Abstract
Antigen was identified histochemically without the use of labeled antibodies by the sequential application of (a) specific rabbit antiserum, (b) sheep antiserum to rabbit immunoglobulin G, (c) specifically purified, soluble horseradish peroxidase-anti-horseradish peroxidase complex (PAP), (d) 3,3'-diaminobenzidine and hydrogen peroxide and (e) osmium tetroxide. A simple method for preparation of high yields of PAP consisted of precipitation of antibody from specific rabbit antiserum with horseradish peroxidase (PO) at equivalence, solubilization of the washed precipitate with excess PO at pH 2.3, 1°C, followed by immediate neutralization and separation of PAP from PO by half-saturation with ammonium sulfate. The ratio of PO to anti-PO in PAP was 3:2 irrespective of the source of antiserum. PAP was heterogeneous on electrophoresis, homogeneous on sedimentation, diffusion and electron microscopy and consisted of pentagons with diameters of 205 Å.