Solid-phase Radioimmunoassay Method Research Articles

Autoantibody to luteinizing hormone in patient with systemic lupus erythematosus (author's transl)

One hundred microgram of luteinizing hormone releasing hormone was intravenously injected into 19 female patients with systemic lupus erythematosus and plasma luteinizing hormone levels before and after the injection were determined with a method of solid phase radioimmunoassay. In 4 patients among them, the basal levels of luteinizing hormone were abnormaly high. A question about the existence of autoantibodies to luteinizing hormone in the patient's plasma raised to explain the reason for the high level of luteinizing hormone. The results from 3 experiments for solution of this question were as follows. 1) The patient's plasma was proved to have a binding activity with labeled luteinizing hormone. 2) This binding activity is specifically inhibited by purified luteinizing hormone. 3) This binding activity was located in gamma-globulin fraction. From the results, the existence of autoantibodies to luteinizing hormone in the 2 patients' plasma was concluded.

Solid phase radioimmunoassay for plasma testosterone using plastic microtiter tray (author's transl)

In order to simply radioimmunoassay for plasma testosterone and to measure many samples at the same time, a method of solid phase radioimmunoassay utilizing a plastic disposable microtiter tray (DMT) by which chromatography can be omitted was investigated. The antiserum was obtained by immunizing rabbits with testosterone-3 BSA which had been synthesized according to the Erlarnger's method. Plasma samples (male: 0.05ml, female: 0.2 ml) were extracted with 1.0 ml of ether. After freezing the plasma layer in an acetone-dryice bath, the ether phase was transfered to a glass tube and evaporated to dryness. These samples and the dried standard testosterone were dissolved with borate buffer containing 3H-testosterone and transfered to plastic DMT which had been precoated with the diluted antiserum, and incubated for 24 hrs. After removal of the incubated solution, the cups of DMT were cut off and were dissolved with toluene scintillator in counting vials. The radioactivity was counted with a liquid scintillation counter. Other steroids except for 5alpha-dihydrotestosterone (5alpha-DHT) had a low degree of cross reactivity with the antiserum. Five alpha-DHT which could be measured together with testosterone in this assay was not a problem clinically because of its strong androgenic activity. The best standard curve was obtained when the antiserum was diluted to 1:1000. The sensitivity of this assay was 10 pg/tube. The maximal adsorption of antibody to plastic DMT was observed when the pH of antiserum was within the range of 6.5-9.5 and the precoating time was 24 hr at room termperature. The best pH of incubation buffer was 8.0, and the antigen-antibody reaction became a plateau when the incubation exceeded 6 hrs. Water blank in this assay was 4.6 +/- 2.1 pg/tube. The recovery of testosterone (50, 100, 200 pg) added to 0.1 ml female plasma was 99 +/- 6.8%. Coefficients of variation within assay and between assay were below 11.2% and 20.0%, respectively. Correlation between this method and the dextran-coated charcoal method was fairly good (r=0.938). Plasma testosterone levels in 10 normal males and 12 normal females were 616 +/- 202 (mean +/- SD) ng/dl and 66 +/- 29 (mean +/- SD) ng/dl, respectively. The levels were low in patients with hypopituitarism, hypogonadism and acromegaly. They were normal in patients with Cushing's syndrome due to adrenal hyperplasia and adenoma, but they were high in a patient with adrenal carcinoma. In a patient with testicular feminization, the level was 632 ng/dl. This increased after the administration of HCG, and decreased to 127.5 ng/dl after castration. This solid phase radioimmunoassay (using plastic DMT) is economically feasible as well as simple because it is possible to separate the bound hormone from the free hormone of all samples at the same time and there is little restriction in time and temperature. According to the above results, this method is suitable for routine clinical use.

The Simultaneous Measurement of Australia-Antigen and Antibody by Solid-Phase Radioimmunoassay Method

The Simultaneous Measurement of Australia-Antigen and Antibody by Solid-Phase Radioimmunoassay Method

Solid-phase radioimmunoassay of ovine prolactin in antibody-coated tubes. Prolactin secretion during estradiol treatment, at parturition, and during milking.

The antibody-coated tube method of solid-phase radioimmunoassay has been applied to the measurement of prolactin in ovine plasma. The sensitivity of the assay was 2 ng prolactin per ml of plasma, and the coefficient of variation over the working range of 4–120 ng/ml plasma was 8–30%. No significant cross-reaction with ovine luteinizing hormone or growth hormone was observed. Non-specific binding of prolactin tracer to plastic tubes was minimized by incubation at 4 C; plasma samples were diluted 1:20, and prolactin standards were incubated in 5% hypophysectomized sheep plasma or horse serum. In anestrous ewes, plasma prolactin showed episodic fluctuations which were unrelated to periods of luteinizing hormone (LH) secretion; administration of estradiol by injection or continuous infusion stimulated the release of both prolactin and LH. At parturition, plasma prolactin levels were extremely high (>400 ng/ml) and remained elevated for 1–2 days after delivery. During lactation, prolactin secretion rose at the time of preparation for milking, and remained high during and after milking, again with extremely large fluctuations in plasma prolactin levels. In these studies, the coated-tube assay method proved to be particularly useful for measurement of serial prolactin levels in large numbers of plasma samples. (Endocrinology91: 1329, 1972)

A simplified radioimmunoassay technique for measuring human IgE

A simplified and sensitive solid-phase radioimmunoassay method for measuring IgE levels in human serum and tissue fluids is described. The test is accurate, reproducible, simple to use, and suitable for research as well as for routine clinical use. By approprate dilutions of the test sera it is possible to achieve a working range from 1 to 10,000 U. of IgE per milliliter with the use of this test. Sera from nonallergic individuals gave a mean value of 80 U. of IgE per milliliter with 90 per cent having levels under 150 U. per milliliter, while an allergic group had a mean value of 290 U. per milliliter with 46 per cent having levels above 150 U. per milliliter.

Assay of Australia Antigen and Antibody Employing Double-Antibody and Solid-Phase Radioimmunoassay Techniques and Comparison with the Passive Hemagglutination Methods

Abstract Australia antigen (Au) and antibody were assayed by double-antibody (RIA-DA) and solid-phase radioimmunoassay methods and the results were compared with those of other serodiagnostic tests. Purified Au (72 µg protein/ml and approximately 1013 particles/ml) was prepared and labeled with 125I. The use of a 2-day RIA-DA procedure (anti-Au + unknown, 6 hr; labeled Au, 18 hr; immunoprecipitating antibody, 24 hr) resulted in Au titers that were usually 5- to 35-fold greater than those obtained by the complement fixation (CF) method, and 5- to 10-fold greater than those resulting from the passive hemagglutination (PHA) inhibition procedure. Shortening the RIA-DA procedure to 1 day or using antibody-coated tubes in a solidphase radioimmunoassay system resulted in Au titers that were two- to threefold less than those obtained by the longer RIA-DA method, although both immunoassays were more sensitive than the CF test. RIA-DA and PHA anti-Au titers were comparable.

A sex difference in the rate of rise of plasma LH in rats following gonadectomy.

SummaryPlasma LH levels were measured in male and female rats soon after gonadectomy by means of a solid phase radioimmunoassay method. Male rats responded to gonadectomy by elevating their plasma LH concentrations within 12 hr postcastration; a continuous rise in plasma LH concentration was observed up to day 20 at which date a plateau level was reached. Female rats showed little elevation in plasma LH concentrations up to 10 days after gonadectomy; at this time, the plasma LH level was considerably lower than that found in the male; the stage of estrus during which gonadectomy was performed had no major effect on the animal's ability to elevate plasma LH concentration. At day 30 following gonadectomy, similar levels of plasma LH were observed in both sexes.

Solid phase radioimmunoassay of human β 1C globulin

In all radioimmunoassay it is necessary to separate free radioactive antigen from that portion which becomes bound to antibody. If the antibody is fixed to a solid surface, partition of free and antibody-bound antigen can easily be accomplished by separation of liquid and solid phases. We have studied a method of solid phase radioimmunoassay first suggested by Catt and Tregear[4] which is based on adsorption of antibody to surfaces such as polystyrene test tubes. Three steps are required. The first, adsorption of specific antibody to a fixed area of polystyrene tube surface, occurs on equilibrium of protein solutions in tubes at room temperature and pH 7.4. Adsorption is much greater and more predictable from globulin fractions than from whole antiserum. The second step is prevention of non-specific adsorption of antigen to sites on the tube. This is done by coating antibody tubes with an inert protein that does not interfere with antigen-antibody interaction and by incorporating the same inert protein into antigen solutions. The last step is equilibration of antibody-coated tubes with solutions of labelled antigen and increasing amounts of unlabelled antigen. The solutions are then removed and radioactivity bound to the tube surface is measured to quantify inhibition of labelled antigen uptake by unlabelled antigen. Solid phase immunoassay presents problems because antibody sites are not saturated with antigen in the practical working range. This sets limits on assay sensitivity because of conflicting requirements of high antigen concentration to approach antibody saturation and low labelled antigen for maximal isotope dilution. The requirements can be met by equilibrating tubes with a minimal antigen solution volume. Detection of 7 ng of β 1C globulin has been achieved. We have shown that the immunoassay results can be predicted from isotope dilution of the antigen mixtures if account is taken of the increased antigen binding that occurs as antigen concentration rises.

Radioimmunoassay of Fibrinogen and Its Proteolysis Products

SummaryThe solid-phase radioimmunoassay method has been applied to the measurement of fibrinogen. The method is extremely sensitive, being able to detect fibrinogen concentrations as low as 10 ng/ml. The immunoreactivity of fibrinogen proteolysis products differs from that of native fibrinogen, early proteolysis products showing enhanced immunoreactivity which decreases progressively with further digestion.

Solid-phase radioimmunoassay in antibody-coated tubes.

The adsorption of antibody to polymeric surfaces has been used to develop a new method of solid-phase radioimmunoassay. Incubation is performed in antibody-coated, disposable tubes that are finally washed-out with water and counted for quantitation of the bound tracer. The method is simple, rapid, inexpensive, and suitable for automation.