Double-Antigen Sandwich ELISA Detects Antibodies to Trypanosoma cruzi

Chagas disease (CD), also known as American trypanosomiasis, is a potentially life-threatening vector-borne tropical zoonosis caused by the protozoan parasite Trypanosoma cruzi. The estimated prevalence of CD in 21 Latin American countries where it is considered endemic exceeds five million individuals; CD accounts for approximately 7,500 deaths annually.

Indirect immunoassays are the recommended method for chronic Chagas disease diagnosis and its performance relies on the employed antigen preparation. Chimeric antigens have been successfully utilized for chronic CD in vitro diagnosis and efficiently address commonly encountered hurdles arising from the use of recombinant and native antigens.

A team of Medical Scientists at the Oswaldo Cruz Foundation (Rio de Janeiro, Brazil) and their colleagues developed and evaluated four chimeric antigens from T. cruzi (IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4) using a double-antigen sandwich ELISA (DAgS-ELISA) as the diagnostic platform. To overcome any limitation, peroxidase-labeled (HRP) antigens can be utilized, diagnosing either acute or chronic infection, in a species and immunoglobulin class-independent manner. The team obtained a total of 412 sera from 207 T. cruzi-positive and 205 T. cruzi-negative individuals. Additionally, to evaluate cross-reactivity, 68 sera from individuals with unrelated diseases, as previously defined by parasitological or serological diagnosis, were acquired.

The optimal dilutions of serum and antigen-enzyme conjugate (HRP) were determined by checkerboard titration at different chimeric antigen coating concentrations. Antigens were diluted at the final concentrations of 400 ng, 200 ng, 100 ng, 50 ng, 25 ng, 12.5 ng, 6.25 ng, 3.125 ng and 1.56 ng in carbonate-bicarbonate buffer (50 mM, pH 9.6). These dilutions (100 μL) were placed on 96-well high-binding microplates. Absorbance was measured on a microplate spectrophotometer at a wavelength of 450 nm on a SPECTRAmax 340PC (Molecular Devices, San Jose, CA, USA).

The investigators reported that in the phase I study, the areas under the curve of IBMP-8.1, IBMP-8.2, IBMP-8.3 and IBMP-8.4 were 98.7%, 99.5%, 98.6% and 98.8%, respectively. Among the positive samples, IBMP-8.1 antigen classified 53 (25.6%) as false negative, IBMP-8.2, 27 (13%), IBMP-8.3, 24 (11.6%) and IBMP-8.4, 43 (20.8%), giving sensitivities of 74.4%, 87%, 88.4% and 79.2%, respectively. The only antigen that did not reach 100% specificity was IBMP-8.3, with 96.6%. IBMP-8.3 was also the only molecule to show cross-reactivity with Human T-cell lymphotropic virus (HTLV).

The authors concluded that that IBMP-DAgS-ELISA is suitable for the detection of anti-T. cruzi antibodies in areas of co-endemicity with Leishmania spp. The findings also demonstrate the notable capability of all four IBMP proteins to distinguish between T. cruzi-positive and -negative samples. The specificity attained under DAgS-ELISA reached 100% using three of the four chimeric antigens evaluated, IBMP-8.1, IBMP-8.2 and IBMP-8.4. The study was published on March 11, 2022 in the journal PLOS Neglected Tropical Diseases.

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