Mazanova A. Development of immunoenzyme test system for the determination of 25OHD as a marker of vitamin D availability in Type 1 diabetes

The thesis is devoted to the development of an immunoenzyme test-system for 25OHD determining in serum for the purpose of characterizing the vitamin D status of the body in a number of pathological conditions, in particular, Type 1 diabetes mellitus. To generate polyclonal antibodies in the rabbits, the 25OHD3 conjugate with keyhole limpet hemocyanin (KLH) was synthesized using a modified carbodiimide method. The conjugates were characterized by gel-filtration and thin-layer chromatography methods. After immunization, in the serum of rabbits, the titer of specific antibodies to 25OHD was 1:10000. In order to concentrate the antibodies, they were salted out with ammonium sulfate ((NH4)2SO4). To construct an immunoassay, the concentration of the competing agent - 25OHD3-LC-biotin 5 ng/ml, was selected. Validation of the created test-system was carried out according to a number of characteristics: a standard calibration curve was constructed in the range of concentrations of calibrators 25OHD3 0-150 ng/ml; detection limit (2.3 ng/ml) and quantitative limit (6.9 ng/ml) were determined; the absence of a "matrix effect" of hemoglobin, bilirubin, and triacylglycerol for analysis was shown; Intra CV and Inter CV defined within the permissible 10%; it has been shown that the cross-reactivity of the system with other metabolites of vitamin D did not exceed 10%. Consequently, the created immunoenzyme test-system can be used to detect 25OHD in serological samples. It has been shown that the development of experimental Type 1 diabetes is accompanied by a significant decrease in the content of 25OHD in experimental animal serum, which leads to disturbances of the synthesis of key elements of the vitamin D-endocrine system in the kidneys, liver, bone marrow and bone marrow. Normalization of vitamin D level in diabetic animals contributes to the correction of vitamin D metabolism and VDR-mediated cell signaling involving 1α,25(OH)2D.