Development of a Multiplex Bead Assay To Detect Immunoglobulin G Antibodies to Babesia duncani in Human Serum

Abstract

Babesia duncani is the causative agent of babesiosis in the western United States. The indirect fluorescent antibody (IFA) assay is the diagnostic test of choice for detection of B. duncani-specific antibodies. However, this test requires parasitized red blood cells harvested from infected hamsters, and test results are often difficult to interpret. To simplify serological testing for B. duncani, a proteomics approach was employed to identify candidate immunodiagnostic antigens. Several proteins were identified by electrospray ionization mass spectrometric analysis, and four recombinant protein constructs were expressed and used in a multiplex bead assay (MBA) to detect B. duncani-specific antibodies. Two antigens, AAY83295.1 and AAY83296.1, performed well with high sensitivities and specificities. AAY83295.1 had a higher sensitivity (100%) but lower specificity (89%) than AAY83296.1, which had a sensitivity of 90% and a specificity of 96%. Combining these two antigens did not improve the performance of the assay. This MBA could be useful for diagnosis, serosurveillance, and blood donor screening for B. duncani infection.

Keywords: Babesia duncani; antibody detection; immunoglobulin G; multiplex bead assay.

FIG 1

Identification of immunodominant antigen candidates using immunoblots and saponized B. duncani-infected parasitized red blood cells. Proteins of saponized parasitized red blood cells were separated using polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. The membranes were then probed with Coomassie blue stain (A), negative serum (B), B. duncani-positive serum (C), B. microti-positive serum (D), and B. divergens-positive serum (E). The antibody-protein complexes were detected with diaminobenzidine (DAB) peroxidase substrate. Lane 1, MW marker; 2, pellet; 3, supernatant. Circled regions were excised in the corresponding Imperial-stained gels for ESI-MS analysis.

FIG 2

Immunoreactivity of four B. duncani recombinant constructs. Purified recombinant proteins were separated using polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membranes. Membranes contained negative serum (A), B. duncani-positive serum (B), B. microti-positive serum (C), B. divergens-positive serum (D), and mouse anti-GST (E). The complex of antibodies-proteins was detected by DAB substrate. The best production yields, as seen in panel E, were for AAY83296.1 and AAY83302.1S. Lane 1, MW marker; 2, AAY83295.1; 3, AAY83296.1; 4, AAY83302.1S; 5, AAY83302.1L. Circles indicate the positions of the expressed proteins.

FIG 3

(A) ROC curves with B. microti samples. (B) ROC curves without B. microti samples. The ROC curves of B. duncani MBA to detect total IgG antibodies were constructed using pROC package of R software version 3.5.3. ROC curves, based on all samples, including B. microti-positive sera, are reported in panel A, whereas ROC curves based on all samples except for B. microti-positive sera are reported in panel B.