Establishment and Assessment of Radioimmunoassay for Follicle Stimulating Hormone in Human Serum

Abstract

Follicle Stimulating Hormone (FSH) is a member of the glycoprotein hormone family that has a central and essential role in reproduction. The measurement of FSH concentration in serum is essential for investigating fertility and especially disorders of the hypothalamic/pituitary gonadal axis. The determination of FSH is fundamental to elucidating reproductive physiology, regulating fertility, and diagnosing and treating disorders of reproduction. This is usually carried out by radioimmunoassay (RIA). With the advent of immunoassay technology almost five decades ago, a powerful analytical tool to become available to the workers medically related areas. The simplicity and cost effectiveness for radioimmunoassay (RIA) has made the method of choice for routine laboratory measurements of numerous substances like hormones, tumor markers, vitamins…….etc. Radioimmunoassay allows for the measurement of wide range of materials of clinical and biological importance. This technique has a significant impact on medical diagnosis due to the ease with which the tests can be carried out, while assuring precision, specificity and sensitivity. The radioimmunoassay technique, as the name implies, achieves sensitivity through the use of radionuclides and specificity that is uniquely associated with immunochemical reactions. RIA is a convenient and simple method which can be used for routine estimations of FSH. The essential requirements for RIA include suitable reactants (specific antibody, standards, labeled antigen and second antibody or solid phase technique for separating the antibody-bound antigen from the free-labeled antigen). The objectives of the present work were designed to achieve the followings: - Preparation of the basic reagents of FSH assay which comprised anti-FSH polyclonal antibody, polyclonal second antibody (goat anti-rabbit IgG) as separating agent, FSH standards or calibrators and radiolabeled of FSH tracer. - The antisera obtained for each (1st and 2nd) were characterized in terms of titre, immunoresponse and displacement percent. - Formulation and optimization of local RIA systems (liquid phase and solid phase cellulose particles RIA techniques) for estimation of FSH in human sera. - Validation of the proposed assays using some of the performance characteristics, such as sensitivity, precision, accuracy and method comparison. The production of polyclonal anti-FSH was undertaken through the immunization of six male mature white New-Zealand rabbits with highly purified human FSH immunogen. Each rabbit primed with 50 µg FSH immunogen with 1 ml FAC. Then boosted with four successive injections with 25 µg FSH immunogen with 1 ml FAI at 4 weeks interval. Completely stable water-in-oil emulsion was achieved by using Hamilton double hub syringes techniques (1:1, aqueous phase: oil phase). Injection route was done subcutaneously (around the rabbit shoulder) and intramuscular. Immunized rabbits were bled after four weeks of the first booster injection and continued until the end of the immunization schedule. Individual bleedings were centrifuged for separating antisera. At the end of immunization schedule individual antisera were characterized for titre and displacement percent. Immunoresponse profile of each immunized rabbit was constructed.