A solid-phase radioimmunoassay of serum dehydroepiandrosterone sulphate using a monoclonal antibody

A monoclonal antibody directed against dehydroepiandrosterone, but with high affinity for dehydroepiandrosterone sulphate (DHA-S), has been used to develop a solid phase radioimmunoassay for measuring serum DHA-S. The antibody was covalently linked to polyacrylamide microbeads with no change in binding characteristics. The procedure requires only the chromatography of serum on anion-exchange cellulose before assaying the equivalent of 0.25 microliter serum. The method is precise, accurate and specific and can detect 19.5 pg of DHA-S. Serum DHA-S levels measured by this method were in good agreement with those found in a validated radioimmunoassay method involving hydrolysis. The method is quick and one operator could assay 50 blood specimens per day. DHA-S levels in serum from 50 men and 86 women were in agreement with those in the literature. With the availability of theoretically limitless quantities of consistently high quality monoclonal antibodies the advantages of developing solid phase radioimmunoassays for steroids is discussed.     

A non-chromatographic radioimmunoassay for serum dehydroepiandrosterone using a mixture of antisera

A simplified method for evaluating serum dehydro-epiandrosterone (DHEA) without chromatography has been developed, using mixtures of two different anti-DHEA antisera, anti-3β-hydroxy-Δ⁵ antiserum and anti-11-deoxy-17-ketosteroid antiserum, in which cross-reactivity of each antiserum is reduced to a negligible amount. Serum (20 μ1) was extracted with 1 ml of n-hexane. One milliliter of 80% methanol was added to the n-hexane extract which was stirred and centrifuged. The n-hexane layer was discarded, and the methanol layer was evaporated to dryness. The residue was incubated with an antiserum mixture containing DHEA-7α-³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEA-7α- ³H. The accuracy, precision, sensitivity and specificity were satisfactory. Good agreement was found between the serum DHEA levels obtained by the present radioimmunoassay and those obtained by radioimmunoassay with paper chromatography, making this method suitable for routine use.     

A rapid radioimmunoassay for serum testosterone

Sekihara et al. have proposed that it is possible to combine two antisera, each of which is unsatisfactory for a clinical radioimmunoassay due to cross-reactivity problems, and obtain an assay which is of sufficiently good specificity for practical application. The present report provides a theoretical analysis of this problem in a "reduced" case of minimal complexity. We assume infinitesimal concentration of tracer, equilibrium of reactants, perfect separation of bound and free, and that each of the two antisera contain only a single class of antibody sites which can bind to the desired ligand or to a cross-reacting species. Numerical methods are used to generate "ideal" dose response curves. The specificity is evaluated by three criteria: 1) as the ratio of ligand concentrations resulting in 10 or 50% reduction of binding of labeled ligand to antibody, i.e. %B/BO = 90 or 50%; 2) the %B/BO or %B/T at an arbitrary dose level; or 3) the apparent amount of ligand present, for an arbitrary dose of crossreacting ligand. Results indicate that mixing of two nonspecific antisera (each cross-reacting with a different ligand) results in a radioimmunoassay system with a specificity intermediate between that obtained with either of the antisera used alone. Whether this will provide a "satisfactory" assay depends on the purposes for which it is intended, the expected concentrations of cross-reacting ligands, etc. Computer simulation studies may be utilized to select the optimal ratio of the two antisera being used for the assay.     

A radioimmunoassay for duck serum ferritin

Development and optimization of a radioimmunoassay for duck serum ferritin described. 125I-labelled ferritin and rabbit anti-ferritin antibody are used together with goat anti-rabbit gamma globulin as separating agent for the bound and free fractions. The assay has a working range of up to 500 micrograms of ferritin per litre and a sample requirement of 50 microliter of serum. The assay requires a 24 h period and has a sensitivity of 10 micrograms of ferritin per litre.     

A radioimmunoassay for measurement of serum thymidine

A simple and sensitive radioimmunoassay for the measurement of serum thymidine has been developed based on the competition between thymidine and an analog (¹²⁵IUdR) for sites on anti-thymidine antibody. Antibody-bound ¹²⁵IUdR was measured in ammonium sulfate precipitates. None of the structurally related compounds that are normal body metabolites showed thymidine's ability to displace the label except at 200-fold higher concentration. A thymidine concentration of slightly greater than 10^?6m has been measured in mouse blood serum. The rate of disappcarance of a thymidine load from the bloodstream has been followed after intravenous administration. The sensitivity and specificity of the technique have been discussed, and applications in radiotherapy and chemotherapy depending on altered DNA synthesis or cell death have been suggested.     

A study of zinc distribution in human serum.

The partition of zinc in human serum between two major zinc-binding proteins, albumin and alpha2-macroglobulin, was studied in 28 control subjects and in 156 hospitalized patients. Albumin-bound zinc was both the major and the more dynamic of the serum zinc components. Over a wide range of values the concentrations of albumin-bound zinc and total serum zinc were highly correlated (r=0.91) with each other, as were concentrations of albumin and albumin-bound zinc (r=0.69). alpha2-Macroglobulin-bound zinc was not strongly correlated either with total serum zinc or with the serum concentration of alpha2-macroglobulin. Twenty-four hour urinary excretion of zinc was not correlated with any of the serum zinc parameters. To a large extent it appears that total serum zinc concentration reflects serum albumin concentration.     

A novel, general radioimmunoassay for acyl carrier proteins

A radioimmunoassay (RIA) for acyl carrier proteins (ACP) is described that is based on the competitive binding between [3H]acyl-ACP and unlabeled ACP of the same species. The radiolabeled antigen, [3H]palmitoyl-ACP, is enzymatically synthesized by Escherichia coli acyl-ACP synthetase. Because acyl-ACP synthetase can specifically radiolabel ACP in crude extracts from several plant sources, the use of this enzyme to prepare [3H]acyl-ACP obviates the need for pure preparations of each ACP. Preparation of [3H]acyl-ACP with a specific activity of 15 Ci/mmol allows RIA detection of total ACP in crude plant extracts at the nanogram level. Because antibodies against spinach ACP partially crossreact with ACP from many plant sources, RIAs for other plant species can be constructed using only one preparation of antibody. ACP preparations from safflower, soybean, avocado, corn, and E. coli show a decreasing order of partial immunocross-reactivity with spinach ACP-specific antiserum, as examined by RIA using spinach [3H]palmitoyl-ACP.     

A non-chromatographic radioimmunoassay of estrone and estradiol-17beta serum

A sensitive and efficient non-chromatographic procedure employing the Girard reagent and solvent-partitioning has been developed for the accurate radioimmunoassay (RIA) of estrone (E1) and estradiol-17β(E2) in a single 1.0 ml specimen of male or female serum. Using standard curves which permitted the discrimination of zero from 0.75–1.5 pg (p=0.05), the following mean procedural blanks (pg ± S.D.) were determined (1.0 ml water, n= 24): estrone, 2. 1 ± 1.1 (range 0–4.1); estradiol 1.0± 1.1 (range 0–3.6).A comparison of RIA of estrogens (1) in serum after separation by the Girard procedure and by TLC yielded correlation coefficients of 0.99 and 0.98 for E1 and E2 respectively. The following results (pg/ml ± S.D.) were obtained on RIA of E1 and E2 in 12 different 1.0 ml specimens of male and female serum using the Girard procedure: male. E1 (32.0 ± 9.2), E2 (24.1 ± 10.9); female, E1 (108.5 ± 60.8), E2 (126.4 ± 63.2).The intra-assay variability (c.v.) was found to be 12.6% for E1 and 9.4% for E2. The interassay variability was 14.2% for both estrogens.Twenty-four assays of E1 and E2 can be completed by one person in 2 working days.     

Vitellogenic ovarian follicles of Drosophila exhibit a charge-dependent distribution of endogenous soluble proteins

In ovarian follicles of Drosophila, soluble endogenous charged proteins are asymmetrically distributed dependent upon their ionic charge. Reversal of the normal ionic difference across the intercellular bridges which connect nurse cells to their oocyte results in a redistribution of these proteins. Twelve soluble endogenous acidic proteins were identified by 2-D gel electrophoresis as being present in both oocytes and nurse cells in samples run on four or more gels. Of these, following osmotically induced reversal of the electrical transbridge gradient the concentration of seven proteins decreased in the oocyte while nurse cell concentrations of all twelve proteins increased. Of seven basic proteins analyzed, following reversal of the electrical gradient the concentration of all seven increased in oocytes. Four of these decreased in nurse cells, while nurse cell concentrations of the remaining three basic proteins also appeared to decrease, but yielded spots too faint for measurement. Data presented here demonstrate that, as in the Saturniidae, the ionic gradient across the nurse cell-oocyte intercellular bridges of the dipteran, Drosophila, can influence the distribution of soluble endogenous charged molecules.     

A study of the auxins in cabbage using countercurrent distribution

The counter-current distribution technique has been applied to the study of the identity of the auxins present in fresh cabbage. The results indicate that the major fraction of the auxin activity is probably due to 3-indoleacetic acid, but this is not established conclusively. In addition to the material presumed to be 3-indoleacetic acid, two other biologically active fractions were obtained from cabbage. Whether these are other auxins or “precursors” of 3-indoleacetic acid has not been established. It has been demonstrated that the colorimetric test which has been used by other workers is not specific for 3-indoleacetic acid, and conclusions which have been based on the use of this test must be reconsidered.     

A radioimmunoassay for lithocholic acid conjugates in human serum and liver tissue

A sensitive and specific radioimmunoassay for glycine and taurine conjugates of lithocholic acid (CLCA) has been developed. ³H-glycolithocholic acid (S.A. = 17Ci/mmol) was used as tracer. Separation of free from antibody-bound bile acid was carried out using ammonium sulphate (saturated solution). The antiserum showed high specificity for both glyco and tauro conjugated lithocholate (100% cross reaction) and lithocholic acid (25% cross reaction). The sensitivity of the assay (1 pmole/tube), was adequate for measuring CLCA in peripheral blood and hepatic tissue in man.