ABSTRACT
We are describing the synthesis and use of biotinamidocaproyl-derivatives of 18-oxocortisol-3-carboxymethoxylamine for the development of enzyme labels using the avidin-peroxidase system for the design of enzyme-linked immunoassays (ELISA). An ELISA for 18-oxocortisol and -hydroxycorticosterone was devised which showed improved sensitivity and specificity in comparison to an RIA using a tritiated tracer. The system is easy to prepare and offers the possibility to design immunoassays when no tritiated tracer is available.
Subject(s)
18-Hydroxycorticosterone/analysis , Biotin/analogs & derivatives , Enzyme-Linked Immunosorbent Assay/methods , Hydrocortisone/analogs & derivatives , 18-Hydroxycorticosterone/analogs & derivatives , 18-Hydroxycorticosterone/immunology , Binding, Competitive , Cross Reactions , Hydrazines , Hydrocortisone/analysis , Hydrocortisone/immunologyABSTRACT
A method for the production of the haptens 18-hydroxy-11-deoxycorticosterone-3-(O-carboxymethyl)-oxime (18-OH-DOC-3-CMO) and 18-hydroxycorticosterone-3-(O-carboxymethyl)-oxime (18-OH-B-3-CMO) is described. The formation of the oximes was studied in kinetic experiments. They were prepared at pH 1.6 in methanol/HCl using a short reaction time. Antisera were raised in rabbits using serum albumin conjugates. The highly specific antisera were used at a final dilution of 1:79 000 (18-OH-DOC) and 1:43 000 (18-OH-B); the affinity constants were 1.2 x 10(10) l/mol and 8.1 x 10(9) l/mol, respectively. The radioimmunoassay procedure for 18-OH-B in serum involves purification by paper chromatography. The intra- and interassay precision was 7.3% and 12.3%, respectively. The mean serum 18-OH-B level (+/- S.D.) for normal male and female ambulatory subjects (n = 40) on a normal sodium diet was 0.802 +/- 0.262 nmol/l. After 60 minutes of recumbency, the serum 18-OH-B level was 0.313 +/- 0.061 nmol/l (n = 6) for men.
Subject(s)
18-Hydroxycorticosterone/immunology , 18-Hydroxydesoxycorticosterone/immunology , Corticosterone/analogs & derivatives , Desoxycorticosterone/analogs & derivatives , 18-Hydroxycorticosterone/blood , Adult , Angiotensin II/pharmacology , Animals , Antibody Formation , Cross Reactions , Female , Humans , Male , Rabbits/immunology , Radioimmunoassay/methodsSubject(s)
18-Hydroxycorticosterone/urine , Aldosterone/urine , Corticosterone/analogs & derivatives , Hyperaldosteronism/diagnosis , Hypertension/diagnosis , 18-Hydroxycorticosterone/immunology , 18-Hydroxydesoxycorticosterone/immunology , 18-Hydroxydesoxycorticosterone/urine , Aldosterone/analogs & derivatives , Aldosterone/immunology , Chromatography , Cross Reactions , Glucuronates/immunology , Glucuronates/urine , Humans , Hyperaldosteronism/immunology , Hyperaldosteronism/urine , Hypertension/immunology , Hypertension/urine , RadioimmunoassayABSTRACT
A sensitive and specific radioimmunoassay has been developed for 18-hydroxy-corticosterone (18-OH-B) and applied to the measurement of this steroid in peripheral plasma. High specific activity label (3H-18-OH-B) was prepared using the incubation of 3H-corticosterone with duck adrenal mitochondria. Antisera were produced by immunisation with 18-OH-B gamma-lactone 3-oxime conjugated to bovine serum albumin. The antibodies examined showed 100% cross-reactivity with 18-hydroxy-deoxycorticosterone gamma-lactone (18-OH-DOC gamma-lactone), but minimal cross-reactivity with other steroids. Paper chromatography was used to separate 18-OH-DOC gamma-lactone from 18-OH-B gamma-lactone. The interassay precision was 7.6% and the intra-assay precision 11.0%. The accuracy of the method was confirmed by showing a linear relationship between amounts of 18-OH-B added and amounts of 18-OH-B gamma-lactone measured (y = 0.854 X +15.1, r = 0.9. p less than 0.001). The mean plasma level in normal subjects on an ad libitum sodium intake was 225 +/- 92.7 (SD) pg/ml (n = 17) when standing, and 99 +/- 38.3 (SD) pg/ml (n = 6) after lying down for 30 minutes.
