Production and characterization of anti-LH polyclonal antibodies for establishment of sepharose solid phase radioimmunoassay

Abstract EN

The present study was designed to achieve the production and characterization of polyclonal antibody of luteinizing hormone (Anti- LH) as a basic component of LH radioimmunoassay (RIA).

The main objective was to improve the immunogenicity of LH by conjugation of LH with bovine serum albumin (LH: BSA) as a protein carrier using Ethyl dimethylaminopropyl Carbodiimide (ECDI).

Production of Anti-LH was described where LH: BSA immunogen was immunized into three male mature white New-Zealand rabbits through primary immunization and four boosters.

The criteria for selecting LH antiserum for liquid phase RIA system were mainly titer, displacement and immunoresponse profile and followed by partial purification of IgG–LH.

The radioiodinated125I–LH tracer was carried out using Chloramine-T as an oxidizing agent and the tracer was purified through PD- 10 column.

The preparation of LH standards was carried out.

Coupling of purified IgG-LH to activated Sepharose particles CL-4B was carried out after activation of Sepharose particles with 1, ʹ1- carbony ldiimidazole.

Extensive studies were carried out to obtain the optimum conditions of using solid phase Sepharose particles to reach the optimum separation efficiency.

The results of validation tests revealed that the local solid phase RIA system is precise and accurate to detect LH concentration in human serum to be used as a diagnostic tool in investigating the infertility in the hypothalamic pituitary gonadal disorders.