Monoclonal antibodies against glutaraldehyde-conjugated histamine: application to immunocytochemistry.
We have developed mouse monoclonal antibodies (AHA-1-5, all IgG1 sub-isotype mAbs) against histamine (HA) conjugated to bovine serum albumin using glutaraldehyde-NaBH4. Among these, AHA-1 mAb was found to be the most useful for HA immunocytochemistry (ICC) in terms of specificity and sensitivity without non-specific immunobinding. AHA-1 was demonstrated to be specific to HA with an enzyme-linked immunosorbent assay (ELISA) binding test, simulating the ICC of tissue sections, and not reactive to any of the other amino acids and peptides with N-terminal histidine tested. By use of this antibody, indirect immunoperoxidase staining was observed in rat stomach fixed with glutaraldehyde (GA) in combination with NaBH4 reduction. In contrast, no immunoreactivity was seen in tissue fixed only with GA. Absorption controls indicated that the immunostaining could be completely inhibited by GA-conjugated HA, which was consistent with the results of an ELISA inhibition test. No cross-reactivity occurred with other GA-conjugated amino acids. ICC staining was dense in the cytoplasm of gastric enterochromaffin-like cells and very weak in mast cells. A new finding was that staining was noticed in some cell bands of the intermediate layer between the stratum lucidum and the stratum corneum of the stratified squamous epithelium of the gastric cardia, esophagus, tongue, and skin in rats. The results strongly suggest that the monoclonal antibody allowed highly specific detection of HA in animal tissues.
Fujiwara
K
K
Faculty of Pharmaceutical Sciences, Nagasaki University, Japan.
Kitagawa
T
T
Inoue
Y
Y
Alonso
G
G
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000917:Antibody Formation / Q000276:immunology; D000918:Antibody Specificity; D002459:Cell Fusion; D004759:Enterochromaffin Cells / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005404:Fixatives; D005976:Glutaral / Q000737:chemistry / Q000276:immunology*; D006632:Histamine / Q000737:chemistry / Q000276:immunology*; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D017208:Rats, Wistar
D000911:Antibodies, Monoclonal; D005404:Fixatives; D006632:Histamine; D005976:Glutaral