An Improved Immunoperoxidase Technique for Identifying SV40 V and T Antigens by Light Microscopy 1

Abstract

Discussion and Summary

Development of the peroxidase reaction for a period of 10 min has consistently resulted in strong nuclear staining of SV40 V and T positive cells. These cells are easily distinguished from antigen negative cells except for some finely granular cytoplasmic staining present in the latter cells. Attempts to eliminate this nonspecific staining by adsorbing either the serum or the conjugate with liver powder or with brain homogenate were unsuccessful. On the contrary, such adsorption weakened the activity of the serum. Incubation in absolute methanol or in 0.5% H₂O₂ (18) before exposure to serum did not eliminate the nonspecific staining but resulted in some nuclear damage and in a general decrease in staining intensity. Lowering the concentration of H₂O₂ (0.1%, 0.01%, 0.001%/) for this pretreatment did not improve the overall staining reaction and also resulted in occasional nuclear damage. Finally, partial purification of the conjugate by removing unbound peroxidase did not improve the staining effect as compared to that of the crude preparation due both to the efficiency of the peroxidase conjugation and to the lack of affinity of free peroxidase for the cells employed.

Throughout the immunoperoxidase test individual staining jars and separate forceps should be used for each cover glass in order to avoid transferring antigen-positive cells or antiserum to control preparations. In addition, three separate sets of staining jars should be used for each cover glass: one for the predevelopment part of the test, another for the development, and a third for the final rinses and dehydration, to avoid formation and deposition on the cover glasses of an intractable dark brown precipitate. (Flecks of dark precipitate may be noted on the nucleus and cytoplasm of some cells in Fig. 3.)

On the basis of our own experience and that of others (19, 20), it can be stated that, in addition to its slightly greater sensitivity, the indirect immunoperoxidase method offers the following major advantages when it is compared to the immunofluorescence technique. First, it allows examination of the preparations under brightfield light microscopy, thus eliminating the requirement for darkfield equipment and an ultraviolet light source. Second, since the procedure is based on a gentle although highly efficient conjugation reaction, neither the antibody nor the enzyme activity appears to be reduced to any appreciable degree while, at the same time, the conjugate remains stable. Third, the color intensity of the staining reaction is not dependent upon narrow pH changes or upon the ratio of peroxidase to antibody in the conjugation reaction, since it depends upon the accumulation of a reaction product. Admittedly, the contrast of a dark deposit against a light background, characteristic of the immunoperoxidase method, is not so marked as that of a few photons of fluorescent light against a black background characteristic of the immunofluorescence technique. Finally and most importantly, since the preparations are permanently mounted, no alterations of the label can occur. As a result, the preparations can be examined repeatedly over prolonged periods for the purpose of differential cell counts, for demonstration, and for photomicrography. In light of these advantages, it appears that the immunoperoxidase procedure is a simple, sensitive technique that can be a useful adjunct to the immunofluorescence procedure in identifying viral and virus-related cellular antigens.