Obtención de conjugados 17 α- Hidroxiprogesterona/ Fosfatasa Alcalina para su empleo en el diagnóstico de Hiperplasia adrenal Congénita.

Abstract

The 17 α-Hydroxyprogesterone is the principal biochemical marker for 21-hydroxilase deficiency, which is responsible for 95 % of Congenital Adrenal Hyperplasia (CAH) cases. 17 α-Hydroxyprogesterone –Phosphatase Alkaline (PA) was prepared for use in a quantification assay of this hormone. The conjugation was carried out with derivatized hormone, 17α-Hydroxy-progesterone 3- (O-Carboxymetil) Oxime (17OHP3OCMO), through the carbodiimide method, using N,N’-diisopropil carbodiimida (EDC). The following parameters were evaluated to establish optimal conjugation conditions: buffer, molar ratio, final concentration of EDC, final volume of reaction environment, pH and mixture composition, and reaction tempera-ture. Its performance in an UMELISA assay was evaluated with plates coated with anti 17 α-Hydroxy-progesterone polyclonal antibodies, using a calibration curve of hormone in dried blood on filter paper (useful range of 15. 6 – 250 nmol/L) and the fluorescent substrate 4- metil-umbelipherilphosphate. The best results were obtained with: PBS 0.05 mol/L pH 7.5 as buffer, PA- 17OHP3OCMO molar ratio 1:4 (4.5 mg - 50 μg), final concentration