| Tree shrew (Tupaia), with the shape of squirrel, was classified into Mammalia, placentalia Scandenti. It had been considered as primate animal, mainly distributed in tropical and subtropical areas of Southeast Asia, and was also inhabitated in Yunnan, Guangxi, Guangdong and Hainan provinces of China. More exactly, trew shrew of China was belonged to Tupaia belangeri, the most distributed subspecies. Tree shrew has small size, grow and reproduce fastly. This animal could be easily captured, domesticated and breed with low cost. Its metabolism and the anatomic characters of were much closer with humans than rodents. Because of the limitation of non-primate animal resource and the miniaturization trend of animal model, the tree shrew, as a possible animal model of human diseases, particularly viral diseases, had attracted the comprehensive attentions.As a novel experimental animal model, the application prospect of tree shrew was desirable. Antibody level is a important parameter to evaluate the ability of immune response of bodies to invaded pathogens. But, the records on the immune response of tree shrew were still scare because of the lake of detection methods of IgG globulin. It is urgent to obtain the anti-IgG monoclonal antibodies, and to establish Enzyme Link Immunosorbent Assay (ELISA) IgG detection method. These will predicatively provide a useful tool to promote the related research on tree shrew.Here, the purified IgG of tree shrew was used to immune female BALB/c mice, followed with artificial cytomixis hybridization of myeloma cell and spleen derived B-cell, and then screening of positive hybridoma, which could secrete anti tree shrew IgG antibody. It was proved that two positive hybridoma (TBS-1, TBS-2) had been obtained. The cell fusion rate was 32.92%, and the positive rate of hybridoma cells was 1.27%. By sub-clony screening of 4 generation, TBS-2 cells still not below of 100% positive rate, mixed with the negative cells. The valence of secreted antibody was also not high enough. All of these indicated that this strain of hybridoma was not productive and bio-stable. In comparison, the positive rate of TBS-1 had increased to 100% after 3 generations sub-clony screening. The higher valence (1:800) of its secreted antibody had been achieved. After abdominal cavity injection with TBS-1, the valence of antibody in collected ascites had been up to 1:25600. The analysis on chromosome karotype of hybridoma had shown that the number of chromosome in different passge fusion cells was normal, and its genetic characters were stable. With Western blot assay, secreated monoclonal have been proved to hybridize with serum derived tree shrew IgG effectively and specifically, no other hybrid band had been found.Uing this prepared anti-IgG monoclonal antibody and previously gotted anti-IgG polyclonal antibody from immuned rabbit, and purified tree shrew IgG taken as positive sample, the ELISA detection method of total IgG. in tree shrew had been established preliminarily. The optimized conditions for detection was listed as below:①Coatting: 100μl 1/400 diluted ascites was used for one hole, incubated at 37℃for 2h, then overnight at 4℃;②seal:200μl 2% BSA was incubated at 37℃for 2h in one hole;③polyclonal antibody concentration:for one hole, 100μl 1/2000 diluted rebbit serum, which containing polyclonal antibody, was incubated at 37℃for 1 hour. PBS was replaced in blank control;④enzyme-labeled antibody was used in 1/8000 diluted goat anti-rabbit IgG (HRP) for 100μl/hole, at 37℃incubation for 1h;⑤TMB substrate was used in 100μl/hole at 37℃dark incubation for 15min;⑥Terminate reaction was performed with 2M H2SO4 for 100μl/hole. The established method was evaluated as high specific, good repeatable. From 0.25μg/ml to 3.0μg/ml of tree shrew IgG, a good linear relationship (R2=0.9912) had been found between OD450 values and IgG concentration.In summary, the anti-IgG monoclonal antibody of tree shrew was prepared succefully, and the ELISA detecte method of tree shrew total IgG was established. These will provide a useful tool to evaluate the antibodies level during immune response of tree shrew. Furthermore, these could be considered as the reference for detection of specific antibody and neutralize antibody. For development of tree shrew animal model of human diseases, these would doubtless to be the necessary foundation. |
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