Antigen retrieval approaches

STANDARDIZATION OF ANTIGEN RETRIEVAL TECHNIQUES BASED ON THE TEST BATTERY APPROACH... 

Shi SR, Cote RJ, Yang C, et al. Development of an optimal protocol for antigen retrieval a test battery approach exemplified with reference to the staining of retinoblastoma protein (pRB) in formalin-fixed paraffin sections. J. Pathol. 1996 179 347-352. 

THE TEST BATTERY APPROACH OF ANTIGEN RETRIEVAL ( AR) TECHNIQUE... 

Antigen antibody recognition is dependent on protein structure. A conformational change in a protein caused by formalin fixation may mask the epitope and thus affect the antigenicity of proteins in formalin-treated tissue (Montero 2003). The antigen retrieval leads to a renaturation or at least partial restoration of the protein structure, with re-establishment of the three-dimensional protein structure to something approaching its native condition (Shi et al. 1991). 

This need may arise when a limited number of slides is available. For this type of study, heat-induced antigen retrieval should be carried out only once. One such treatment lasts for many months. Repeated use of antigen retrieval tends to cause background staining. Such an approach has been successfully used for staining epithelial membrane antigens and cytokeratin in the tonsil tissue fixed with Bouin s fixative (Roche et al 2000). 

Frozen sections, as well as sections of formaldehyde-fixed and paraffin-embedded tissues, can be used for direct or indirect immunofluorescence staining after antigen retrieval using microwave heating. Although frozen sections yield higher sensitivity than that obtained with paraffin sections, the latter approach is necessary for retrospective studies of archival specimens. Moreover, fresh tissue is not always available. 

Shi, S.-R., Gu, J., Kalra, K. L., Chen, T., Cote, R. J., and Taylor, C. R. 1995b. Antigen retrieval technique A novel approach to immunohistochemistry on routinely processed tissue sections. Cell Vision 2 6-22. 

Legions of investigators, and many manufacturers, have addressed different aspects ofthe problem, focusing upon better sample preparation (fixation), more effective methods of antigen retrieval, improved reagents, more sensitive detection methods, and the development of reference standards or controls (2-8). To date, these approaches have failed to produce an overall system of IHC that assures uniform high quality, with a level of reproducibility and reliability sufficient to allow robust comparison of IHC results across laboratories, especially where semi-quantitative results are sought (as for Her 2, or ER) (4, 7, 8). 

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