Production of a Monoclonal Antibody to Human a-Fetoprotein and Development of Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assays for Human α-Fetoprotein
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This study was attempted to generate a monoclonal antibody against human α-fetoprotein (AFP) and to produce an immunoassay, recognizing AFP in plasma and amniotic fluid. AFP was purified from human amniotic fluid and used to immunize mice. Spleens were taken from the mice and the cells were fused with mouse myeloma cells (Sp2/O-Ag-14) for the production of monoclonal antibodies by employing the hybridoma technology. As a result, a hybridoma cell line producing anti-AFP monoclonal antibody was cloned out and designated as MabF22. From isotyping analysis, it was found that monoclonal antibody MabF22 was IgG type with IgG1 heavy chain and κ light chain. The binding specificity of MabF22 was analyzed by immunoblotting as well as by ELISA. MabF22 was highly specific, reacting with only AFP-containing samples. The binding affinity was determined by ELISA (free-capture mode) and Scatchard analysis. As a result, the value of Kd was 0.8×10?¹? M. The validity of the MabF22 for AFP assay was examined by two kinds of ELISAs, i.e., non-competitive and competitive ELISA. Both assays revealed that MabF22 reacted well with AFP in sample in a concentration-dependent manner. Standard curve and antibody titration curve were obtained by using purified AFP and MabF22. These results indicate that the monoclonal antibody produced in this study would be useful not only for research purposes but also for further development of immunodiagnostic kit for the measurement of AFP concentration.Keywords:
Hybridoma technology
Alpha-fetoprotein
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Objective: To prepare monoclonal antibody of carbohydrate antigen 19-9( CA19-9). Methods: Based on the titer test results of mouse ascites and its IC50 values,the mouse that prepare for fusion was identified. Positive monoclonal cell strains were established by cell fusing and screening. Monoclonal antibody from ascites was produced by peritoneal injection monoclonal cell,and then purified by octoic acid-ammonium sulfate precipitation method. After determine the protein concentrations by UV-spectrophotometry,the monoclonal antibody against CA19-9 was labelled with horseradish peroxidase. Based on antibody pairing test,DAS-ELISA method was established. To compared with abroad kit,analyzing performance of this method. Results: Three strains of monoclonal antibody were obtained. And the optimal working concentrations of mAb( ZJY3-1G9),as coated antibody,McAb( ZJY2-7F10),as HRPIgG,were assured. Limit of detection was 26. 4 U /ml. Linear range was 30-300 U /ml. By detecting patients with serum 33,confirmed the correlation coefficient of r = 0. 950 4,compared with abroad kit that measure simultaneously. Conclusion: Monoclonal antibody prepared for CA19-9 can be used to develop a kit.
Horseradish peroxidase
Ammonium sulfate precipitation
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Polyclonal antibodies
Horseradish peroxidase
Conjugate
Chimera (genetics)
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Objective To prepare monoclone antibody against recombinant human Cytoglobin(rhCygb) and establish a double antibody sandwich ELISA for the investigation of pharmacokinetic of rhCygb.Methods BALB/c mice were immunized with rhCygb.Methyl cellulose semi-solid culture medium was used for the screening of the hybirdoma cell lines.The characteristics of these monoclonal antibodies were determined by ELISA.A double antibody sandwich ELISA was established.Results Cell lines secreting monoclonal antibody against rhCygb were obtained and mouse ascites of five hybridoma cell lines were prepared and purified.Purity of these antibodies was 90%.The specificity was confirmed by Western blotting and the antibodies could not bind to another PET28a-BL21 recombinant protein by indirect ELISA.The sensitivity of this assay was 1.25 ng/ml(range 10～1250 ng/ml,R2=0.9931).Intra-assay and inter-assay CV was 6.2% and 10.92%,respectively.Conclusion Monoclonal antibodies against rhCygb were obtained.A double antibody sandwich ELISA was established with satisfactory sensitivity and specificity.This work laid the foundation for further study of pharmacokinetic of rhCygb.
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[Preparation of monoclonal antibody against cystatin C and establishment of its immunoassay system].
To prepare human cystatin C( CysC) recombinant protein and produce monoclonal antibodies with high affinity and specificity. Develop a competitive ELISA detection system to detect of CysC in human serum.The CysC gene sequence was found on NCBI. The optimized gene fragments were synthesized and the recombinant CysC protein was expressed in Escherichia coli then used to immunize Balb/c mice. The positive hybridoma cell lines were obtained by hybridoma cell fusion techniques and ascites monoclonal antibody was prepared and purified. Affinity of the antibody was measured by indirect ELISA. Then competitive ELISA detection system was established, and 52 cases of human serum samples were detected by the detection system.Four stable cell lines secreting CysC monoclonal antibodies were obtained. Antibody Ab3 was used as a detection antibody and HRP labeling was performed. Its affinity constant was 4. 26 × 10~6L/mol. The linear range of detection was 0. 011-1. 924 μg/mL. The detection limit was 4. 598 ng/mL and IC_(50) was 0. 145 μg/mL. The established competitiveELISA serum detection system could accurately detect those 52 serum samples.The monoclonal antibody against CysC with high affinity and specificity has been successfully obtained. A reliable competitive ELISA serum detection system is established. The method provides a basis for the development of CysC rapid immunoassay kit.
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Polyclonal antibodies
Alpha-fetoprotein
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Polyclonal antibodies
Interferon alfa
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Four anti-alphafoetoprotein (AFP) monoclonal antibodies (MAb) were raised in the laboratory and characterized using ELISA and immunodot assays. The affinity constants of the MAbs, analysed by scatchard plots, ranged from 3.1 X 10(8) to 2.15 X 10(9) M/l. Epitope analysis using competition RIA indicated that MAb 5E2D7 and 5E2E3 recognize different epitopes on AFP. This combination was used to set up a two site sandwich ELISA with HRPO conjugated 5E2D7. AFP values in sera of hepatocellular carcinoma patients and pregnant women were quantitated using sandwich ELISA. The anti-AFP MAbs showed strong reactivity when tested on hepatoma tissue sections using immunoperoxidase technique.
Immunoperoxidase
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The aim of this study was to test how the affinity of monoclonal antibodies, raised against a vaccine candidate, influences the results obtained in a quantitative antigen-specific enzyme-linked immunosorbent assay (ELISA). The vaccine candidate is a fusion protein consisting of the N-terminal parts of two bacterial cell surface proteins, Rib and Alpha-C. To determine binding protein (Rib or Alpha C) the plates were coated with Rib-N and Alpha C-N separately. The quantity of antibodies was determined in U/ml against a mouse serum standard. By dividing the values obtained for each monoclonal antibody in this antigen specific ELISA with the concentration of IgG for each of the hybridoma supernatants, the specific activity of each monoclonal antibody was determined and expressed as units (U)/µg monoclonal antibody. Kinetic parameters and affinity constants were then determined for the 5 Rib-N specific and 5 of the Alp-N specific antibodies by surface plasmon resonance (SPR), using a Biacore 3000 instrument. The monoclonal antibodies were captured by an anti-mouse antibody and the vaccine candidate was injected over the captured monoclonal antibody in different concentrations. The results showed that the same quantity of different monoclonal antibodies gave different results when quantified by the antigen specific ELISA. Furthermore, the results indicate that differences in affinity for the antigen explain the difference observed between the antibodies in the quantitative ELISA analysis. These results also show that studies like this are of importance for the understanding of what is measured in an ELISA. (Less)
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