The Preparation Of Mouse SP Kit

Objective: Immunohistochemistry(IHC) technique has been widely used in the research work of basic medicine and the diagnosis of clinical diseases, because it has many advantages, such as simple and convenient operation, high sensitivity and specificity, etc. Moreover, the pathological diagnosis methods have also been improved in a certain extent by the application of IHC assay. But there are many problems in practice. Many scholars and clinical checkers detected many factors that can influence the result of IHC, such as tissue fixation, epitope retrieval, the endogenous avidin-binding activity(EABA) and the quality of antibody, etc. Among the factors above, the quality of secondary antibody is a very important factor to the result of IHC. In this experiment, our objective was to prepare a kind of top-quality secondary antibody with a new method and to obtain a stable result of IHC. At the same time, we also prepared HRP-SA (streptavidion-horseradish peroxidase) composite by coupling SA with HRP, and SA was prepared and purified by our laboratory. Then we compared the quality of secondary antibody and HRP-SA complex that prepared by ourselves with that bought from ZhongShan Company. Methods: 1 In our study, 12 hybridoma cell lines secreting McAbs of mouse anti-RSA(rabbit serum albumin) were obtained by cell fusion technique. Among them, 8 hybridoma cell lines could stably secret McAbs after having been stored at -196℃, then we prepared ascites fluid with BALB/c mice. Sub-classes and the titer of McAbs were identified by indirect ELISA. 2 McAbs were extracted and purified from ascites fluid by Sepharose 4B conjugated with RSA. We collected and condensed the eluate of McAbs from Sepharose 4B agarose. The concentration of protein and purity of McAbs were evaluated by double-diffusion assays in gels, spectrophotometer and immunoelectrophoresis (IEP). Then we conjugated Sepharose 4B with McAbs, which acted as antigen in order to purify goat anti-mouse IgG. 3 KM mice were immunized with RSA and mouse anti-RSA serum was gotten, then mouse anti-RSA IgG was purified by affinity chromatography. Its purity and titer were determined by double-diffusion assays in gels. 4 Goat anti-mouse IgG serum was obtained by immunizing goat with purified mouse anti-RSA IgG. The titer of the serum was determined by double-diffusion assays in gels, its specificity was estimated by IEP. 5 Biotin-labeling secondary antibody was obtained by coupling goat anti-mouse IgG with biotin. Then IHC was done with different Biotin-IgG products prepared by our laboratory and bought from ZhongShan Company or HuaMei Company.6 The HRP-SA complex was obtained by coupling SA with HRP by routine method. Then the HRP-SA composite was used in IHC at different concentration. Different HRP-SA complexes prepared by our lab and bought from ZhongShan Company were compared by the results of staining. Results: 1 Preparation of monoclonal antibody against RSA and identification of its specificity 8 hybridoma cell lines stably secreting anti-RSA McAbs were obtained by cell fusion. 5 cell lines could secret IgG1, 1 could secret IgG2a, and 2 could secret IgG2b. The titer of all the antibodies was high (1:2¹⁴¹:2²⁰). 2 The results of McAb purification There was high concentration of protein in the eluate of 2B₃, 2E₃, 2A₉ and 5D₄, and deposited line was observed in IEP, while there was low concentration of protein in other eluate and deposited line wasn't observed. 3 The purity of 2B₃ McAb By the method of IEP, a single deposited line was observed between 2B3 McAb and goat anti-mouse serum. 4 The titer and the purity of mouse anti-RSA IgG The titer of mouse anti-RSA IgG was 1:16, and there were more than one deposited line in double-diffusion assays, which indicated that there were other antibodies except mouse anti-RSA antibody. 5 The titer of goat anti-mouse IgG serum One deposited line was observed between goat anti-mouse IgG serum dilutedat 1:128 and mouse IgG in double-diffusion assays, while one faint deposited line between goat anti-mouse IgG serum diluted at 1:256 and mouse IgG was detected, then the titer of goat anti-mouse IgG serum was 1:256. 6 The purity of goat anti-mouse IgG serum The specificity of goat anti-mouse IgG serum was poor when normal mouse serum was purified with the method of DEAE diethylaminoethyl-cellulose, and several deposited lines were observed in IEP. However the specificity of anti-serum prepared by immunizing goat with mouse anti-RSA IgG was excellent. 7 Comparison of secondary antibodies produced by us and other companies Through comparing the ratio of immunity, there was not significant statistical difference between different secondary antibodies (P>0.05). The results indicated our product had the same level of quality as those bought from different companies. 8 Comparison of HRP-SA at different concentration Through comparing the ratio of immunity, it was found that the lower HRP-SA concentration was, the fewer non-specific staining was. In addition, when the HRP-SA prepared by our laboratory was diluted to 5μg/ml, there was not significant statistical difference between HRP-SA prepared by our lab and that from ZhongShan (P>0.05). Conclusion: 1 If pure McAb is used as antigen, the purity of secondary...