用纯化的抗体包被微孔板，制成固相载体，往包被抗Insulin抗体的微孔中依次加入标本或标准品、生物素化的抗Insulin抗体、HRP标记的亲和素，经过彻底洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的Insulin呈正相关。用酶标仪在450nm波长下测定吸光度（OD值），计算样品浓度。
Intended use
This immunoassay kit allows for the for the in vitro quantitative determination of canine Insulin concentrations in cell culture supernates, serum, plasma and other biological fluids.
Introduction
Insulin is a polypeptide hormone originating in the beta cells of the pancreas and serving as a principal regulator for the storage and production of carbohydhumanes. Its secretion is normally stimulated by increases in the amount of glucose in circulation. This leads to higher insulin levels and more rapid tissueassimilation of glucose followed by a decline in the insulin level as the glucose level subsides. In a number of conditions, notably insulinoma and diabetes, this relationship is impaired. Insulin tends to circulate at inappropriately high levels in patients with insulin-secreting pancreatic tumors; such tumors can thus be a cause of hypoglycemia. Accordingly, insulin immunoassays used sometimes in connection with provocative doses of tolbutamide or calcium play an essential role in the identification (and localization) of insulinomas. The finding of fasting hypoglycemia in association with an inappropriately high serum insulin concenthumanion is considered diagnostic. Insulin levels do not figure in the subclassification of diabetes worked out by the National Diabetes Data Group. Nevertheless, when obtained in the course of a glucose tolerance test, they appear to be of some prognostic value in predicting the benefits of insulin therapy and the likelihood of progression to insulin-dependence and the complications (such as retinopathy) characteristic of diabetes.
Test principle
The microtiter plate provided in this kit has been pre-coated with an antibody specific to Insulin. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for Insulin and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain Insulin, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Insulin in the samples is then determined by comparing the O.D. of the samples to the standard curve.
