Quantitative Radioimmunoassay Research Articles

A radioimmunoassay for quantitation of secretory component.

Secretory component (SC) isolated from human colostrum was radioiodinated with chloramin-T method and it was subjected to a radioimmunoassay for quantitation of SC. A monospecific rabbit anti-sera against human SC which could react with free SC as well as immunoglobulin bound SC was used for the assay. The present method can measure as low as 300 ng/ml of SC. The level (mean +/- 2 S.D.) of serum SC in healthy subjects was determined to be 31.3 +/- 7.2 micrograms/ml. Studies with Sephadex G-200 gel filtration demonstrated that SC in the normal sera existed mostly as a form of SC-IgA. In contrast, free SC was never detected in them.

A sensitive radioimmunoassay for quantitation of IgM rheumatoid factor.

A solid-phase radioimmunoassay capable of detecting nanogram quantities of human IgM rheumatoid factor (RF) in biologic fluids has been developed. Binding curves for monoclonal IgM RF and polyclonal IgM rheumatoid factors were similar under the conditions utilized for the assay. Human IgG did not interfere with the detection of IgM RF by this method. Small quantities (less than or equal to 0.2%) of nonspecific binding by nonRF IgM to the human IgG coated tubes utilized in the assay were corrected for by assaying samples in parallel bovine serum albumin coated control tubes. As expected, patients with seropositive rheumatoid arthritis (RA) had significantly higher concentrations of IgM RF than seronegative RA patients (mean +/- 1 SD = 652 +/- 553 microgram/ml versus 11.3 +/- 13.3 microgram/ml, P less than 0.001). In contrast, all normal control sera assayed to date contained less than 0.1 microgram/ml of IgM RF. The capacity of the assay to detect nanogram quantities of IgM RF should permit investigation of the cellular mechanisms underlying RF production.

The phospholipid-binding and immunochemical properties of amidinated, guanidinated and acetylated apolipoprotein A-II

To determine the importance of lysine groups in the sites for lipid-protein interaction and antigenic reactivity, reduced, carboxymethylated apoliprotein A-II and apolipoprotein A-II were modified by either amidination, guanidination or acetylation. The conformation, phospholipid-binding properties and immunochemical reactivity of each derivative were then determined. Retaining the positive charges on CM-apolipoprotein A-II by either amidination or guanidination had little or no effect on the conformation of the apoprotein; each had approximately 35% α-helical structure in the absence of dimyristoylphosphatidylcholine (DMPC) and 68% in the presence. In the absence of lipid, acetylated apolipoprotein A-II contained 17% α-helicity; the α-helicity of the protein increased to 48% in the presence of DMPC. Furthermore, each derivative formed complexes with vesicles of DMPC which are similar to that of CM-apolipoprotein A-II. The molar ratio of DMPC to protein for CM-apolipoprotein A-II, amidinated apolipoprotein A-II, guanidinated apolipoprotein A-II and acetylated apolipoprotein A-II were 43, 52, 62, and 69: 1 respectively. By quantitative radioimmunoassay, the immunoreactivity of CM-apolipoprotein A-II and its amidinated, guanidinated and acetylated derivatives were indistinguishable. Based on these results, we conclude that retaining the positive charges by amidination or guanidination or neutralizing the positive charge by acetylation does not affect the lipid-binding or immunochemical properties of apolipoprotein A-II.

Detection and Quantitation of the β-Subunit of Human Chorionic Gonadotropin in Serum by Radioimmunoassay

Qualitative and quantitative radioimmunoassays (RIA) specific for the beta-subunit of human chorionic gonadotropin (HCG) produced by Wampole Laboratories and Monitor Science Corporation were compared in a study involving the sera from 213 individuals. Qualitative assays from both manufacturers were found to be equivalent in clinical specificity (99.2%). However, the clinical sensitivity of the Wampole beta-Tec qualitative RIA (95.6%) was greater than that of the Monitor Science beta-CG qualitative RIA (89%) in detecting hCG in serum. In quantitative assays, the Wampole beta-Tec RIA gave serum values 1- to 10-fold higher than those obtained using the Monitor Science beta-hCG system when the same patient or control serum was assayed. Preliminary studies comparing two additional quantitative RIA kits (Serno hCG-beta and Bio-RIA hCG-beta rapid RIA) indicate that the results obtained with the quantitative Wampole and Monitor Science reagents are not peculiar to these two assay systems. Possible explanations of the cause of such aberrant results are presented. It is concluded that variable results can be expected when assaying the same serum sample with different RIA reagents for beta-hCG owing to the lack of an acceptable reference preparation for hCG that can be utilized by all commercial or private producers of such reagents.

Hepatitis B surface antigen production as a growth cycle-related terminal event in PLC/PRF/5 hepatoma cells.

The dynamics of hepatitis B surface antigen production of PLC/PRF/5 hepatoma cells were studied using a quantitative radioimmunoassay method. Maximum rates of antigen production were found in nutrient-depleted, non-dividing cultures and were temporally related to cytological changes preceding cell death. These results indicate that antigen production may be cell-cycle related and represent a terminal event.

Characterization of a Circulating Subpopulation of Spontaneous Antitetanus Toxoid Antibody Producing B Cells Following in Vivo Booster Immunization

Abstract The appearance of circulating B lymphocyte subpopulations responsible for the in vitro synthesis of IgG or IgM antitetanus toxoid antibody after in vivo booster immunization has been analyzed. Before booster immunization, only background levels of IgG antitetanus toxoid antibody were synthesized by B lymphocytes alone, or in combination with T cells and pokeweed mitogen, as measured by quantitative radioimmunoassay. Within 5 days after booster immunization, a population of B lymphocytes appeared within the circulation that had the capacity to synthesize IgG antitetanus toxoid antibody in the absence of T lymphocytes or pokeweed mitogen. These cells could be detected in the circulation until 10 to 12 days after immunization, and the antibody synthesized was inhibited by the inclusion of 50 µg/ml cyclohexamide in the culture medium. At the peak of this response, between 10 and 60% of the total IgG synthesized was IgG directed against tetanus toxoid. The cells responsible had a precursor frequency of up to 10-3 in the non-E-rosetting cell fraction obtained from peripheral blood and sedimented at 1 × G with velocities ranging from 4 to 11 mm/hr. The synthesis of IgM antitetanus toxoid antibody by this cell fraction was not detected. In contrast to the above B cells, lymphocytes capable of responding to pokeweed mitogen and T lymphocytes with the synthesis of IgG antitetanus toxoid antibody did not appear in the circulation until 10 days after immunization. Limiting dilution analysis of the pokeweed mitogen responsive B lymphocytes 2 to 4 weeks after booster immunization gave a precursor frequency of 1 × 10-4. In the presence of T cells and PWM, the synthesis of IgM anti-Tet remained at constant levels before and for 3 weeks post-booster immunization. These results indicate that multiple subsets of circulating B cells can be functionally defined in humans.

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 < 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.

Rapid and sensitive radioimmunoassays for human myoglobin.

A radioimmunoassay for quantitation of serum myoglobin in healthy individuals and patients with different diseases is described. Purified myoglobin was labelled by an 125I-labelled ester (N-succinimidyl 3-(-4 hydroxy, 5-[125I]iodophenyl) propionate), a commercially available antiserum was used, and the antigen-antibody complex was precipitated with polyethylene glycol 6000. The rapid assay can be performed within 1 h at 37 degrees C with a detection limit of 45 micrograms/l. Prolonged incubation at 4 degrees C for 18 or 72 h gives a detection limit of 6 and 2 micrograms/l, respectively. The mean coefficient of variation of the routine assay was 11%. In healthy human subjects a significant difference in mean serum myoglobin concentration was found between 43 women (34 +/- 17 micrograms/l) and 51 mean 47 +/- 15 micrograms/l). In twenty patients admitted to hospital with the clinical diagnosis acute myocardial infarction, the serum myoglobin concentration profiles were in close agreement with the final diagnosis. In three patients with myocardial infarction serum samples were taken every 2 h after the acute episode, and serum myoglobin levels were compared with the levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and creatine kinase isoenzyme-MB.

Enkephalins and Endorphins

This chapter discusses the identification and distribution of enkephalins and endorphins. The two main methods of detecting and quantitating enkephalins and endorphins used so far are radioimmunoassay and bioassay. In addition, opioid receptor binding assays were originally utilized to detect general opioid activity. An area of the brain in which the localization of enkephalin seems to tie in well with certain effects of narcotics is the hypothalamus. Immunohistochemistry reveals that both enkephalin-containing terminals and cell bodies are localized within the hypothalamus. In particular, enkephalinergic terminals are localized in the external layer of the median eminence. Quantitative radioimmunoassay data have been reported by two groups for β-endorphin, both using the same antibody. Concentrations of β-endorphin and β-lipotropin in the pituitary are extremely high. The main localization of this peptide by radioimmunoassay appears to be the neurohypophysis/pars intermedia area rather than in the adenohypophysis. β-endorphin can also be degraded by an enzyme in synaptosomal membranes. The products formed depend on the pH at which the experiment is performed and also on whether bacitracin is present.

Expression and specificity of FcIgG receptor sites on neoplastic lymphocytes

We investigated the ability of an FcIgG receptor marker to discriminate between subtypes of malignant lymphoproliferative diseases that differed in their clinical presentations. A quantitative radioimmuno assay was established that enabled us to evaluate average receptor densities on a population basis. Surface receptors were first saturated with IgG complexes. The number of membrane associated IgG molecules was subsequently determined with 125I-staphylococcal protein A. Results obtained with this assay on a battery of malignant lymphocytes suggested that the range of receptor densities of malignant B and T cells might overlap each other but would correlate with the degree of tumor cell differentiation and the clinical stage of the underlying disease. This behavior limits the use of this marker in the characterization of the derivation of malignant lymphocytes; these findings, however, may be useful in the prognostic classification of lymphomas of known origin.

Immunoglobulin profiles of the chronic antibody response: discussion in relation to brucellosis infections

SummaryThe use of a sensitive and quantitative radioimmunoassay has enabled much finer dissection of the immunoglobulin-antibody profiles for an individual immune response. The kinetics of the response are considered in relation to the switch from IgM to IgG antibody production. In the domestic fowl, the kinetics of this switch varied with different antigens: whereas the response to a thymus-dependent antigen proceeded through a brief 19S response to a declining 7S response, the response to a thymus-independent antigen failed to switch from 19S to 7S for several weeks and consisted of repeated excursions of 19S antibodies. When injected intravenously and simultaneously, Salmonella adelaide O (killed) organisms (thymus-independent) and sheep red cells (thymus-dependent) interact so that the response to the latter fails to switch from 19S to 7S and consists of repeated excursions of 19S antibody. The changed character of the sheep red cell response is interpreted as being due to lack of 7S antibody. Passive antibody to either sheep red cells or to S. adelaide produced an inhibition of the sheep red rell response so that only one excursion of 19S antibody was observed.The use of the radio-immunoassay enables an independent measurement of all IgM, IgG and IgA antibody to the surface antigen of Brucella obortus. The test, when applied to forty-six sera from individuals with various types of brucellosis, successfully detected antibody in many instances in which conventional serological tests were negative, and such antibody (if IgM) was associated with acute or (if IgG or IgA) with chronic cases of brucellosis. The radioassay test should prove highly valuable effectively to eliminate, in individual patients, the diagnosis of brucellosis based on the inability of conventional tests to detect significant antibody levels.

Measurement of IgE Antibody by an Antigen-Binding Assay: Correlation with PK Activity and IgG and IgA Antibodies to Allergens

Abstract A quantitative radioimmunoassay for IgE antibodies (ab) is described. The assay measures IgE antigen-binding activity (BA) by using purified allergens radiolabeled with 125I, IgE myeloma serum as a source of carrier protein, and monospecific goat anti-IgE (anti-IgE) to precipitate the IgE. IgE BA for the Group I protein of rye grass pollen (Rye I) was measured in the serum of patients with grass pollen hay fever who had never received immunotherapy. Values for IgE BA were found to correlate well with radioallergosorbent technique (RAST) results (rs = 0.89, p<0.001). By assaying IgE-rich fractions, pure IgG, and whole serum, we found a good correlation between IgE BA, Prausnitz-Küstner (PK) activity, and total IgE. A positive PK test could be elicited with 0.05 ml of dilutions containing as little as 1 or 2 units IgE BA for Rye I/ml. PK testing was more sensitive than either RAST or the antigen-binding assay. Estimates based on the quantity of antigen bound in antigen excess suggested that 1 unit IgE BA represented 0.1 ng IgE ab. With higher concentrations of antigen (up to 1 µg/ml), however, estimates suggested that 1 unit IgE BA represented as much as 0.4 ng IgE ab. These higher estimates appeared to correlate reasonably well with estimates based on RAST elution or RAST absorption. The assay for IgE BA was directly comparable with assays for IgG BA and IgA BA. Results of these assays on sera from the patients showed that IgG BA for Rye I was detectable in 25 of 25 IgE BA in 24 of 25, and IgA BA in 21 of 25. Quantitatively we found a characteristic pattern IgG BA > IgE BA > IgA BA (geometric mean values 862, 214, and 60.2 units/ml, respectively). In keeping with this there was a highly significant quantitative correlation between IgG BA and IgE BA (r = 0.72, p<0.001). None of the sera from nonallergic persons contained detectable IgA or IgE BA for Rye I; however, 20% of these sera (10 of 48) contained IgG BA.

521 HUMAN HEMOGLOBIN IN VIVO SOMATIC CELL MUTATION ASSAY

A codon-specific somatic cell mutation assay model system is being developed, using human erythrocytes and antisera which will distinguish human hemoglobins differing by a single amino acid residue. A goat anti-hemoglobin S antiserum (“anti-β6val)has been obtained which specifically distinguishes the single amino acid difference between HbS(α2 β26val)and HbA(α2 β26glu).Unabsorbed goat anti-HbS antiserum bound nearly equivalent amounts of HbS and HbA, but after absorption with an excess of HbA, was rendered specific for HbS. A quantitative radioimmunoassay for HbS has been developed which is highly specific and sensitive. With constructed mixtures of purified hemoglobins A and S, our current assay is capable of specifically detecting <10-9 gram of hemoglobin S in the presence of more than a million-fold excess of hemoglobin A. We have documented equivalent sensitivity and specificity in human whole blood lysates. Results to date allow us to conclude that in “normal” AA individuals the sum of errors of replication (mutation) plus errors of expression(transcription plus translation)may be less than one molecule of Hb S per 107 molecules of HbA synthesized. We are currently determining whether there are detectable quantities of Hb S in bloods of AA individuals exposed to toxic environmental agents and whether these individuals differ significantly from unexposed populations

Radioimmunoassay of polyacrylamide.

Antiserum to polyacrylamide has been produced in rabbits. It can be used in a sensitive radioimmunoassay for quantitation of polyacrylamide at very low concentrations. This novel approach to the analysis of a synthetic, biologically inactive, hydrocarbon polymer has potentially widespread implications, for example, in the downstream measurement of flocculating agents used in water purification treatments.

Effects of Ionic and Nonionic Detergents on Antigen-Antibody Reactions

Using highly sensitive and quantitative radioimmunoassay procedures we have measured the effects of different concentrations of three commonly used detergents, SDS, DOC, and Triton X-100, on antibody-antigen reactions. Triton X-100, had a relatively mild effect on primary antigen-antibody bindings, the precipitin reaction, and a double antibody RIA as evidenced by only an 8 to 10% inhibition of binding or precipitation. These results were not detergent concentration dependent, as Triton concentrations ranging from 5 to 0.1% had virtually no differential effects. Sodium deoxycholate (DOC) had a more profound effect on both primary antigen-antibody binding and the precipitin reaction than did Triton X-100, and its effects, unlike those of Triton X-100, were concentration dependent. There was a direct relationship between concentration of DOC and degree of inhibition of both primary binding and immune precepitation especially in antigen excess. Sodium dodecylsulfate (SDS), at concentrations 10- to 100-fold less than either Triton X-100 or DOC, had profound inhibitory effects on primary antigen-antibody binding, the precipitin reaction, and a double antibody radioimmunoassay. Generally, at concentrations greater that 0.01% SDS, almost all immunochemical reactivity is destroyed.

Quantitative serological analysis of a rabbit anti-rat lymphocyte serum and preliminary biochemical characterisation of the major antigen recognised.

Rabbit antisera to rat thoracic duct lymphocytes were examined by absorption analysis with quantitative radioimmunoassays to determine the tissue distribution of the antigens recognised and the amount of antibody against different specificities. Seven of eight sera were remarkably specific for leucocytes and one serum was analysed in detail. In this serum about 70 per cent of the antibody was leucocyte specific and most was against determinants on thymocytes, bone marrow cells, peripheral lymphocytes, and macrophages. This specificity is referred to as the leucocyte-common antigen. Smaller amounts of antibody were specific for antigens on peripheral lymphocytes but not thymocytes, and this could be subdivided into two antigens, one present on bone marrow cells and the other not. For preliminary biochemical analysis of the leucocyte-common antigen, thymocyte membrane was solubilized in deoxycholate. The homogeneity and apparent size of the antigen was determined by gel filtration and zone sedimentation on sucrose gradients. The activity behaved as a single component with the following hydrodynamic properties: sedimentation coefficient, 5.9S; partial specific volume, 0.71 ml/g; and Stokes radius, 7.5 nm. The molecular weight (including any bound deoxycholate) was calculated to be 172,000 and the frictional ratio 2.05. All activity bound to a lentil lectin affinity column and was eluted by methyl alpha-D-glucopyranoside, suggesting that the antigen is a glycoprotein.

The development of a radioimmunoassay procedure for the estimation of Tamm-Horsfall glycoprotein in human serum.

1. A quantitative radioimmunoassay was developed for the measurement of nanogram amounts of Tamm-Horsfall (TH) glycoprotein in the presence of serum proteins at concentrations above 30 mg/ml. 2. Specific anti-(TH-glycoprotein) antibodies were labelled with 125I and these were usable for a period of 8 weeks. 3. Agarose beads (Sepharose 4B), to which TH-glycoprotein had been coupled via cyanogen bromide activation of the Sepharose, were used as the solid phase in the assay. This proved stable upon storage at 4 degrees C for periods in excess of 4 months. 4. The dissociation of the glycoprotein in the presence of serum proteins that was necessary for quantification was achieved by subjecting the sample to ultrasonication. 5. Assays conducted on a small sample of normal serum produced evidence that normal serum contained amounts (50-180 ng/ml) of a substance that reacted similarly to TH-glycoprotein in the assay procedure and in a series of experiments conducted to confirm the presence of this substance in human serum.

Radioimmunoassay of beta2-microglobulin.

A radioimmunoassay for quantitation of beta2-microglobulin in serum and urine is described. Polyethylene glycol 6000 was used to precipitate the antigen-antibody complex. The assay can be performed within 5 hr. In healthy human subjects (mean age, 30.6 years and 42.0 years for serum and urine determinations, respectively) the mean concentration of beta2-microglobulin in serum was 125 nmol/l (range, 49-190 nmol/l). In men the mean 24-hr urinary excretion was 12.9 nmol (range, 8.6-19.2 nmol), and in women it was 5.5 nmol (range, 2.9-8.7 nmol). The assay range was 0.23-19.40 nmol/l, and the detection limit was 0.042 nmol/l, using a prolonged incubation time. The coefficient of variation based on 20 determinations in serum dilutions with a concentration of beta2-microglobulin of 8.14 and 1.92 nmol/l was 8.1 per cent and 9.0 per cent, respectively.

Heterogeneity of the BALB/c antiphosphorylcholine antibody response at the precursor cell level.

Immune responsiveness to phosphorylcholine (PC) in BALB/c mice has been characterized by combining (a) usuage of highly sensitive radioimmunoassays for quantitation of antibody, heavy-chain class, and idiotype on a weight basis; (b) isolation of PC-specific B cells in fragment cultures; and (c) stimulation in a carrier-primed environment with the PC hapten coupled to carrier through a tripeptide spacer in order to maximize carrier recognition. The specificity and accuracy of the radioimmunoassays have veen verified by specific inhibition, lack of nonspecific binding, and excellent concordance of values for monoclonal antibody concentration obtained independently for Fab and idiotype content. The latter evidence also serves as strong confirmation of the monoclonality of in vitro monofocal responses as well as the preservation of the idiotype on antibodies of differing immunoglobulin classes. The results indicate that while B cells expressing the TEPC 15 idiotype predominate, other idiotypes may be represented by 2-50% of PC-specific precursors, and monoclonal antibodies even of the TEPC 15 idiotype are produced in both the IgM and IgG1 immunoglobulin classes. These findings are confirmed by the analysis of serum antibodies produced in carrier-primed mice immunized with hapten coupled through a tripeptide spacer, thus re-emphasizint the enhancement of primary responsiveness, particularly IgG1 production, by maximizing carrier recognition. The finding of idiotype diversity in the PC response, as well as diversity of expression in terms of quantity and immunoglobulin class of antibody synthesized by the clonal progeny of B cells within the TEPC 15 clonotype, emphasize the heterogeneity of the B-cell population both in terms of specificity repertoire and the physiological state of cells even within a single clonotype.

Effects of maleylation on the lipid-binding and immunochemical properties of human plasma high density apolipoprotein-A-II

Chemical modification by maleylation of the human plasma high density lipoprotein-protein, apoA-II, causes significant changes in its properties. These changes are as follows: 1) a shift from a helical to a more disordered secondary structure; 2) a decreased ability to bind egg phosphatidylcholine vesicles; and 3) a decrease in immunoreactivity as measured by a quantitative radioimmunoassay. Each of these changes was reversed by demaleylation. These results are discussed in relation to a molecular theory of lipid binding by the plasma lipoproteins.