PURIFICATION OF SERUM IGM FROM GRASS CARP (CIENOYHARYNGODONI DELLUS) AND PREPARATION OF RABBIT SERA ANTI-IGM

Immunoglobulins are the primary humoral component of the acquired immune system. In order to know the ImmunoglobulinM (IgM) of grass carp (Cienoyharyngodoni dellus), two different isolating methods were applied to find the best way to purify serum IgM in the present study. The purity and molecular weight of the serum IgM were determined, and then the rabbit polyclonal antisera were prepared with the purified IgM. In this study, blood was collected with a syringe from the caudal sinus of 30 fish, and allowed to clot at 20 for 20 ℃ min and at 4℃ for 30 min. Serum was obtained by centrifugation at 500 g to remove cells and at 15000 g to remove particulate matter, and stored frozen at ?80℃. Firstly, 33% ammonium sulfate precipitation was used to purify the serum, the result showed that most proteins in grass carp were precipitated and could not get high purity IgM, so it could only be a crude method. At the same time, HiTrap rProteinA Sepharose affinity was performed at 10℃ to purify IgM. The partial characteristics of purified proteins were then analyzed and compared by SDS-PAGE and Western-blot. The results revealed that grass carp serum immunoglobulin purified by HiTrap rProtein A affinity chromatography had only two bands of 78 kD and 28 kD, the same two bands as those in serum. Western-blot analysis showed that the mouse anti-human Ig antibody can recognize bands of 78 kD and 28 kD. Sera anti-IgM of grass carp had been prepared by repeatedly immunized New Zealand rabbits with purified IgM. and the titers of anti-sera obtained up to 1:25600 examined by indirect ELISA. Total serum protein and the concentration of IgM of the normal sera of grass carp were determined by using the Coomassie blue dye binding method of Bradford and sandwich ELISA, the values being 25.87 mg/mL and 4.5 mg/mL, respectively. The percentage of IgM in the total serum protein was 17.39%. Measurements of IgM levels in serum from several fish species have been surveyed and the results point to a range between 0.25 and 23.5 mg/mL. The studies also pointed to considerable individual variations in serum IgM levels among fish, as occurs with other fish immune activities. These changes may be related to size and/or age, environmental conditions or disease status. It must be emphasized, therefore, that the serum IgM values obtained in the present work represent the mean of at least thirty healthy and non-immunized individuals of similar size kept under same conditions. In present work, the 13mg amount of purified IgM from 10 mL serum was not larger than other fish because the grass carp was not immunized with antigen such as BSA in the research. In conclude, the results indicated that protein A-sepharose affinity chromatography was feasible in purification of IgM of grass crap. The polyclonal antibody obtained could be used in correlative studies in future.