| Objective: IHC is a common used labeled immunoassay technique in the research work and the clinical practice of medicine today. It has many advantages, such as the easily operated steps, the conveniently observed result, etc. Because of many influencing factors IHC doesn't have a good reproducibility in practice, so its result isn't satisfying. To stabilize the IHC result, scientists made a lot of experiments to seek the causes and to try to resolve the problems in each step from specimen stabilization, antigen restoration, substrate concentration to stain-time control etc. The objective of our experiment is to observe whether secondary antibody is one of the influencing factors of IHC, and to compare IHC result stained by the secondary antibody prepared by different sub-class mouse IgG in order to summarize a method by which we can prepare secondary antibody having steady quality.Methods: In this research, 10 breast cancer tissues were made paraffin sections in routine. Each tissue was stained by the S-P method of IHC with monoclonal antibodies (McAbs) of two sub-class (IgG1,IgG2b) and secondary antibodies purchased from three companies. All the reagents were the product of S-P 9002 kit except the secondary antibody. Images were chosen randomly and 300 cells were drawn also randomly for each section with the image analysis system. The analysis target was cell-average-optical-density. The positive cell was that the cell-average-optical-density was larger than the optical density which was the tissue negative control's mean plus duplicate standard deviation (+2s). Positive intensity and positive rate were the positive sample's two targets which were named stain intensity. Positive intensity was the mean of cell-average-optical-density of all the positive cells. Positive rate was the percent of positive cell in 300 cells. The positive cell was cytoplasm-stained under the microscope. Mice were immunized by two sub-class McAbs(IgG1, IgG2b) prepared by our lab to acquire secondary antibody. The two secondary antibodies were named No.1 secondary antibody and No.2 secondary antibody separately. The two secondary antibodies were purified and labeled with biotin. Then the two secondary antibodies and the product of Zymed company were compared in the same way as above. Results: 1.Comparion of the stain intensity of the secondary antibodies from three companies Positive intensity of Zymed's secondary antibody was highest (P<0.05) with both IgG1 and IgG2b. Positive rate of HuaMei's secondary antibody was higher than BoShide's with IgG1(P<0.05). Positive rate had no statistical difference among the three secondary antibodies with IgG2b(P>0.05).2.Comparison of the ratio (IgG1:IgG2b) of stain intensity of the secondary antibodies from three companies The ratio of positive intensity of Zymed's secondary antibody was different from BoShide's(P<0.05). The ratio of positive rate had no statistical difference among the three secondary antibodies (P>0.05).3.Comparison of the stain intensity among the two self-preparing secondary antibody and Zymed's Positive intensity of Zymed's secondary antibody was higher than No.2 with IgG1 (P<0.01). Positive intensity of Zymed's secondary antibody was higher than No.1 with IgG2b (P<0.01). Positive rate of Zymed's secondary antibody was higher than No.2 with IgG1 (P<0.01). Positive rate of Zymed's secondary antibody was higher than No.2 with IgG2b (P<0.01).4. Comparison of the ratio (IgG1:IgG2b) of stain intensity among the two self-preparing secondary antibody and Zymed's Both the ratio of positive intensity and positive rate , Zymed's secondary antibody had statistical difference from the two self-preparing secondary antibody (P<0.01). Conclusions: 1. The difference of IHC stain among the three companies indicates that the secondary antibody affects IHC. The concentration, affinity and specificity etc. of secondary antibody all can affect the antibody's quality.2. We chose two McAbs of different sub-class to remove the effect of the reagent's... |
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