Preparation Of Antibodies Against Human Serum Albumin And The Establishment Of Immunoassays

The quantitative detection of Microalbuminuria (MA), as earliest diagnostic criteria for diabetes, renal disease and cardiovascular, have great significance for clinical trials. It is important to develop methods with good specificity and sensitivity for quantitative detection of MA.The immunofluorescence technique and enzyme-linked immunosorbent assay for quantitative detection of MA with polyclonal antibody from rabbit and monoclonal antibody from mice were initially established in this paper.Polyclonal antibody against human serum albumin with a 1×10~6 titer were prepared from immunized white rabbits and purified by protein A affinity column. After immunization of BALB/c mice with human serum albumin, six hybridoma cell strains against human serum albumin were developed by fusion between SP2/0 myeloma cell and the stimulated splenocytes. McAbs ascites were prepared by three hybridoma cell strains (3F4,6F2 and 8B5) with higher titer. McAbs of 3F4,6F2 and 8B5 had the same epitopes on HSA molecule and the Ig subclass belonged to IgG2b(k). McAbs with 1×10~7 titer were purified by protein G affinity column fromascites.The immunofluorescence technique assay with a detection range from 50 to 1000ng/ml and enzyme-linked immunosorbent assay with a detection range from 10 to 400ng/ml for quantitative detection of MA with polyclonal antibody from rabbit and monoclonal antibody were initially established. The results indicate that the methods have good specificity, sensitivity and repeatability for quantitative detection of MA suggesting potential efficacy in future clinical trials.