SUMMARY An enzyme linked immunosorbent assay (ELISA) based on the antibody class capture method for the detection of specific IgM against Toxoplasma gondii, using the microtitre plate format, was developed. Antigen binding was detected using a monoclonal antibody, CIE3, conjugated to horseradish peroxidase. Prior mixing of the conjugate and antigen improved the stability of these reagents as well as removing an incubation stage from the assay. The incubation time of less than four hours permits a rapid throughput of specimens.Using the assay, a total of 163 sera were examined in a three centre study and good agreement was found. Results were expressed as arbitrary enzyme immunoassay units (EIUs) against a freeze dried standard. Throughout the study the standard serum showed a coefficient of variation less than 10% across the microtitre plate. By measuring IgM titres in patients having toxoplasmic lymphadenopathy with a known date of onset, IgM class antibodies were shown to peak at two months, persisting for about six months. In addition, a case of laboratory acquired toxoplasmosis was monitored. Sera shown to contain rheumatoid factor and antinuclear factor did not give false positive results.This rapid, robust, and simplified assay is used by the Public Health Laboratory Service Toxoplasma Reference Units and will provide a standard with which other assays can be compared.
SUMMARY Two monoclonal antibodies CH6 and Cl E3 were used in an antibody class capture assay for the detection of IgM antibodies specific for Toxoplasma gondii. CH6 was used on the solid phase to capture human IgM. After a Toxoplasma gondii antigen had been added, specifically bound material was detected using Cl E3 coupled to horseradish peroxidase. The assay was compared with an established system using polyclonal antisera at both the capture and antigen detection stages. A good correlation was found, with 97 3% (125 of 128) of sera giving the same classification in both assays. Three sera were positive only in the polyclonal system. No false positive results were found when 118 negative sera were examined.The These problems can be overcome by the antibody class capture assay (ACCA).6 Antisera bound to a solid phase is used to capture IgM from the test serum and the remaining serum components washed away. The specificity of the bound IgM can then be assayed by the addition of the antigen of interest, followed by an enzyme labelled antibody against this antigen. Standardisation of both the capture antibody and the enzyme labelled antibody of specific interest is important to ensure that the sensitivity, specificity, and reproducibility of the assay are optimised.Although an antihuman IgM monoclonal antibody Tibi 82 has been used in a radioimmunoassay and in Accepted for publication 9 March 1987 two reverse immunosorbent methods for specific IgM detection, it was not used in an enzyme linked immunosorbent assay (ELISA) system.7 8 In this study an antihuman IgM monoclonal antibody CH6 was used in an ACCA ELISA to detect IgM antibodies specific for Toxoplasma gondii and a second monoclonal antibody, C1E3, used at the antigen detection stage. Cl E3 recognises a major membrane protein antigen of Tgondii and will induce complement mediated cell lysis of the organism. This assay was evaluated and compared with an established ACCA ELISA in which polyclonal antisera were used. Material and methodsSera routinely submitted for toxoplasma serology were titrated in both the dye test and the indirect haemagglutination test (IHAT).9 A total of 128 sera with a dye test titre of > 32 were subsequently tested in the two ACCA enzyme linked immunoabsorbent assay (ELISA) systems for specific IgM. A further 118 sera with no detectable antibody were used to test the specificity of the monoclonal assay.
Aims: Lenticules TM consist of control-dried plano convex discs in which biologically-active materials are contained within a water-soluble matrix. They can be produced to contain stable numbers of bacteria from 10 cfu lenticule ±1 to 10 8 cfu lenticule ±1 with a wide variety of organisms. These experiments were carried out to validate their use as a tool for internal quality control in quantitative microbiology. Methods and Results: The Lenticules TM were used routinely in standard quantitative microbiological procedures across ®ve laboratories. Results showed the materials to be stable, homogenous and capable of identifying systematic errors. Conclusions: The Lenticules TM provide suitable, stable control materials. Signi®cance and Impact of the Study: Routine internal quality control of quantitative measurements is greatly improved; the materials are easy to use and enable comparisons between laboratories to be made.
A total of 91 food and environmental samples were examined for the presence of salmonellae using a commercially available enzyme immunoassay kit (EIA) and a conventional culture technique. A 78% agreement was obtained, but reexamination of culture-negative, EIA-positive samples gave agreement of 86%. The problem of comparing EIA and culture results is discussed. A partially selective pre-enrichment broth was tested in 37 samples and gave better EIA ratios. Artificially contaminated cooked foods gave 100% agreement.
Material and methodsHuman serum samples routinely submitted from suspected cases of toxoplasmosis were used for this study; they were selected to cover the full range of dye test titres. Samples with a minimum volume of 300 ,ul and a shorter storage time were used in preference. Samples were stored at -20°C until required. Thirty six samples from 15 patients from whom more than one specimen had been submitted were also investigated.
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