DEVELOPMENT AND VALIDATION OF IMMUNOENZYME TEST-SYSTEM FOR DETERMINATION OF 25-HYDROXYVITAMIN D IN BLOOD SERUM
Journal: Biotechnologia Acta (Vol.9, No. 2)Publication Date: 2016-04-30
Authors : A. O. Mazanova; I. O. Shymanskyy; M. M. Veliky;
Page : 28-36
Keywords : 25-Hydroxyvitamin D3; immunoenzyme analysis; polyclonal antibodies.;
Abstract
The present study was aimed at designing of immunoenzyme test-system for competitive determination of 25OHD in blood serum using polyclonal antibodies against 25OHD and biotin-streptavidin visualization technique. The validation parameters of test-system were determined. For development of test-system we used the preparation of specific antibodies against 25OHD with the titer 1:3000. The optimal concentration of competing agent (25OHD3-LC-biotin) was found to be equal to 5 ng/ml. The standard calibration curve was built by using a set of seven calibrators. For the validation of test-system the following parameters were established: limit of detection, quantitative limit, cross-reactivity, intra- and inter-system coefficients of variation (Intra.CV, Inter.CV) and “matrix effect”. Constructed immunoenzyme test-system can be used successfully for 25OHD determination in blood serum samples.
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