Characterization of NSE monoclonal antibodies and establishment of a double-antibody sandwich ELISA assay
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Objective:Preparation and characterization of monoclonal antibodies against NSE protein,and establishment of a double-antibody sandwich ELISA assay.Methods:BALB/c mice were immunized by using purified recombinant NSE,and monoclonal antibodies were generated by hydridoma technique.These antibodies were characterized with ELISA,Western blot,Immunofluorescent and Immunohistochemical staining.The isotypes of these antibodies were determined with an antibody isotyping kit.With Horseradish Peroxidase labelled NSE monoclonal antibody,we were able to establish a double-antibody sandwich ELISA to detect NSE protein.Results:Two positive hybridoma cell lines were selected for test,the titers of these two monoclonal antibodies could reach 4.2×107-6.5×107,and their isotypes were IgG2b.Our NSE antibodies could detect not only endogernous NSE protein from cells,but also secreted NSE protein from cells in culture medium by Western blot,in addition,they could be used for immunofluorescent and immunohistochemical staining.The minimum amount of NSE protein could be detected by this double-antibody sandwich ELISA was 8.85 ng/ml.Conclusion:Our NSE monoclonal antibodies achieved good sensitivity and specificity with high titers,and we established a double-antibody sandwich ELISA assay which could be used for clinical test in future.Keywords:
Horseradish peroxidase
Immunofluorescence
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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.
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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To prepare the monoclonal antibodies against HBsAg epitopes and establish a sandwich ELISA system for HBV serotype detection.The BALB/c mice were immunized with HBsAg subtype proteins. The hybridoma cells were cultured in serum-free medium after cell fusion and high throughput screening ELISA (HTS-ELISA). Monoclonal antibodies were purified with protein A. The specificity, affinity, isotype, and epitope of the mAbs were characterized respectively and the sandwich ELISA system was established.The titers of mouse anti-sera reached 1:10(5);. After the cell fusion and HTS-ELISA, the four hybridoma cell lines were obtained. The mAbs were purified and named #2-4, #18-23, #7-7, #56-71, respectively. The mAbs had a high affinity (over 10(9); L/mol). Indirect ELISA showed that #2-4, #18-23, #7-7 and #56-71 could recognize HBsAg "d, y, r, w" epitopes, respectively. The sandwich ELISA was established through using #3-11 (Anti-HBsAg "a" epitope) as the coating antibody while the HRP labeled mAb as the secondary antibody. The optimized sandwich ELISA was proved to have a good specificity by testing different antibody-antigen reactions.The mAbs against HBsAg epitopes we prepared had a good affinity and specificity. The sandwich ELISA for HBV serotype detection we established successfully provided a basis for HBV serotype detection and disease diagnosis.
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Objective: To establish an antibody-sandwich ELISA for detection of HIV-1 gp41 antigen and evaluate its value for clinical application. Methods: Based on gp41-5 monoclonal antibody(mAb) HRP-conjugation, the sandwich ELISA detecting gp41 was established. The specific and sensibility was tested. And then 40 HIV positive serums were examed by this method. Results: In antibody-sandwich ELISA, the optimal capture mAb was E12(5 μgmL),and the optimal anzyme-conjugate mAb was 2H6(1∶900). The minimal gp41-5 antigen which could be detected by this method was 100 pgmL. By antibody-sandwich ELISA, the positive rates of HIV positive serums were 67.5%(2740). Conclusion: A sensitive and specific antibody-sandwich ELISA for detection of gp41 antigen is established.
Conjugate
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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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OBJECTIVE To prepare specific antibodies against the recombinant Eml8 antigen (ReEml8), establish a sandwich ELISA and detect the Eml8 circulating antigen in patients' sera. METHODS Rabbits and BALB/c mice were immunized with the ReEml8 antigen, which was purified by affinity method for preparation of the specific poly-clonal and monoclonal antibodies. A sandwich ELISA was established by the specific antibodies. RESULTS By immunizing with the ReEml8 antigen, high antibody level was reached with a serum dilution of 1: 204 800 and above in the immunized rabbits. After double selection by ELISA using the ReEml8 antigen and block ELISA using both AE-positive and negative control sera, 14 positive cell clones were obtained with an inhibition rate of more than 50%. Those mono-and poly-clonal antibodies were matched freely in sandwich ELISA tests for detecting the ReEml8 antigen. A combination of monoclonal antibody No.9 and polyclonal antibody showed the best result. The sensitivity to detect ReEml8 antigen was at 3 ng/ml. Six of 11 AE sera were positive when tested with the sandwich ELISA system. CONCLUSION Highly specific polyclonal and monoclonal antibodies have been prepared, and a sensitive sandwich ELISA established. Preliminary result is suggested that a detectable level of Eml8 circulating antigen is present in AE patients' sera.
Polyclonal antibodies
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Polyclonal antibodies
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OBJECTIVE: To establish a double antibody sandwich ELISA method for detection of nucleoside triphosphate hydrolase-II (NTPase-II) protein of Toxoplasma gondii. METHODS: BALB/c mice were immunized with recombinant NTPase-II (rTgNTPase-II) protein of T. gondii. The hybridomas that secreted high titer of monoclonal antibodies (mAbs) with high specificity were screened and used to establish the double antibody sandwich ELISA for the detection of rTgNTPase-II. In order to evaluate the sensitivity of the method, the concentration of whole-tachyzoite lysate and rTgNTPase-II was detected, respectively. Serum samples from patients with malaria (7 cases), schistosomiasis (12 cases), paragonimiasis (14 cases) and cysticercosis (10 cases) were examined by the same method. RESULTS: Two hybridoma cell lines, MNTI and MNT2, were developed for secreting mAbs against rTgNTPase-II. Western blotting analysis showed that the two mAbs specifically recognized rTgNTPase-II and whole-tachyzoite lysate. The MNT1 was used as coating antibody, and HRP-labeled MNT2 as secondary antibody. The double antibody sandwich ELISA detecting rTgNTPase-II was developed with a minimum concentration of 6 microg/ml for whole-tachyzoite lysate and 1.5 microg/ml for rTgNTPase-II. An overall specificity of 100% was determined. CONCLUSION: The double antibody sandwich ELISA based on MNT1 as coating antibody and MNT2 as secondary antibody has a high specificity.
Toxoplasmosis
Antibody titer
Nucleoside triphosphate
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The study was aimed to prepare monoclonal antibody against BEV-2 and develop ELISA for the detection of antigen of bovine enterovirus type 2(BEV-2)based on the monoclonal antibody.The purified BEV-2VP1+recombinant protein was used to immunize BALB/c mice,of which splenocytes were fused with SP2/0cells by PEG4000.The selection of positive hybridomas was conducted.Two hybridomas(designated as 1G1 and 5G6)secreting monoclonal antibodies(mAb)against BEV-2 VP1+protein were selected.Based on the monoclonal antibodies against BEV-2,a sandwich ELISA was established to detect BEV-2through a series of optimization.The sensitivity of the sandwich ELISA was 100TCID50·0.1mL-1,and this method had no cross-reaction with BVDV,IBRV and BLV.The results showed that the sandwich ELISA was specific,sensitive and reproducible.This is the first domestic report of establishment of sandwich ELISA for the detection of BEV-2based on the preparation of the monoclonal antibodies,and it provided a basis for development of kit for rapid diagnosing BEV-2.
Splenocyte
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