APAAP complex: Production and usage in immunocytochemical and immunohistochemical staining

Abstract

Background:

The affinity of antibody to antigen, in addition to providing the possibility of measuring the antigen in tissue extracts through methods such as RIA (Radioimmunoassay) and EIA (Enzymimmunoassay) and possibility of isolating and analyzing dispersed cell colonies using flowcytometry, makes it possible to determine the site of antigen in tissues (Immunohistochemistry) or in cells (Immunocytochemistry).

Objective:

Production of APAAP complexes and comparing them with similar foreign products to determine the site of antigen in tissues or in cells.

Methods:

Secreted antibodies of the two hybridomas (A₁G₉G₃ and A₁G₈F₇ produced in our laboratory) were concentrated, purified and characterized. Then the monoclonal antibodies were mixed with alkaline phosphatase enzyme (ALP) to use in immunocytochemistry (ICC) and immunohistochemistry (IHC) staining.

Results:

Both of the cell colonies had the potentiality of producing anti- alkaline phosphatase monoclonal antibody with high affinity. The complex from mAb and enzyme – for the third phase of APAAP technique – was very effective and its sensitivity was comparable to that of the similar foreign kit.

Conclusion:

Considering the high affinity of the mAb of the two hybridomas and the stability of the complex resulted from mixing mAb and the enzyme ALP for a long time, it is possible to use the obtained APAAP complex in the immunocyto (or histo) chemistry – as the third phase.