Monoclonal antibody to rat uterine peroxidase and its use in identification of the peroxidase as being of eosinophil origin.
Peroxidase was purified from uteri of estrogen-treated rats by calcium chloride extraction, affinity chromatography on concanavalin A-Sepharose and hydrophobic interaction chromatography on phenyl-Sepharose. An overall purification of greater than 1700-fold was achieved with a final recovery of 27%. Monoclonal antibodies to peroxidase were subsequently prepared by immunization of male C57BL/10J mice with the highly purified peroxidase from rat uterus. Spleen and lymph node cells from the mice were fused with Sp2/0-Ag 14 mouse myeloma cells. The resultant hybrid cells were screened for production of antibody using a solid-phase, double antibody radioimmunoassay. The mature rat spleen, shown previously to be abundant in eosinophils, contains high peroxidase activity. Spleen peroxidase purified by the same procedure as the uterine enzyme cross-reacted with a monoclonal antibody, designated IgG-107B, used in all subsequent studies. Peroxidase extracted from isolated rat eosinophils also cross-reacted with the antibody and yielded identical titers as the spleen and uterine peroxidases. Spleen, uterine and horse eosinophil peroxidase had the same apparent molecular weight, 57000, as determined by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis. Following electrophoretic transfer to nitrocellulose, spleen, uterine and eosinophil peroxidase reacted with monoclonal antibody, using an immunoblotting technique. These results provide biochemical and immunological evidence that the majority of the calcium chloride-extractable peroxidase activity from the uteri of estrogen-treated rats is derived from infiltrating eosinophils.
D000818:Animals; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002122:Calcium Chloride / Q000494:pharmacology; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004804:Eosinophils / Q000201:enzymology*; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008970:Molecular Weight; D010544:Peroxidases / Q000032:analysis* / Q000302:isolation & purification; D051381:Rats; D014599:Uterus / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D010544:Peroxidases; D002122:Calcium Chloride