Antigen retrieval studies

Rhodes A, Jasani B, Balaton AJ, et al. Study of interlaboratory reliability and reproducibility of estrogen and progesterone receptor assays in Europe documentation of poor reliability and identification of insufficient microwave antigen retrieval time as a major contributory element of unreliable assays. Am. J. Clin. Pathol. 2001 115 44-58. 

Figure 5.3 Diagram depicts the further-designed studies to test our hypothesis with respect to standardization of immunohistochemistry based on the antigen retrieval technique exemplified in a multiple direction to draw a conclusion, (a) Periods of formalin fixation, (b) Variable delay of fixation, (c) Storage of FFPE tissue blocks or sections, (d) Variable thickness of FFPE tissue sections, (e) Other variable conditions of processing FFPE tissue blocks. The stereoscopic frame of a cube represents the reliable limitation of quantitative IFIC demonstrated by serial studies as recommended in the text. Reproduced with permission from Shi et al., J. Histochem. Cytochem. 2007 55 105-109.

O Leary TJ, Fowler CB, Evers DL, et al. Protein fixation and antigen retrieval chemical studies. Biotech. Histochem. 2009 84 217-221. 

STUDY OF FORMALIN FIXATION AND HEAT-INDUCED ANTIGEN RETRIEVAL... 

Taylor CR, Shi S-R, Chen C, et al. Comparative study of antigen retrieval heating methods microwave, microwave and pressure cooker, autoclave, and steamer. Biotech. Histochem. 1996 71 263-270. 

These initial findings do not exclude other possible formaldehyde-induced reactions with tissue proteins. Notably, this first model system was not designed to detect the role of lysine residues. Lysine has a propensity to react with and form a variety of different types of cross-links with other amino acids in the presence of formaldehyde.1,3 417 Therefore, it is likely to also be important in reactions with formaldehyde. In fact, peptides with internal lysine residues were purposefully excluded from this initial study for technical reasons. To explore the importance of lysine residues in antigen retrieval, an alternative method was employed. 

Chemical treatment with formaldehyde may not necessarily result in significant denaturation. A recent study of RNase A indicated that treatment with formaldehyde does not significantly alter secondary structure.21,22 Although formalin treatment may induce subtle changes in secondary structure, alpha helices and beta-pleated sheets are left essentially intact after formalin treatment. It is reasonable to assume, however, that boiling (as per antigen retrieval protocols) will significantly alter secondary structure. 

Although antigen retrieval techniques including HIAR continue to be regarded as enigmatic techniques, recent studies are rapidly clarifying the mechanisms of HIAR. In this article, the effects of pH and the ion concentrations of retrieval solutions on HIAR will be reviewed, and the probable mechanisms of HIAR and other antigen retrieval techniques will be discussed based on these results. 

The above discussion indicates that although 0.01 M sodium citrate buffer (pH 6.0) is commonly used, it is not a universally ideal antigen retrieval fluid for all types of tissues and antigens. If published information is not available with regard to the best antigen retrieval fluid for the antigen under study, the ideal retrieval fluid for each type of epitope must be determined by trial and error. 

Also, different forms of an antigen require different concentrations of the antibody for their maximal detection. This is exemplified by the PC-10 primary antibody, which identifies PCNA antigen at a dilution of 1 1000 in epithelial cells in normal colon tissue, whereas a dilution of 1 400 is required to localize these proliferating cells in adenomatous polyps (Holt et al., 1997). In contrast, some types of antigens (e.g., Ki-67) can be optimally detected in various tissue types at the MIB-1 dilution of 1 50, using the microwave heating antigen retrieval method. However, in a few studies MIB-1 dilutions of 1 20 to 1 100 have been used. 

There are many reasons for the lack of consensus on this highly complex phenomenon, and they are discussed below. Various studies mentioned above were conducted using different parameters of antigen retrieval methods, including antigen retrieval fluids, pH, heat source, temperature, and duration of treatments for detecting different antigens. The type of fixation and duration of fixation also varied in these studies. Other variants were the type of epitope and antibody and source of antibodies used. 

Determine the optimal pH of antigen retrieval solution for each antigen. Citrate buffer (0.01 M) adjusted to pH 6.0 with HC1 is used widely. Determine the desired temperature based on the type of tissue and antigen under study. For fatty tissues, 90°C is recommended adjust the duration of heating accordingly. Place slides in plastic Coplin jars containing the... 

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