Validation of Multiplex Serology for human hepatitis viruses B and C, human T-lymphotropic virus 1 and Toxoplasma gondii

Abstract

Multiplex Serology is a high-throughput technology developed to simultaneously measure specific serum antibodies against multiple pathogens in one reaction vessel. Serological assays for hepatitis B (HBV) and C (HCV) viruses, human T-lymphotropic virus 1 (HTLV-1) and the protozoan parasite Toxoplasma gondii (T. gondii) were developed and validated against established reference assays. For each pathogen, between 3 and 5 specific antigens were recombinantly expressed as GST-tag fusion proteins in Escherichia coli and tested in Monoplex Serology, i.e. assays restricted to the antigens from one particular pathogen. For each of the four pathogen-specific Monoplex assays, overall seropositivity was defined using two pathogen-specific antigens. In the case of HBV Monoplex Serology, the detection of past natural HBV infection was validated based on two independent reference panels resulting in sensitivities of 92.3% and 93.0%, and specificities of 100% in both panels. Validation of HCV and HTLV-1 Monoplex Serology resulted in sensitivities of 98.0% and 95.0%, and specificities of 96.2% and 100.0%, respectively. The Monoplex Serology assay for T. gondii was validated with a sensitivity of 91.2% and specificity of 92.0%. The developed Monoplex Serology assays largely retained their characteristics when they were included in a multiplex panel (i.e. Multiplex Serology), containing additional antigens from a broad range of other pathogens. Thus HBV, HCV, HTLV-1 and T. gondii Monoplex Serology assays can efficiently be incorporated into Multiplex Serology panels tailored for application in seroepidemiological studies.

Expression of in vivo biotinylated recombinant antigens SAG1 and SAG2A from Toxoplasma gondii for improved seroepidemiological bead-based multiplex assays

“…Using SAG2A bio in BBMA resulted in higher MFI and thus an IU/mL with similar discriminatory power between seropositive and seronegative sera ( Fig. 6), as was used successfully in prior studies [10,11]. The speci city and sensitivity (accuracy) was in our hands slightly lower, however, as compared to SAG1 bio .…”

“…Thus, MBP or GST protein tags could hinder antibody access. This notion is also supported by a recent BBMA study that included GST-SAG1 fusion protein bound with its N-terminus to beads and which reported sensitivity and speci city of less than 87% towards human sera [11].…”

“…Combining both proteins into a single BBMA was shown recently to be essential for satisfactory accuracy [11]. In some humans with overall low anti-T. gondii antibody titers, the immune response might be directed toward more than one antigen, as has been reported for other T. gondii antigens [50].…”

“…However, surprisingly little attention has been paid in the past to their potential in uence on antigenicity of the recombinant proteins used for diagnostic purposes when the N-and Cterminal signal sequences are left intact (see e.g. [11,[25][26][27][28][29]). Additionally, deletions or N-terminal fusions with relatively large glutathione-S-transferase (GST) protein tags have both been used, which may impact antigenicity.…”

“…While a few studies have reported the application of bead-based multiplex assays that include T. gondii antigens, such assays differ substantially in detail [9][10][11][12][13] and none are commercially available. Given our interest in the epidemiology of T. gondii [14], we developed a bead-based multiplex assay based on the recombinant T. gondii antigens SAG1 (SRS29B) and SAG2A (SRS34A) -two of the most widely used diagnostic antigens [15].…”