Antigen Retrieval

One of the earliest methods of antigen retrieval was proteolytic digestion of tissue sections employed prior to the application of the primary antibodies. A number of proteolytic enzymes served this purpose, including trypsin, proteinase K, pronase, pepsin, ficin, DNase, and others. Not only are the enzymes different but there is also variation in concentration, duration, and temperature of digestion.

Furthermore, not all antigens benefit from proteolytic digestion, and some show deleterious effects with loss of staining. In addition, inappropriate protocols result in tissue breakdown and loss of morphology with high levels of background and falsepositive staining.

The introduction of heat-induced antigen retrieval signified a major milestone in immunohistology, greatly enhancing our ability to demonstrate antigens in FFPT with greater consistency.

The initial technique was achieved with MWs, which has remained the most convenient method for antigen retrieval, but a variety of other methods of generating heat have since been spawned including water baths, hot plates, wet autoclaves, pressure cookers, and vegetable steamers. Shi et al (1991) described MW heating of FFPT in the presence of heavy metal solutions, such as lead thiocyanate, up to temperatures of 100o C to “unmask” a wide variety of antigens for immunostaining. It was subsequently shown that MW-irradiation of deparaffinised-rehydrated sections in 10 mmol citrate buffer at pH 6.0 produced, with few exceptions, increased intensity and extent of immunostaining of a wide variety of tissue antigens. The use of citrate buffer eleminated the need to employ heavy metal solutions which, ehen heated, generate toxic fumes. Several commercial antigen retrieval reagents are available but they mostly do not produce any better results than that obtained with citrate buffer.

MWs are a form of non-ionizing radiation with a typically standard frequency of 2.45 GHz, a wavelength of 12.2 cm and photon energy of 10-5 electron volts. When dipolar molecules such as water or the polar side chains of proteins are exposed to the rapidly alternating electromagnetic fields, they oscillate through 180o at the rate of 2.45 billion cycles per second. The molecular movement or kinetics so induced results in the generation of instantaneous heat that is proportional to the energy flux and continues until radiation ceases.

It was only recently recognized that molecules other than water and the polar side chains of proteins may oscillate in the electromagnetic field generated by MWs. Molecules with an uneven distribution of electrical charge such as inorganic material and copper oxides can also be rotated.

All methods of heat generation listed above suffer from problems with accurate temperature and time control. These two variables have been shown to be critical to the process of heat-induced antigen retrieval. They are inversely related so that antigen retrieval at lower temperatures requires longer durations to achieve the same results as that obtained with higher temperatures. The time taken to attain the desired temperature from variable starting temperatures, time required to cool to room temperature, and actual temperature attained are variables that cannot be controlled with most methods of heating. Furthermore, there is the problem of unevenness of heating within microwave cavities, making the entire process impossible to standardize with resulting inconsistencies in methodology. Computerized control of time and temperature that is available with some commercial MW instruments takes the guesswork out of heat-induced antigen retrieval.

Accurate time and temperature control not only produces superior antigen retrieval across the spectrum of diagnostic antigens but accurate heating to 120oC or “superheating” has proven to produce notably better antigen staining.

Our understanding of the effects of formaldehyde on proteins dates back to work done in the 1940s (Fraenkel-Conrat et al, 1947; Fraenkel-Conrat and Olcott, 1948a, 1948b). A recent comprehensive review of the fixative action of formaldehyde and antigen masking is available. The amino acid side chain of proteins includes many groups that may react with aldehydes that contribute to the stabilization of proteins. However, despite the vast

literature on the subject of protein modification by formaldehyde, there is no clear consensus as to which are the predominant molecular species resulting from this method of fixation.

There is no doubt that some of the cross-linked adducts are very stable and remain irreversibly changed even after extensive washing, while others revert under varying conditions to free formaldehyde and the amino acid. Without a complete understanding of the actions of formaldehyde on proteins, it is not surprising that we do not fully understand the mechanisms of antigen retrieval.

Heat appears to be a common denominator in antigen retrieval produced by a variety of methods including MWs. Heat is hypothesized to cause protein denaturation based on the observation that some antigens or endogenous enzymatic activities may be lost after heat treatment and heat induces reversal of various chemical modifications of the protein structure that result from formalin fixation. Other actions that produce antigen retrieval include the loosening or breakage of the cross-linkages caused by formalin fixation, hydrolysis of schiff bases, and multiple pathways including extraction of diffusible blocking proteins, precipitation of proteins and dehydration of the tissue sections to allow better penetration of antibody and increased accesibility to epitopes, all or some of which may be achieved by other methods of retrieval including enzyme digestion and change in pH. MW energy may itself mobilize the last traces of paraffin that may not have been extractable by standard techniques, there by improving antibody penetration.

It was proposed that the calcium complex formation that occurs with formalin fixation may mask antigens and that the release of calcium from this cage-like complex may require a considerable amount of energy such as high temperature heating and calcium chelation by citrate. However, it has been argued that while this calcium effect may be acting with some antigens it may not be sufficient to explain the loss of immunoreactivity for many other antigens and is unlikely to represent the general mechanism of antigen retrieval.

The role of kinetics in antigen retrieval is also not known. While the focus has been on heat as the responsible factor in MW retrieval, the rapidly oscillating electromagnetic field of MWs may itself have an effect on chemical reactions and proteins. While heat or thermal energy will increase molecular kinetics and hasten chemical reactions, the rapid rotation of molecules directly induced by the MWs will give rise to greatly increased collision of molecules, which will in turn accelerate chemical reactions. The heat generated may represent only an epiphenomenon secondary only to the kinetics. One study that examined MW stimulation of CEA/anti-CEA reaction in an enzyme-linked immunosorbent assay system found that despite continuous cooling by ice MW stimulation increased reaction rates by a factor of 1000, allowing the investigators to conclude that such rate increases were far too large to be explained solely by the modest increase in temperature.

Another study went further to elucidate the existence of a “microwave effect” (Choi et al,1997) The authors showed that the rate of droplet temperature increase obtained in a thermal cycler was similar to that achieved by MW irradiation. However, the immunostaining obtained from a 3-minute incubation at 37oC in the thermal cycler followed by 2-minute incubation without heating was much weaker than that seen with MWs. Similarly it was demonstrated that 7-s MW irradiation followed by 5-min room temperature incubation for each step of the avidin-biotin peroxidase complex procedure produced good immunolabelling.

The droplet temperature raised no more than 5oC following 7-s irradiation at 100% power in a 850-watt oven so that temperature was not a significant component of the accelerated reaction. Others have argued that there is no significant MW effect and the accelerated reactions are a function of heat. It has been concluded that MW irradiation did not produce cleavage or polymerisation of proteins and irradiation resulted an electrophoretic pattern that was similar to that obtained when lysozyme and hemoglobin was heated in formaldehyde to 60o C for 30 min. Interestingly, results to the contrary have been shown in a study of S-adenosylhomocysteine hydrolase and 5’-methylthioadenosine phosphorylase, two thermophilic and thermostable enzymes, where exposure to MWs caused a non-thermal, irreversible and time-dependent inactivation of both enzymes, In a model immunostaining system using short synthetic peptides to mimic the antibodybinding site of common diagnostic protein targets, Sompuran et al (2006), found that not all of the peptides studies exhibited the formalin-fixation and antigen retrieval phenomenon. One group of peptides was recognized by antibody even after prolonged exposure to formalin while another group exhibited the formalin-fixation and antigen-retrieval phenomenon only after another irrelevant protein was mixed with the peptide before fixation. Amino acid sequence analysis indicated that fixation and antigen retrieval were associated with a tyrosine in or near the antibody-binding site bound covalently to a nearby arginine implicating the Mannich reaction as an important factor in the process.

The Mannich reaction is a complicated, multistep interaction, which firstly involves a reaction between formaldehyde and an amine to produce an iminium ion. The iminium ion may then react with another carbonyl-containing molecule to form an intermediate product and for this to occur, the carbonyl-containing molecule must be in an enol configuration. In the final step of the reaction, the iminium ion and the enol react together to form a stable product. The findings of Sompuran et al (2004) concurred with those of Fraenkel-Conrat et al (1947, 1948) who had indicated that of all the protein cross-linking reactions that occur as a result of formalin fixation, the Mannich reaction is different, in that the cross-linkages can be hydrolysed with heat or alkaline treatment. Antibodies appear to recognize linear protein epitopes in FFPT and antigen retrieval may simply remove cross-linked proteins that are sterically interfering with antibody binding. The recent demonstration that antigen retrieval produces immunohistological staining results in FFPT that are comparable or better than that in acetone-fixed fresh frozen section and that heat-induced antigen retrieval enhances immunostaining in unfixed fresh frozen sections and dot-blot protein extracts is further support of the concept that intrinsic natural steric barriers exist and interfere with antibody binding. The demonstration that MWs can also be employed to enhance the demonstration of HER2/neu gene in chromogenic in-situ hybridization (CISH) is particularly interesting and it suggests that similar mechanisms may be operative in the ‘masking’ of DNA.

However, it would seem that these observations provide some insights into the action of antigen retrieval with some peptides but answers to the majority still remain unknown.

The demonstration that exposure to ultrasound can significantly increase antibody-antigen reaction in immunostaining lends further support to the relevance of molecular movement as an important factor in the acceleration of the chemical reaction as the amount of heat generated by this physical modality is negligible. A number of other hypothetical physical mechanisms may also play a role in the actions of MWs.

Although the proton energy generated in MW fields is too small to alter covalent bonding, they may readily affect the integrity of non-covalent secondary bonding, including hydrophobic interactions, hydrogen bonds and van der Waal’s interactions that make up the precise steric interactions at the cell membrane.

The combination of heat retrieval with enzymatic digestion has allowed enhanced demonstration and localization of a number of antigens including the immunoglobulins. Proteolytic digestion can be performed with a number of enzymes, and at varying concentrations and for varying durations. It can precede or follow heat induced antigen retrieval with different results. For optimal outcomes, it is necessary to explore all possible combinations and permutations of these variables with the realization that excesses can result in loss of antigen and cell morphology.

The chemical composition of the retrieval solution may affect the efficacy of the process and a wide variety of solutions have been advocated including citrate buffer, Tris buffer, glycine-hydrochloric acid, EDTA, urea, heavy metal solutions, and other proprietary reagents.

The molarity of the solution may also significantly influence immunostaining. The pH of the retrieval solution has been shown to be one of the most important factors in antigen retrieval. Three patterns of staining reflect the influence of pH.

Some antigens (CD20, AE1, EMA, NSE and PCNA) showed no variation at pH values ranging from 1.0 to 10.0, other antigens (MIB1, ER) displayed a dramatic decrease in staining intensity at middle pH values (pH 3.0-6.0) with strong staining above and below the range, and a third pattern was demonstrated by other antigens (CD43, HMB45) which were weakly stained at low pH (1.0-2.0) and displayed a sharp rise in intensity with increasing pH.

Use of MWs to accelerate antibody-antigen reactions in the staining of labile lymphocyte membrane antigens in cryostat sections and the exposure of cryostat sections briefly to MWs before the commencement of immunolabelling produced better quality cytomorphology and staining. A similar procedure has been adopted for freshly frozen brain sections with notable enhancement of immunostaining, without affecting the integrity of cytomorphology. MWs have been successfully used to accelerate immunolabelling in paraffin-embedded sections and the same technique has been applied for immunofluorescence labeling.

MWs have been applied between sequential rounds of a three-layer immunoenzyme staining (mouse Mab, goat anti-mouse IgG and mouse PAP or mouse APAAP) and color development technique for multiple antigen detection. The MWs denatured bound antibody molecules resulting in the blocking of cross reactivity between the sequential staining steps, allowing the use of primary and other antibodies raised in the same species.

Besides serving a role in antigen retrieval, MWs also inactivated peroxidase and alkaline phosphatase enzymes present in PAP and APAAP complexes, which would otherwise have led to inappropriate color development.

Source :

Biochemistry and Hystocytochemistry Research Developments : Editor by Stefan Fuchs and Max Auer.