Double-antibody Assay Research Articles

Allergological examination of cystic fibrosis patients with skin reactions during carbenicillin treatment.

Sixteen of 84 cystic fibrosis patients developed skin manifestations during intensive carbenicillin therapy. The possibility of allergic drug reactions was examined. None of the patients showed any significant levels of antibodies of IgE, IgG or IgM classes against carbenicillin and different penicilloyl determinants as recorded with RAST, sandwich-radioimmunoassay and double antibody assay. The leukocytes of the patients did not release histamine on in vitro provocation with carbenicillin. Furthermore, none of the patients responded to prick test or to peroral penicillin provocation. The negative findings indicated that the recorded reactions were most probably not due to antibody-mediated allergy of type I or type III.

Antigens in Penicillin Allergy

The effects of the different acyl side chains of azidocillin, ampicillin and benzylpenicillin on the immunogenic potency of penicilloylated antigens as well as on the specificity of the developed antibodies were investigated in CBA mice. The antigens used were penicilloylated bovine gammaglobulm (BGG), bovine serum albumin (BSA) and human serum albumin (HSA). Immunization was performed with injection of high doses of antigens together with adjuvant or by injection of minute amounts of antigen over periods of 10 days. IgE antibodies were recorded with PCA in rats and IgG antibodies were measured with a double antibody assay. The nature of the carrier as well as the number of epitopes was found to influence the development of antibodies irrespective of the immunization schedule used. The immunogenic activity of the PO-BSA antigens was related to the epitope density. The PO-BSA antigens were, in contrast to the PO-BGG antigens, weak immunogens in the CBA mice. The acyl side chains of the different penicillins influenced the induction and specificity of the IgE antibody responses obtained after daily treatment.

The utility of a rapid assay for human chorionic gonadotropin in the management of trophoblastic disease

The diagnosis and treatment of trophoblastic disease depend upon the reliable measurement of human chorionic gonadotropin (hCG) in serum or urine. The double-antibody hCG beta-subunit assay for serum hCG combines great sensitivity, specificity, and precision but requires 36 hours to yield results. This investigation sought to increase the efficiency and total assay volume capabilities of our laboratory through the development of a rapid hCG assay to augment the slower beta-subunit system. Two separate rapid hCG assays were examined—a double-antibody radioimmunoassay and a radioreceptor assay. The double-antibody assay took 5 hours to complete, while the radioreceptor assay could be completed within 3½ hours. One hundred serum samples from patients with trophoblastic disease were assayed for hCG in the beta-subunit radioimmunoassay and in both rapid assay systems. No differences in results produced by the rapid double-antibody and the hCG beta-subunit assays were noted; however, results from the radioreceptor assay were significantly different (p < 0.05) from those obtained in the beta-subunit system.

Radioimmunoassay of rat liver metallothionein

A simple, inexpensive and convenient radioimmunoassay for rat liver metallothionein has been developed. The double-antibody assay involves the labeling of homogeneous, rat liver zinc thionein with trace amounts of 109Cd(II) to a specific activity of 1–2 × 10 6 cpm/μg protein; the binding of this antigen by rabbit anti-rat liver metallothionein antiserum; the displacement of this antigen by unlabeled zinc thionein or cadmium, zinc-thionein; the precipitation of the rabbit antibody-rat antigen complex by goat anti-rabbit IgG immunoglobulins; and the binding of this precipitate to cellulose acetate filters. The radioimmunoassay is useful in the range of concentration of metallothionein of 10–500 ng protein. The assay is accurate as compared with a previous technique of quantitating metallothionein in extracts of rat liver. A radial immunodiffusion assay for metallothionein is also described.

Separation of bound from free hormone in radioimmunoassay of lutropin and follitropin.

A new technique in which polyethylene glycol is added to double-antibody assays of lutropin and follitropin eliminates the need for an incubation period during the separatory phase. After the antigen-first-antibody equilibrium reaction, the second antibody and polyethylene glycol (30 g/L final dilution) are added, the tubes are immediately centrifuged, each supernate is aspirated, and the precipitated antibody-bound hormone is counted. With this combined polyethylene glycol-second-antibody separatory method, the incubation time is shorter than the usual double-antibody separation procedure, but avoids the problem of high nonspecific precipitation, which occurs with polyethylene glycol alone. In addition, the combined separation technique allows the use of sera containing low titers of second antibody and use of smaller volumes of sera containing high titers of second antibodies, thus conserving second-antibody reagent.

Solid Phase Radioimmunoassay for Quantitation of Antigen-Specific IgG in Human Sera with 125I-Protein A from Staphylococcus Aureus

Abstract Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 hr during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 hr with 125I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, results of the Staph A assay correlated highly (r = 0.981, p &amp;lt; 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of 125I-Staph A in the solid phase radioimmunoassay was supperior to 125I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the 125I Staph A assay may be applied to IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.

Comparison of double antibody and solid-phase radioimmunoassays

Two commonly used radioimmunoassay (RIA) procedures, double antibody assay and solid-phase assay, have been compared using the same reagents and conditions. Mouse hemoglobulin—horse anti-mouse hemoglobulin system was chosen for this study. The antibody was found to have more binding capacity in soluble form than in the Sepharose-bound form. The inhibition curves in both the assay systems were shifted to higher inhibitor concentration at higher control binding levels. At 40% control binding level, the double antibody RIA was found to be 2.7 times more sensitive than the solid-phase RIA. The reason for this most probably lies in the lower binding capacity of the Sepharose-bound antibody which necessitates the use of higher amounts of the antibody to reach 40% control binding.

A method for the radioimmunoassay of large numbers of samples using quantitative autoradiography of multiple-well plates

A double antibody assay for thyroxine using 125I as label was carried out on 10-μl samples in Microtiter V-plates. After an additional centrifugation to compact the precipitates the plates were placed in contact with X-ray film overnight and the spots were scanned. In the 20 to 160 ng/ml rangs the average coefficient of variation for thyroxine concentration determined on the basis of film spot optical density was 11% compared to 4.8% obtained using a standard gamma counter. Eliminating the need for each sample to spend on the order of 1 min in a crystal well detector makes the method convenient for largescale applications involving more than 3000 samples per day.

Comparison of Radioimmunoassay Techniques in the Detection of IgE and IgG Antibody Activity against Aspergillus Fumigatus Antigens

Abstract Radioimmunoassays suitable for detection of antibodies against mixtures of antigens were used to provide quantitative estimation of antibody activity against Aspergillus fumigatus (Af) antigens. Sera from subjects with allergic bronchopulmonary aspergillosis, aspergillomas, and asthma with immediate-type cutaneous reactivity were studied. With 125I IgE and 125I IgG, a solid phase polystyrene tube multilayered type radioimmunoassay detected IgE and IgG antibodies in the same serum samples. With 125I Af, a solid phase polystyrene tube and a double antibody assay detected IgE and IgG antibodies, respectively, in the same serum samples. We conclude that either type of assay may be used for detection of antibody activity and that demonstration of elevated IgE and IgG antibody levels in the same serum may be useful in characterization and differentiation of disease states in which Af is involved.

Nicotinamide mononucleotide adenylyltransferase, a nonhistone chromatin protein: Immunochemical comparative studies

Rabbit antibodies to partially purified nicotinamide mononucleotide adenylyltransferase precipitated the enzyme, which remained fully active in the insoluble complexes. Precipitation of antigen-excess soluble complexes with sheep anti-rabbit γ globulin increased the sensitivity of the immunoassay. With this double-antibody assay, the enzymes from chicken erythrocytes, liver, kidney, and thymus showed nearly identical reactivity. Goose, pheasant, and turkey enzymes were highly cross-reactive with the chicken form; pigeon liver enzyme was markedly less reactive. There was no cross-reactivity with fish, amphibian, or mammalian enzymes. The specificity of the antiserum was increased by absorption of antibodies to nonenzyme proteins. The absorbed serum still precipitated the enzyme; in complement fixation assays, it reacted with an antigen that behaved like the enzyme. This antigen was detectable in whole chromatin and in the proteins extracted from chromatin by high salt or urea concentrations. Its immunological reactivity survived exposure to 0.5 m urea, but was reduced by exposure to 6.0 m urea plus 0.4 m guanidine. The enzyme was present as an inactive, partially denatured protein in nonhistone chromatin proteins prepared with these reagents.

Production of specific antisera for radioimmunoassay of human luteinizing hormone (LH) in the presence of human chorionic gonadotropin (hCG).

A specific radioimmunoassay for LH, which measures plasma LH in the presence of human chorionic gonadotropin (hCG) is described. Rabbits were immunized with highly purified native LH. One of the antisera with a difference in its reactivity against LH and hCG was further purified by affinity chromatography on a column with hCG coupled to Sepharose 4B. The adsorbed antiserum and 125I-LH was used in a double antibody assay. The LH standard (MRC 68/40) efficiently inhibited the binding of 125I-LH, and the standard curve showed a sensitivity of 0.5 ng/ml in the sample. hCG up to 10 000 ng/ml did not inhibit the binding of 125I-LH. The plasma level of LH in pregnant women in the first trimester was low (1.3 +/- 0.1 ng/ml). When LH was measured in fertile or menopausal women with or without stimulation with LH/FSH releasing hormone (LH-RH)X the results agreed to those found with our conventional LH-assay based on antiserum against hCG.

The Relative Paucity of IgE in Human Milk

The levels of IgE were determined in paired samples of serum and milk when whey obtained 3 to 8 days of postpartum, from 16 human lactating mothers who had reported a history of allergy to a variety of common allergens. Two assay procedures were employed to measure total IgE, a double-antibody assay and a commercially available solid phase assay (RIST). In addition, each sample of serum and whey was tested for specific IgE antibodies to a variety of allergens by the RAST test. The levels of total serum IgE were between 30 and 2300 I.U./ml and relatively good agreement was observed for both the double-antibody and RIST methods. In contrast, total IgE levels in milk whey were either undetectable (less than 3.0 I.U./ml in 14 of 16 subjects) or very low when analyzed by the double-antibody method, but were very high (400 to 1650 I.U./ml when analyzed by the RIST method. However, IgE added to milk whey could be measured by the double-antibody procedure indicating that the low levels detected in milk were not a fault of the double-antibody assay. It was assumed that the RIST test was subject to nonspecific interference by factors in milk whey which caused the determination of high, but incorrect, levels of IgE. Specific IgE antibodies were detected in the serum of 10 of 16 subjects but were not present in milk whey. A comparison of the whey/serum ratios of albumin, IgA, and IgE suggested that little, if any, IgE is selectively synthesized or secreted in the mammary gland.

A detailed investigation of circulating IgE levels in a normal population.

Total circulating IgE levels were measured in a carefully selected normal population, using a highly sensitive double antibody assay. The mean level of 36.3 u/ml found in this group is much lower than that reported in all previous studies. No circadian rhythm was evident, but IgE levels varied slightly from day-to-day. IgE levels in 100 serum samples were compared using the double antibody assay and the Phadebas test kit, based on a solid phase technique. A poor correlation was obtained with values below 100 u/ml, however, there was an excellent correlation for samples with IgE levels above this figure.

Differences in immunologic activity among preparations of human insulin

Two preparations of human insulin (Lilly 1064B-27 and Medical Research Council 66 304 ) recently tested in this laboratory are more immunoreactive to certain guinea pig antiinsulin antisera than an older preparation of human insulin (Lilly 33) widely used as a standard in the assay for serum insulin. All three preparations have approximately the same biologic activities. The difference in immunologic activity appears not only with the double-antibody assay, but also with the enzyme-partition assay; the difference is greater with the latter assay. The quantitative value obtained in the measurement of serum insulin is lower when the newer standards are used in these assay; similar changes may occur in other assays when changing from one standard preparation to another. A need exists for a reference preparation of human insulin.

A multiple ligand-binding radioimmunoassay of diiodotyrosine.

A radioimmunoassay has been developed for the measurement of 3,5-diiodo-L-tyrosine (DIT) in serum. DIT was coupled to porcine thyroglobulin (PTg) with a molar ratio of 205:1. Rabbits were immunized with 1 mg of immunogen emulsified in complete Freund's adjuvant. Sera were screened for their ability to bind trace amounts of [125I]DIT. A serum that bound 40% of the tracer at a final dilution of 1:1,750 was used in the assay. Assay specificity was improved by the use of thyroxine (T4)-binding globulin as a second ligand-binding protein to decrease T4 and triiodothyronine (T3) cross-reactivity with the antibody. Double antibody and polyethylene glycol radioimmunoassays were compared. DIT present in the second antiserum shifted the double antibody assay standard curve and altered estimates of assay specificity and assay sensitivity. By using the polyethylene glycol system and butanol:ethanol extracts of serum, DIT was measured in human serum. In 35 apparently healthy young adult controls DIT levels averaged 156 ng/100 ml. Random DIT levels averaged 158 ng/100 ml in 11 untreated hyperthyroid patients and 84 ng/100 ml in 15 untreated primary hypothyroid patients. No diurnal pattern in DIT levels could be demonstrated. Thyroid-stimulating hormone administration led to a variable but small rise in DIT levels, but short term T3 suppression was not associated with a measurable fall in DIT concentrations. Paired serum samples from the carotid artery and thyroid vein of 10 euthyroid goiter patients and one patient with a toxic solitary adenoma all showed a positive transthyroidal gradient indicating the thyroidal release of DIT in each patient. Measurable DIT levels of 45, 47, 68, and 80 ng/100 ml, respectively, were found in four fasting athyrotic patients indicating that the thyroid is not the only source of serum DIT.

A semiautomated technic for radioimmunoassay. Double antibody assay of insulin.

A semiautomated modification of the double antibody assay for immunoreactive insulin, using commercial equipment and easily constructed accessories, has been devised. It has been found to be a convenient and reliable method for performing insulin assay upon large numbers of samples.

A double antibody assay for glucagon

A two-antibody immunoassay for glucagon has been described. The method is sensitive to as little as 0.1 ng of glucagon, permitting reliable measurements of glucagon in 1 ml of human serum. The use of a proteolytic enzyme inhibitor (Trasylol) made satisfactory recoveries of added glucagon possible. The average fasting serum immuno-reactive glucagon (IRG) concentration of 5 normal subjects was 0.44 ng/ml (0.40 to 0.46; 10 determinations). The oral administration of 100 g of glucose caused a small, but statistically significant increase in serum IRG, while the intravenous injection of glucose was followed by a prompt and statistically significant decline. The insulinogenic response to oral glucose was greater than the response to intravenous glucose. The significance of these results is discussed.

Urine insulin in normal subjects

Immunochemical insulin is measurable in urine by the double-antibody assay procedure. Its excretion per hour correlates with the mean serum level and its “clearance” does not vary with changes in serum insulin concentrations. In 28 normal subjects the “insulin clearance”, measured during an oral GTT, was low (mean 0.42 ml./min.; range 0.23–0.87 ml./min.) and varied with the “creatinine clearance” and less with urinary albumin. This low “clearance” suggests that either serum insulin is bound to a larger protein or polymerized, or that it is avidly reabsorbed by the renal tubules after glomerular filtration. The log-mean basal (overnight) urine insulin level in 28 normal subjects was 215 μU/hr. (range 89–499). During the 2 hr. following 50 Gm. oral dextrose the level rose to a log-mean of 834 μU/hr. (range 330–2370).