Development and application of CK-MB specific monoclonal antibodies / 生物工程学报

The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche's CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche's was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche's.

Development, identification and application of 33 monoclonal antibodies against cardiac troponin T / 生物工程学报

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

Development of monoclonal antibodies against PD-L1 and preliminary investigation on potential application in treatment of chronic hepatitis B virus in-fection / 中国免疫学杂志

Objective:To get specific monoclonal antibodies ( mAbs) against PD-L1 which can block PD-1/PD-L1 binding, and explore the feasibility of its application on the treatment of chronic HBV infection preliminarily by in vitro and in vivo model. Methods:E. coli expression and series chromatography purification system were employed to get human and mouse PD-1/PD-L1 that had binding activity in vitro. By immunizing BALB/c mouse with purified recombination proteins of PD-L1,mAb hybridoma cell lines against PD-L1 were obtained. The reactivity with human/mice PD-L1 of individual antibody and the interaction blocking activity of the mAbs to PD-1/PD-L1 in vitro were examined by indirect chemiluminescence immune assay. Results: 8 cell lines against PD-L1 were obtained and 2 Anti-PDL1 mAbs (Ab5 &Ab6) performed strong immune activity to human/mice PD-L1 and blocking activity to PD-1/PD-L1. In the PBMC stimulation experiment of chronic HBV patient,Ab5 and Ab6 could promote theγ-IFN levels. With HBV in-fecting mice model,intravenous injections of these mAbs induced dramatically decrease of HBV DNA copies about 20 times, HBsAg levels in serum reduced to 30% of the baseline level. Conclusion:We obtained 2 PD-L1 mAbs with the reactivity to human/mice PD-L1 and blocking activity to PD-1/PD-L1. The 2 mAbs can promote T cell function in PBMC stimulation culture of chronic HBV patient, have significant antiviral effect in HBV transgenic mice and can be candidates for immunotherapy applications.