New rapid flow-through immunofiltration assay system for rapid detection of Trichomonas vaginalis in ThinPrep specimens

Abstract number: r2070

Alderete  J., Chang  T.

Objectives: 

The ThinPrep Pap Test (TP) is preferred over the traditional Papanicolaou (Pap) smear, and the liquid-based cytology is being increasingly employed for detection of the sexually transmitted HPV. Such fixative renders the number one, non-viral sexually transmitted parasite, Trichomonas vaginalis, undetectable. Given the association of trichomonosis with cervical cancer, we wanted to develop a rapid flow-through (Filtration) assay system to detect T. vaginalis in TP specimens.

Methods: 

Spleen lymphocytes of mice immunized with trichomonads after treatment with TP fixative were fused with NS1 myeloma cells to generate hybridomas producing mAbs. Supernatant of hybridomas were tested for reactivity with organisms exposed to TP fixative coating microtitre wells. Immunoreactive mAbs of single cell-cloned hybridomas were tested for detection of trichomonad antigen in ThinPrep specimens under optimized conditions. Finally, mAbs were examined using a Filtration assay system on disposable porous filter-membranes with bound antigen. Finally, TP specimens from patients with trichomonosis and uninfected individuals were tested for reactivity by mAbs using the Filtration assay.

Results: 

The mAbs were identified by standard colorimetric ELISA at 405 nm that immunoreacted with trichomonads exposed to TP liquid fixative coated onto microtitre wells. Then, mAbs detected trichomonad antigen immobilized onto membranes in a Filtration assay system, and this was done by both colorimetric ELISA and emission chemiluminescence (ECL). The Filtration ECL assay was very sensitive and detected antigen from as little as 10 organisms immobilized on membranes. Filtration ECL yielded rapid, accurate results with fewer fixed organisms within 30 sec compared to colorimetric ELISA. The mAbs reacted with organisms added to the ThinPrep fixative and with antigen in samples of patients with trichomonosis. No immuno-crossreactivity was ever detected with the mAbs with other trichomonad species, in the absence of T. vaginalis antigen, and in samples with materials from uninfected individuals.

Conclusion: 

A rapid flow-through immunofiltration assay system was developed for detecting T. vaginalis antigen. Individual mAbs readily detect in a sensitive and specific fashion trichomonad antigen in TP specimens. This demonstrates the ability to now detect this sexually transmitted infection in the liquid-based ThinPrep specimens.