Assessment of immunocytochemical techniques with particular reference to the mixed-aggregation immunocytochemical...

Antibody-mediated assays are believed to be the most sensitive and specific techniques for antigen localization available to date, but they need extensive controls if specific and quantitative results are to be obtained. Problems arise from both the antibody source and the antigen in the tissue preparation. Specificity of the antibody is often difficult to prove in any direct or indirect antibody technique. When markers are coupled to antibody, binding and diffusion properties of the antibody are changed. Diffusion of antigen from its site and loss of antigenic determinants due to procedures for preserving morphology are two other main problems. The mixed-aggregation immunocytochemical technique provides another way of checking the selectivity of the antibody, since a cross-reactive antigen introduced into the section and not the antibody itself is what is seen. Quantitative studies using localization of lactic dehydrogenase isoenzymes in human skeletal muscle fibres show that the technique fulfils most of the present criteria on precision, specificity and validity for determination of enzymes. It also ensures that more than 80% native antigen in frozen sections is fixed and is two to three times more sensitive and selective than the direct antibody technique.