Colorimetric and fluorometric substrate immunoassays for detection of Salmonella in all foods: comparative study.

Two modified fluorescent enzyme immunoassays for the detection of Salmonella in food have been developed. Both of the new procedures, which substitute a colorimetric substrate for the fluorescent substrate and in which results are read visually or with a photometer, are modifications of AOAC method 989.15. The visually read procedure uses the same antibody-coated wells as in method 989.15. The colorimetric end point of the assay is determined by comparing the solution color to a color chart. The assay result may also be read in a photometer, if the solution is first transferred to a transparent microtiter well. The second procedure designed to be read in a photometer substitutes clear, antibody-coated wells for those used in the fluorescent assay. The colorimetric assays employ identical monoclonal antibodies for capture and detection of Salmonella as used in the fluorescent assay. In this comparative study, the performance of each new assay was consistent with the performance of method 989.15. These methods have been adopted official first action by AOAC as alternative methods for the detection of Salmonella in foods.

Fluorescent enzyme immunoassay for rapid screening of Salmonella in foods: collaborative study.

A collaborative study was performed in 13 laboratories to validate an enzyme immunoassay (EIA) procedure for rapid detection of Salmonella in foods. The EIA was compared with the standard culture procedure for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole eggs, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by the EIA and culture procedures at the 5% level for any of the 6 foods. The enzyme immunoassay screening method has been adopted official first action as a rapid screening method for detection of Salmonella.

Comparison of several test systems used for determination of rubella immune status.

Hemagglutination inhibition (HAI) is currently the most widely used technique for the determination of rubella immune status. However, two new methods, enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (FIAX), have also been adapted for this purpose. In comparing a commercially available ELISA system (BIO-BEAD, Litton Bionetics) with an HAI system (RUBA-tect, Abbott Laboratories), some ELISA-positive sera were found to be rubella antibody negative by the HAI system. To determine which of these results more accurately reflected the immune status of the patient, 74 RUBA-tect-negative sera were retested by ELISA BIO-BEAD, FIAX (International Diagnostic Technology) and by modified HAI, employing fresh erythrocytes (using Flow Laboratories reagents). Eleven RUBA-tect-negative sera (15%) were positive by ELISA, FIAX, and modified HAI. Two sera were positive only by ELISA and FIAX, two sera were positive by ELISA and HAI, four sera were positive by ELISA only, and one serum was positive by FIAX only. Neutralization assays were subsequently performed on sera positive by only one or two of the procedures to determine the presence of protective rubella antibodies in these sera; all but three of the sera were positive for neutralizing antibody. Commercial ELISA and FIAX systems appear to be more sensitive indicators of rubella immune status than are commercial HAI kits which use stabilized erythrocytes. Neither ELISA nor FIAX require extraction of serum; moreover, the ELISA BIO-BEAD test assay can be performed without an expensive instrument for reading.

Rubella immunity: comparison of hemagglutination inhibition and radioimmunoassay antibody methods.

A threshold (1:10) rubella antibody hemagglutination inhibition (HAI) titer was obtained for 288 of 6537 (4.4%) obstetric patients. Random sera from 84 of these patients were compared for rubella antibody by both HAI and a very sensitive radioimmunoassay (RIA) technique. By RIA, 17% of the sera had no detectable rubella antibody, suggesting that those patients were truly susceptible to rubella. In addition, 8 of 55 paired prevaccine and postvaccine sera with titer increases demonstrated by HAI were analyzed by RIA. Seven of the 8 prevaccine sera were shown to have no rubella virus antibody by RIA, and RIA showed seroconversion in all 8 postvaccine sera.

Simultaneous radioimmunoassay for specific antibodies to members of the human herpesvirus group.

A method of described for the simultaneous radioimmunoassay (RIA) for antibody to members of the human herpesvirus group. The RIA is compared with some of the conventional serologic techniques used to quantitate antibody to these viruses (Epstein-Barr virus, cytomegalovirus, herpesvirus type 1 and varicella-zoster virus). Color-coded beads, each coated with the antigens of a different herpesvirus, were similtaneously placed in a well which contained a human serum to be assayed for antibody to each of these 4 viruses. The results of this test were compared with the results obtained when the serum was assayed for antibody to the 4 viruses in 4 separate tests. We conclude that the antigen-antibody reactions do not significantly interfere with each other when a serum is assayed for antibody to the 4 viruses simultaneously. A comparison of the RIA with conventional serologic techniques shows excellent correlation in the antibody titers obtained. Features of the solid-phase RIA allow significant savings of time, reagents and space, and thus make it feasible for the small laboratory to screen large numbers of sera for antibody to a variety of antigens.

Magnetic transfer devices for use in solid-phase radioimmunoassays and enzyme-linked immunosorbent assays.

A method was developed for the simultaneous transfer of large numbers of solid-phase adsorbents in radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Specially coated ferromagnetic spheres (beads) were used as the solid phase. These beads were transferred from a reaction mixture through a rinse bath to another reaction mixture by magnetic probes. The quality of results obtained with this new methodology compared favorably with that obtained when solid-phase adsorbents were handled individually. The magnetic transfer system provided a high degree of mechanization, thus permitting many more tests to be performed at one time, under almost identical conditions. Certain mechanical features of the test and the micro scale of the reactions provided substantial economy in time and consumption of valuable reagents. The sensitivities of the RIA and ELISA for detection of viral antibody were about the same. An important advantage in using magnetic devices for transfer of beads is that the immune and enzyme-substrate reactions can be started and stopped instantly in either system.

A solid phase radioimmunoassay for detection of serum autoantibodies in systemic lupus erythematosus.

Solid phase radioimmunoassay (RIA) methods for measuring autoantibodies in systemic lupus erythematosus (SLE) patients' serum were developed to improve the sensitivity and quantitative precision of these determinations. Two mechanical systems were studied: (1) acetone fixed cell monolayers in glass tubes and (2) antigen coated plastic beads. Both systems were sensitive and reproducible, giving serum dilution end-points between two and four orders of magnitude (100-10,000 times) greater than those obtained by fluorescence microscopy. The most sensitive, versatile system involves the coating of plastic beads with nuclear antigen(s), incubation overnight with sera and labelling with 125I conjugated antihuman globulin. Linear binding of this radioactive tag is obtained over a wide range of SLE serum dilutions and the slopes of the serum dilution titration curves are almost identical for all SLE patients' sera we have tested. Therefore, a standard titration curve can be constructed from the results with a positive serum, and end-point dilutions of unknown sera estimated from results obtained with a single serum dilution. Alternatively, binding ratios of unknown sera can be usefully compared at fixed dilutions with standard positive and negative sera. For example, high binding ratios (greater than 3.0) were obtained with 19/20 SLE sera and 0/20 control sera. Antigens used in these systems include crude, whole-cell lysates and lysates from purified nuclei. These RIA methods appear to provide certain advantages over existing autoantibody assay methods because they are relatively simple, sensitive, reproducible and potentially capable of measuring a variety of autoantibody specificities.

Radioimmunoassay for toxoplasmosis.

A solid-phase radioimmunoassay for toxoplasmosis has been developed, and the results show good correlation with the indirect hemagglutination test.

An approach to the serodiagnosis of human lung cancer. Cell culture lines reactive as antigens with tumor patients' sera.

A solid-phase radioimmunoassay technique was used to quantitate antigen-antibody reactions between various human cell lines and lung cancer patients' sera. Four human fetal lung cell lines and four human tumor cell lines were more or less reactive as antigens. Failure to obtain exact correspondence between reactions with these cell lines indicates that more than one antigen may be required for detecting specific antibodies to the various lung tumor types. These results suggest that serum antibody detection might be a feasible approach to the immunodiagnosis of lung cancer at stages when the tumor masses are relatively small.