Characteristics of the m2000 automated sample preparation and multiplex real-time PCR system for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

We evaluated a new real-time PCR-based prototype assay for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae developed by Abbott Molecular Inc. This assay is designed to be performed on an Abbott m2000 real-time instrument system, which consists of an m2000sp instrument for sample preparation and an m2000rt instrument for real-time PCR amplification and detection. The limit of detection of this prototype assay was determined to be 20 copies of target DNA for both C. trachomatis and N. gonorrhoeae, using serially diluted linearized plasmids. No cross-reactivity could be detected when 55 nongonococcal Neisseria isolates and 3 non-C. trachomatis Chlamydia isolates were tested at 1 million genome equivalents per reaction. Concordance with the Roche Amplicor, BDProbeTec ET, and Gen-Probe APTIMA Combo 2 tests was assessed using unlinked/deidentified surplus clinical specimens previously analyzed with these tests. For C. trachomatis, concordance for positive results ranged from 93.7% to 100%, while concordance for negative results ranged from 98.2% to 100%. For N. gonorrhoeae, concordance for positive and negative results ranged from 91.4% to 100% and 99.3% to 100%, respectively. A workflow analysis of the prototype assay was conducted to obtain information on throughput under laboratory conditions. At 48 samples/run, the time to first result for both C. trachomatis and N. gonorrhoeae was 4.5 h. A total of 135 patient specimens could be analyzed in 8.9 h, with 75 min of hands-on time. This study demonstrated the technical and clinical feasibility of the new Abbott real-time PCR C. trachomatis/N. gonorrhoeae assay.

Recombinant mouse-human chimeric antibodies as calibrators in immunoassays that measure antibodies to Toxoplasma gondii.

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was > or =0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

Fluorescence polarization immunoassay of urinary 5-hydroxy-3-indoleacetic acid.

Fluorescence polarization immunoassay of 5-hydroxy-3-indoleacetic acid in urine is described and compared with liquid chromatography (electrochemical detection) and colorimetry. Reports of in-house performance data and results of clinical trials are included to emphasize the usefulness of the assay for routine work.

The POSCH Information Management System: experience with alternative approaches.

An unconventional approach to the information management system for a clinical trial is presented in this paper: employment of a large shared computer utility, use of existing packaged software, and use of a generalized data base management system. An easily maintained system was developed at a very economical cost. The paper is designed to present our experiences and include descriptions of a series of problems that were faced and solved: design problems, problems with error corrections, and problems unique to our operational procedures. The good results are due as much to careful management as to the initial decision to use the unconventional approach. Special attention has been paid to cost controls, patient safety, and data security. The approach has been successful, and others are encouraged to consider a similar approach.