A monoclonal antibody to lactogenic receptors from female rat liver.
Affinity-purified lactogenic receptors from female rat liver microsomal membranes were used to raise antibodies in female Balb/c mice. Mouse spleen and myeloma cells were fused and hybridoma-derived monoclonal antibodies (Mabs) were produced by in-vitro cell culture. Mab from a selected clone was sequentially purified by chromatography on a thiophilic gel and on agarose-bound protein A. The Mab was found to be of IgG1 subclass and of kappa type. The Mab recognized membrane-bound and solubilized (by the detergent heptaoxyethylene lauryl ether; G3707) receptors as well as receptors purified by affinity chromatography and subsequent sodium dodecyl sulphate (SDS) electrophoresis from female rat liver. The Mab bound to receptors from several other female rat tissues, such as ovary, kidney and adrenal, whereas there was no binding to liver receptors from cow and rabbit. Displacement experiments showed that the Mab was specific for a lactogenic type of receptor, in agreement with the finding that the Mab did not recognize receptors from male rat liver. The Mab also bound to cytoplasmic receptors (present in the supernatant after centrifugation at 100,000 g) from female rat liver, suggesting a structural similarity between the cytoplasmic and the microsomal receptors. Analysis of purified receptors by SDS electrophoresis and subsequent Western blot with 125I-labelled Mab as a probe showed one band corresponding to an Mr of 45,500 +/- 2500 (n = 5). The same band was obtained with 125I-labelled human GH, showing that the Mab binds to the unit which accommodates the hormone-binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Emtner
M
M
Department of Biochemistry, University of Uppsala, Sweden.
Brandt
J
J
Johansson
U
U
Jouper
B
B
Fryklund
L
L
Roos
P
P
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008024:Ligands; D008099:Liver / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D018000:Receptors, Peptide; D011981:Receptors, Prolactin / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D008024:Ligands; D018000:Receptors, Peptide; D011981:Receptors, Prolactin; C028202:placental lactogen receptor