A new antibody capture enzyme linked immunoassay specific for transforming growth factor beta 1.
Previous studies which examined Transforming Growth Factor beta 1 (TGF-beta 1) generation have relied on the identification of TGF-beta 1 mRNA or measurement of TGF-beta 1 by bioassay. Quantitation of TGF-beta 1 message alone however is inadequate since the regulation of TGF-beta 1 synthesis is often post-transcriptional. TGF-beta 1 is poorly immunogenic, and sensitive and specific immunoassays for this peptide have proved difficult to develop. Bioassays depend on stimulation or inhibition of cell proliferation in a TGF-beta 1 dependent manner, and are very rigid in their requirements for optimal performance. The aims of this work was therefore to develop a sensitive and reproducible immunoassay for TGF-beta 1. Microtitre plates were coated with human recombinant TGF-beta 1, unbound protein was discarded from the wells prior to blocking with bovine serum albumin. Chicken anti-human TGF-beta 1 antibody was incubated with the test solution overnight at 4 degrees C and then added to the coated wells. Bound antibody was detected with alkaline phosphatase conjugated anti-chicken antibody. The assay is sensitive to 0.2 ng/ml with a range to 100 ng/ml. The assay detects the mature form of human recombinant TGF-beta 1, natural platelet extracted TGF-beta 1, and TGF-beta 1 derived from human monocytes stimulated with Phorbol myristate acetate (PMA). Active TGF-beta 1 is measured directly and latent TGF-beta 1 can be measured indirectly following acid activation of samples. Inter-assay precision ranged from 4.3 to 9.6%, (coefficient of variation, %CV) and intraassay precision ranged from 2.8 to 8.6% (CV).(ABSTRACT TRUNCATED AT 250 WORDS)
Phillips
A O
AO
Institute of Nephrology, Cardiff Royal Infirmary, U.K.
Steadman
R
R
Donovan
K D
KD
Williams
J D
JD
D000818:Animals; D000918:Antibody Specificity; D001681:Biological Assay; D002417:Cattle; D002645:Chickens / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D010586:Phagocytes / Q000276:immunology / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012044:Regression Analysis; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000032:analysis* / Q000096:biosynthesis*
D012333:RNA, Messenger; D016212:Transforming Growth Factor beta