitro ADCC activity and became activated as demonstrated by the induction of cell surface expression of CD107a. To assess the role of NK cells on antitumor efficacy in vivo, the EphA2 antibodies were evaluated in xenograft models in severe compromised immunodeficient (SCID) mice (which have functional NK cells and monocytes) and SCID nonobese diabetic (NOD) mice (which largely lack functional NK cells and monocytes). Dosing of EphA2 antibody in the SCID murine tumor model resulted in a 6.2-fold reduction in tumor volume, whereas the SCID/nonobese diabetic model showed a 1.6-fold reduction over the isotype controls. Together, these results demonstrate that the anti-EphA2 monoclonal antibodies may function through at least two mechanisms of action: EphA2 receptor activation and ADCC-mediated activity. These novel EphA2 monoclonal antibodies provide additional means by which host effector mechanisms can be activated for selective destruction of EphA2-expressing tumor cells. PMID:19484140

Correlation of monoclonal and polyclonal somatostatin receptor 5 antibodies in pancreatic neuroendocrine tumors

PubMed Central

Kaemmerer, Daniel; Lupp, Amelie; Peter, Luisa; Fischer, Elke; Schulz, Stefan; Klöppel, Günter; Hommann, Merten

2013-01-01

Aims: To evaluate the frequency of somatostatin-receptor 5 (SSTR 5) in pancreatic neuroendocrine tumors by using monoclonal and polyclonal antibodies. Material and Method: we analyzed 66 proven pancreatic neuroendocrine tumors immunohistochemically with monoclonal (clone UMB-4) and polyclonal SSTR 5-antibodies. Immunoreactive score (IRS) and DAKO-score Her2/neu were evaluated. Results: Immunohistochemistry analysis demonstrated for the IRS a significant higher staining of all specimen using the monoclonal antibodies ( IRS SSTR5 poly vs IRS SSTR 5 mono; 20.0% vs 30.3% p < 0.001) by a correlation of 0.21; p = 0.04. For the HER2 score there was also a significant higher staining in the monoclonal group (Her2 SSTR 5 poly vs Her2 SSTR 5 mono; 21.5% vs 28.8% p < 0.001) by a correlation of 0.20; p = 0.08. Conclusion: Both antibodies are useful in staining of SSTR, although UMB-4 demonstrated a 10% higher SSTR 5 staining. Due to the previous underestimated expression rate of SSTR 5, current standards in diagnostics and therapy should be reconsidered. The increasing usage of long-acting pansomatostatin receptor analogues will rise the adverse effects connected to SSTR5 binding. PMID:23236542

Immunolocalization of type X collagen in normal fetal and adult osteoarthritic cartilage with monoclonal antibodies.

PubMed

Girkontaite, I; Frischholz, S; Lammi, P; Wagner, K; Swoboda, B; Aigner, T; Von der Mark, K

1996-09-01

For studies on processing and tissue distribution of type X collagen, monoclonal antibodies were prepared against human recombinant collagen type X (hrCol X) and tested by ELISA, immunoblotting and immunohistology. Forty-two clones were obtained which were grouped into four different subsets based on their reactivity against native and denatured hrCol X, pepsin-treated hrCol X, and the C-terminal NC-1 domain. Here we present results obtained with four monoclonal antibodies: Clone X 53, a representative of group I, binds with high affinity to both native and pepsin-digested hrCol X but with low affinity to the NC-1 dimer; monoclonal antibodies of group II and III recognized native and denatured hrCol X but not NC-1; antibodies of group II, but not III, reacted to some extent with pepsin treated hrCol X; one antibody (X 34) was obtained that reacted strongly with the isolated NC-1 dimer and native hrCol X but not with the NC-1 monomer or pepsin-digested hrCol X (group IV). Antibodies of all groups stained specifically the hypertrophic zone of fetal human epiphyseal cartilage. Mab X 53 stained the peri- and extracellular matrix of hypertrophic chondrocytes in the lower hypertrophic zone and in the calcified cartilage core in endochondral bone trabecules, while clone X 34 stained intracellularly and the pericellular matrix. All other tissues or cells of the epiphysis were negative. Antibody X 53 reacted also with canine, murine and guinea pig hypertrophic cartilage in tissue sections, but not with bovine or porcine type X collagen. In sections of osteoarthritic cartilage, clusters of hypertrophic chondrocytes in the deep zone were stained, confirming previous observations on enhanced chondrocyte hypertrophy and type X collagen expression in osteoarthritic articular cartilage.

Analysis of lymphopoietic stem cells with a monoclonal antibody to the rat transferrin receptor.

PubMed Central

Jefferies, W A; Brandon, M R; Williams, A F; Hunt, S V

1985-01-01

A mouse monoclonal IgG2a antibody, designated MRC OX-26, is shown to be specific for the rat transferrin receptor, but does not block transferrin binding. The antibody labelled a myeloma, three leukaemia cell lines and normal dividing cells of various types, but also bound to a number of nondividing normal tissues. No labelling of lymphopoietic stem cells could be detected, even though approximately 25% of bone marrow and over 95% of fetal liver cells were clearly labelled. Images Figure 1 Figure 3 PMID:2981766

Monoclonal Antibodies to Ferric Pseudobactin, the Siderophore of Plant Growth-Promoting Pseudomonas putida B10

PubMed Central

Buyer, Jeffrey S.; Sikora, Lawrence J.; Kratzke, Marian G.

1990-01-01

Monoclonal antibodies to ferric pseudobactin, the siderophore (microbial iron transport agent) of plant growth-promoting Pseudomonas putida B10, have been developed. Three immunoglobulin G subclass 1-type monoclonal antibodies have been characterized. Each antibody appears to be unique on the basis of their reactions with ferric pseudobactin and with culture supernatants from other pseudomonads. None of the three cross-reacts with ferric pseudobactin-type siderophores produced by seven other pseudomonads. However, P. aeruginosa ATCC 15692 and P. fluorescens ATCC 17400 produced relatively high-molecular-mass compounds (mass greater than approximately 30,000 daltons) that did react with the antibodies. The compound from P. aeruginosa was not iron regulated, while the compound from P. fluorescens was produced only under iron-limiting conditions. A competitive assay using these antibodies has a detection limit of 5 × 10−12 mol of ferric pseudobactin. This is, to our knowledge, the first report of monoclonal antibodies reactive with siderophores. PMID:16348116

Insulin Action is Blocked by a Monoclonal Antibody That Inhibits the Insulin Receptor Kinase

NASA Astrophysics Data System (ADS)

Morgan, David O.; Ho, Lisa; Korn, Laurence J.; Roth, Richard A.

1986-01-01

Thirty-six monoclonal antibodies to the human insulin receptor were produced. Thirty-four bound the intracellular domain of the receptor β subunit, the domain containing the tyrosine-specific kinase activity. Of these 34 antibodies, 33 recognized the rat receptor and 1 was shown to precipitate the receptors from mice, chickens, and frogs with high affinity. Another of the antibodies inhibited the kinase activities of the human and frog receptors with equal potencies. This antibody inhibited the kinase activities of these receptors by more than 90%, whereas others had no effect on either kinase activity. Microinjection of the inhibiting antibody into Xenopus oocytes blocked the ability of insulin to stimulate oocyte maturation. In contrast, this inhibiting antibody did not block the ability of progesterone to stimulate the same response. Furthermore, control immunoglobulin and a noninhibiting antibody to the receptor β subunit did not block this response to insulin. These results strongly support a role for the tyrosine-specific kinase activity of the insulin receptor in mediating this biological effect of insulin.

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP

NASA Technical Reports Server (NTRS)

Fattaey, A.; Lenz, L.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.

Qualification of a Quantitative Method for Monitoring Aspartate Isomerization of a Monoclonal Antibody by Focused Peptide Mapping.

PubMed

Cao, Mingyan; Mo, Wenjun David; Shannon, Anthony; Wei, Ziping; Washabaugh, Michael; Cash, Patricia

Aspartate (Asp) isomerization is a common post-translational modification of recombinant therapeutic proteins that can occur during manufacturing, storage, or administration. Asp isomerization in the complementarity-determining regions of a monoclonal antibody may affect the target binding and thus a sufficiently robust quality control method for routine monitoring is desirable. In this work, we utilized a liquid chromatography-mass spectrometry (LC/MS)-based approach to identify the Asp isomerization in the complementarity-determining regions of a therapeutic monoclonal antibody. To quantitate the site-specific Asp isomerization of the monoclonal antibody, a UV detection-based quantitation assay utilizing the same LC platform was developed. The assay was qualified and implemented for routine monitoring of this product-specific modification. Compared with existing methods, this analytical paradigm is applicable to identify Asp isomerization (or other modifications) and subsequently develop a rapid, sufficiently robust quality control method for routine site-specific monitoring and quantitation to ensure product quality. This approach first identifies and locates a product-related impurity (a critical quality attribute) caused by isomerization, deamidation, oxidation, or other post-translational modifications, and then utilizes synthetic peptides and MS to assist the development of a LC-UV-based chromatographic method that separates and quantifies the product-related impurities by UV peaks. The established LC-UV method has acceptable peak specificity, precision, linearity, and accuracy; it can be validated and used in a good manufacturing practice environment for lot release and stability testing. Aspartate isomerization is a common post-translational modification of recombinant proteins during manufacture process and storage. Isomerization in the complementarity-determining regions (CDRs) of a monoclonal antibody A (mAb-A) has been detected and has been shown to

[Techniques for rapid production of monoclonal antibodies for use with antibody technology].

PubMed

Kamada, Haruhiko

2012-01-01

A monoclonal antibody (Mab), due to its specific binding ability to a target protein, can potentially be one of the most useful tools for the functional analysis of proteins in recent proteomics-based research. However, the production of Mab is a very time-consuming and laborious process (i.e., preparation of recombinant antigens, immunization of animals, preparation of hybridomas), making it the rate-limiting step in using Mabs in high-throughput proteomics research, which heavily relies on comprehensive and rapid methods. Therefore, there is a great demand for new methods to efficiently generate Mabs against a group of proteins identified by proteome analysis. Here, we describe a useful method called "Antibody proteomic technique" for the rapid generations of Mabs to pharmaceutical target, which were identified by proteomic analyses of disease samples (ex. tumor tissue, etc.). We also introduce another method to find profitable targets on vasculature, which is called "Vascular proteomic technique". Our results suggest that this method for the rapid generation of Mabs to proteins may be very useful in proteomics-based research as well as in clinical applications.

A novel micro-emulsion and micelle assembling method to prepare DEC205 monoclonal antibody coupled cationic nanoliposomes for simulating exosomes to target dendritic cells.

PubMed

Li, Kexin; Chang, Shasha; Wang, Zhongyan; Zhao, Xiuli; Chen, Dawei

2015-08-01

Cationic biomimetic exosomes were prepared using a novel micro-emulsion and micelle assembling method by introducing DEC205 monoclonal antibody as specific ligand to target dendritic cells (DCs). The Box-Behnken experimental design was applied for optimization of nanoliposomes (NLip) and DEC205 monoclonal antibody was then conjugated on the surface of NLip (DEC205-NLip). NLip and DEC205-NLip respectively had an average size of 62.7 ± 6.33 nm and 81.64 ± 4.25 nm, zeta potential of +30.5 ± 2.3 mV and +19.8 ± 1.8 mV and encapsulation efficiency of 91.02 ± 3.1% and 93.10 ± 2.2%. In addition, the toxicity studies confirmed DEC205 monoclonal antibody could significantly reduce the cytotoxicity of the cationic lipid against DCs. And the cellular uptake experiment evaluated the significant targeting effect of the DEC205 monoclonal antibody on DC cells. In conclusion, the novel method presented here to prepare biomimetic exosomes was an efficient approach to develop antigen carriers for specific DCs targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Monoclonal Antibodies Against Tyrosyl-tRNA Synthetase and Its Isolated Cytokine-Like Domain

PubMed Central

Khoruzenko, Antonina; Cherednyk, Olga; Filonenko, Valeriy; Kornelyuk, Aleksander

2013-01-01

Tyrosyl-tRNA synthetase (TyrRS) is one of the key enzymes of protein biosynthesis. In addition to its basic role, this enzyme reveals some important non-canonical functions. Under apoptotic conditions, the full-length enzyme splits into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. The NH2-terminal catalytic fragment, known as miniTyrRS, binds strongly to the CXC-chemokine receptor CXCR1 and, like interleukin 8, functions as a chemoattractant for polymorphonuclear leukocytes. On the other hand, an extra COOH-terminal domain of human TyrRS has cytokine activities like those of a mature human endothelial monocyte-activating polypeptide II (EMAP II). Moreover, the etiology of specific diseases (cancer, neuronal pathologies, autoimmune disorders, and disrupted metabolic conditions) is connected to specific aminoacyl-tRNA synthetases. Here we report the generation and characterization of monoclonal antibodies specific to N- and C-terminal domains of TyrRS. Recombinant TyrRS and its N- and C-terminal domains were expressed as His-tag fusion proteins in bacteria. Affinity purified proteins have been used as antigens for immunization and hybridoma cell screening. Monoclonal antibodies specific to catalytic N-terminal module and C-terminal EMAP II-like domain of TyrRS may be useful as tools in various aspects of TyrRS function and cellular localization. PMID:23750478

Dashboard systems: Pharmacokinetic/pharmacodynamic mediated dose optimization for monoclonal antibodies.

PubMed

Mould, Diane R; Dubinsky, Marla C

2015-03-01

Many marketed drugs exhibit high variability in exposure and response. While these drugs are efficacious in their approved indications, finding appropriate dose regimens for individual patients is not straightforward. Similar dose adjustment problems are also seen with drugs that have a complex relationship between exposure and response and/or a narrow therapeutic window. This is particularly true for monoclonal antibodies, where prolonged dosing at a sub-therapeutic dose can also elicit anti-drug antibodies which will further compromise safety and efficacy. Thus, finding appropriate doses quickly would represent a substantial improvement in healthcare. Dashboard systems, which are decision-support tools, offer an improved, convenient means of tailoring treatment for individual patients. This article reviews the clinical need for this approach, particularly with monoclonal antibodies, the design, development, and testing of such systems, and the likely benefits of dashboard systems in clinical practice. We focus on infliximab for reference. © 2015, The American College of Clinical Pharmacology.

Development of immunoglobulin class-specific enzyme-linked immunosorbent assays for measuring antibodies against avian rotavirus.

PubMed

Myers, T J; Schat, K A; Mockett, A P

1989-01-01

Immunoglobulin class-specific enzyme-linked immunosorbent assays were developed for detecting antibodies against avian rotavirus in serum, intestinal contents, and bile from experimentally infected specific-pathogen-free (SPF) chickens. Both indirect and antibody-capture (AbC) assays were developed based on monoclonal antibodies specific for chicken IgG, IgM, and IgA. Treatment of purified rotavirus with sodium thiocyanate before coating the plate improved the rotavirus-specific reading in the indirect assay. Use of Immunolon 2 plates facilitated attachment of monoclonal antibodies to the plate in the AbC assay. Addition of 5% powdered skim milk to the diluent buffer reduced nonspecific background readings. The indirect assay was superior for detecting rotavirus-specific IgG, whereas the AbC assay was better for detecting rotavirus-specific IgM and IgA. The presence of intestinal contents in the assay wells did not reduce the measurable titers of IgG, IgM, or IgA. These assays showed that SPF chickens produced systemic and mucosal antibodies against avian rotavirus.

Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

PubMed

Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G; Chiang, Leslie A; Pan, Yun; Moran, Jennifer L; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G

2015-01-01

Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target. © FASEB.

Functional single-walled carbon nanotubes based on an integrin αvβ3 monoclonal antibody for highly efficient cancer cell targeting

NASA Astrophysics Data System (ADS)

Ou, Zhongmin; Wu, Baoyan; Xing, Da; Zhou, Feifan; Wang, Huiying; Tang, Yonghong

2009-03-01

The application of single-walled carbon nanotubes (SWNTs) in the field of biomedicine is becoming an entirely new and exciting topic. In this study, a novel functional SWNT based on an integrin αvβ3 monoclonal antibody was developed and was used for cancer cell targeting in vitro. SWNTs were first modified by phospholipid-bearing polyethylene glycol (PL-PEG). The PL-PEG functionalized SWNTs were then conjugated with protein A. A SWNT-integrin αvβ3 monoclonal antibody system (SWNT-PEG-mAb) was thus constructed by conjugating protein A with the fluorescein labeled integrin αvβ3 monoclonal antibody. In vitro study revealed that SWNT-PEG-mAb presented a high targeting efficiency on integrin αvβ3-positive U87MG cells with low cellular toxicity, while for integrin αvβ3-negative MCF-7 cells, the system had a low targeting efficiency, indicating that the high targeting to U87MG cells was due to the specific integrin targeting of the monoclonal antibody. In conclusion, SWNT-PEG-mAb developed in this research is a potential candidate for cancer imaging and drug delivery in cancer targeting therapy.

Identifying risk factors for exposure to culturable allergenic moulds in energy efficient homes by using highly specific monoclonal antibodies

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

Sharpe, Richard A.; Cocq, Kate Le; Nikolaou, Vasilis

The aim of this study was to determine the accuracy of monoclonal antibodies (mAbs) in identifying culturable allergenic fungi present in visible mould growth in energy efficient homes, and to identify risk factors for exposure to these known allergenic fungi. Swabs were taken from fungal contaminated surfaces and culturable yeasts and moulds isolated by using mycological culture. Soluble antigens from cultures were tested by ELISA using mAbs specific to the culturable allergenic fungi Aspergillus and Penicillium spp., Ulocladium, Alternaria, and Epicoccum spp., Cladosporium spp., Fusarium spp., and Trichoderma spp. Diagnostic accuracies of the ELISA tests were determined by sequencing ofmore » the internally transcribed spacer 1 (ITS1)-5.8S-ITS2-encoding regions of recovered fungi following ELISA. There was 100% concordance between the two methods, with ELISAs providing genus-level identity and ITS sequencing providing species-level identities (210 out of 210 tested). Species of Aspergillus/Penicillium, Cladosporium, Ulocladium/Alternaria/Epicoccum, Fusarium and Trichoderma were detected in 82% of the samples. The presence of condensation was associated with an increased risk of surfaces being contaminated by Aspergillus/Penicillium spp. and Cladosporium spp., whereas moisture within the building fabric (water ingress/rising damp) was only associated with increased risk of Aspergillus/Penicillium spp. Property type and energy efficiency levels were found to moderate the risk of indoor surfaces becoming contaminated with Aspergillus/Penicillium and Cladosporium which in turn was modified by the presence of condensation, water ingress and rising damp, consistent with previous literature. - Highlights: • Monoclonal antibodies were used to track culturable allergenic moulds in homes. • Allergenic moulds were recovered from 82% of swabs from contaminated surfaces. • The mAbs were highly specific with 100% agreement to PCR of recovered fungi. • Improvements to

Establishment of a sandwich enzyme-linked immunosorbent assay for specific detection of Bacillus thuringiensis (Bt) Cry1Ab toxin utilizing a monoclonal antibody produced with a novel hapten designed with molecular model.

PubMed

Dong, Sa; Zhang, Xiao; Liu, Yuan; Zhang, Cunzheng; Xie, Yajing; Zhong, Jianfeng; Xu, Chongxin; Liu, Xianjin

2017-03-01

Cry1Ab toxin is commonly expressed in genetically modified crops in order to control chewing pests. At present, the detection method with enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibody cannot specifically detect Cry1Ab toxin for Cry1Ab's amino acid sequence and spatial structure are highly similar to Cry1Ac toxin. In this study, based on molecular design, a novel hapten polypeptide was synthesized and conjugated to keyhole limpet hemocyanin (KLH). Then, through animal immunization with this antigen, a monoclonal antibody named 2C12, showing high affinity to Cry1Ab and having no cross reaction with Cry1Ac, was produced. The equilibrium dissociation constant (K D ) value of Cry1Ab toxin with MAb 2C12 was 1.947 × 10 -8  M. Based on this specific monoclonal antibody, a sandwich enzyme-linked immunosorbent assay (DAS-ELISA) was developed for the specific determination of Cry1Ab toxin and the LOD and LOQ values were determined as 0.47 ± 0.11 and 2.43 ± 0.19 ng mL -1 , respectively. The average recoveries of Cry1Ab from spiked rice leaf and rice flour samples ranged from 75 to 115%, with coefficient of variation (CV) less than 8.6% within the quantitation range (2.5-100 ng mL -1 ), showing good accuracy for the quantitative detection of Cry1Ab toxin in agricultural samples. In conclusion, this study provides a new approach for the production of high specific antibody and the newly developed DAS-ELISA is a useful method for Cry1Ab monitoring in agriculture products. Graphical Abstract Establishment of a DAS-ELISA for the specific detecting of Bacillus thuringiensis (Bt) Cry1Ab toxin.

Monoclonal antibody disulfide reduction during manufacturing

PubMed Central

Hutterer, Katariina M.; Hong, Robert W.; Lull, Jonathon; Zhao, Xiaoyang; Wang, Tian; Pei, Rex; Le, M. Eleanor; Borisov, Oleg; Piper, Rob; Liu, Yaoqing Diana; Petty, Krista; Apostol, Izydor; Flynn, Gregory C.

2013-01-01

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production. PMID:23751615

DEVELOPMENT OF MONOCLONAL ANTIBODIES AGAINST FATHEAD MINNOW (PIMEPHALES PROMELAS) VITELLOGENIN

EPA Science Inventory

We have obtained a panel of monoclonal antibodies directed against fathead minnow vitellogenin (Vtg) for use in sensitive ELISAs to quantify the response of exposure in vivo to estrogen or estrogen mimics.

Myeloma-Derived Light Chain Paired with a Diagnostic Monoclonal Antibody Hinders Immunoassay Performance.

PubMed

Tu, Bailin; Tieman, Bryan; Moore, Jeffrey; Pan, You; Muerhoff, A Scott

2017-06-01

Monoclonal antibodies are widely used as the capture and detection reagents in diagnostic immunoassays. In the past, myeloma fusion partners expressing endogenous heavy and/or light chains were often used to generate hybridoma cell lines. As a result, mixed populations of antibodies were produced that can cause inaccurate test results, poor antibody stability, and significant lot-to-lot variability. We describe one such scenario where the P3U1 (P3X63Ag8U.1) myeloma fusion partner was used in the generation of a hybridoma producing protein induced vitamin K absence/antagonist-II (PIVKA II) antibody. The hybridoma produces three subpopulations of immunoglobulin as determined by ion exchange (IEx) chromatography that exhibit varying degrees of immunoreactivity (0%, 50%, or 100%) to the target antigen as determined by Surface Plasmon Resonance. To produce an antibody with the highest possible sensitivity and specificity, the antigen-specific heavy and light chain variable domains (VH and VL) were cloned from the hybridoma and tethered to murine IgG1 and kappa scaffolds. The resulting recombinant antibody was expressed in Chinese hamster ovary cells and is compatible for use in a diagnostic immunoassay.

A Monoclonal–Monoclonal Antibody Based Capture ELISA for Abrin

PubMed Central

Tam, Christina C.; Cheng, Luisa W.; He, Xiaohua; Merrill, Paul; Hodge, David; Stanker, Larry H.

2017-01-01

Abrin, one of the most highly potent toxins in the world, is derived from the plant, Abrus precatorius. Because of its high toxicity, it poses potential bioterror risks. Therefore, a need exists for new reagents and technologies that would be able to rapidly detect abrin contamination as well as lead to new therapeutics. We report here a group of abrin-specific monoclonal antibodies (mAbs) that recognize abrin A-chain, intact A–B chain toxin, and agglutinin by Western blot. Additionally, these mAbs were evaluated for their ability to serve as capture antibodies for a sandwich (capture) ELISA. All possible capture–detector pairs were evaluated and the best antibody pair identified and optimized for a capture ELISA. The capture ELISA based on this capture–detector mAb pair had a limit of detection (L.O.D) of ≈1 ng/mL measured using three independent experiments. The assay did not reveal any false positives with extracts containing other potential ribosome-inactivating proteins (RIPs). Thus, this new capture ELISA uses mAbs for both capture and detection; has no cross-reactivity against other plant RIPs; and has a sensitivity comparable to other reported capture ELISAs using polyclonal antibodies as either capture or detector. PMID:29057799

Monoclonal antibodies passively protect BALB/c mice against Burkholderia mallei aerosol challenge.

PubMed

Treviño, Sylvia R; Permenter, Amy R; England, Marilyn J; Parthasarathy, Narayanan; Gibbs, Paul H; Waag, David M; Chanh, Tran C

2006-03-01

Glanders is a debilitating disease with no vaccine available. Murine monoclonal antibodies were produced against Burkholderia mallei, the etiologic agent of glanders, and were shown to be effective in passively protecting mice against a lethal aerosol challenge. The antibodies appeared to target lipopolysaccharide. Humoral antibodies may be important for immune protection against B. mallei infection.

Immunotherapy of Alzheimer's disease (AD): from murine models to anti-amyloid beta (Abeta) human monoclonal antibodies.

PubMed

Geylis, Valeria; Steinitz, Michael

2006-01-01

The deposition of amyloid beta (Abeta) protein is a key pathological feature in Alzheimer's disease (AD). In murine models of AD, both active and passive immunization against Abeta induce a marked reduction in amyloid brain burden and an improvement in cognitive functions. Preliminary results of a prematurely terminated clinical trial where AD patients were actively vaccinated with aggregated Abeta bear resemblance to those documented in murine models. Passive immunization of AD patients with anti-Abeta antibodies, in particular human antibodies, is a strategy that provides a more cautious management and control of any undesired side effects. Sera of all healthy adults contain anti-Abeta IgG autoimmune antibodies. Hence antigen-committed human B-cells are easily immortalized by Epstein-Barr virus (EBV) into anti-Abeta secreting cell lines. Two anti-Abeta human monoclonal antibodies which we recently prepared bind to the N-terminus of Abeta peptide and were shown to stain amyloid plaques in non-fixed brain sections from an AD patient. It is anticipated that specifically selected anti-Abeta human monoclonal antibodies could reduce and inhibit deposits of amyloid in brain while avoiding the cognitive decline that characterizes AD. In the future, this type of antibody may prove to be a promising immune therapy for the disease.

[Diagnostic and therapeutic use of human anti-D (Rho) monoclonal antibodies. Evaluation and perspectives].

PubMed

Rouger, P; Goossens, D; Champomier, F; Tsikas, G; Liberge, G; Leblanc, J; Richard, C; Bailleul, C; Salmon, C

1985-12-01

Human monoclonal antibodies will be essential in medicine. They are valuable tools for biological diagnosis and therapeutics. Our model, human monoclonal antibodies directed against the Rhesus D antigen can be used for the determination of the Rhesus D phenotype and for the suppression of Rh(D) immunisation in women. These new products require new procedures of preparation, new regulations for the quality controls, which will be discussed in this paper.

Generation and Partial Characterization of Rabbit Monoclonal Antibody to Amyloid-β Peptide 1-37 (Aβ37).

PubMed

Mehta, Pankaj D; Blain, Jean-Francois; Freeman, Emily A; Patrick, Bruce A; Barshatzky, Marc; Hrdlicka, Lori A; Mehta, Sangita P; Frackowiak, Janusz; Mazur-Kolecka, Bozena; Wegiel, Jerzy; Patzke, Holger; Miller, David L

2017-01-01

Secreted soluble amyloid-β 1-37 (Aβ37) peptide is one of the prominent Aβ forms next to Aβ40, and is found in cerebrospinal fluid (CSF) and blood. Recent studies have shown the importance of quantitation of CSF Aβ37 levels in combination with Aβ38, Aβ40, and Aβ42 to support the diagnosis of patients with probable Alzheimer's disease (AD), and the value of antibody to Aβ37 to facilitate drug discovery studies. However, the availability of reliable and specific monoclonal antibody to Aβ37 is very limited. Our aims were: 1) to generate and partially characterize rabbit monoclonal antibody (RabmAb) to Aβ37, and 2) to determine whether the antibody detects changes in Aβ37 levels produced by a γ-secretase modulator (GSM). Our generated RabmAb to Aβ37 was found to be specific to Aβ37, since it did not react with Aβ36, Aβ38, Aβ39, Aβ40, and Aβ42 in an ELISA or immunoblotting. The epitope of the antibody was contained in the seven C-terminal residues of Aβ37. The antibody was sensitive enough to measure CSF and plasma Aβ37 levels in ELISA. Immunohistological studies showed the presence of Aβ37-positive deposits in the brain of AD, and Down syndrome persons diagnosed with AD. Our studies also showed that the antibody detected Aβ37 increases in CSF and brains of rodents following treatment with a GSM. Thus, our antibody can be widely applied to AD research, and in a panel based approach it may have potential to support the diagnosis of probable AD, and in testing the effect of GSMs to target AD.

Effect of cell culture medium components on color of formulated monoclonal antibody drug substance.

PubMed

Vijayasankaran, Natarajan; Varma, Sharat; Yang, Yi; Mun, Melissa; Arevalo, Silvana; Gawlitzek, Martin; Swartz, Trevor; Lim, Amy; Li, Feng; Zhang, Boyan; Meier, Steve; Kiss, Robert

2013-01-01

As the industry moves toward subcutaneous delivery as a preferred route of drug administration, high drug substance concentrations are becoming the norm for monoclonal antibodies. At such high concentrations, the drug substance may display a more intense color than at the historically lower concentrations. The effect of process conditions and/or changes on color is more readily observed in the higher color, high concentration formulations. Since color is a product quality attribute that needs to be controlled, it is useful to study the impact of process conditions and/or modifications on color. This manuscript summarizes cell culture experiments and reports on findings regarding the effect of various media components that contribute to drug substance color for a specific monoclonal antibody. In this work, lower drug substance color was achieved via optimization of the cell culture medium. Specifically, lowering the concentrations of B-vitamins in the cell culture medium has the effect of reducing color intensity by as much as 25%. In addition, decreasing concentration of iron was also directly correlated color intensity decrease of as much as 37%. It was also shown that the color of the drug substance directly correlates with increased acidic variants, especially when increased iron levels cause increased color. Potential mechanisms that could lead to antibody coloration are briefly discussed. © 2013 American Institute of Chemical Engineers.

Anti-EGFR monoclonal antibody in cancer treatment: in vitro and in vivo evidence

PubMed

Quatrale, Anna Elisa; Petriella, Daniela; Porcelli, Letizia; Tommasi, Stefania; Silvestris, Nicola; Colucci, Giuseppe; Angelo, Angelo; Azzariti, Amalia

2011-01-01

The complexity of EGFR signaling network suggests that the receptor could be promising targets for new personalised therapy. In clinical practice two strategies targeting the receptor are available; they utilise monoclonal antibodies, directed towards the extracellular domain of EGFR, and small molecule tyrosine kinase inhibitors, which bind the catalytic kinase domain of the receptor. In this review, we summarise currently known pre-clinical data on the antitumor effects of monoclonal antibodies, which bind to EGFR in its inactive configuration, competing for ligand binding and thereby blocking ligand-induced EGFR tyrosine kinase activation. As a consequence of treatment, key EGFR-dependent intracellular signals in cancer cells are affected. Data explaining the mechanisms of action of anti-EGFR monoclonal antibodies, currently used in clinical setting and under development for the treatment of solid tumors, are revised with the aim to provide an overview of the most important preclinical studies showing the impact of this class of EGFR targeted agents on tumor biology.

The generation of monoclonal antibodies and their use in rapid diagnostic tests

USDA-ARS?s Scientific Manuscript database

Antibodies are the most important component of an immunoassay. In these proceedings we outline novel methods used to generate and select monoclonal antibodies that meet performance criteria for use in rapid lateral flow and microfluidic immunoassay tests for the detection of agricultural pathogens ...

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

PubMed Central

Dotsey, Emmanuel Y.; Gorlani, Andrea; Ingale, Sampat; Achenbach, Chad J.; Forthal, Donald N.; Felgner, Philip L.; Gach, Johannes S.

2015-01-01

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV. PMID:25938510

Generation and characterization of monoclonal antibodies specific for 18 kDa antigen from Taenia solium cysticerci.

PubMed

Zhang, Shaohua; Luo, Xuenong; Guo, Aijiang; Zhu, Xueliang; Cai, Xuepeng

2016-07-01

The gene encoding a mature 18 kDa glycoprotein of Taenia solium cysticerci (Ts18) was cloned and bacterially expressed with a His-tagged fusion protein. Monoclonal antibodies (MAbs) against the recombinant Ts18 antigen were generated in vitro by routine murine hybridoma technique of fusing splenocytes, from BALB/c mice immunized with the vesicular fluid of T. solium cysticerci (TsVF), with mouse myeloma cells (SP2/0). The reactivity and specificity of these MAbs were evaluated by indirect ELISA and immunoblotting techniques. Three stable hybridoma clones, namely 3B11, 6C5, and 6G4, were screened using His-Ts18-based ELISA, and these showed two IgG1 isotypes and one IgM isotype. All MAbs reacted with His-Ts18 at molecular weight (MW) 12.8 kDa and the native antigen at MW 18 kDa in TsVF and whole larval extracts (WLE). In a dot blotting test, MAbs 6C5 and 6G4 showed no obvious cross-reactivity with heterologous vesicular fluids from other taeniid species, including Taenia saginata (TsaVF), Taenia pisiformis (TpVF), Taenia hydatigena (ThVF), Taenia multiceps (TmVF), and Echinococcus granulosus (EgVF). Immunofluorescent assays showed that MAb 6C5 specifically reacted with the Ts18 expressed from pEGFP-N1-Ts18-transfected HeLa cells. Immunolocalization analysis, using MAb 6C5 as a probe, indicated that Ts18 was present at high concentrations in the region of the larval sucker and spiral canal. The results indicate that the Ts18 protein is an abundantly secreted parasite protein and MAbs against it might provide a step forward for improving the diagnosis of porcine cysticercosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Unmasking the anti-La/SSB response in sera from patients with Sjogren's syndrome by specific blocking of anti-idiotypic antibodies to La/SSB antigenic determinants.

PubMed

Routsias, John G; Touloupi, Evgenia; Dotsika, Eleni; Moulia, Avrilia; Tsikaris, Vassilios; Sakarellos, Constantinos; Sakarellos-Daitsiotis, Maria; Moutsopoulos, Haralampos M; Tzioufas, Athanasios G

2002-06-01

Autoantigen La/SSB is molecular target of humoral autoimmunity in patients with primary Sjogren's Syndrome (pSS) and systemic lupus erythematosus (SLE). In this study, we investigated the existence and possible influence of anti-idiotypic response to anti-La/SSB antibodies. Synthetic peptide analogs (pep) of the major antigenic determinants of La/SSB (289-308 aa and 349-364 aa) were prepared. Based on "molecular recognition" theory, complementary peptides (cpep), derived by anti-parallel readings of the noncoding strand of La/SSB DNA encoding for its antigenic determinants, were constructed. Sera from 150 patients with anti-La/SSB antibodies, 30 patients without anti-La/SSB antibodies, and 42 normal individuals were tested against all four peptides. F(ab')(2) fragments from anti-peptide IgG were prepared and F(ab')(2) - IgG interactions were evaluated using a specific anti-idiotypic ELISA. All four peptides were recognized by anti-La positive sera (83% and 51% for pep and cpep 349-364 and 51% and 28% for pep and cpep289-308, respectively). Anti-cpep F(ab')(2 )bound to a common idiotype (Id) located within or spatially close to the antigen combining site of anti La/SSB (anti-pep) antibodies. Homologous and cross-inhibition experiments further confirmed this relation. The anti-idiotypic antibodies inhibited the anti-La/SSB antibody binding to recombinant La/SSB by 91%. To overcome the anti-idiotypic interference in anti-La/SSB detection, a specific assay was developed. Sera were heated for dissociation of Id-anti-Id complexes, anti-Id antibodies blocked with cpep, and anti-La/SSB reactivity was recovered. Application of this method to anti-Ro positive-anti-La/SSB "negative" sera showed that all anti-Ro/SSA positive autoimmune sera also possess anti-La/SSB antibodies. This reaction was not observed in 14 anti-Ro negative- anti-Sm/RNP positive sera from patients with SLE. Autoimmune sera from patients with pSS and SLE contain anti-idiotypic antibodies targeting a

Production and characterization of monoclonal antibodies specific for canine CD138 (syndecan-1) for nuclear medicine preclinical trials on spontaneous tumours.

PubMed

Diab, M; Nguyen, F; Berthaud, M; Maurel, C; Gaschet, J; Verger, E; Ibisch, C; Rousseau, C; Chérel, M; Abadie, J; Davodeau, F

2017-09-01

We isolated 11 antibodies specific for canine CD138 (cCD138) to validate the interest of CD138 antigen targeting in dogs with spontaneous mammary carcinoma. The affinity of the monoclonal antibodies in the nanomolar range is suitable for immunohistochemistry and nuclear medicine applications. Four distinct epitopes were recognized on cCD138 by this panel of antibodies. CD138 expression in canine healthy tissues is comparable to that reported in humans. CD138 is frequently expressed in canine mammary carcinomas corresponding to the human triple negative breast cancer subtype, with cytoplasmic and membranous expression. In canine diffuse large B-cell lymphoma, CD138 expression is associated with the 'non-germinal center' phenotype corresponding to the most aggressive subtype in humans. This homology of CD138 expression between dogs and humans confirms the relevance of tumour-bearing dogs as spontaneous models for nuclear medicine applications, especially for the evaluation of new tumour targeting strategies for diagnosis by phenotypic imaging and radio-immunotherapy. © 2016 John Wiley & Sons Ltd.

Production of Monoclonal Antibody Against Excretory-Secretory Antigen of Fasciola hepatica and Evaluation of Its Efficacy in the Diagnosis of Fascioliasis.

PubMed

Abdolahi Khabisi, Samaneh; Sarkari, Bahador; Moshfe, Abdolali; Jalali, Sedigheh

2017-02-01

Parasitological methods are not helpful for the diagnosis of fascioliasis in acute and invasive periods of the disease. Detection of coproantigens seems to be a suitable alternative approach in the diagnosis of fascioliasis. The present study aimed to develop a reliable antigen detection system, using monoclonal antibodies raised against excretory-secretory (ES) antigen of Fasciola hepatica, for the diagnosis of fascioliasis. Fasciola adult worms were collected from the bile ducts of infected animals. Species of the fluke was determined by polymerase chain reaction-restriction fragment length polymorphism (RFLP-PCR). ES antigen of F. hepatica was prepared. For production of monoclonal antibodies, mice were immunized with ES antigens of F. hepatica. Spleen cells from the immunized mice were fused with NS-1 myeloma cells, using polyethylene glycol. Hybridoma cells secreting specific antibody were expanded and cloned by limiting dilution. Moreover, polyclonal antibody was produced against F. hepatica ES antigen in rabbits. A capture enzyme-linked immunosorbent assay (ELISA) system, using produced monoclonal antibody, was designed and stool samples of infected animals along with control samples were tested by the system. The capture ELISA detected the coproantigen in 27 of 30 (90%) parasitologically confirmed fascioliasis cases, while 4 of 39 (10.25%) samples infected with other parasitic infections showed a positive reaction in this system. No positive reactivity was found with healthy control samples. Accordingly, sensitivity of 90% and specificity of 94.2% were obtained for the capture ELISA system. The results were compared with those obtained with commercial BIO-X ELISA, and a very good (kappa = 0.9) agreement was found between the commercial kit and the developed capture ELISA. Findings of this study showed that the produced monoclonal antibody has appropriate performance for the detection of Fasciola coproantigen in stool samples and can be appropriately

A monoclonal antibody to inclusion body disease of cranes virus enabling specific immunohistochemistry and competitive ELISA

USGS Publications Warehouse

Letchworth, G.J.; Fishel, J.R.; Hansen, W.R.

1997-01-01

Inclusion body disease of cranes (IBDC) herpesvirus kills some infected cranes and persists in convalescent animals. To enable further study and rapid identification of carrier animals, we developed a monoclonal antibody (MAb) to IBDC virus and used it in immunohistochemistry and a competitive enzyme-linked immunosorbent assay (ELISA). We used conventional techniques to make murine MAbs directed against IBDC virus purified from infected duck embryo cells. Hybridomas reacting in an ELISA with IBDC virus but not uninfected duck embryo cells were characterized by radioimmunoprecipitation, in situ immunohistochemistry, and competitive ELISA with neutralizing and nonneutralizing crane sera. MAb 2C11 immunoprecipitated 59-, 61-, and 110-kD proteins from IBDC virus-infected but not uninfected cells and stained glutaraldehyde-fixed IBDC virus plaques but not surrounding uninfected duck embryo cells in vitro. Antibody 2C11 did not react with duck embryo cells infected with falcon herpesvirus, psittacine herpesvirus, infectious laryngotracheitis, pigeon herpesvirus, or duck plague virus. A competitive ELISA using antibody 2C11 identified most sera that were positive in the neutralization test. This antibody will be useful in further characterizing IBDC virus, its pathogenesis, and its natural history.

A monoclonal antibody that recognizes B cells and B cell precursors in mice

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

Coffman, R.L.; Weissman, I.L.

1981-02-01

The monoclonal antibody, RA3-2C2, appears to be specific for cells within the B cell lineage. This antibody does not recognize thymocytes, peripheral T cells, or nonlymphoid hematopoietic cells in the spleen or bone marrow. Nor does it recognize the pluripotent hematopoietic stem cells, the spleen colony-forming unit, All sIg+ B cells and most plasma cells are RA3-2C2+. In addition, approximately 20% of nucleated bone marrow cells are RA3-2C2+ but sIg-. This population contains B cell precursors that can give rise to sIg+ cells within 2 d in vitro.

Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

USGS Publications Warehouse

Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

1991-01-01

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.

Generation of monoclonal antibodies to recombinant vascular endothelial growth factor.

PubMed

Shein, S A; Gurina, O I; Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Grinenko, N F; Ivanova, N V; Volgina, N E; Ryabukhin, I A; Chekhonin, V P

2012-05-01

Female BALB/c mice were subcutaneously immunized with recombinant VEGF-164. After 3 immunization cycles, splenic B cells from immunized mouse were fused with immortalized myeloma culture SP2/0-Ag14 cells. Screening of hybrid cells producing anti-VEGF antibodies was performed by ELISA and immunocytochemical analysis on cultured C6 glioma cells. Subsequent cloning yielded hybridoma stably expressing monoclonal anti-VEGF antibodies recognizing recombinant and native VEGF.

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies

PubMed Central

von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Moldt, Brian; Le, Khoa; Robinson, James E.; Burton, Dennis R.

2016-01-01

ABSTRACT Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO. ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. IMPORTANCE This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. PMID:27122574

Comparison of Antibody-Dependent Cell-Mediated Cytotoxicity and Virus Neutralization by HIV-1 Env-Specific Monoclonal Antibodies.

PubMed

von Bredow, Benjamin; Arias, Juan F; Heyer, Lisa N; Moldt, Brian; Le, Khoa; Robinson, James E; Zolla-Pazner, Susan; Burton, Dennis R; Evans, David T

2016-07-01

Although antibodies to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein have been studied extensively for their ability to block viral infectivity, little data are currently available on nonneutralizing functions of these antibodies, such as their ability to eliminate virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 Env-specific antibodies of diverse specificities, including potent broadly neutralizing and nonneutralizing antibodies, were therefore tested for ADCC against cells infected with a lab-adapted HIV-1 isolate (HIV-1NL4-3), a primary HIV-1 isolate (HIV-1JR-FL), and a simian-human immunodeficiency virus (SHIV) adapted for pathogenic infection of rhesus macaques (SHIVAD8-EO). In accordance with the sensitivity of these viruses to neutralization, HIV-1NL4-3-infected cells were considerably more sensitive to ADCC, both in terms of the number of antibodies and magnitude of responses, than cells infected with HIV-1JR-FL or SHIVAD8-EO ADCC activity generally correlated with antibody binding to Env on the surfaces of virus-infected cells and with viral neutralization; however, neutralization was not always predictive of ADCC, as instances of ADCC in the absence of detectable neutralization, and vice versa, were observed. These results reveal incomplete overlap in the specificities of antibodies that mediate these antiviral activities and provide insights into the relationship between ADCC and neutralization important for the development of antibody-based vaccines and therapies for combating HIV-1 infection. This study provides fundamental insights into the relationship between antibody-dependent cell-mediated cytotoxicity (ADCC) and virus neutralization that may help to guide the development of antibody-based vaccines and immunotherapies for the prevention and treatment of HIV-1 infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Passive immunization with Leptospira LPS-specific agglutinating but not non-agglutinating monoclonal antibodies protect guinea pigs from fatal pulmonary hemorrhages induced by serovar Copenhageni challenge.

PubMed

Challa, Sreerupa; Nally, Jarlath E; Jones, Carroll; Sheoran, Abhineet S

2011-06-15

Leptospira interrogans serovar Copenhageni causes pulmonary hemorrhages with respiratory failure, a major cause of death in leptospirosis patients. Protective immunity to Leptospira is known to correlate with the production of leptospiral lipopolysaccharide (L-LPS)-specific agglutinating antibodies. We generated L-LPS-specific mouse monoclonal antibodies (MAbs) and investigated if these MAbs can protect guinea pigs against fatal pulmonary hemorrhages caused by serovar Copenhageni. The MAbs L8H4 and L9B11 against 22kDa L-LPS agglutinated leptospires and completely protected guinea pigs from the development of fatal pulmonary hemorrhages by serovar Copenhageni, whereas the MAb L4C1 against 8kDa L-LPS neither agglutinated the bacteria nor protected the animals against the fatal pulmonary hemorrhages. Copyright © 2011 Elsevier Ltd. All rights reserved.

A monoclonal antibody specific for Δ12-prostaglandin J2 and its utilization in the immunological assay in cell culture system of adipocytes.

PubMed

Syeda, Pinky Karim; Hossain, Mohammad Salim; Chowdhury, Abu Asad; Rahman, Mohammad Sharifur; Khan, Ferdous; Nishimura, Kohji; Jisaka, Mitsuo; Nagaya, Tsutomu; Shono, Fumiaki; Yokota, Kazushige

2012-10-01

Prostaglandin (PG) D(2) can be produced in adipocytes and dehydrated to PGs of J(2) series, including Δ(12)-PGJ(2) and 15-deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), which serve as pro-adipogenic prostanoids through the activation of peroxisome proliferator-activated receptor γ. To accomplish the quantification of Δ(12)-PGJ(2) in the cell culture system of adipocytes, the present study aimed to develop a sensitive and specific immunological assay for Δ(12)-PGJ(2). Here, we established a cloned hybridoma cell line secreting a monoclonal antibody specifically recognizing Δ(12)-PGJ(2) and utilized for the development of its solid-phase enzyme-linked immunosorbent assay (ELISA). The immobilized antigen using a conjugate of Δ(12)-PGJ(2) and γ-globulin was competitively allowed to react with the monoclonal antibody in the presence of free Δ(12)-PGJ(2). The assay provided a sensitive calibration curve for Δ(12)-PGJ(2), allowing us to determine a range from 0.16 pg to 0.99 ng with a value of 13 pg at 50% displacement in one assay. The monoclonal antibody showed almost no cross-reactivity with other related prostanoids since PGJ(2) and 15d-PGJ(2) were only recognized with much lower values of 0.5% and 0.2%, respectively. The accuracy for determining Δ(12)-PGJ(2) in the culture medium of adipocytes was confirmed by measurement after the culture medium was fortified with known amounts of authentic Δ(12)-PGJ(2) in a range from 10 to 200 pg/mL. The application of our ELISA revealed that the formation of Δ(12)-PGJ(2) became more pronounced after several hours of incubation of PGD(2) at 37°C in fresh maturation medium of cultured adipocytes. Furthermore, we provide evidence for the increased ability of cultured adipocytes to synthesize endogenous Δ(12)-PGJ(2) during the progression of adipogenesis. These results indicate the reliability and usefulness of our solid-phase ELISA for stable Δ(12)-PGJ(2), reflecting the biosynthesis of unstable PGD(2) in the culture

Monoclonal antibody passive hemagglutination and capture enzyme-linked immunosorbent assays for direct detection and quantitation of F41 and K99 fimbrial antigens in enterotoxigenic Escherichia coli.

PubMed Central

Raybould, T J; Crouch, C F; Acres, S D

1987-01-01

Production of diarrhea in neonatal calves by enterotoxigenic Escherichia coli depends on its ability to attach to the epithelial cells of the intestine via surface adhesins called pili or fimbriae and to secrete enterotoxins. The most important of these fimbriae are designated K99 and F41. We produced and characterized a murine monoclonal antibody specific to F41. This monoclonal antibody and a K99-specific monoclonal antibody were used to develop sensitive and specific passive hemagglutination and capture enzyme-linked immunosorbent assays (ELISAs) for detection and quantitation of F41 and K99 antigens in E. coli cultures and culture supernatants. The capture ELISA systems exhibited excellent sensitivity and specificity, whereas the passive hemagglutination systems appeared to be oversensitive. The ability of the capture ELISAs to detect K99 and F41 fimbrial antigens in fecal specimens from calves was evaluated. Fimbrial antigens were detected in six of six specimens from scouring calves but not in four of four specimens from nonscouring calves. PMID:2880866

Functional Transplant of a Dengue Virus Serotype 3 (DENV3)-Specific Human Monoclonal Antibody Epitope into DENV1.

PubMed

Messer, William B; Yount, Boyd L; Royal, Scott R; de Alwis, Ruklanthi; Widman, Douglas G; Smith, Scott A; Crowe, James E; Pfaff, Jennifer M; Kahle, Kristen M; Doranz, Benjamin J; Ibarra, Kristie D; Harris, Eva; de Silva, Aravinda M; Baric, Ralph S

2016-05-15

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus

Monoclonal Antibodies Attached to Carbon Nanotube Transistors for Paclitaxel Detection

NASA Astrophysics Data System (ADS)

Lee, Wonbae; Lau, Calvin; Richardson, Mark; Rajapakse, Arith; Weiss, Gregory; Collins, Philip; UCI, Molecular Biology; Biochemistry Collaboration; UCI, Departments of Physics; Astronomy Collaboration

Paclitaxel is a naturally-occurring pharmaceutical used in numerous cancer treatments, despite its toxic side effects. Partial inhibition of this toxicity has been demonstrated using weakly interacting monoclonal antibodies (3C6 and 8A10), but accurate monitoring of antibody and paclitaxel concentrations remains challenging. Here, single-molecule studies of the kinetics of antibody-paclitaxel interactions have been performed using single-walled carbon nanotube field-effect transistors. The devices were sensitized with single antibody attachments to record the single-molecule binding dynamics of paclitaxel. This label-free technique recorded a range of dynamic interactions between the antibody and paclitaxel, and it provided sensitive paclitaxel detection for pM to nM concentrations. Measurements with two different antibodies suggest ways of extending this working range and uncovering the mechanistic differences among different antibodies.

Production of human monoclonal IgG antibodies against Rhesus (D) antigen.

PubMed Central

Bron, D; Feinberg, M B; Teng, N N; Kaplan, H S

1984-01-01

An Epstein-Barr virus (EBV)-transformed human B-cell line ( LB4r ) producing anti-Rhesus [Rho(D) antigen] antibody was fused with a non-immunoglobulin-producing mouse-human heteromyeloma ( SHM - D33 ) and selected in hypoxanthine/aminopterin/thymidine medium containing 0.5 microM ouabain. Surviving hybrids found to secrete specific anti-Rho(D) antibody were cloned by limiting dilution. Two clones (D4-B2 and E10-C1) producing high levels (12 and 20 micrograms/ml per 10(6) cells per 24 hr, respectively) of monospecific antibody (IgG3, lambda chain) were selected for expansion and further characterization. Compared to the parental cell line ( LB4r ), these hybridoma cell lines presented several advantages: antibody production was increased 10-fold, cloning efficiency was improved, and the EBV genome was not retained. Antibody production has been stable for greater than 8 months. These human monoclonal anti-Rho(D) antibodies have demonstrated utility in routine blood-group typing. They may also prove useful in the biochemical and genetic characterization of the Rh antigen system. Most important, they offer a source of Rh-immune globulin for the prevention of Rh immunization and alloimmune hemolytic disease of the newborn. Images PMID:6427767

Generation of monoclonal antibodies to vertebrate albumins for analysis of arthropod blood meals.

PubMed

Schwab, Lori Kae; Nardi, James B; Holly, Theresa; Wang, Liping; Frye, Janie; Novak, Robert J

2011-06-01

An immunoassay using monoclonal antibodies (MAbs) that are specific for different vertebrate taxa (from class to species) has been developed that simplifies and facilitates analysis of vertebrate blood meals from arthropod vectors. The MAbs have been prepared against the single protein albumin, the most abundant protein in vertebrate sera. A panel of these antibodies has been generated against albumins from 33 species of vertebrates, representing four classes, 15 orders, and 25 families. Immunoreactivity of albumin in mosquito blood meals can be detected as late as 48 h after feeding. Immunoassays with MAbs can be carried out in the field as well as the laboratory. Used in conjunction with nucleic acid assays or used alone with an appropriate assortment of antibodies, the assay is simple, sensitive, and unambiguous. © 2011 The Society for Vector Ecology.

Applicability of predictive toxicology methods for monoclonal antibody therapeutics: status Quo and scope.

PubMed

Kizhedath, Arathi; Wilkinson, Simon; Glassey, Jarka

2017-04-01

Biopharmaceuticals, monoclonal antibody (mAb)-based therapeutics in particular, have positively impacted millions of lives. MAbs and related therapeutics are highly desirable from a biopharmaceutical perspective as they are highly target specific and well tolerated within the human system. Nevertheless, several mAbs have been discontinued or withdrawn based either on their inability to demonstrate efficacy and/or due to adverse effects. Approved monoclonal antibodies and derived therapeutics have been associated with adverse effects such as immunogenicity, cytokine release syndrome, progressive multifocal leukoencephalopathy, intravascular haemolysis, cardiac arrhythmias, abnormal liver function, gastrointestinal perforation, bronchospasm, intraocular inflammation, urticaria, nephritis, neuropathy, birth defects, fever and cough to name a few. The advances made in this field are also impeded by a lack of progress in bioprocess development strategies as well as increasing costs owing to attrition, wherein the lack of efficacy and safety accounts for nearly 60 % of all factors contributing to attrition. This reiterates the need for smarter preclinical development using quality by design-based approaches encompassing carefully designed predictive models during early stages of drug development. Different in vitro and in silico methods are extensively used for predicting biological activity as well as toxicity during small molecule drug development; however, their full potential has not been utilized for biological drug development. The scope of in vitro and in silico tools in early developmental stages of monoclonal antibody-based therapeutics production and how it contributes to lower attrition rates leading to faster development of potential drug candidates has been evaluated. The applicability of computational toxicology approaches in this context as well as the pitfalls and promises of extending such techniques to biopharmaceutical development has been highlighted.

Characterization of recombinant monoclonal antibody variants detected by hydrophobic interaction chromatography and imaged capillary isoelectric focusing electrophoresis.

PubMed

King, Cory; Patel, Rekha; Ponniah, Gomathinayagam; Nowak, Christine; Neill, Alyssa; Gu, Zhenyu; Liu, Hongcheng

2018-05-15

In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen. Copyright © 2018 Elsevier B.V. All rights reserved.

H2Mab-77 is a Sensitive and Specific Anti-HER2 Monoclonal Antibody Against Breast Cancer.

PubMed

Itai, Shunsuke; Fujii, Yuki; Kaneko, Mika K; Yamada, Shinji; Nakamura, Takuro; Yanaka, Miyuki; Saidoh, Noriko; Chang, Yao-Wen; Handa, Saori; Takahashi, Maki; Suzuki, Hiroyoshi; Harada, Hiroyuki; Kato, Yukinari

2017-08-01

Human epidermal growth factor receptor 2 (HER2) plays a critical role in the progression of breast cancers, and HER2 overexpression is associated with poor clinical outcomes. Trastuzumab is an anti-HER2 humanized antibody that leads to significant survival benefits in patients with HER2-positive metastatic breast cancers. In this study, we developed novel anti-HER2 monoclonal antibodies (mAbs) and characterized their efficacy in flow cytometry, Western blot, and immunohistochemical analyses. Initially, we expressed the full length or ectodomain of HER2 in LN229 glioblastoma cells and then immunized mice with ectodomain of HER2 or LN229/HER2, and performed the first screening by enzyme-linked immunosorbent assays using ectodomain of HER2. Subsequently, we selected mAbs according to their efficacy in flow cytometry (second screening), Western blot (third screening), and immunohistochemical analyses (fourth screening). Among 100 mAb clones, only three mAbs reacted with HER2 in Western blot, and clone H 2 Mab-77 (IgG 1 , kappa) was selected. Finally, immunohistochemical analyses with H 2 Mab-77 showed sensitive and specific reactions against breast cancer cells, warranting the use of H 2 Mab-77 to detect HER2 in pathological analyses of breast cancers.

Production and characterization of monoclonal antibodies specific to pangasius catfish, basa, and tra.

PubMed

Gajewski, K G; Chen, Y-T; Hsieh, Y-H P

2009-04-01

Four IgG (subclass IgG1) class monoclonal antibodies (MAbs) strongly reactive to Asian farm-raised Pangasius catfish, tra (Pangasius hypophthalmus) and basa (Pangasius bocourti), have been developed. These MAbs were raised by immunizing an animal with thermal-stable crude sarcoplasmic protein extract of cooked tra. The MAbs were selected by screening hybridoma clones against more than 70 common fish and meat protein extracts. Two MAbs, T7E10 and T1G11, were found to be specific to the Asian Pangasius catfish, tra, and basa, with no cross-reactions with any of the common fish and meat species or with the food additive proteins (bovine serum albumin, soy proteins, milk proteins, egg proteins, and gelatin) tested. MAb T7E10 recognized 2 antigenic proteins (molecular weight approximately 36 and 75 kDa) in raw and cooked tra and basa extracts, while T1G11 bound to several proteins (molecular weight between 13 and 18 kDa) in tra and basa extracts. Two other MAbs, F7B8 and F1G11, recognized a common protein (36 KDa) and cross-reacted with all the fish extracts tested and with several mammalian species. These MAbs can be employed individually or in combination in various formats of immunoassays for rapid identification of Pangasius catfish, either raw or cooked. They can also be used to study the biological, biochemical, and physiological aspects of thermal-stable antigenic proteins. This is the first study identifying these thermal-stable antigenic proteins present in Pangasius catfish as species-specific biomarkers.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions

PubMed Central

Bradbury, Andrew R. M.; Trinklein, Nathan D.; Wilkinson, Ian C.; Tandon, Atul K.; Anderson, Stephen; Bladen, Catherine L.; Jones, Brittany; Aldred, Shelley Force; Bestagno, Marco; Burrone, Oscar; Maynard, Jennifer; Ferrara, Fortunato; Görnemann, Janina; Glanville, Jacob; Wolf, Philipp; Frenzel, Andre; Wong, Julin; Koh, Xin Yu; Eng, Hui-Yan; Lane, David; Lefranc, Marie-Paule; Clark, Mike

2018-01-01

ABSTRACT Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use. PMID:29485921

Kinetic Characterisation of a Single Chain Antibody against the Hormone Abscisic Acid: Comparison with Its Parental Monoclonal

PubMed Central

Badescu, George O.; Marsh, Andrew; Smith, Timothy R.; Thompson, Andrew J.; Napier, Richard M.

2016-01-01

A single-chain Fv fragment antibody (scFv) specific for the plant hormone abscisic acid (ABA) has been expressed in the bacterium Escherichia coli as a fusion protein. The kinetics of ABA binding have been measured using surface plasmon resonance spectrometry (BIAcore 2000) using surface and solution assays. Care was taken to calculate the concentration of active protein in each sample using initial rate measurements under conditions of partial mass transport limitation. The fusion product, parental monoclonal antibody and the free scFv all have low nanomolar affinity constants, but there is a lower dissociation rate constant for the parental monoclonal resulting in a three-fold greater affinity. Analogue specificity was tested and structure-activity binding preferences measured. The biologically-active (+)-ABA enantiomer is recognised with an affinity three orders of magnitude higher than the inactive (-)-ABA. Metabolites of ABA including phaseic acid, dihydrophaseic acid and deoxy-ABA have affinities over 100-fold lower than that for (+)-ABA. These properties of the scFv make it suitable as a sensor domain in bioreporters specific for the naturally occurring form of ABA. PMID:27023768

Host virus and pneumococcus-specific immune responses in high-count monoclonal B-cell lymphocytosis and chronic lymphocytic leukemia: implications for disease progression

PubMed Central

Criado, Ignacio; Muñoz-Criado, Santiago; Rodríguez-Caballero, Arancha; Nieto, Wendy G.; Romero, Alfonso; Fernández-Navarro, Paulino; Alcoceba, Miguel; Contreras, Teresa; González, Marcos; Orfao, Alberto; Almeida, Julia

2017-01-01

Patients diagnosed with chronic lymphocytic leukemia (CLL) display a high incidence of infections due to an associated immunodeficiency that includes hypogammaglobulinemia. A higher risk of infections has also been recently reported for high-count monoclonal B-cell lymphocytosis, while no information is available in low-count monoclonal B-cell lymphocytosis. Here, we evaluated the status of the humoral immune system in patients with chronic lymphocytic leukemia (n=58), as well as in low- (n=71) and high- (n=29) count monoclonal B-cell lymphocytosis versus healthy donors (n=91). Total free plasma immunoglobulin titers and specific levels of antibodies against cytomegalovirus, Epstein-Barr virus, influenza and S.pneumoniae were measured by nephelometry and ELISA-based techniques, respectively. Overall, our results show that both CLL and high-count monoclonal B-cell lymphocytosis patients, but not low-count monoclonal B-cell lymphocytosis subjects, present with relatively high levels of antibodies specific for the latent viruses investigated, associated with progressively lower levels of S.pneumoniae-specific immunoglobulins. These findings probably reflect asymptomatic chronic reactivation of humoral immune responses against host viruses associated with expanded virus-specific antibody levels and progressively decreased protection against other micro-organisms, denoting a severe humoral immunodeficiency state not reflected by the overall plasma immunoglobulin levels. Alternatively, these results could reflect a potential role of ubiquitous viruses in the pathogenesis of the disease. Further analyses are necessary to establish the relevance of such asymptomatic humoral immune responses against host viruses in the expansion of the tumor B-cell clone and progression from monoclonal B-cell lymphocytosis to CLL. PMID:28385786

RAPID CLONING OF HIGH AFFINITY HUMAN MONOCLONAL ANTIBODIES AGAINST INFLUENZA VIRUS

PubMed Central

Wrammert, Jens; Smith, Kenneth; Miller, Joe; Langley, Trey; Kokko, Kenneth; Larsen, Christian; Zheng, Nai-Ying; Mays, Israel; Garman, Lori; Helms, Christina; James, Judith; Air, Gillian M.; Capra, J. Donald; Ahmed, Rafi; Wilson, Patrick C.

2008-01-01

Pre-existing neutralizing antibody provides the first line of defense against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14 to 21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B cell receptor (BCR) repertoire that in some donors were dominated by only a few B cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over fifty human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high affinity mAbs from humans within a month after vaccination. The panel of influenza virus specific human mAbs allowed us to address the issue of original antigenic sin (OAS) - the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared to the virus strain present in the vaccine1. However, we found that the vast majority of the influenza virus specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal healthy adults receiving influenza vaccination. PMID:18449194

Rapid cloning of high-affinity human monoclonal antibodies against influenza virus.

PubMed

Wrammert, Jens; Smith, Kenneth; Miller, Joe; Langley, William A; Kokko, Kenneth; Larsen, Christian; Zheng, Nai-Ying; Mays, Israel; Garman, Lori; Helms, Christina; James, Judith; Air, Gillian M; Capra, J Donald; Ahmed, Rafi; Wilson, Patrick C

2008-05-29

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14-21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Cathepsin B Cleavage of vcMMAE-Based Antibody-Drug Conjugate Is Not Drug Location or Monoclonal Antibody Carrier Specific.

PubMed

Gikanga, Benson; Adeniji, Nia S; Patapoff, Thomas W; Chih, Hung-Wei; Yi, Li

2016-04-20

Antibody-drug conjugates (ADCs) require thorough characterization and understanding of product quality attributes. The framework of many ADCs comprises one molecule of antibody that is usually conjugated with multiple drug molecules at various locations. It is unknown whether the drug release rate from the ADC is dependent on drug location, and/or local environment, dictated by the sequence and structure of the antibody carrier. This study addresses these issues with valine-citrulline-monomethylauristatin E (vc-MMAE)-based ADC molecules conjugated at reduced disulfide bonds, by evaluating the cathepsin B catalyzed drug release rate of ADC molecules with different drug distributions or antibody carriers. MMAE drug release rates at different locations on ADC I were compared to evaluate the impact of drug location. No difference in rates was observed for drug released from the V(H), V(L), or C(H)2 domains of ADC I. Furthermore, four vc-MMAE ADC molecules were chosen as substrates for cathepsin B for evaluation of Michaelis-Menten parameters. There was no significant difference in K(M) or k(cat) values, suggesting that different sequences of the antibody carrier do not result in different drug release rates. Comparison between ADCs and small molecules containing vc-MMAE moieties as substrates for cathepsin B suggests that the presence of IgG1 antibody carrier, regardless of its bulkiness, does not impact drug release rate. Finally, a molecular dynamics simulation on ADC II revealed that the val-cit moiety at each of the eight possible conjugation sites was, on average, solvent accessible over 50% of its maximum solvent accessible surface area (SASA) during a 500 ns trajectory. Combined, these results suggest that the cathepsin cleavage sites for conjugated drugs are exposed enough for the enzyme to access and that the drug release rate is rather independent of drug location or monoclonal antibody carrier. Therefore, the distribution of drug conjugation at different

Microchip assays for screening monoclonal antibody product quality.

PubMed

Chen, Xiaoyu; Tang, Kaiyan; Lee, Maximilian; Flynn, Gregory C

2008-12-01

Microchip CE-SDS was evaluated as a high-throughput alternative to conventional CE-SDS for monitoring monoclonal antibody protein quality. A commercial instrument (LabChip) 90) was used to separate dodecyl sulfate coated proteins through a sieving polymer based on the proteins' sizes. Under reducing conditions, the microchip CE-SDS separation was similar to that of conventional CE-SDS, providing reasonable resolution of the non-glycosylated and the glycosylated heavy chains. The fluorescence detection on LabChip 90 using non-covalent fluorescent labeling method was about as sensitive as the 220 nm UV detection used in a conventional CE instrument. A simple glycan typing assay was developed for the reducing microchip CE-SDS format. Antibodies, either pure or in crude cell culture media are treated with Endoglycosidase H, which specifically cleaves the hybrid and high mannose type glycans. A heavy chain migration shift on reducing CE-SDS resulting from the loss of glycan is used to measure the level of high mannose/hybrid type glycans as a percentage of the total glycans. Microchip CE-SDS, under both non-reducing and reducing conditions, can be used in a variety of antibody product screening assays. The microchip analyses provide sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s versus 50 min per sample) than conventional CE separation under typical operational conditions.

Monoclonal antibodies against human angiotensinogen, their characterization and use in an angiotensinogen enzyme linked immunosorbent assay.

PubMed

Rubin, I; Lykkegaard, S; Olsen, A A; Selmer, J; Ballegaard, M

1988-01-01

Monoclonal antibodies were produced against human angiotensinogen. An enzyme linked immunosorbent assay (ELISA) was developed using a high affinity monoclonal antibody as catching antibody and a polyclonal rabbit anti human angiotensinogen antibody as detecting antibody in a "sandwich" ELISA. Linear range of the ELISA was 15-450 pmol/l of human angiotensinogen. Intra- and inter- assay variation coefficients were in the range of 2% to 8%. A correlation coefficient, r = 0.97, (n = 20), with values obtained by radioimmunoassay. This correlation coefficient, obtained by using both normal and pregnant sera, confirmed that the ELISA fulfill the requirements for clinical useful assay. Characterization of the antibodies were performed with respect to affinity constant and epitopes.

Human Monoclonal Antibodies Targeting Glypican-2 in Neuroblastoma | NCI Technology Transfer Center | TTC

Cancer.gov

Researchers at the National Cancer Institute’s Laboratory of Molecular Biology (NCI LMB) have developed and isolated several single domain monoclonal human antibodies against GPC2. NCI seeks parties interested in licensing or co-developing GPC2 antibodies and/or conjugates.

Expression of deleted, atoxic atypical recombinant beta2 toxin in a baculovirus system and production of polyclonal and monoclonal antibodies.

PubMed

Serroni, Anna; Magistrali, Chiara Francesca; Pezzotti, Giovanni; Bano, Luca; Pellegrini, Martina; Severi, Giulio; Di Pancrazio, Chiara; Luciani, Mirella; Tittarelli, Manuela; Tofani, Silvia; De Giuseppe, Antonio

2017-05-25

Clostridium perfringens is an important animal and human pathogen that can produce more than 16 different major and minor toxins. The beta-2 minor toxin (CPB2), comprising atypical and consensus variants, appears to be involved in both human and animal enterotoxaemia syndrome. The exact role of CPB2 in pathogenesis is poorly investigated, and its mechanism of action at the molecular level is still unknown because of the lack of specific reagents such as monoclonal antibodies against the CPB2 protein and/or the availability of a highly purified antigen. Previous studies have reported that purified wild-type or recombinant CPB2 toxin, expressed in a heterologous system, presented cytotoxic effects on human intestinal cell lines. Undoubtedly, for this reason, to date, these purified proteins have not yet been used for the production of monoclonal antibodies (MAbs). Recently, monoclonal antibodies against CPB2 were generated using peptides designed on predicted antigenic epitopes of this toxin. In this paper we report, for the first time, the expression in a baculovirus system of a deleted recombinant C-terminal 6xHis-tagged atypical CPB2 toxin (rCPB2 Δ1-25 -His 6 ) lacking the 25 amino acids (aa) of the N-terminal putative signal sequence. A high level of purified recombinant rCPB2 Δ1-25 -His 6 was obtained after purification by Ni 2+ affinity chromatography. The purified product showed no in vitro and in vivo toxicity. Polyclonal antibodies and twenty hybridoma-secreting Mabs were generated using purified rCPB2 Δ1-25 -His 6 . Finally, the reactivity and specificity of the new antibodies were tested against both recombinant and wild-type CPB2 toxins. The high-throughput of purified atoxic recombinant CPB2 produced in insect cells, allowed to obtain monoclonal and polyclonal antibodies. The availability of these molecules could contribute to develop immunoenzymatic methods and/or to perform studies about the biological activity of CPB2 toxin.

Immunoscintigraphy and radioimmunotherapy in Cuba: experiences with labeled monoclonal antibodies for cancer diagnosis and treatment (1993-2013).

PubMed

Peña, Yamilé; Perera, Alejandro; Batista, Juan F

2014-01-01

INTRODUCTION The availability of monoclonal antibodies in Cuba has facilitated development and application of innovative techniques (immunoscintigraphy and radioimmunotherapy) for cancer diagnosis and treatment. Objective Review immunoscintigraphy and radioimmunotherapy techniques and analyze their use in Cuba, based on the published literature. In this context, we describe the experience of Havana's Clinical Research Center with labeled monoclonal antibodies for cancer diagnosis and treatment during the period 1993-2013. EVIDENCE ACQUISITION Basic concepts concerning cancer and monoclonal antibodies were reviewed, as well as relevant international and Cuban data. Forty-nine documents were reviewed, among them 2 textbooks, 34 articles by Cuban authors and 13 by international authors. All works published by the Clinical Research Center from 1993 through 2013 were included. Bibliography was obtained from the library of the Clinical Research Center and Infomed, Cuba's national health telematics network, using the following keywords: monoclonal antibodies, immunoscintigraphy and radioimmunotherapy. RESULTS Labeling the antibodies (ior t3, ior t1, ior cea 1, ior egf/r3, ior c5, h-R3, 14F7 and rituximab) with radioactive isotopes was a basic line of research in Cuba and has fostered their use as diagnostic and therapeutic tools. The studies conducted demonstrated the good sensitivity and diagnostic precision of immunoscintigraphy for detecting various types of tumors (head and neck, ovarian, colon, breast, lymphoma, brain). Obtaining different radioimmune conjugates with radioactive isotopes such as 99mTc and 188Re made it possible to administer radioimmunotherapy to patients with several types of cancer (brain, lymphoma, breast). The objective of 60% of the clinical trials was to determine pharmacokinetics, internal dosimetry and adverse effects of monoclonal antibodies, as well as tumor response; there were few adverse effects, no damage to vital organs, and a positive

Recovery and purification process development for monoclonal antibody production

PubMed Central

Ma, Junfen; Winter, Charles; Bayer, Robert

2010-01-01

Hundreds of therapeutic monoclonal antibodies (mAbs) are currently in development, and many companies have multiple antibodies in their pipelines. Current methodology used in recovery processes for these molecules are reviewed here. Basic unit operations such as harvest, Protein A affinity chromatography and additional polishing steps are surveyed. Alternative processes such as flocculation, precipitation and membrane chromatography are discussed. We also cover platform approaches to purification methods development, use of high throughput screening methods, and offer a view on future developments in purification methodology as applied to mAbs. PMID:20647768

Development and evaluation of a competitive ELISA using a monoclonal antibody for antibody detection after goose parvovirus virus-like particles (VLPs) and vaccine immunization in goose sera.

PubMed

Wang, Qian; Ju, Huanyu; Li, Yanwei; Jing, Zhiqiang; Guo, Lu; Zhao, Yu; Ma, Bo; Gao, Mingchun; Zhang, Wenlong; Wang, Junwei

2014-12-01

An assay protocol based on a monoclonal antibody-based competitive enzyme-linked immunosorbent assay (MAb-based C-ELISA) for detecting antibodies against goose parvovirus (GPV) and its virus-like particles (VLPs) is described. The assay was developed using baculovirus-expressed recombinant VP2 virus-like particles (rVP2-VLPs) as antigens and a monoclonal antibody against GPV as the competitive antibody. Of the four anti-GPV MAbs that were screened, MAb 1G3 was selected as it was blocked by the GPV positive serum. Based on the distribution of percent inhibition (PI) of the known negative sera (n=225), a cut-off value was set at 36% inhibition. Using this cut-off value, the sensitivity of the assay was 93.3% and the specificity was 95.8%, as compared with the gold standard (virus neutralization assay). The rVP2-VLPs did not react with anti-sera to other goose pathogens, indicating that it is specific for the recognization of goose parvovirus antibodies. The assay was then validated with serum samples from goslings vaccinated with several VLPs (rVP1-VLPs, rVP2-VLPs, rVP3-VLPs, and rCGV-VLPs) and other vaccines (inactivated and attenuated). The C-ELISA described in this study is a sensitive and specific diagnostic test and should have wide applications for the sero-diagnosis and immunologic surveillance of GPV. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of monoclonal antibodies as an effective strategy for treatment of ciguatera poisoning.

PubMed

Inoue, Masayuki; Lee, Nayoung; Tsumuraya, Takeshi; Fujii, Ikuo; Hirama, Masahiro

2009-06-01

Ciguatera is a global food poisoning caused by the consumption of fish that have accumulated sodium channel activator toxins, ciguatoxins. At present, most diagnosed cases of ciguatera are treated with symptomatic and supportive remedies, and no specific therapy has been devised. Here we report that ciguatoxin CTX3C can be effectively neutralized in vitro and in vivo by simultaneous use of two anti-ciguatoxin monoclonal antibodies, providing the first rational approach toward directly preventing and treating ciguatera.

Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.

PubMed Central

Bodmer, J; Bodmer, W; Heyes, J; So, A; Tonks, S; Trowsdale, J; Young, J

1987-01-01

Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies. Images PMID:2885841

Development of an enzyme-linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti-glycyrrhizic acid monoclonal antibody.

PubMed

Zhang, Yue; Qu, Huihua; Zeng, Wenhao; Zhao, Yan; Shan, Wenchao; Wang, Xueqian; Wang, Qingguo; Zhao, Yan

2015-07-01

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti-glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line (Sp2/0-Ag14). Subsequently, an indirect, competitive enzyme-linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12-2500 ng/mL. Both intra-assay and inter-assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of reduced monoclonal antibodies using size exclusion chromatography coupled with mass spectrometry

NASA Astrophysics Data System (ADS)

Liu, Hongcheng; Gaza-Bulseco, Georgeen; Chumsae, Chris

2009-12-01

Size-exclusion chromatography (SEC) has been widely used to detect antibody aggregates, monomer, and fragments. SEC coupled to mass spectrometry has been reported to measure the molecular weights of antibody; antibody conjugates, and antibody light chain and heavy chain. In this study, separation of antibody light chain and heavy chain by SEC and direct coupling to a mass spectrometer was further studied. It was determined that employing mobile phases containing acetonitrile, trifluoroacetic acid, and formic acid allowed the separation of antibody light chain and heavy chain after reduction by SEC. In addition, this mobile phase allowed the coupling of SEC to a mass spectrometer to obtain a direct molecular weight measurement. The application of the SEC-MS method was demonstrated by the separation of the light chain and the heavy chain of multiple recombinant monoclonal antibodies. In addition, separation of a thioether linked light chain and heavy chain from the free light chain and the free heavy chain of a recombinant monoclonal antibody after reduction was also achieved. This optimized method provided a separation of antibody light chain and heavy chain based on size and allowed a direct measurement of molecular weights by mass spectrometry. In addition, this method may help to identify peaks eluting from SEC column directly.

Analysis of Monoclonal Antibodies in Human Serum as a Model for Clinical Monoclonal Gammopathy by Use of 21 Tesla FT-ICR Top-Down and Middle-Down MS/MS

NASA Astrophysics Data System (ADS)

He, Lidong; Anderson, Lissa C.; Barnidge, David R.; Murray, David L.; Hendrickson, Christopher L.; Marshall, Alan G.

2017-05-01

With the rapid growth of therapeutic monoclonal antibodies (mAbs), stringent quality control is needed to ensure clinical safety and efficacy. Monoclonal antibody primary sequence and post-translational modifications (PTM) are conventionally analyzed with labor-intensive, bottom-up tandem mass spectrometry (MS/MS), which is limited by incomplete peptide sequence coverage and introduction of artifacts during the lengthy analysis procedure. Here, we describe top-down and middle-down approaches with the advantages of fast sample preparation with minimal artifacts, ultrahigh mass accuracy, and extensive residue cleavages by use of 21 tesla FT-ICR MS/MS. The ultrahigh mass accuracy yields an RMS error of 0.2-0.4 ppm for antibody light chain, heavy chain, heavy chain Fc/2, and Fd subunits. The corresponding sequence coverages are 81%, 38%, 72%, and 65% with MS/MS RMS error 4 ppm. Extension to a monoclonal antibody in human serum as a monoclonal gammopathy model yielded 53% sequence coverage from two nano-LC MS/MS runs. A blind analysis of five therapeutic monoclonal antibodies at clinically relevant concentrations in human serum resulted in correct identification of all five antibodies. Nano-LC 21 T FT-ICR MS/MS provides nonpareil mass resolution, mass accuracy, and sequence coverage for mAbs, and sets a benchmark for MS/MS analysis of multiple mAbs in serum. This is the first time that extensive cleavages for both variable and constant regions have been achieved for mAbs in a human serum background.

Evaluation of immunoreactivity of normal tissues from dogs, using monoclonal antibody B72.3.

PubMed