Screening of specific monoclonal antibodies for different binding sites of connexin 43 and development and verification of double antibody sandwich ELISA for Cx43
10.13200/j.cnki.cjb.004564
- VernacularTitle:人缝隙连接蛋白43不同结合位点单克隆抗体的筛选及双抗体夹心ELISA检测方法的建立及验证
- Author:
ZHANG Xiaogang
- Publication Type:Journal Article
- Keywords:
Connexin 43(Cx43);
Peptide immunity;
Monoclonal antibodies;
Double antibody sandwich ELISA method
- From:
Chinese Journal of Biologicals
2025;38(09):1094-1100
- CountryChina
- Language:Chinese
-
Abstract:
Objective To screen monoclonal antibodies against different binding sites of connexin 43(Cx43) and establish a double antibody sandwich ELISA method for quantitative detection of Cx43 protein in order to further study the structure,function and subcellular localization of full-length and truncated Cx43 protein.Methods The sequence of Cx43 protein was analyzed by bioinformatics method. The peptide fragments were designed and synthesized for different domains, and coupled with keyhole limpet hemocyanin(KLH) protein, which were used as immunogen to immunize 15 female BALB/c mice.Hybridoma cells were prepared by cell fusion technology, and cell lines stably secreting monoclonal antibodies against Cx43 protein were screened. The antibodies were purified by Protein G affinity chromatography, and its subtype, affinity and specificity were identified. Two high affinity monoclonal antibodies were used as capture antibody and detection antibody respectively(labeled with HRP) to establish a double antibody sandwich ELISA method. The sealing solution(5% milk-PBS, 3%BSA-PBS), washing solution [0. 01 mol/L PBS(pH 7. 2), 0. 01 mol/L PBS-0. 5% Tween 20(pH 7. 2)(i. e., PBST)] and sample diluent [3% BSA-PBS and 0. 01 mol/L PBS(pH 7. 2)]were optimized, and the method was verified for the linear range, sensitivity, specificity, precision and accuracy. The content of Cx43 protein in mouse brain tissue, intestine tissue, femur tissue and MC3T3-E1 mouse embryonic osteoblast lysate was detected by the established method.Results Three hybridoma cell lines secreting monoclonal antibodies against Cx43 were screened and named as 1-1E2, 2-1G11 and 3-2G12 respectively. The antibody subtypes were IgG1, IgG1 and IgG2b respectively, and the antibody sensitivity reached 0. 002 μg/mL, among which 2-1G11 and 3-2G12 had higher affinity. A double antibody sandwich ELISA was developed with 2-1G11 as capture antibody and HRP-labeled 3-2G12 as detection antibody. The optimum sealing solution was 3% BSA-PBS, washing solution was PBST, and sample diluent was 0. 01 mol/L PBS(pH 7. 2). Cx43 protein standard showed a good linear relationship with the mean value of A_(450)in the concentration range of 3. 12-200 ng/mL, R~2> 0. 98. Cx43 protein could be specifically detected by this method, and there was no cross reaction with KLH protein, bone morphogenetic protein-2(BMP-2) and human recombinant TGM2 protein. The CV of precision verification was 7. 72%. The recovery rates of high, medium and low concentrations of test samples were all in the range of 85%-115%. The content of Cx43 protein in MC3T3-E1 mouse embryonic osteoblasts was higher(58. 03 ng/mL), while the content in osteogenic tissue was lower(38. 4 ng/mL).Conclusion Specific monoclonal antibodies against different epitopes of human Cx43 were successfully prepared, and a double antibody sandwich ELISA method with high sensitivity and strong specificity for quantitative detection of Cx43 protein was established, which provided a reliable detection method for the study of structure and function of Cx43 protein.
