Preformed, soluble complexes of enzyme with polyclonal or monoclonal antibodies

Preformed, soluble complexes of enzyme with polyclonal or monoclonal antibodies 

An elegant method for the preparation of PAP from polyclonal antisera was developed by Sternberger et al. (1970 for review, see Stemberger, 1979). Soluble PAP complexes are useful as tracers in EIA, but particularly in EIH. In general, their activity is superior, sometimes by several orders of magnitude, to that of chemically linked conjugates. 

This procedure is based on the observation that the immune precipitate, obtained after the addition of POase to an anti-POase antiserum, can be solubilized, after the removal of non-specific proteins 

It is necessary to determine the equivalence zone of the anti-POase antiserum to obtain optimum results (Table 11.22). If antiserum is used in excess, the yield of POase is higher, if POase is used in excess the yield of antibody is higher. At equivalence, the yield of PAP is 30%, both for the antibody and the antigen. PAP in good yields is obtained if anti-POase is precipitated with POase at 1.5 times the amount of equivalence (Table 11.23). In all cases PAP will be of the same composition (3 2 molecular ratio). 

The composition of PAP can be determined spectrophotometrically by measuring the absorbance (A) at 401 nm (Soret band of POase a = 2.25 cmVmg Table 10.4) and at 278 nm [protein absorbance by both POase (a = 0.75 cm mg) and IgG (a = 1.4 cm /mg)] of a sample diluted with a neutral buffer. The POase content of the PAP (in mg per ml) is calculated according to Section 9.2.3.4 . 4401 x dilution factor x 2.25 . Using this value, the contribution of POase (P) to the absorbance of the PAP at 278 nm (mg POase x 0.75) can be calculated and, thence, the contribution of the anti-POase 

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