How to Detect Antibodies Against Babesia divergens in Human Blood Samples

Abstract

Background: Today only indirect fluorescent antibody assays (IFAs) are commercially available to detect antibodies against Babesia divergens in humans. IFA is subjective and requires highly experienced staff. We have therefore developed an enzyme-linked immunosorbent assay (ELISA)-based method for measuring anti-B. divergens immunoglobulin G antibodies in human blood samples.

Methods: Crude merozoite extract from in vitro cultures of a new B. divergens isolate was used in ELISA to detect antibodies in different sets of samples: Borrelia burgdorferi-positive samples, healthy individuals, tick-bitten individuals including follow-up samples 3 months later, positive control samples from patients with an active Babesia infection, and samples from malaria-endemic regions. As a reference, IFA was used to detect antibodies in the tick-bitten samples. Western blot was used to evaluate reactions against specific bands in extracts with/without parasites.

Results: Using IFA as the reference method, the sensitivity and specificity of the ELISA were 86% (12/14) and 100% (52/52). There was a very high correlation (r = -0.84; P = .0004) between IFA dilution factors and ELISA absorbances among the samples classified as positive. Five percent of the B. burgdorferi-positive samples were judged as weakly positive and 5% as strongly positive in our ELISA. Western blot showed that the immunodominant antigens (∼120 kDa) were from merozoites and not from erythrocytes.

Conclusions: This ELISA can detect antibodies directed against B. divergens, and it can be a useful and easy assay to handle compared with IFA. The ELISA can also measure high and low levels of antibodies, which could give insight into the recency of a B. divergens infection.

Keywords: Babesia; ELISA; antibody; blood transfusion; divergens.

Figure 1.

Suspension of purified B. divergens merozoites in PBS (A). Acridine orange-stained B. divergens merozoites purified from culture supernatants (B). Abbreviation: PBS, phosphate-buffered saline.

Figure 2.

Spearman's correlation between IFA dilution factors and absorbance (antibody levels) obtained from ELISA (r = 0.84; P = .0004). Abbreviations: ELISA, enzyme-linked immunosorbent assay; IFA, indirect fluorescent antibody assay.

Figure 3.

Samples were run on a 4%–12% SDS-polyacrylamide gel, which was electroblotted onto a PVDF membrane. Lanes 1 and 3, B. divergens merozoite extract; lanes 2 and 4, extract from O+RBC; lane 5, molecular size marker. The membrane was cut in 2 and incubated with serum from 2 different samples that were positive on ELISA. Abbreviations: ELISA, enzyme-linked immunosorbent assay; RBC, red blood cells; PVDF, polyvinylidene difluoride.