Validation of immunoassays for the Chlamydia trachomatis antigen Pgp3 using a chimeric monoclonal antibody

Abstract

Seroepidemiology, or measuring antibodies to pathogens to estimate population-level exposure, can provide useful public health data. The tests used, however, often lack sufficient validation data due to absence of a gold standard. For many pathogens, serum antibodies can be detected long after resolution of infection, but infection status is often used as a gold standard for antibody positivity. To ensure that recently developed antibody tests for seroepidemiology of Chlamydia trachomatis (Ct), the causative agent of urogenital chlamydia and the blinding eye disease trachoma, have high performance, we generated a chimeric antibody to the immunodominant Ct antigen Pgp3. Two clones were selected to evaluate the test performance of three assays to measure antibodies to Pgp3: multiplex bead assay (MBA), enzyme-linked immunosorbent assay (ELISA), and lateral flow assay (LFA). Overall, each assay demonstrated high accuracy and precision when tested using either clone, and the clones were stable when stored at - 20 °C and 4 °C for almost 2 years. The limit of detection was similar for MBA and LFA, but almost a log-fold higher (i.e. less sensitive) using ELISA. Overall, the chimeric antibodies represent stable control reagents for tests with robust performance and will facilitate deployment of these tests to other laboratories.

Figure 1

Precision samples for MBA and ELISA by operator. MFI (for MBA testing) and Abs (for ELISA) testing by sample concentration for each clone. Samples tested by operator 1 are in blue and operator 2 are in red. Each point is an average of all replicates tested (15/sample) by each operator. Error bars on each point represent standard deviation. MFI data are presented on a log/log scale to better display the linear range of the assay. MFI = Median fluorescence intensity, Abs = Absorbance, MBA = Multiplex bead assay.

Figure 2

Limit of detection standard curve samples by clone, assay, and operator. MFI (for MBA testing) and Abs (for ELISA) testing by sample concentration (in ng/mL) for each clone. Samples tested by operator 1 are in blue and operator 2 are in red. Each point is an average of all replicates tested (12/sample) by each operator. Error bars on each point represent standard deviation. MFI = Median fluorescence intensity, Abs = Absorbance, MBA = Multiplex bead assay.