Analysis of purified antigen

If the antigen is to be used for immunization it may be sufficient to test that the required epitopes are present, but often it will be necessary to assay for recovery of biological activity. The antigen may be denatured or misfolded in some way that would not show up by these techniques. It is useful therefore to have some form of bioassay by means of which the purified ligand and the original starting material can be compared, this will of course depend on the nature of the antigen itself. 

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Poly(ADP-ribose) polymerase from different species appears to be structurally conserved in eucaryotes with regard to molecular mass, amino acid composition and enzyme activity as detected by gel electrophoresis (1-3). In addition, analysis of the antigenic structure by inununoblotting with polyclonal antibodies (4, 5) provides evidence that some epitopes are common to various animal cells. We have recently developed monoclonal antibodies against the main functiomd domains of calf thymus poly(ADP-ribose) polymerase (6). The purified monoclonal antibodies have been used to map three epitopes along the domains described by Kameshita etal. (7), to examine the presence of these epitopes in the enzyme from different species by immunoblot analysis, and to inhibit enzyme activity in vitro. 

Affinity chromatography (12) has become an important tool in the isolation of purified fractions of such substances as enzymes. Advantage is taken of specific interactions such as antigen-antibody interactions. One substance of the pair (e.g. antigen) is bonded to a support. When a mixture is passed through the column, the specific interaction retains the corresponding antibody relative to other substances. A change of mobile phase conditions then elutes the pure antibody. This method has a real potential for analysis of specific proteins in body fluids. 

If quantitative analysis of antibody-phage binding is desirable, it is possible to perform an ELISA procedure using purified phage as antigen. 

All the anti-carbohydrate antibodies purified by affinity chromatography are isomers of a different number of isoforms. The forms can be separated by electrofocusing and all have been found to have combining activity for the immunodeterminant of the same antigen by the method of coupled analysis of electrofocusing and agar diffusion [88], The isoforms of antimonosaccharide antibodies are shown in Fig. (48). 

Polyclonal antibodies that detect both the 204- and 200-kDa HCs were raised in rabbits using purified bovine aortic smooth muscle myosin, which is a 1 1 mixture of both isoforms as antigens. These antibodies have approximately equal affinity for HC204 and HC200 (Kelley and Adelstein, 1990). Immunoblot analysis of smooth muscle HMM and LMM using the 50% ammonium sulfate immunoglobulin (IgG) fraction revealed that practically all the epitopes were located in the LMM region (Christine A. Kelley, unpublished results, NHLBI). 

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