Isolation And Purification Of IgG Subclasses From Bactrian Camel And Preparation Of IgG Subclasses Polyclonal Antibodies

Bactrian camel in the arid and desert environment in the formation of its unique organ structure and unique ability to adapt to the environment.Of the three subtypes of IgG in the Bactrian camel serum,IgG1 is a tetramer consisting of two homologous light chains and two homologous heavy chains,whereas IgG2 and IgG3 are composed of two homologous heavy Chain structure of the dimer,due to the lack of natural light chain is called the heavy chain antibody.Heavy chain antibody accounts for 75% of the Bactrian camel cycle antibody,because of its unique structure and many unique biological functions,in the Bactrian camel body fluid play an important role.The study was aimed to isolate and purify IgG subclasses from the serum of Bactrian camel(IgG1,IgG2 and IgG3),to prepare polyclonal antibodies of IgG subclasses,and to investigate specificity of the polyclonal antibodies between the three subclasses.By a combination of affinity chromatography on protein A and protein G,three subclasses of IgG can be isolated from the serum of Bactrian camel.SDS-PAGE analyze different IgG subclasses under reduce and non-reduced condition.IgG subclasses were used as antigen to immunize male rabbits for prepare polyclonal antibodies.By using ELISA to evaluate the titers of polyclonal antibodies and specificity of the polyclonal antibodies between the three subclasses in a preliminary estimate by ELISA;By using Western blotting to evaluate specificity of the three subclasses polyclonal antibodies in the serum of Bactrian camel and the polyclonal antibodies between the three subclasses.The plasma cells secreted by IgG were studied by immunohistochemical method.In the results of polyacrylamide gel electrophoresis under non-reducing and reducing conditions,the three subtypes of Bactrian camel IgG were in accordance with their molecular weight and structure and the results showed no other proteins were eligible for polyclonal antibody.The results of ELISA showed that the antibody titers were consistent with the experimental purpose and that the polyclonal antibodies of these three subtypes were initially judged to have cross-reactivity.Western blotting results showed that the three subtypes of polyclonal antibodies had cross-reactivity.In the immunohistochemical results,the distribution of positive cells in the spleen of Bactrian camel was observed by light microscopy and statistical analysis.It was found that the positive cells in the spleen were higher in the frontal and red pulp than in the white pulp.There was no significant difference in the density of positive cells in the red pulp(P> 0.05)by using the immunohistochemical results of the three subtypes of IgG.The positive cell density of the polyclonal antibody using IgG1 in the white pulp was significantly higher than that of the polyclonal antibody using the IgG2 polyclonal antibody(P <0.01).The positive cell density using IgGl polyclonal antibody was significantly increased compared to the positive cell density using IgG2 and IgG3 polyclonal antibodies(P <0.05),The positive cell density using IgG2 polyclonal antibody was not significantly different from the positive cell density using IgG3 polyclonal antibody(P> 0.05).The IgG subclasses(IgG1,IgG2 and IgG3)were isolated and purified by Protein A and Protein G affinity chromatography.The results showed that the three subtypes were consistent with their structures and molecular weight by polyacrylamide gel electrophoresis under reducing and non-reducing conditions.The polyclonal antibodies of the two subtypes of Bactrian Camel IgG were prepared by immunizing rabbits with two subspecies of Bactrian camel IgG.The specificity of the polyclonal antibody was detected by ELISA and Western blotting.It was found that the three subtypes had cross-reactivity.Then the positive cells in the spleen were analyzed and analyzed.It was found that the positive cells were mainly distributed in the marginal zone and the red pulp.The use of IgG1 polyclonal antibody-positive cell density in the white pulp was significantly greater than that of the other two polyclonal antibodies.This paper provides the basic support for the future research of Bactrian camel IgG.