Establishment And Application Of A New CLIA Assay For Detection Of Human Serum Insulin

Diabetes mellitus is one of the most common occurring chronic disease, over 40 million people are affected in China. Obesity, western dietary habits, sedentary lifestyles, and aging will continue to drive dramatic growth of diabetes for decades.Diabetes is progressively debilitating and cause severe complications throughout the body because of lacks effective treatments. A rapid and reliable laboratory test for detection of serum insulin level can be helpful to diagnose diabetes early. Radioimmunoassay (RIA) is now the routine method for quantitative insulin detection. However, this assay has significant practical disadvantages, the most notable of which is not accuracy for human net insulin in serum. We have developed a new assays chemiluminescence immunoassay (CLIA) insulin kit, which can meet the challenging demands of the clinical applications .Materials and Methods: We put together the techniques of chemiluminescent assay and immunoassay and established a method, CLIA kit, for insulin determination using sandwhich procedure with the horseradish peroxidase(HRP) as label of antibody and using the detection system of enhanced luminol-HRP chemiluminescence.Results: 1. A new enhanced chemiluminescence enzyme immunoassay (CLIA) was developed, which combined luminol-H^CVHRP system enhanced by para-iodophenol (PIP). The optimal experimental conditions of the CLIA , which obtained by orthogonal design and analysis of variance, were found as follows: luminol buffer pH was 8.2,the concentrations of luminol, H2O2, PIP, protector A and B were 5mmol/L mol/L, 3 mmol/L , 7mmol/L, 5mmol/L and 1.5mg/mL respectively,and the reaction time was 5-10 minites. Under theseconditions, the working range of HRP was 10 to 10 mol/L, and regression equation. Y= 0.4446X + 8.7728(R=0.9769) was obtained. Theminimum detection limit was 10 mol/L. 2. A diagnostic kit of CLIA insulin ,which composes of 48-well-plate, No.l enzyme solution, No.2 washing buffer, No.3 and No.4 substrate solutions,a serial standards, was developed and applied. Its sensitivity, specificity, consistency, and stability were tested respectively.The detection limit is 0.8mU/L, the assay coverd a working range of 1.5-160 mU/L,and did not cross-react with high dosage of human proinsulin (2000pmol/L),C-peptide (5000pmol/L) and IGF-l(2000pmol/L).the mean recovery was 101.7% and the intra-assay CV and inter-assay CV were 4.7% and 7.0%,respectively. 1572 serum insulin levels both in normal and diabetes, as detected by CLIA ,showed a good correlation with those measured by RIA.R being 0.9502.Conclusions: All the technical parameters of CLIA in measuring serum insulin gain an advantage over those of RIA. These results suggest that the CLIA has more advantages than the routine RIA in estimating insulin. Our CLIA for human insulin is thus very sensitive, specific and easy to perform on a large scale, and can provide a new method to evaluate the islet-B-cell founction for clinical research and diagnosis.