The processes of fixation, dehydration, and embedding of tissues can result in changes in the conformation and accessibility of epitopes on proteins within the sample. These changes can result in the loss of epitope binding by an antibody.  Limited enzymatic digestion of formalin fixed, paraffin embedded tissues was commonly utilized to unmask epitopes that had become inaccessible. While this approach was successful for many polyclonal antibodies, its usefulness was more limited with monoclonal antibodies. The ability to unmask epitopes was revolutionized by Antigen Retrieval (AR) method developed by Shi et al. in 1991. This is a high-temperature heating method which recovers the antigenicity of tissue sections masked by formalin fixation. Microwave Oven, Pressure Cooker and Steamer are the most commonly used heating methods.

 

The introduction of antigen retrieval has enabled immunohistochemistry to become an integral component of morphologic diagnosis, routinely employed in cancer diagnosis, and for the identification of therapeutic and prognostic markers. The mechanism of antigen retrieval, as reviewed by Leong and Leong (1) however, remains speculative with the key to our understanding embedded in the actions of formaldehyde on proteins. One commonly held concept is that heat primarily breaks down protein cross-linkages that occur with aldehyde fixation, thus "unmasking" protein epitopes of interest. Enzymatic pretreatment is also thought to have a similar action whereas such "breakages" are the result of extremely rapid molecular movement induced by microwaves and ultrasound. The formation of rigid cage-like calcium complexes during formaldehyde fixation is another suggested mechanism of antigen masking requiring chelating agents for reversal. A more recent suggestion for the antigen retrieval phenomenon has evoked the Mannich reaction, which occurs with the cross-linking of some proteins. Such cross-linkages can be hydrolysed by heat or alkalis so that the process of antigen retrieval may be the simple removal of such cross-linked proteins that are sterically interfering with the binding of antibodies to linear protein epitopes in the tissue section. We are clearly not yet in possession of all the answers to the problem.

 

 

Thomas Boenisch (2) has demonstrated that electrostatic charges, i.e., net negative on antigens and net positive on antibodies, play an important part in immune reactions and therefore should be given greater attention.

 

Boenisch (2) and his team at DAKO stained 15 antigens both with and without the use of AR at the diluent pH of 6.0 and 8.6, respectively, and with increasing concentrations of cations. Without the use of AR, they found that antigens reactive with monoclonal antibodies of subtype IgG1 stained most intensely at the slightly acidic pH, whereas antigens reacting with monoclonal antibodies of subtypes IgG2a and IgG3 stained strongest at the alkaline pH of 8.6. When they did use AR, the IgG2a antibodies also stained strongest at pH 6, whereas optimum staining with a subclass IgG3 antibody remained at pH 8.6. They believe that this reversal of optimal staining from pH 8.6 to 6 with antibodies of subclass IgG2a is an indication of the antigen’s recovery of lost negative charges during AR.

 

Varying the concentration of Na⁺ ions (in the form of NaCl) can markedly influence staining intensities at all antibody concentrations. Larsson (2) referred to the presence of inorganic cations as forming a ‘‘shield’’ around the negatively charged antigens, thereby reducing or obstructing the attraction of the positively charged antibodies. (Boenisch also adds that adding an identical concentration of K⁺ ions had an even stronger detrimental influence, but it is not known why.) It is also noteworthy to mention that phosphate buffered saline, a widely used antibody diluent, had one of the most pronounced negative effects on staining intensities. This makes sense because this diluent not only contains Na⁺ ions from NaC⁺ but also the monosodium (NaH₂⁺) and disodium (Na₂H⁺) cations of the phosphate buffer. Increasing additions of NaCl to Tris buffers also had an increasingly suppressive effect on staining intensities (2).

 

 

Mittelbronn et al (3) studied the effect that albumin had on immunohistochemistry. They found that the most important effect concerning background staining reduction could be significantly attributed to the omission of albumin which usually is recommended as a reducer of background stainings.

 

However, in contrast to this negative effect, albumin could also increase specific staining intensity. Mittelbronn et al (3) recommend the careful use of albumin in immunohistochemistry because of the dichotomous effects mentioned above. Furthermore, their results imply that in case of a good specific staining pattern, the use of albumin in immunohistochemical solutions merely exerts significant negative background staining effects.

 

 

Namimatsu et al (4) from Tokyo have developed an antigen retrieval method based on the use of citraconic anhydride (a reversible protein cross-linking agent).

 

In general, formalin-fixed tissues demonstrated specific immunostainings comparable to that in fresh frozen tissues and significantly more enhanced than after conventional antigen retrieval methods. In particular, even difficult to-detect antigens such as CD4, cyclin D1, granzyme β, bcl-6, CD25, and lambda chain revealed distinct immunostainings. Different classes of antigens such as cellular markers and receptors, as well as cytoplasmic and nuclear proteins, consistently produced enhanced reactions.

 

 

1.      Leong and Leong (2007) Advances in Anatomic Pathology. 14(2):129-131.

2.      Boenisch (2006) J Histochem Cytochem 54:961–964

3.      Mittelbronn et al (2006) Appl. Immunohist Molec Morph 14(4):441-444.

4.      Namimatsu et al (2005) J Histochem Cytochem 53:3–11.

 

Submitted by

Tony Henwood MSc, BAppSc, GradDipSysAnalys, CT(ASC)

Laboratory Manager & Senior Scientist, Histopathology, The Children's Hospital at Westmead, Sydney, Australia