The Preparation And Applications Of Monoclonal Antibody Against Heart Fatty Acid-binding Protein

Heart-type fatty acid-binding protein (H-FABP) is a low molecular mass cytoplasmic protein (15 kD) abundant in heart muscle cells. Due to its small molecular weight characteristics, serum concentration starts rising in 2-3 hours when myocardial damage. Detection of serum H-FABP content so that it can determine the incidence of myocardial infarction. H-FABP has a high potential as a sensitive biomarker for early diagnosis of AMI. Global annual number of deaths due to cardiovascular disease accounts for a third of global deaths. And in the implementation of reperfusion therapy of myocardial infarction occuring within 6 hours, the mortality rate down to 1%, which is of great significance for this early diagnosis of AMI. The H-FABP as an early biomarker for early diagnosis of AMI will have important significance.BALB/c mice were immunized by recombinant H-FABP and B cell epitope peptide of H-FABP. Choosing splenocytes of the immunized mice were fused with SP2/0 cells. The hybridoma was subcloned by limited dilution to obtain three specific antibodies named 2C06, 2F08 and 6G03, and then prepare monoclonal antibody by inducing ascites in vivo. Ascites induced from mice belly cavity is purified by protein G affinity column. All the monoclonal antibody is IgG subtype and the titer was more than 1:106, affinity reach more than 108. The specific detection and western blot showed that three antibodies are better specificity. Using double-antibody sandwich ELISA antibody pairs were selected for the package to the 6G03 is labeled with the 2F08 pairing can detect H-FABP antigen. We had established the ELISA and GICA to detect H-FABP.By optimizing conditions, the detection range of the established ELISA method is from 0 ng·mL-1 to 200 ng·mL-1, a standard curve of R2 is 0.9935, there is a good linear correlation. Clinical samples was determined and give the average content of H-FABP in normal human serum was 2.6621 ± 0.1321 ng·mL-1, AMI patients was 22.4897 ± 1.8217 ng·mL-1, P<0.05. It showed that H-FABP content in serum has significant differences between normal human and patients with AMI. Minimum detectable concentration by GICA method is 10ng · mL-1. In the 131 cases of clinical samples, the sensitivity and the specificity were 86.96% and 97.65%. Kappa was 0.8633 and consistency test showed no significant difference between results of the clinical diagnosis and colloidal gold test strip. Therefore, the establishment of the ELISA and GICA method is suitable for the detection of H-FABP.