南方鲇免疫球蛋白单克隆抗体的制备及特性

采用PEG沉淀结合Sepharose-4B柱层析法分离纯化了健康非免疫状态下南方鲇血清免疫球蛋白,在SDS-PAGE电泳条件下血清免疫球蛋白重链和轻链的分子量分别约为77 kD和27 kD。应用杂交瘤单克隆抗体技术制备了4个南方鲇免疫球蛋白特异性的单克隆抗体细胞株,并对这些单克隆抗体的特性进行了分析。经抗体亚级份测定,其中IgG1有2株,IgG2a有1株,IgG2b有1株;抗体滴度为104—106,有三株单抗具有Western-blot反应特性,识别南方鲇免疫球蛋白的重链。4株单抗都能特异地识别南方鲇、鲇的免疫球蛋白,而与鲫、草鱼、罗非鱼、斑点叉尾、光泽黄颡鱼血清以及水产动物常见病原菌如气单胞菌、爱德华氏菌、弧菌、柱状屈桡杆菌、沙门氏菌及大肠杆菌等无任何交叉反应。单克隆抗体F4-A12对纯化的南方鲇免疫球蛋白的检测灵敏度为31 ng。实验结果证明这些单抗具有高度特异、高度灵敏等特点,可用于南方鲇免疫球蛋白的结构分析、免疫应答水平监测和病原诊断,具有广阔的应用前景。

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES AGAINST SILURUS MERIDIONALIS CHEN SERUM IMMUNOGLOBULIN

Monoclonal antibodies(MAb) to serum immunoglobulins(Ig) of fish are of immense use in understanding on the fish immune system and rapid identification of pathogens.Southern catfish(Silurus meridionalis Chen) is a unique and important culture fish in China.Literature review indicated that MAbs have not been developed against the Ig of Southern catfish.This paper described the purification and characterization of serum Ig on southern catfish,along with the development and characterization of MAbs against Ig on southern catfish.Serum Ig from healthy southern catfish was purified by 9% polyethylene glycol(PEG6000) precipitation and followed by Sepharose-4B gel column chromatography.SDS-PAGE analysis showed that molecular weight of heavy chain and light chain was approximately 77 kD and 27 kD respectively.The purified serum Ig of southern catfish was used as antigen to immunize Balb/c mice.After four times immunization,the spleen of immunized mouse was removed,and the spleenocytes were obtained and fused with myeloma cells by using PEG 4000.The hybrids were plated into 96 well tissue culture plates and observed for colony growth.After two weeks,wells with hybridomas were tested by ELISA for screening the antibody secreting cell.Four hybridoma cell lines which secrete MAbs against the Ig of southern catfish had been established.Culture supernatant of the four positive hybirdomas were collected and tested using the IsoStripTM Mouse Monoclonal Antibody Isotyping Kit,the results showed that two of them were IgG1,one was IgG2a and one belonged to IgG2b.Ascites of four MAbs were obtained and titers ranged from 104 to 107 tested by ELISA.Standard procedures of Western blotting were used to determine the reactivity of the MAbs in hybridoma culture supernatants to reduced southern catfish serum Ig on a 10% gradient gel.Three of four Mabs were able to recognize the heavy chain of the Ig on Western blotting;the last one did have no reaction with reduced southern catfish Ig under low dilution condition,although it showed strong reactivity in ELISA under high dilution condition.Specificity of four MAbs was investigated by ELISA and Western blotting using different fish serum as antigen.Results from ELISA showed that all of them only specifically reacted with serum of Silurus meridionalis Chen and Silurus asotus,and did not have any cross reaction with serum of Ietalurus Punetaus,Peheobagrus nitidus,Cienoyharyngodoni dellus,Carrassius auratus and Oreochromis niloticu.Western blotting further proved that three of four MAbs were able to bind the heavy chain of Ig only from Silurus meridionalis Chen and Silurus asotus,and all MAbs had no reaction with serum Ig of the other fish.The reactivity of MAbs with the fish common bacterial pathogen was tested by ELISA using bacteria,such as Aeromonas,Edwardsiella,Vibrio,Flexibacter columnaris,Salmonella and Escherichia coli,as coating antigen,and all four MAbs had no reaction with those tested bacterial pathogen.The measuring sensitivity of the Mab MaF4-A12 to purified southern catfish Ig was as low as 31 ng.All of these results demonstrated that Mabs were highly specific and sensitive to the Ig of southern catfish,and had the great potential to be used for analyzing the structure of southern catfish Ig,monitoring the immunity level and pathogen diagnosis.