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Classic diagnostics: immunofluorescence

Immunofluorescence using polyclonal antisera had been used since the 1960s for the detection of chlamydial antigen, both in cell culture and in clinical material. Such antisera were, however, generally the property of a limited number of chlamydial research laboratories. They also tended to be individually prepared and of inconsistent quality. A quantum leap in the use of immunofluorescence came in the early 1980s with the advent of monoclonal antibody technology. This enabled the production of highly reproducible antisera of defined specificity and it lead to the elucidation of the relationship between the newly discovered MOMP and the serotypes of C. trachomatis [see: MOMP and serological classification]. Commercial companies were quick to see the potential for C. trachomatis infections, of the new technology that Cesar Milstein at Cambridge University had unleashed. 

The first product was the Syva Microtrak^® based on a fluorescein-conjugated and species specific monoclonal antibody to C. trachomatis major outer membrane protein, which was used for the direct detection of chlamydial elementary bodies in clinical specimens. The test gave good results where people had good fluorescence microscopy equipment and had taken the trouble to learn how to use it properly. Thus, right at the start, Uyeda et al., 1984 assessed  401 paired, endocervical specimens by Syva Microtrak immunofluorescence or by traditional cell culture, and found that 398 (99.3%) gave identical immunofluorescence and culture results. Overall sensitivity of the direct test was 96.3% (26 of 27), and the specificity was 99.5% against culture. More than four-fifths of the direct smears were read within two minutes and there was no doubt that the rapid turnaround time and elimination of the need for cell culture or a cold transport chain gave the test significant advantages. While Syva cautiously recommended 10 apple green 'pin pricks' of elementary bodies as the end point, some highly competent laboratories argued that a single such object was sufficient.

Direct immunofluorescence with monoclonal antibodies was the start of the present day trend towards the use of non viability-dependent methods for the laboratory diagnosis of C. trachomatis infection. Other companies brought out genus specific monoclonal antibodies, directed against chlamydial lipopolysaccharide which, while they gave less clear staining than those directed against MOMP, extended the reach of the new methodology to what we would now regard as the Chlamydophila species. However, it soon became clear that the new methodology required careful, painstaking microscopists. Throughput for screening purposes was limited, with few people able to tolerate the 'eye-sucking' strain of looking at fluorescing 'pin pricks' for hours on end, however fetching the shade of green! What was needed was to harness the antigen detection capability of antibodies to the new technique of enzyme immunoassay (EIA). Enter the Abbott Chlamydiazyme^® test.

[MEW] May 2002.

NEXT: Antigen detection EIA.

Reference

Uyeda, C. T., Welborn, P., Ellison-Birang, N., Shunk, K. & Tsaouse, B. (1984). Rapid diagnosis of chlamydial infections with the MicroTrak direct test. Journal of Clinical Microbiology 20, 948 -  950.