Development of a Novel Rapid Immunoassay for Chlamydia Trachomatis

Background:  There are approximately 3 million cases of Chlamydia trachomatis (CT) in the US each year, costing in excess of $2.8 billion for diagnosis and treatment.  FDA-approved rapid immunoassays for CT utilize a colorimetric sandwich capture mechanism on a nitrocellulose (NC) substrate, but such mechanisms are insufficiently sensitive for clearance for urine (currently cleared for cervical swabs), thus limiting their use for point-of-care testing (POCT). A rapid assay utilizing a fluorescent compound and fluorometer for detection should offer much greater sensitivity.

Objectives:  To develop of a POCT substrate-based rapid immunoassay utilizing anti-LPS, fluorescent compounds and a small, portable fluorometer.

Methods:  NC, PVDF and FL-PVDF (PVDF modified for fluorescent probing) were illuminated at four different fluorescent wavelengths to determine the substrate-wavelength combination with the lowest autofluorescence. A DyLight-fluorochrome (fc) corresponding to that emittance was then covalently bound to anti-LPS. Anti-LPS-fc conjugates were then incubated with chlamydial elementary bodies (EBs), and binding efficiency determined by flow cytometry.  The anti-LPS-fc-EB complexes were serially diluted onto the substrate to examine detection limits.

Results:  Though FL-PVDF had the lowest autofluorescence, the need for methanol pre-wetting precluded its utility and NC was chosen. DyLight-488 most closely matched the minimal absorbance/emission characteristics of the blue filter module with NC. Anti-LPS-488 labeling of EBs was >97% efficient as measured by flow cytometry (MFI = ~5.7 fold above background). LPS-labeled-EBs were consistently detectable with fluorescent standard units (FSUs) = ~10,000; however, serial dilutions produced inconsistent fluorometer readings.

Conclusions:   We identified two substrates with low background autofluorescence (FL-PVDF, NC) and successfully labeled EBs with LPS-conjugated antibodies.  Preliminary results suggest technical issues with fluorescent detection of EBs need to be overcome for a successful POCT assay to be developed.

Implications for Programs, Policy, and Research:   Development of a fluorescent rapid immunoassay, inexpensive to perform, could greatly improve detection and expand locations where opportunistic POCT be performed.