Insulin, a hormone primarily concerned with glucose homeostasis, plays a critical role in several physiological processes, such as lipid metabolism, cell proliferation, differentiation, aging, reproduction and growth (1, 3). Impairment in insulin secretion and/or downstream signaling results in the deregulation of carbohydrate and lipid metabolism, a hallmark of diabetes. An increase or decrease in the level of insulin is pathophysiologically linked with type 2 or type 1 diabetes. Apart from glucose homeostasis, the hormone has growth-stimulating effects and has been directly and/or indirectly involved in the pathogenesis of several diseases, such as poly cystic ovary disease (PCOD), obesity, atherosclerosis, prostatic hyperplasia, hypertension, and cancer. To investigate the physiological role of insulin and its involvement in the pathogenesis of different diseases, the estimation of insulin level is required.
The Rat/Mouse Insulin ELISA Kit from Millipore offers an easy and sensitive method to quantify insulin in biological samples (4,8). Estimation of insulin with this kit generally takes 3.5 to 4 hours. The 96-well microtiter plate is pre-coated with pre-titered amounts of monoclonal mouse anti-rat insulin antibodies, providing binding sites for the insulin present in the samples. After the sample has been applied, biotinylated polyclonal antibodies bind to the captured insulin. (The uncaptured components of the sample are washed away.) The streptavidin conjugated horseradish peroxidase (HRP) enzyme is then applied and selectively binds with the immobilized biotinylated immunocomplex. Any unbound enzyme is washed away with washing buffer. 3,3’, 5,5’ tetramethylbenzidine (TMB) is then used as a substrate; this gets converted to a blue, insoluble precipitate in the presence of HRP enzyme. The quantity of blue colored precipitate depends on the quantity of HRP enzyme present in the microtiter well which in turn reflects the amount of insulin in the sample. Thus, this assay provides an easy to perform and convenient means to estimate the concentration of insulin. In order to quantify results, the blue precipitate is dissolved in HCl to give a soluble yellow color which is read at 450 nm.
Fig. 1 Schematic representation for the estimation of insulin concentration in the samples using Rat/mouse insulin ELISA Kit, of Millipore®
For accurate determination of the insulin concentration, it is preferred to run the test in duplicate for samples as well as standards. The insulin ELISA kit includes two quality control samples (Q1 and Q2), one in the lower and the other in the higher range of insulin concentration. Inclusion of these two quality control samples provides an estimate of the accuracy of the test and allows inter-assay comparison.
In general, insulin estimation with this kit is an easy-to-perform, but a few precautions can further enhance its accuracy. Heavily hemolysed and heparinized samples should be avoided as red blood cell contaminants and heparin can interfere with the binding of insulin to the antibodies and may affect the sensitivity and accuracy of the assay. Washing, the least respected part, is the most critical step in the insulin estimation and should be carefully performed as many times as indicated in the manufacturer's data sheet. Washing is involved at two points: first, after the 2 hr incubation of the sample/standard and antibody solution (3X), second, after the 30 minute incubation with the enzyme solution (6X). Improper washing can lead to false positive results. Further, presence of air bubbles can affect the reading and hence the accuracy of the assay.
Color development can be monitored by determining the absorbance at 370nm. Terminate
the reaction by adding stop solution when the absorbance is in the range of 1.2 to 2.8. Although the development of color is subject to the temperature of the room, in general, it takes
15-30 minutes. If it is not possible to monitor the optical density continuously, observe
the color development in the wells where known concentrations of insulin (standards) have been added. Add stop solution when a light blue tinge appears in the well with lowest concentration of insulin. After adding stop solution, read the absorbance at 450nm and 590nm. The absorbance at 590 nm is a measure of optical imperfections in the plate and therefore, this is subtracted from the absorbance at 450 nM. This provides an accurate and more correct reading. Although the reading at 450 alone can also be used, it will be less accurate as compared to the corrected one. Use the corrected absorbance at 450nm and draw an x-y scatter-plot between insulin concentration and absorbance. Based upon the standard curve, calculate the concentration of insulin in the test samples.
Although detailed instructions are provided with the kit, the importance of sample collection/preparation and the washing step should be emphasized. I have used this kit to examine the effect of dietary manipulation (high-fat diet feeding), selective b-cell toxin streptozotocin and insulin-receptor antagonist S961treatment on the insulin level in rat serum. The best results are obtained if the serum sample is free from sever hemolysis, washing is done properly, standards and samples are run in duplicate, and wells do not contain any air bubbles. In my experience, the rat/mouse insulin ELISA kit provides good reproducible results.
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