ACTH adrenal cell bioassay: improved sensitivity (12 ng/L) achieved by immunoextraction of ACTH from human plasma by a monoclonal antibody.
We have previously reported a bioassay for human plasma ACTH based upon trypsin dispersed guinea-pig adrenal cells which was sensitive to 100 ng/L ACTH in unextracted human plasma when measured against human pituitary ACTH (1-39) standard 74/555. We now present a bioassay of increased sensitivity (12 ng/L) which incorporates three major changes. The trypsin/trypsin inhibitor step in the cell dispersion protocol has been replaced with collagenase, donor calf serum (3%) has been incorporated into the standard curve and ACTH has been extracted from human plasma and dilutions of standard hormone by a sephacryl bound monoclonal antibody (2A3) directed towards the 25-39 sequence. The extracted standard curve has a detection limit of 6 ng/L and the cells can tolerate up to 50% plasma equivalent concentration. Thus, the improved assay has a detection limit of 12 ng/L ACTH in plasma. The assay can now measure bioactive plasma ACTH levels reliably in the normal range.
Mitchell
R
R
Department of Clinical Biochemistry (Medicine), University of Manchester, Hope Hospital, Salford, UK.
Lambert
A
A
Crosby
S R
SR
White
A
A
Robertson
W R
WR
D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism*; D000324:Adrenocorticotropic Hormone / Q000097:blood* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal; D001681:Biological Assay; D002478:Cells, Cultured; D003907:Dexamethasone; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D003012:Microbial Collagenase; D011786:Quality Control; D011863:Radioimmunoassay; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D014361:Trypsin Inhibitors; D003907:Dexamethasone; D000324:Adrenocorticotropic Hormone; D003012:Microbial Collagenase