Production and Purification of Equine Chorionic Gonadotropin Hormone Using Polyclonal Antibody

Equine chorionic gonadotropin (eCG) is commonly used in association with a progestagen treatment to synchronize estrus of goats and ewes during breeding and non-breeding seasons. Classical purification of the eCG from serum includes pH fractionation with metaphosphoric acid two ethanol precipitation steps as well as dialysis followed by fixed-bed chromatography. The aim of the current study was to develop an accurate and fast method for production and purification of eCG using a polyclonal antibody assay. The blood samples (300 mL) were taken from the jugular vein of 17 mares on days 50, 70 and 90 of pregnancy. Plasma of the samples was siphoned and phenol solution was added to the plasma and stored in the refrigerator until eCG extraction. To prepare the polyclonal antibody against eCG, four male rabbits, about four months old with 2 kg weight, were chosen. A basic immunization was done by injecting 25 IU of eCG to the rabbits. Ouchterlony assay or double immunodiffusion test was used to assess the immunization and the titer of antiserum against eCG. eCG was purified from the plasma via the solid-phase extraction (SPE) method. Results based on agarose-gel double immunodiffusion test showed that rabbits were completely immunized. SDS-PAGE analysis showed purified eCG is extracted without any significant contamination. The extraction of eCG with polyclonal antibody using the SPE method and production of anti- eCG antiserum in rabbits is suitable and may be a cost effective method for large scale production of eCG and anti-eCG antiserum in Iran.