Epitope Recognition in the Human–Pig Comparison Model on Fixed and Embedded Material

Abstract

The conditions and the specificity by which an antibody binds to its target protein in routinely fixed and embedded tissues are unknown. Direct methods, such as staining in a knock-out animal or in vitro peptide scanning of the epitope, are costly and impractical. We aimed to elucidate antibody specificity and binding conditions using tissue staining and public genomic and immunological databases by comparing human and pig—the farmed mammal evolutionarily closest to humans besides apes. We used a database of 146 anti-human antibodies and found that antibodies tolerate partially conserved amino acid substitutions but not changes in target accessibility, as defined by epitope prediction algorithms. Some epitopes are sensitive to fixation and embedding in a species-specific fashion. We also find that half of the antibodies stain porcine tissue epitopes that have 60% to 100% similarity to human tissue at the amino acid sequence level. The reason why the remaining antibodies fail to stain the tissues remains elusive. Because of its similarity with the human, pig tissue offers a convenient tissue for quality control in immunohistochemistry, within and across laboratories, and an interesting model to investigate antibody specificity.

NOT ALL ANTIBODIES HAVE BEEN CREATED EQUAL: Antibodies validated for routinely processed tissue seldom apply to frozen tissue sections

“…This may have occurred because of the fine requirement for some Abs of the conformation of the target side peptide chains not involved in the antigen binding (16) or because of non-identical juxtaposed adjacent proteins in the fixed complex. Of the Abs reacting with both species, we had evidence of species-specific negative effect of formalin fixation of BCL6, restricted to one antibody and not to others, while all did reacted on acetone-fixed frozen sections (15). In a separate investigation (11), we found that the epitopes for some antibodies were selectively destroyed by antigen retrieval (AR), possibly because of the conformational nature of the epitope.…”

“…-Independent Antibody: A) two antibodies directed against two separate epitopes of the same protein and having an identical staining pattern and/or co-localizing by double IF or B) an antibody uniquely identifying a cell in the tissue whose high-dimensional phenotype corresponds to a cell whose phenotype is defined by a multidimensional flow cytometry panel, are considered validated -Genetic: an antibody whose staining or absence of staining corresponds to a genetically engineered ectopic expression or absence of the target is considered validated -Peptide Microarray: an antibody recognizing its unique peptide target on peptide microarrays (20,21) is considered validated -Cross-species: an antibody whose reactivity is conserved across genomic sequence variations in another species (15)…”

“…Because of the antibody panel choice, biased toward leukocyte antigens, most Abs did not react with swine FFPE tissue (15), thus no epitope variation information could be obtained.…”

“…We have previously investigated the epitope specificity and binding requirements of a large number of antibodies for human FFPE material by applying them to swine routinely fixed material (15). The results showed that about 50% did not react, despite complete identity of the epitope in both species.…”

Optic nerve injury upregulates retinoic acid signaling in the adult frog visual system

“…(4) Appropriately-sized band(s) in Western blots. (5) BLAST sequence similarity of the likely immunogenic region(s) (see Supplemental file), using a cutoff of >65% similarity of positive scores (Scalia et al, 2015). Likely immunogenic regions were located within the given amino acid range by eliminating regions that were identical in closely-related but different proteins, and also eliminating regions with a low immunogenicity score as determined by the program AbDesigner (Pisitkun et al, 2012) (see Supplemental Excel file).…”