Application of the Critical Molar Concentration Concept to Heat-mediated Antigen Retrieval in Immunohistochemistry

Overview

Journal Histochem J

Specialty Biochemistry

Date 2000 May 18

PMID 10816076

Authors

C Montero

E Chi-Ahumada

M Gutierrez

Affiliations

Soon will be listed here.

Abstract

Previously we have applied Scott's critical molar concentration concept to show that divalent cations, especially Mg2+ may be used to measure the affinity of a known monoclonal antibody for its antigen. In this paper we report the application of this same procedure to a study of a series of antigens (three globular proteins and three intermediate filaments). The concept was applied to samples without any previous treatment or after the application of heat-mediated antigen retrieval (using a pressure cooker). Our findings suggest that heat-mediated antigen retrieval sets free protein side-chain(s) that have been masked by formaldehyde fixation. This is reflected in a higher affinity of the antibody for the antigen in question.

References

Rini J, Wilson I . Structural evidence for induced fit as a mechanism for antibody-antigen recognition. Science. 1992; 255(5047):959-65. DOI: 10.1126/science.1546293. View

Shi S, Key M, Kalra K . Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991; 39(6):741-8. DOI: 10.1177/39.6.1709656. View

FRAENKEL-CONRAT H, Olcott H . Reaction of formaldehyde with proteins; cross-linking of amino groups with phenol, imidazole, or indole groups. J Biol Chem. 1948; 174(3):827-43. View

FRAENKEL-CONRAT H, Olcott H . The reaction of formaldehyde with proteins; cross-linking between amino and primary amide or guanidyl groups. J Am Chem Soc. 1948; 70(8):2673-84. DOI: 10.1021/ja01188a018. View

Fox C, Johnson F, Whiting J, Roller P . Formaldehyde fixation. J Histochem Cytochem. 1985; 33(8):845-53. DOI: 10.1177/33.8.3894502. View

PUCHTLER H, Meloan S . On the chemistry of formaldehyde fixation and its effects on immunohistochemical reactions. Histochemistry. 1985; 82(3):201-4. DOI: 10.1007/BF00501395. View

Pappas P . The use of a chrome alum-gelatin (subbing) solution as a general adhesive for paraffin sections. Stain Technol. 1971; 46(3):121-4. DOI: 10.3109/10520297109067835. View

Scott J, Dorling J . Differential staining of acid glycosaminoglycans (mucopolysaccharides) by alcian blue in salt solutions. Histochemie. 1965; 5(3):221-33. DOI: 10.1007/BF00306130. View

Scott J, Willett I . Binding of cationic dyes to nucleic acids and ther biological polyanions. Nature. 1966; 209(5027):985-7. DOI: 10.1038/209985a0. View

10.

Berod A, Hartman B, Pujol J . Importance of fixation in immunohistochemistry: use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem. 1981; 29(7):844-50. DOI: 10.1177/29.7.6167611. View

11.

Braden B, POLJAK R . Structural features of the reactions between antibodies and protein antigens. FASEB J. 1995; 9(1):9-16. DOI: 10.1096/fasebj.9.1.7821765. View

12.

Lan H, Mu W, Nikolic-Paterson D, Atkins R . A novel, simple, reliable, and sensitive method for multiple immunoenzyme staining: use of microwave oven heating to block antibody crossreactivity and retrieve antigens. J Histochem Cytochem. 1995; 43(1):97-102. DOI: 10.1177/43.1.7822770. View

13.

Montero C, Segura D, Gutierrez M . The influence of salts on the reaction between hGH and several of its monoclonal antibodies in fixed, paraffin-embedded human pituitary as a model system. Eur J Histochem. 1996; 40(2):153-8. View

14.

Shi S, Cote R, Taylor C . Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem. 1997; 45(3):327-43. DOI: 10.1177/002215549704500301. View