Constructive Expression of Creatine Kinase Isoenzyme CK-MB and Antibody Production and Preliminary Validation
Objective Construct heterodimeric CK-MB recombinant protein and prepare its specific gene-engineered antibody as the diagnostic raw material of CK-MB quantitative detection kit,so as to assist the early diagnosis of acute myocardial infarction in clinic,and to improve the accuracy of diagnosis.Method Mouse plasma cells were obtained by obtaining heterodimeric CK-MB through co-expression vector and animal immunization,further,single mouse B cells specific for antigen were isolated using single-cell photoconductive system,and genetically engineered antibodies were constructed after obtaining the gene sequences of the variable regions of the light and heavy chains of the antibodies through single-cell PCR.Candidate antibodies were screened based on preliminary functional validation,and the optimal pairing scheme was further explored by checkerboard titration,and a CK-MB quantitative detection kit was initially assembled using the principle of double-antibody sandwich ELISA.Result CK-MB immunogen was successfully obtained;single plasma cells specific for CK-MB antigen were screened by single B-cell photoconductivity and two strains of mouse-derived genetically engineered antibodies were prepared,and both of them had the ability of recognizing the natural CK-MB;two pairwise protocols were screened by double-antibody sandwich ELISA,and the R2 of the fitted curves were both greater than 0.99.Conclusion Mouse-derived genetically engineered antibody targeting CK-MB was successfully prepared,and the specificity and affinity of the antibody was preliminarily verified by ELISA,laying a solid foundation for the further development of a new type of CK-MB quantitative detection kit with high specificity and high sensitivity.
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