Characterization of monoclonal antibodies against ovine prolactin: suitability for use in immunocytochemical analysis of rat prolactin

The aim of this study was to identify a monoclonal antibody (MAb) suitable for use in the immunocytochemical localization of prolactin in rat tissues. We took advantage of the conservation of certain amino acid sequences in prolactin among species by examining the crossreactivity patterns of five MA...

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Bibliographic Details
Published in:	The journal of histochemistry and cytochemistry Vol. 38; no. 1; pp. 117 - 122
Main Authors:	Scammell, JG, Scott, MG, Outlaw, KK, Thompson, ME, O, SJ, Belen, RB
Format:	Journal Article
Language:	English
Published:	Los Angeles, CA Histochemical Soc 01-01-1990 SAGE Publications Histochemical Society
Subjects:	Analytical, structural and metabolic biochemistry Animals Antibodies, Monoclonal - analysis Antigen-Antibody Reactions Biological and medical sciences Blotting, Western Cells, Cultured Cross Reactions Enzyme-Linked Immunosorbent Assay Fluorescent Antibody Technique Fundamental and applied biological sciences. Psychology Immunoglobulin G - analysis Prolactin - immunology Protein hormones. Growth factors. Cytokines Proteins Rats Sheep Species Specificity Cell culture Protein hormone Vertebrata Mammalia Rat Immunocytochemistry Prolactin Rodentia Immunoblotting assay Monoclonal antibody Gonadotropin ELISA assay
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Summary:	The aim of this study was to identify a monoclonal antibody (MAb) suitable for use in the immunocytochemical localization of prolactin in rat tissues. We took advantage of the conservation of certain amino acid sequences in prolactin among species by examining the crossreactivity patterns of five MAb, originally generated to ovine prolactin, with rat prolactin by enzyme-linked immunoassay (ELISA), Western blot analysis, and immunocytochemistry. Two of five antibodies (17D9 and 6F11) showed reactivity with 100 ng of immobilized rat prolactin (NIH RP-3) by ELISA, 6F11 reacting more strongly than 17D9. Only 6F11 reacted with prolactin in lysates of GH4C1 rat pituitary tumor cells by Western blot analysis. When we examined the crossreactivity of the MAb with rat prolactin in monolayer cultures of GH4C1 cells by indirect immunofluorescence, we found that both 17D9 and 6F11 reacted strongly with the cultures. The distribution of staining with 17D9 or 6F11 was coincident with staining with a polyclonal antiserum to rat prolactin. Preabsorption of the antibodies with a 20-fold excess of purified rat prolactin abolished the staining of GH4C1 cell cultures with either antibody. Therefore, we have selected from a series of MAb raised to ovine prolactin two antibodies (17D9 and 6F11) that react specifically with rat prolactin in immunocytochemical studies, whereas 6F11 also reacts strongly with rat prolactin by ELISA and Western blot analysis.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	0022-1554 1551-5044
DOI:	10.1177/38.1.2403576