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Correspondence to: Claudio Montero, Plaza Rafael Salgado 30-3°D, 41013 Sevilla, Spain. E-mail: montclau@supercable.es
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Summary |
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The problems of major concern in immunohistochemical practice are discussed in the following order: (a) the mechanism of the AgAb reaction in fixed tissue as opposed to the in vitro reaction; (b) the chemistry of fixation and its influence on the final result of the immunohistochemical reaction; (c) the various procedures used for antigen retrieval in formaldehyde-fixed tissue; and (d) the consideration of the possible mechanism underlying heat-induced antigen retrieval. Suggestions for further work to attempt a clarification of the mechanism involved in the AgAb reaction in immunohistochemistry resorting to existing histochemical methods for the demonstration of protein side groups are presented, together with some examples already published.
(J Histochem Cytochem 51:14, 2003)
Key Words: antigenantibody reaction, antigen retrieval, fixation, immunohistochemistry
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Introduction |
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Mechanism of the AgAb Reaction in Formaldehyde-fixed Specimens |
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THE ANTIGEN–ANTIBODY (AgAb) reaction in immunohistochemistry (IHC) usually takes place generally between two protein macromolecules: the antigen, which may also be a glycoprotein, a lipoprotein, or just a protein, and the antibody, which is a glycoprotein. It must be emphasized, however, that in this case one of the macromolecules, the antigen, is located in a section of formaldehyde-fixed, paraffin-embedded tissue. Obviously the antigen protein is immobilized, and is more or less altered in its constitution and conformation, in the tissue section. However, according to the findings of
The antibody macromolecule that is diluted in the buffer covering the tissue section is free, during the incubation period, to wander in that buffer, obeying the laws of Brownian kinetics. Of course, the final result must be a consequence of the various factors intervening in these kinetics, e.g., pH, concentration, buffer constituents, salts added, dilution used, temperature. One of the most critical parameters is pH because the isoelectric point of the IgG used in this procedure is in a range of pH between 6.5 and 8.5 (
Whereas the conformation of the immunoglobulin protein used in the AgAb reaction in tissues is well preserved, usually in its native form, the conformation of the antigen protein, located in the tissue, cannot be considered intact. It has suffered the effect of the formaldehyde fixative and may have been modified in its constitution or its conformation. Even when, according to
The kinetics of two macromolecules, such as the antigen and the antibody, as described by
Summarizing, in IHC the AgAb reaction is chiefly conditioned by the following facts: (a) the state of fixation of the antigen in the tissue section, with the existence of chemical bridges binding reactive side groups of proteins; (b) the relative freedom of the antibody macromolecules free to move in the dilution buffer covering the sections; and (c) the pH of the buffer used for dilution of the primary antibody. The range of best pH for IgG reaction used in the IHC procedure is between 6.5 and 8.5, as will be shown below.
The complex action of each one of the above factors may make it very difficult to interpret the results of the antigen retrieval (AR) procedure that is now so frequently used as a way to increase the final intensity of the color reaction or, even, to make a reaction that was negative without heat positive after its application.
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The Chemistry of Fixation |
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Formaldehyde is the universal fixative in anatomic pathology. Consequently most of the research efforts have concentrated on this method of fixation. As stated by
Formaldehyde is usually used in a 10% solution of the commercial one, usually in neutral phosphate buffer. Formaldehyde exists in this solution in the form of methylene glycol, CH2(OH)2.
According to
The groups that may be involved in these bindings are amino, imino, amido, peptide, guanidyl, and carboxyl, SH, and aromatic rings. The investigations of
In tissue samples heavily fixed with formaldehyde and stained with hematoxylineosin, it is not easy to retain the eosin staining of most histological structures presumed to be bound to the basic components in the tissue (e.g., amino groups of proteins) after ethanol dehydration. Sections treated by heat, as in the AR technique, stain more strongly with eosin and this staining is not easily lost by dehydration. These are only casual observations but are very suggestive of the increased number of protein side groups after heat retrieval procedures.
Of course, the AgAb binding is stronger than the binding between a stain and tissue structures. In the AgAb binding, as has been repeatedly said, there is not only the binding of complementary side groups of the two proteins but also a conformational fitting of two small areas, one each in the antigen and the antibody, the epitope and the paratope.
It is likely that a blockade of the pertinent side groups with existing histochemical methods may help in the confirmation of the above hypothesis. A detailed explanation of all these possibilities is not possible here but it can easily be foreseen.
An attempt to evaluate these binding forces has been attempted by
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Antigen Retrieval |
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We cannot help but agree with
Studying the influence of pH on the antigen-retrieval IHC (
Considering that there is a time interval between treatment with the AR solution at a predetermined pH and the incubation with the primary antibody, used at a pH not stated but presumed to be the pH usually used in routine histochemical procedures, the results of IHC appear to depend chiefly on the modifications introduced, not only of heat but also of the pH, in the protein macromolecules of the tissue. Their findings have been summarized recently (
The pH-dependent modifications of the macromolecules must be different in the various kinds of proteins, something that appears obvious when we consider that those proteins do have different degrees of polarization (i.e., either acidic or basic). This is a point that seems to have received little attention although it is outstanding in the reaction with the antibody.
In dealing with these problems it is always assumed that we are using monoclonal antibodies. Obviously, the reaction of polyclonal antibodies, with so many subsets of antibodies against different specific epitopes in the same solution, is more difficult to block. The mechanism of blockade cannot affect different epitopes in a similar way. On the other hand, monoclonal antibodies with only one possibility of reaction with an epitope are prone to give clear-cut results.
The comment of
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Heat Retrieval and the Side Groups of Proteins |
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The statement by
These and other explanations for negative results occasionally obtained when the IHC procedure is used for the demonstration of tissue proteins further demonstrate the complex problem of elucidating the mechanism of the AgAb reaction in formaldehyde-fixed, paraffin-embedded tissues. Moreover, they tell us that the availability of chemical groups after AR that are responsible for the increased reactivity with the antibody cannot be easily explained in simple and direct terms. The only alternative that appears possible is a detailed study of each one of the many possibilities that may exist in the extensive variety of protein macromolecules existing in organic tissues.
An unpretentious possibility is given, in a report by
The use of different monoclonal antibodies for the same macromolecule can also be recommended because not all the epitopes in the same molecules have the same amino acid constitution. At the same time, the use of the corresponding positive and negative controls and of the blocking procedures suitable in each case is advised.
The judicious use of the above-mentioned histochemical procedures for the demonstration of protein side groups, together with the antigen retrieval technique, might be helpful in the interpretation of the results of these procedures.
Received for publication October 3, 2001; accepted September 11, 2002.
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