The quantification of insulin is a key factor in the study of diabetes mellitus and other conditions involving abnormal glucose metabolism. One of the most popular animal models of human Type I diabetes is the streptozotocin (STZ) treated rat, which develops hyperglycemia as a result of reducing the number of beta-cells of the Langergans islets. In many studies, blood glucose concentration measurements are made using an indirect index of insulin level. However, blood glucose concentration is not always a good indicator of diabetic injury.
Our lab has tested several techniques for determining insulin concentrations in the blood of normal and STZ diabetic rats using survivor experiments. We expected a 10-fold concentration difference between these groups of animals but we were limited by the volume of blood that can be taken from a living animal. Under these circumstances, a detection system that requires only a small volume of sample and can be used for analysis without an additional concentration step, is critical. The Ultra Sensitive Rat Insulin ELISA Kit from Crystal Chem Inc. complies with the above restriction. Furthermore, antibodies that we were using for immunoreactions with this kit were developed specially against rat insulin and exhibit minimum cross-reactivity with other rat plasma proteins.
The Ultra Sensitive Rat Insulin ELISA Kit contains reagent for 96 wells, which is sufficient for 42 samples and one standard curve in duplicate. It has twelve 8-well antibody coated strips that fit into 2 frame racks. The kit contains stock solutions for all sample buffer solutions, wash and stop buffers and color reactions. After dilution, the working solution may be stored at 4Cº for up to two weeks. Also contained within the kit, is an insulin standard in freeze-dried form. Perhaps the most significant parts of the kit were the detailed protocol sheets, describing each step of blood plasma/serum preparation, the detection process, and the interpretation of results. This clear description of all steps makes it possible for a novice to use the Insulin ELISA Kit without additional supervision. Additional materials required, but not provided, are 10, 100, 1000 ul micropipettes with disposable tips, 1000 ml volumetric glassware, milli-Q or redistilled water and an ELISA plate reader with 460 nm filter.
In conclusion, we have used this kit for the last two years in our investigations to compare the concentration of plasma insulin with peripheral nerve neuropathy in animal models of insulin-dependant diabetes. Insulin has been successfully measured in plasma control and diabetic rats (we needed just 5 ul and 25 ul of plasma, respectively; Romanovsky et. al., JPNS 2004). Ease-of-use and reproducible results are the main reasons why we are going to use the Ultra Sensitive Rat Insulin ELISA Kit from Crystal Chem Inc. in our future experiments.
Dmitriy Romanovsky, Ph.D.
Postdoctoral fellow
Department of Anesthesiology
University of Arkansas for Medical Sciences