Relationship Between Insulin Resistance And Cardiovascular Risk Factors: Clinical Epidemiological Studies

It is well established that dyslipidemia, hypertension, hyperinsulinemia and obesity (special central obesity) have been recognized as potent risk factors for coronary heart disease in adults, the clustering of above cardiovascular risk factors often occurs in adults, such a condition has been termed insulin resistance syndrome, and insulin resistance emerges as a common pathogenetic denominator underlying the above risk factor clustering. In the earlier studies, insulin concentration was measured by radioimmunoassays with polyclonal antibodies, which cross-react with largely inactive insulin precursor molecules such as proinsulin, so that it was called immunoreactive insulin. Several groups reported that proinsulin was more strongly associated with cardiovascular disease than true insulin. Hence, it is necessary to explore the relationship between true insulin-based insulin resistance index versus Proinsulin and cardiovascular risk factors.Part I Monoclonal antibody-based sandwich enzyme immunoassay for human serum true insulin and proinsulinTwo highly sensitive and specific monoclonal antibody-based sandwich enzyme immunoassays were developed to measure true insulin and proinsulin in human serum and plasma, and in these assays, thebiotin-avidin system was employed to amplify the detecting signal.1 Monoclonal antibody-based sandwich enzyme immunoassay for human serum true insulinThis assay method for insulin is based on the enzyme immunoassay principle and it is constructed as a sandwich immunoassay. Two monoclonal murine antibodies specific for insulin are employed. One of the antibodies (HUI-018), which bind to an epitope on one side of the insulin-molecule, is used for coated on the ELISA plate wells. The other antibody (OXI-005), which binds to another epitope on the other side of the insulin-molecule, is covalently bound to biotin. The biotin-antibody reagent and sample or calibrator is added to the precoated-wells followed by incubation. During the incubation is formed a complex between plastic-surface, coated-antibody, insulin and the biotinylated-antibody. This complex will not be removed by the washing procedure, which follows the incubation. After the wash is added Avidin-peroxidase. This will bind to the biotin on the biotinylated-antibody and extends the complex with the enzyme peroxidase. The amount of enzyme is visualized by addition of the substrate peroxid and TMB, which is converted to a soluble coloured product. The developed colour is proportional with the amount of insulin in den sample. The colour-development progresses with time and it is interrupted after a fixed time by addition of phosphoric acid. The colouris now stable ad absorbance can be measured in an ELISA-plate photometer. A calibrator curve is constructed based on the absorbance values and the insulin concentration in the serum samples can be found.With a detection limit of 0.6306 mIU/L, the ELISA covered linear calibrator rang was 0.6306-100 mIU/L, human proinsulin and human C-peptide did not cross-react at 142 and 3960 pmol/L respectively. The mean recovery rate was 97.12% and intra-assay coefficient of variation and inter-assay coefficient of variation of the ELISA was less than 11.65% and 5.95% respectively.2, Monoclonal antibody-based sandwich enzyme immunoassay for human serum proinsulinThe analysis for human proinsulin is an enzyme immunoassay constructed as a sandwich immunoassay. Two monoclonal mouse antibodies are used: one with specificity for human C-peptide (PEP-001) is used for coating, the other antibody (HUI-001) with specificity for insulin is biotin labelled and acts as detecting antibody. The plasma samples are diluted 1:2 during analysis to eliminate the plasma effect. In the first reaction, after the samples are applied to the coated wells, proinsulin in the samples will bind to the coating antibody. In the second reaction the bound proinsulin is detected by the use of another monoclonal antibody directed against human insulin and biotin labelled. In a third reaction the a...