Indirect Solid-phase Radioimmunoassay Research Articles

Isolation and Characterization of Monoclonal Antibody Directed Against Bovine αs2-Casein

A monoclonal antibody 62-1A of isotype IgM, directed against bovine αs2-casein, was isolated and characterized. Monoclonal antibody 62-1A recognized bovine αs2-11P- and αs2-9P-caseins by indirect solid phase radioimmunoassay and Western blot analysis. Crossreactivity toward native genetic variants (A, B, and C) of αs1-casein was similar but lower (˜60 to 70%) than that for αs2-11P-casein). Little or no crossreactivity was observed for other bovine milk or serum proteins. Antibody affinity for αs2-11P-casein was 1.3 × 109/M. As little as 25ng/ml (.5 ng/well) of αs2-11P-casein was detected by solid phase radioimmunoassay.

Neuropathogenesis of pseudorabies: leakage of anti-viral antibody and serum constituents into cerebrospinal fluid of infected pigs

Antibody levels to pseudorabies virus (PRV) in cerebrospinal fluid (CSF) were compared to serum levels from immunized and infected pigs. Antibody was measured by single-dilution indirect solid-phase radioimmunoassay (IRIA). There was significantly higher CSF anti-PRV IgG relative to serum anti-PRV IgG (anti-PRV index, %) from infected pigs (1.390%, n = 14) than from vaccinated ones (0.141−0.149%, n = 5 and 7). The index from vaccinated and challenged pigs was intermediate (0.627%, n = 16), suggesting that vaccination cannot abrogate but can reduce the severity of encephalitis. Piglets with maternal antibody contained minimal CSF antibody similar to that of vaccinated animals. The CSF anti-PRV antibody was detected in piglets infected with as low as 10 2 TCID 50 at 15 days postinfection. In infected pigs, the elevated CSF anti-PRV level was due to a leakage of serum antibody through a possible blood-brain barrier (BBB) impairment but not due to intrathecal antibody synthesis. Multiple regression analysis showed that the leakage was more time dependent than dose dependent. Leakage was detected until at least 4 weeks after disappearance of acute clinical symptoms. We have associated different levels of CSF anti-viral antibody with various infection or vaccination conditions.

Serum antibody against Tamm-Horsfall protein in patients with renal cell carcinoma.

We assayed sera from 24 patients with metastatic renal cell carcinoma for class specific antibody against urinary Tamm-Horsfall protein (THP) using indirect solid-phase radioimmunoassay techniques. Overall, 16 patients had elevated antibody levels (greater than 2 SD above the mean levels of control subjects). IgG against THP was elevated in eight of the patients, IgA in 12, and IgM in five. However, THP was not identifiable by immunohistochemical techniques in the sections of 15 of 24 tumors available for study. Nine of the 15 tumors were from patients with elevated antibody levels. Serum antibody against THP in patients with renal cell carcinoma appears to result from antigenic stimulation by THP derived from the urine rather than from the neoplasm.

Comparison of a DNA-DNA dot-blot hybridisation assay with light microscopy and radioimmunoassay for the detection of a nuclear polyhedrosis virus

A dot-blot hybridisation assay was developed for the detection of a nuclear polyhedrosis virus (NPV) and was compared to light microscopy and radioimmunoassay (RIA). Using cloned NPV DNA labelled with 32P as a probe, a number of hybridisation assay procedures was examined. The assay was found to be more sensitive than differential staining, phase-contrast microscopy, or indirect solid-phase RIA with as few as 20 occlusion bodies (150 pg DNA) being detected. Samples do not require prior purification or DNA extraction. The assay was shown to be specific for NPV and has the potential to detect and discriminate between strains of the virus. With little modification the assay may be used to detect other insect viruses.

Dissection of the Human Antigammaglobulin Idiotype System with Monoclonal Antibodies

Murine monoclonal antibodies (MoAb) were prepared by immunizing mice with human monoclonal rheumatoid factors from patients with mixed cryoglobulinaemia. Indirect solid phase radioimmunoassay and haemagglutination inhibition were used to screen the MoAb. Reactivity patterns of MoAb were determined using (a) polyclonal and monoclonal IgM proteins, (b) monoclonal IgM proteins from patients with neuropathy, (c) monoclonal and polyclonal IgM antigammaglobulins, and (d) various isolated VkIIIb-positive immunoglobulins. Several patterns were obtained: MoAb reacting with private idiotypic determinants, with VkIIIb-related determinants, and with cross-reactive idiotypes (CRI). Two MoAb of the second type reacted with VkIIIb-positive immunoglobulins and light chains regardless of their antigenic activity. Another MoAb reacted with VkIII light chains only when in association with mu heavy chains. MoAb of the third type defined distinct CRI systems. One of these was restricted to antigammaglobulins and another also involved neuropathy-associated monoclonal IgM proteins. All MoAb clearly reacted with a minor population of normal polyclonal IgM, except for the MoAb detecting private idiotypic determinants. Studies using inhibition of binding by enzyme-linked immunosorbent assay showed that polyclonal IgM antigammaglobulins may carry a CRI determinant detected by one of the MoAb. This CRI system, defined by the MoAb Glo 86.3, was similar to but not identical with the previously described Wa CRI, which is widely prevalent among IgM antigammaglobulins of rheumatoid arthritis.

Use of chicken and rabbit antibodies in a solid phase protein a radioimmunoassay for virus detection

A new rapid indirect solid phase radioimmunoassay was developed for the detection of Sindbis virus. Chicken antibodies were adsorbed onto wells in microplates to serve as ‘capture antibodies’ and rabbit antibodies were used as the second antibody. 125I-labelled protein A that does not bind to chicken antibodies, but binds firmly to rabbit antibodies was used as the tracer. All the steps necessary for the development of the assay are described. The minimal amount of Sindbis virus detectable was around 3 × 10 5 PFU/ml and the interassay reproducibility was about ± 30%.

Immunological analysis of auto-antibodies against the adenine nucleotide translocator in dilated cardiomyopathy

We have previously identified the adenine nucleotide translocator (ANT), an intrinsic protein of the inner mitochondrial membrane, as an auto-antigen in dilated cardiomyopathy (DCM). Further immunochemical characterization by crossed immunoelectrophoresis, indirect solid phase radioimmunoassay and immunoadsorption studies on the isolated translocator protein and mitochondria from heart, kidney and liver showed the existence of organ-specific antigenic determinants although partial crossreactivity between the three proteins was observed. Sera from 18 patients with histologically proven dilated cardiomyopathy were studied for their capacity to bind to the translocator protein. Seventeen of 18 patients showed significant binding, while in the sera of patients with coronary heart disease, suspected alcoholic heart disease or healthy blood donors, no anti-ANT antibodies were observed. Further studies showed organ-specific and functionally active autoantibodies, which decreased the ADP/ATP exchange rate from heart mitochondria. A close correlation was found between the antibody-titer and the hemodynamic function. These results give new evidence for autoimmunological events in dilated cardiomyopathy.

Production and characterization of monoclonal antibody to a 60-kD glycoprotein in ovarian carcinoma.

Monoclonal IgG1 antibodies 2C8 and 2F7, derived by immunization of mice with a glycoprotein-enriched fraction of human ovarian adenocarcinoma, recognized a 60 kD glycoprotein in the ovarian tumor but not in normal ovary. Survey of other normal adult tissues by an indirect solid-phase radioimmunoassay (RIA) revealed the presence of the antigen in trace amounts in various normal organs such as small intestine, liver colon and urinary bladder, except in lung where its concentration was as high as in tumors. Among fetal tissues tested, intestine and placenta had the highest activities. By RIA, about 50% of ovarian and colonic tumors had elevated levels of the antigen. All ovarian cyst fluids, both benign as well as malignant, also contained a high level of the antigen. Immunodepletion studies indicated that the antigen was distinct from carcinoembryonic antigen and the ovarian cancer antigens described in our laboratory with other monoclonal antibodies. The antigen bound to Con A-Sepharose and was eluted with 2% alpha-D-mannoside, was soluble in 0.6 M perchloric acid and stable at 100 degrees C for 30 min. The antigenic activity in isolated plasma membrane enriched fractions of ovarian adenocarcinomas was sensitive to trypsin, chymotrypsin or protease treatment but unaffected by neuraminidase, beta-galactosidase, periodate or methanol treatment. By immunoperoxidase staining, the antigen was localized in a variety of human tumors showing widespread distribution.

Lewis blood group antigens defined by monoclonal anti-colon carcinoma antibodies

Monoclonal antibodies directed against human cancer cells were prepared by the murine hybridoma technique. These antibodies detect Lewis blood group antigens as determined by indirect solid-phase radioimmunoassay, hapten inhibition studies, and chromatogram binding assay. One monoclonal antibody is specific for the Le a terminal carbohydrate of Galβ1 → 3Glc NAc(4 ← 1α Fuc)β1 → 3LacCer. Five monoclonal antibodies react with the Le b terminal carbohydrate sequence of Fucα1 → 2Galβ1 → 3GlcNAc(4 ← 1αFuc)β1 → 3LacCer, and four of these antibodies are highly specific for this glycolipid and do not react with other similar di- and monofucosylated glycolipids. One of the anti-Le b antibodies cross-reacts with blood group H glycolipid and has binding properties similar to those of the previously described antibody NS-10-17 [ M. Brockhaus, J. L. Magnani, M. Blaszczyk, Z. Steplewski, H. Koprowski, K.-A. Karlsson, G. Larson, and V. Ginsburg (1981), J. Biol. Chem., 256, 13223–13225]. Two antibodies react with both the Le a and Le b antigens, though both bind preferentially to Le b.

Bacterial Infection and Male Infertility: Absence of Immunoglobulin A with Specificity for Common Escherichia Coli 0-Serotypes in Seminal Fluid of Infertile Men

We used an indirect solid phase radioimmunoassay to detect and quantitate immunoglobulin A (IgA) in male genital secretions with specificity for Escherichia coli (E. coli) antigen. We assayed the seminal fluid and expressed prostatic secretion (EPS) of a patient with chronic bacterial prostatitis (CBP) due to an 01 E. coli, the seminal fluid of 24 fertile men, and the seminal fluid of 62 men of infertile marriages. IgA directed apparently against the 0-antigen of the infecting E. coli was measurable in both the seminal fluid and EPS of the CBP patient. IgA in these specimens also bound to antigen in a mix of 8 common E. coli 0-serotypes that included an 01 E. coli. IgA with specificity for antigen in the mix of common E. coli 0-serotypes could not be detected in the seminal fluid of the fertile men or the men of infertile marriages. These data suggest that subclinical E. coli infections of the male reproductive tract are not commonly associated with infertility.

Immunologic Response of the Prostate to Bacteriuria and bacterial Prostatitis I. Immunoglobulin Concentrations in Prostatic Fluid

We investigated the impact of bacteriuria and bacterial prostatitis on the immunoglobulin content of prostatic fluid. Using an indirect solid-phase radioimmunoassay, we assayed samples of expressed prostatic secretion (EPS) from 20 men with no histories of bacteriuria and from 13 men with histories of culture proven bacteriuria for IgG, IgA and IgM concentrations. Five of the bacteriuric men had culture proven bacterial prostatitis. The concentrations of each immunoglobulin varied widely and were unrelated to age, race, history of urologic instrumentation, presence of benign prostatic hyperplasia, leukocyte content of EPS or EPS aerobic bacterial culture results. The mean concentration of IgG for samples from the bacteriuric patients, 44.3mg./ml., was similar to that of the uninfected patients, 58.4mg./dl. (p < 0.5). The mean concentrations of IgA and IgM for samples from the bacteriuric patients, 89.2 and 1.44mg./dl., respectively, were greater than those of the uninfected patients, 25.2 and 0.95mg./dl., respectively. These differences, however, did not achieve statistical significance, p < 0.025 and p < 0.3, respectively. However, the mean ratio of IgA:IgG for samples from the bacteriuric patients, 1.67, was significantly greater than that of the uninfected patients, 0.34 (p < 0.005). The fraction of IgA in the form of secretory IgA was x> 60 per cent for samples obtained from both the uninfected and bacteriuric patients. These data suggest that bacteriuria is associated with increased secretion of IgA in the prostatic fluid.

Immunologic Response of the Prostate to Bacteriuria and Bacterial Prostatitis II. Antigen Specific Immunoglobulin in Prostatic Fluid

We used an indirect solid-phase radioimmunoassay to quantitate immunoglobulin binding to formalin-fixed Escherichia coli in expressed prostatic secretion (EPS). EPS samples from 20 men with no histories of bacteriuria, from eight men with histories of culture proven E. coli bacteriuria but with no clinical or bacteriologic evidence of prostatic infection, and from 5 men with culture proven E. coli bacterial prostatitis were assayed. Measurable binding of IgA to E. coli was observed for EPS samples from all the infected patients but from only 1 of the uninfected controls. Measurable binding of IgG to E. coli was observed for samples from 7 of 8 infected patients and from 2 of 10 uninfected controls. In no cases was there measurable IgM binding to E. coli. There was no measurable binding of IgA to formalin-fixed S. epidermidis; and no measurable binding of IgA to formalin-fixed K. pneumoniae, P. mirabilis or P. aeruginosa for samples from patients who had not been previously infected with these gram negative bacilli. Absorption of immune EPS with suspensions of these bacteria did not alter the binding of IgA to E. coli but absorption with a suspension of E. coli completely eliminated this binding. The magnitude and specificity of EPS immunoglobulin binding to E. coli was similar for the bacteriuric and bacterial prostatitis patients.Serum from 8 infected patients was assayed for immunoglobulin binding to E. coli. The per cent of serum IgG binding to E. coli for these patients was similar to that of EPS IgG, but the per cent of EPS IgA binding to E. coli was 3.5 to 147.5 times greater than that of serum IgA. In 1 patient with bacteriuria due to bacterial prostatitis, the concentrations of EPS IgG and EPS IgA binding to E. coli were 53 and 217 times greater, respectively, than those of a 1st voided urine specimen. Absorption of immune EPS with rabbit anti-human-secretory component indicated that>70 per cent of the IgA directed against E. coli was in the form of secretory-IgA.These data indicate that E. coli bacteriuria and E. coli bacterial prostatitis are associated with the local secretion of antigen specific IgA in prostatic fluid that appears to be independent of the systemic immune response. Further, these observations raise the possibility that the prostate gland is usually colonized during episodes of urinary tract infection but that symptomatic or chronic infection is prevented by the secretion of antigen specific IgA.

Factors influencing the quantitation of antibodies to influenza virus by indirect solid-phase radioimmunoassay

An indirect solid-phase radioimmunoassay involving purified influenza haemagglutinin bound to polyvinyl microtitre plates has been used to quantitate the humoral response to influenza infection in ferrets. Sialic acid residues, present on normal ferret immunoglobulin, caused non-specific binding of immunoglobulin to the haemagglutinin, resulting in a non-linear relationship between serum dilution and primary antibody bound to antigen. Direct proportionality was achieved by removal of sialic acid residues from serum immunoglobulin or by inclusion of free sialic acid in the incubation buffer. Absolute quantitation of IgG and IgM antibodies was achieved by determining the relationship between primary antibody bound to antigen and secondary anti-immunoglobulin antibodies using 131I-IgG or -IgM and the homologous 25I-labelled monospecific antiserum. Examples of antibody quantitations in sequential post-infection sera are presented.

Analysis of a solid-phase radioimmunoassay for antibodies to cytoplasmic antigen fractions of Candida albicans

An indirect solid-phase radioimmunoassay (SPRIA) in individual polystyrene microtiter cu cups has been adapted for measurement of antibody to various cytoplasmic and carbohydrate antigen fractions of Candida albicans. The assay was optimized for sensitivity, precision and linearization of serum dilution curves. The optimized procedure allows computerized measurement of anti- Candida antibodies and can be used for measurement of antibody over a wide concentration range. The procedure obviates variation due to changes in day-to-day counts as a result of isotope decay and end-point antibody dilutions. The assay has been used to demonstrate a Poisson-like distribution of antibody levels in the sera of persons showing no symptoms of candidiasis. The minimum antibody level detectable by the assay is about two orders of magnitude lower than the lowest level found in human serum and 4 orders of magnitude lower than the most sensitive test used hitherto, the hemmgglutination test.

Detection of enterovirus specific IgG and IgM antibodies in humans by an indirect solid phase radioimmunoassay.

The development of a solid phase radioimmunoassay which is able to detect virus-specific IgG and IgM antibodies in serum specimens from patients with enterovirus infections is described. Viral antigen partially purified from infected tissue culture fluid was absorbed passively to individual polystyrene microtiter wells. Dilutions of sera were incubated on these antigens and bound anti-viral antibodies were monitored by the addition of 125-iodine labeled anti-human-IgG or anti-human-IgM antibody. Specificity of the assay to detect virus-specific IgM antibody was ensured by highly specific anti-IgM antibody which did not cross react with IgG and 2-mercaptoethanol sensitivity of IgM antibody titers. Changes of IgM antibody titers clearly indicated a current infection by that virus strain which was isoalted as etiological agent. Advantages and restrictions of the introduced radioimmunoassay are discussed.

A Solid-Phase Radioimmunoassay to Detect Anti-Tuberculin Antibodies in the Guinea Pig

Despite the importance of the guinea pig as a model test animal for studying the humoral immune response in comparison to the development of cellular immunity after BCG or tuberculosis infection, no precise and sensitive method for the determination of such antibodies is available. We developed an indirect solid-phase radioimmunoassay on microtiter plates for this purpose. All assay conditions have been optimized such as selection of suitable plastic material, use of carrier substances, concentrations and amount of the antigen as well as the serum and second antibody, buffer, incubation times and all steps of the procedures for coating, washing, and measuring. The assay gives a linear relationship between antibody concentration in the serum to be tested and binding of the radiolabelled second antibody. A comparison with four conventional antibody titration methods reveals a 128- to 512-fold higher sensitivity of the RIA. Favorable aspects of this RIA as opposed to the methods hitherto used are discussed.

Serologic response in human hepatitis A: detection of antibody by radioimmunoassay and immune adherence hemagglutination.

An indirect solid-phase radioimmunoassay (RIA) for detection of antibody to the hepatitis A antigen (anti-HAV) was developed using polystyrene pearls as the solid phase and hepatitis A antigen (HAAg) extracted from marmoset livers. This RIA was compared to an immune adherence hemagglutination assay (IAHA) which employed HAAg derived from the stools of chimpanzees collected during acute hepatitis A. Anti-HAV was detected in the sera of 15 humans with naturally acquired hepatitis A infection. Sensitivity and specificity were greater using the RIA, permitting the detection of anti-HAV as early as the time of onset of jaundice. Either seroconversion or a significant increase in the titer of anti-HAV was demonstrated following hepatitis A exposure in paired sera from six patients by both techniques. No significant difference in anti-HAV responses was noted between patients with icteric compared to anicteric hepatitis A or between children and adults with hepatitis A.

Detection of double-stranded RNA in Semliki Forest virus-infected cells by an indirect solid phase radioimmunoassay: an assay for interferon.

An indirect solid phase radioimmunoassay using rabbit antibodies to the synthetic double-stranded RNA poly rI-rC and 125I-labelled sheep anti-rabbit IgG could detect 1 ng of poly rI-rC. This assay could also detect the presence of double-stranded RNA in Semliki Forest virus-infected chick fibroblasts and can be used as an assay for interferon activity.

Solid-phase radioimmunoassay as a method for evaluating antigenic differences in type A influenza viruses

An indirect solid-phase radioimmunoassay (RIA) procedure was developed to determine its usefulness in assessing antigenic variation in the surface antigens of type A influenza virus strains. The importance of several test variables was examined, and those having a significant effect on the binding ratios were identified. The reproducibility of the RIA procedure was investigated. Maximum variation of the mean binding ratios encountered in repetitive tests was found to be approximately 20%. The antigenic characteristics of the A/Aichi/68 virus strain were compared with several different type A virus strains. Utilizing anti-A/Aichi/68 immune serum together with specific anti-hemagglutinin and antineuraminidase immune sera, the RIA method was shown to quantitatively differentiate the surface antigens of the A/Aichi/68 virus strain from the surface antigens of the type A strains that preceded, as well as from those that succeeded, the introduction of the Aichi virus strain in 1968. Using antigen-specific serum, both the hemagglutinin and neuraminidase antigens can be independently characterized in one test system. This advantage, together with the ease and greater sensitivity of the RIA, should make it a useful serological test for evaluating antigenic variation of type A influenza viruses.

Sensitivity of a radioimmunoassay method for detection of certain viral antibodies in sera and cerebrospinal fluids

An indirect solid-phase radioimmunoassay (RIA) was applied to titration of serum and cerebrospinal fluid (CSF) antibodies against a variety of viruses including rubella, mumps, measles, herpes simplex, varicella-zoster, and vaccinia. The test used fixed, virus-infected cells as a source of antigen, and conditions for optimal production of viral antigen were determined for each virus-host cell system. In acute, uncomplicated viral infections, sera taken 2 to 5 days after onset generally had low homotypic RIA titers ranging from less than 1:100 to 1:500, whereas convalescent-phase titers ranged from 1:128,000 to 1:512,000. Rubella and measles antibody titers as high as 1:256,000 were demonstrated by RIA in CSF from patients with chronic panencephalitis, whereas homologous antibody titers of 1:4,000 were detected in CSF from acute mumps, herpes simplex, and varicella-zoster virus infections with central nervous system involvement. Some heterotypic antibody was demonstrable by RIA in CSF, but, with the exception of herpes simplex antibody in a mumps virus infection, titers were markedly lower than those to the infecting virus type. RIA generally demonstrated titers at least 1,000 times higher than those obtained by conventional assays such as complement fixation, hemagglutination inhibition, neutralization, and immunofluorescent staining.