Microtiter Solid-phase Radioimmunoassay Research Articles

IgG4 Autoantibodies to DNA in Systemic Lupus erythematosus Patients

The presence of DNA-specific IgG4 antibodies was demonstrated in the sera of patients with systemic lupus erythematosus (SLE) by a microtiter solid-phase radioimmunoassay. A patient with distal interphalangeal swelling and extensive ulcers in the oral cavity, seronegative for anti-DNA antibodies of the IgG isotype, was found to have anti-DNA autoantibodies exclusively of the IgG4 subclass. These autoantibodies directed against the dsDNA conformation cross-reacted with chondroitin sulfate, dermatan sulfate and heparin.

Direct quantitation of activated C3 in human plasma with monoclonal anti-iC3b-C3d-neoantigen

We developed a microtiter solid-phase radioimmunoassay for quantitating C3 breakdown products (iC3b, C3dg, C3d) in human plasma with a unique monoclonal antibody specific for a neoantigen present on iC3b and C3d (MoAb 130). This monoclonal antibody reacts with a neoantigen which appears when C3b is converted to iC3b. The neoantigen is also present on the C3dg and C3d fragments derived from iC3b. The concentration of the neoantigen is elevated in the plasma of most patients with rheumatoid arthritis and systemic lupus erythematosus as compared to normal volunteers. Some patients with glomerulonephritis also had elevated concentration of the neoantigen in their plasma.

Immunoassay technique for specific immunoglobulin E

Using monoclonal mouse anti-human IgE antibodies, the microtiter solid phase radioimmunoassay (MSPRIA) and the enzyme-linked immunoabsorbent assay (EILSA) were modified to quantitatively measure honeybee venom (HBV) and perennial rye grass (PRG) specific immunoglobulin E (sIgE). By using a novel dilution, serial transfer and cummulative counting technique, the inhibiting and interfering effects of specific immunoglobulin G (sIgG) in the assay of (sIgE) can be reduced and the quantitative determination of (sIgE) accomplished with precision and reliability.

IgE and IgG Antibodies to Staphylococcus aureus Solubilized Cell Wall Proteins and Teichoic Acid in Patients with the Hyper‐IgE Syndrome

AbstractThe anti‐staphylococcal IgE and IgG antibody levels of patients with hyper‐IgE syndrome (HIE) were measured by microtiter solid phase radioimmunoassay (MSPRIA) and enzyme‐linked immunosorbent assay (ELISA) using solubilized cell wall proteins (SCWP) and teichoic acid extracted from Staphylococcus aureus.Using MSPRIA, specific IgE antibodies to SCWP were detected at significant levels in all 5 patients with HIE, but not in 23 patients with atopic allergy or Wiskott‐Aldrich syndrome with or without staphylococcal infections. IgG antibodies to SCWP and teichoic acid were lower in the patients with HIE than those in non‐allergic infectious controls. These findings suggest an abnormal immunologic response to Staphylococcus aureus in patients with HIE.

Reduction of interference by specific IgG with a modified microtiter solid-phase radioimmunoassay to measure honeybee venom IgE

By means of monoclonal mouse anti-human IgE antibody, the microtiter solid-phase radioimmunoassay (MSPRIA) was modified to measure honeybee venom (HBV) IgE. HBV IgG was measured by MSPRIA(G) using affinity column purified goat anti-human IgG antibody and correlated with results obtained with staphylococcal protein A-SPRIA. Nonlinear specific IgE dilution curves were associated with high HBV IgG content. By serial transfer and assay of the supernatant, the interference of IgE by specific IgG could be reduced. The degree of interference was correlated with the specific IgG content. The principle of serial supernatant transfer and assay was applied to the standardization of reference HBV IgE serum, and the quantitation of HBV IgE contents in patients and controls. HBV IgE levels in patients allergic to honeybee stings correlated with the skin-test endpoint titrations. While 75% of the allergic patients were skin-test positive, 83% of them were IgE positive by the MSPRIA and 67% were IgE positive by the RAST.

Platelet-associated immunoglobulin G in childhood idiopathic thrombocytopenic purpura

Platelet-associated IgG was studied in children with acute and chronic ITP and in patients with thrombocytopenic SLE, using the microtiter solid-phase radioimmunoassay. Of the children with acute ITP, 85% had elevated PAIgG levels. The degree of elevation of PAIgG at onset of disease did not correlate with the development of chronicity. Of the children with acute ITP, clinically and hematologically indistinguishable from the rest, 15% had normal PAIgG values. All of 22 children with chronic ITP had elevated PAIgG values. Although there was good correlation between the platelet count and the PAIgG value in children with chronic ITP, the association was not as striking in those with acute ITP; thus, factors in addition to the level of PAIgG may contribute to the thrombocytopenia in the latter group. Patients with SLE and thrombocytopenia had higher values of PAIgG than would be predicted from the platelet count; the PAIgG value is probably not the only factor determining the degree of immune thrombocytopenia.

Detection of Human Rotavirus by Reversed Passive Hemagglutination (RPHA) Using Antibody against a Cultivable Human Rotavirus as Compared with Electron Microscopy (EM) and Enzyme‐Linked Immunosorbent Assay (ELISA)

Detection of Human Rotavirus by Reversed Passive Hemagglutination (RPHA) Using Antibody against a Cultivable Human Rotavirus as Compared with Electron Microscopy (EM) and Enzyme‐Linked Immunosorbent Assay (ELISA)

A radioimmunoassay for HBcAg in the sera of HBsAg carriers: Serum HBcAg, serum DNA polymerase activity, and liver HBcAg immunofluorescence as markers of chronic liver disease

A procedure was developed to detect Dane particle-associated HBcAg in blood of HBsAg carriers with high titers of anti-HBc. Dane particles were separated from anti-HBc by gel filtration (Sepharose 4B) and centrifugation of the eluate through 20% (wt/ vol) sucrose at 40,000 rpm for 5 h, and the pellet was assayed for HBcAg by a microtiter solid-phase radio-immunoassay (RIA). Despite a significant loss of antigenic activity throughout the procedure, the RIA for serum HBcAg was a more sensitive and specific indicator of productive HBV infection than DNA polymerase assay of blood or 20-fold Dane particle concentrates from blood. HBc antigenemia correlated positively with HBcAg in the liver by immunofluorescence and prevailed in patients with chronic hepatitis of the aggressive and persistent type; HBsAg membrane fluorescence was prominent in the liver of patients with high serum concentrations of HBcAg. HBc antigenemia was undetectable in 7 patients with chronic hepatitis and HBcAg in the liver. This finding and lack of correlation between the amount of HBcAg expressed in the liver and that circulating in the blood, indicates that under circumstances related to development of chronic liver disease, HB antigens synthetized in the liver may not be released into the blood. A negative serum HBcAg by RIA is of limited clinical value, but a positive test is significant and can provide useful information on synthesis of HBcAg in the absence of a liver biopsy data.

Microtiter solid-phase radioimmunoassay for specific immunoglobulin E

A microtiter solid phase-radioimmunoassay (MSPRIA) for perennial rye grass-specific IgE is described. The assay is carried out inflexible polyvinyl chloride “u” microtiter plates by sequentially incubating antigen, albumin, test sera, and finally radiolabeled antihuman IgE. Wells are cut free of the plate with a hot wire-cutting apparatus and counted individually in a gamma counter. The amount of radioactivity bound is proportional to the amount of specific IgE in the serum. The MSPRIA is shown to be antigen and antibody specific, reproducible, and easily done. Rye-specific IgE levels assayed with the MSPRIA correlated with quantitative end. point titration skin testing using perennial rye grass antigen performed on 31 volunteers with a maximum correlation coefficient of 0.92. The MSPRIA was compared with the radioallergosorbent test (RAST) method using sera from the same 31 volunteers. The rye-specific IgE levels assayed by MSPRIA correlated with those assayed by RAST with a correlation coefficient of 0.95. The MSPRIA is well suited for mass screening and represents a useful method for measuring specific IgE.

The hepatitis B virus-associated delta antigen: isolation from liver, development of solid-phase radioimmunoassays for delta antigen and anti-delta and partial characterization of delta antigen.

The delta antigen-antibody (delta Ag/anti-delta) system is associated with type B hepatitis in man. The delta Ag was extracted and solubilized by sonication and treatment with 6 M GuHCl from the nuclei of human liver obtained at autopsy of a delta-positive subject. A microtiter solid-phase radioimmunoassay was developed and used for the partial characterization of delta Ag. Equilibrium centrifugation, gel filtration and chemical and enzymatic treatments revealed delta Ag to be a protein of approximately 68,000 m.w. with a buoyant density of 1.28 g/cm3 in CsCl, stable to heat, acid, nucleases, and glycosidases, and inactivated by alkali and proteases. A standard preparation of delta Ag was used to develop a blocking solid-phase radioimmunoassay for anti-delta.

Application of microtiter solid-phase radioimmunoassay to the determination of hepatitis B e antigen.

Microtiter solid-phase radioimmunoassay (micro-SPRIA) was applied to the detection of hepatitis B e antigen (HBeAg) in the serum. Antibody against HBeAg to coat the microtiter plate and for radiolabeling was obtained from the serum of asymptomatic carriers of hepatitis B surface antigen by an affinity column of partially purified HBeAg. The micro-SPRIA was sensitive and could be performed without prior concentration of the test serum. HBeAg was detected in 21 (35%) out of 60 serum samples positive for hepatitis B surface antigen, all of which were negative by the conventional immunodiffusion method even after they had been concentrated threefold.

Simplified Radioimmunoassay for Detection of Human Rotavirus in Stools

A simplified radioimmunoassay (RIA) technique was developed to facilitate the diagnosis of human rotavirus in stools of infants with diarrhea. This microtiter solid-phase RIA utilizes as a critical reagent hyperimmune serum against a tissue culture-grown simian rotavirus that is immunologically closely related to the human agent. One hundred thirteen pediatric stool samples were tested. Thiry-five specimens known to be positive for rotavirus by electron microscopy were also positive by RIA. All RIA-positive reactions (36) were shown to be specific for rotavirus by means of an antibody-blocking test. Nonspecific RIA-positive reactions were not encountered. This accurate, sensitive RIA technique is practical for use in diagnostic laboratories because critical serum reagents are prepared against culture-grown virus and not against rotavirus derived from carefully selected stools of humans or experimentally infected gnotobiotic animals.

Microtiter solid-phase radioimmunoassay for detection of Escherichia coli heat-labile enterotoxin

The development of a microtiter solid-phase radioimmunoassay for the detection of Vibrio cholerae enterotoxin and heat-labile Escherichia coli enterotoxin is described. The test is based on the immunological similarity between V. cholerae toxin and E. coli heat-labile toxin. The assay is easy to perform, quantitative, and at least as sensitive and specific as the Y-1 adrenal cell system.

A Microtiter Solid Phase Radioimmunoassay for Detection of the Human Reovirus-Like Agent in Stools

The development of a microtiter solid-phase radioimmunoassay for detection of the human reovirus-like (RVL) agent is described. The test is simple to perform and uses small amounts of reagents; a large number of specimens can rapidly be tested in a single test. Both filtered and unfiltered stool suspensions can be employed. The test was as sensitive as immune electron microscopy, and with an appropriate blocking test, was specific for the human RVL agent.

Purification of hepatitis A antigen from feces and detection of antigen and antibody by immune adherence hemagglutination

Hepatitis A antigen (HA Ag) was purified from feces collected during acute illness from patients with naturally occurring viral hepatitis, type A. Positive fecal specimens were identified by immune electron microscopy, but for detection of HA Agduring purification immune adherence hemagglutination (IAHA) and microtiter solid-phase radioimmunoassay were used. Isopycnic banding in cesium chloride, rate-zonal separation in sucrose, and preparative zonal electrophoresis were used in various combinations for successive purification, and the purified antigen was successfully used in a test for antibody by IAHA. Seronconversions to HA Ag were demonstrated by IAHA in 20 instances of hepatitis A virus infection, but in none of six cases of type B hepatitis or three cases of post-transfusion hepatitis unrelated to heaptitis A or B viruses, nor in two individuals without hepatitis. In addition, the temporal pattern of antibody development during type A hepatitis was studied in serial sera from an experimentally infected chimpanzee. Antibody titers by IAHA correlated well with antibody ratings determined by immune electron microscopy.