A novel bead-based assay to detect specific antibody responses against Toxoplasma gondii and Trichinella spiralis simultaneously in sera of experimentally infected swine

Abstract

Background: A novel, bead-based flow cytometric assay was developed for simultaneous determination of antibody responses against Toxoplasma gondii and Trichinella spiralis in pig serum. This high throughput screening assay could be an alternative for well known indirect tests like ELISA. One of the advantages of a bead-based assay over ELISA is the possibility to determine multiple specific antibody responses per single sample run facilitated by a series of antigens coupled to identifiable bead-levels. Furthermore, inclusion of a non-coupled bead-level in the same run facilitates the determination of, and correction for non-specific binding. The performance of this bead-based assay was compared to one T. spiralis and three T. gondii ELISAs. For this purpose, sera from T. gondii and T. spiralis experimentally infected pigs were used. With the experimental infection status as gold standard, the area under the curve, Youden Index, sensitivity and specificity were determined through receiver operator curve analysis. Marginal homogeneity and inter-rater agreement between bead-based assay and ELISAs were evaluated using McNemar's Test and Cohen's kappa, respectively.

Results: Results indicated that the areas under the curve of the bead-based assay were 0.911 and 0.885 for T. gondii and T. spiralis, respectively, while that of the T. gondii ELISAs ranged between 0.837 and 0.930 and the T. spiralis ELISA was 0.879. Bead-based T. gondii assay had a sensitivity of 86% and specificity of 96%, while the ELISAs ranged between 64-84% and 93-99%, respectively. The bead-based T. spiralis assay had a sensitivity of 68% and specificity of 100% while the ELISA scored 72% and 95%, respectively. Marginal homogeneity was found between the T. gondii bead-based test and one of the T. gondii ELISAs. Moreover, in this test combination and between T. spiralis bead-based assay and respective ELISA, an excellent inter-rater agreement was found. When results of samples before expected seroconversion were removed from evaluation, notably higher test specifications were found.

Conclusions: This new bead-based test, which detects T. gondii and T. spiralis antibodies simultaneously within each sample, can replace two indirect tests for the determination of respective antibodies separately, while performing equally well or better.

Figure 1

Estimation of T. gondii non-specific binding correction factor. Log transformed responses of T. gondii negative swine field sera on non-coupled beads (x-axis) versus T. gondii coupled beads (y-axis). Linear regression line (log y = 0.404* log x + 2.818), 95%CI lines and the linear R² are presented.

Figure 2

Estimation of T. spiralis non-specific binding correction factor. Log transformed responses of T. spiralis negative swine field sera on non-coupled beads (x-axis) versus T. spiralis coupled beads (y-axis). Linear regression line (log y = 0.646*log x + 1.863), 95%CI lines and the linear R² are presented

Figure 3

ROC curves of T. gondii assays calculated with a limited serum set from experimental infection. ROC analysis of a T. gondii bead-based assay and three ELISAs using responses from a set of serum samples, consisting of sera from experimentally infected pigs minus sera drawn on day 5 after inoculation with T. gondii, against their infection status. AUC, area under the curve; 95%CI, interval of AUC at 95% confidence; BBA-TOX, T. gondii antigen coupled bead-based assay; E1-TOX, RIVM in-house T. gondii ELISA, E2-TOX: Safepath T. gondii ELISA, E3-TOX, ID-VET T. gondii ELISA.

Figure 4

ROC curves of T. spiralis assays calculated with a limited serum set from experimental infection. ROC analysis of a T. spiralis bead-based assay and ELISA using responses from a set of serum samples, consisting of sera from experimentally infected pigs minus sera drawn on days 5, 12 and 19 after inoculation with T. spiralis, against their infection status. AUC, area under the curve; 95%CI, interval of AUC at 95% confidence; BBA-TRI, T. spiralis antigen coupled bead-based assay; E-TRI: Safepath T. gondii ELISA.