An enzyme immunoassay for rat growth hormone: validation and application to the determination of plasma levels and in vitro release.
A competitive enzyme immunoassay for rat growth hormone (rGH) has been developed using polyclonal anti-rGH antibodies and an acetylcholinesterase (EC 3.1.1.7.) enzymatic tracer coupled covalently with rGH. The assay was performed in 96-well microtiter plates coated with rabbit polyclonal anti-goat immunoglobulin antibodies. Molecular sieve filtration and Western blot analysis revealed a single immunoreactive peak for rat plasma or pituitary extracts. Cross-reactivity with other rat pituitary hormones or human GH was less than 1%. Assay of samples in a concentration range of 0.7 to 69 ng/ml by enzyme immunoassay and radioimmunoassay were well correlated (r = 0.87 and 0.85 respectively for plasma and culture medium samples). Intra- and inter-assay variations in plasma were 4 (n = 24) and 14% (n = 9) respectively. Minimal detectable amounts of rGH were 0.6 ng/ml. A two-site immunometric assay also developed with the same antibodies allowed a detection threshold of 0.25 ng/ml.
Ezan
E
E
CEA, Service de Pharmacologie et d'Immunologie, Gif-sur-Yvette, France.
Laplante
E
E
Bluet-Pajot
M T
MT
Mounier
F
F
Mamas
S
S
Grouselle
D
D
Grognet
J M
JM
Kordon
C
C
D000110:Acetylcholinesterase / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D003429:Cross Reactions; D003470:Culture Media / Q000737:chemistry; D013006:Growth Hormone / Q000032:analysis* / Q000097:blood / Q000276:immunology; D019382:Human Growth Hormone / Q000032:analysis / Q000276:immunology; D006801:Humans; D007124:Immunoenzyme Techniques; D008970:Molecular Weight; D010903:Pituitary Gland, Anterior / Q000737:chemistry; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis; D012680:Sensitivity and Specificity
D003470:Culture Media; D011994:Recombinant Proteins; D019382:Human Growth Hormone; D013006:Growth Hormone; D000110:Acetylcholinesterase