Enzyme-lectin histochemistry

Simultaneous detection of two antigens by peroxidase-anti-peroxidase antibody (PAP) and alkaline phosphatase-anti-alkaline phosphatase (APAAP) complexes 

Apply primary antibodies (100-1000 times diluted in isotonic buffer) to both antigens. Incubation for 1 h at room temperature in a humid chamber. 

Wash as in step 3 (Table 17.3), apply both anti-IgG as in step 4 (Table 17.3), wash again. 

Apply enzyme-antibody complexes (PAP and APAAP at a concentration of about 40 /Jg/ml). Alternatively, instead of APAAP, anti-APase antiserum (1 400) plus APase (5.0 U/ml of intestinal enzyme) may be applied. 

Enzymatic revelation. First POase is revealed with DAB (brown color) and then APase with naphthol AS phosphate plus Fast Blue BBN, according to Section 17.3.3.2. 

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Enzyme-lectin histochemistry has many features in common with EIH. Lectins are of non-immune origin but recognize fine differences in complex saccharide structures. Usually lectin specificity is expressed as reactivity to a given monosaccharide, but it has been demonstrated repeatedly (e.g.. Debray et al., 1981) that this is an oversimplification (Section 3.4). It is becoming increasingly clear that cellular differentiation, maturation and neoplastic transformation are associated with changes of carbohydrate composition of the cell membrane (Ponder, 1983). 

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