Development and validation of a blocking ELISA for measurement of rabies virus neutralizing antibody

Abstract

Enzyme-linked immunosorbent assay (ELISA) is an effective approach for monitoring herd immunity of animals against rabies but not recommended to detect neutralizing antibody response of individual animals in the framework of international travel since its insufficient agreement with the globally recognized rabies virus (RABV) neutralizing tests. In this study, a blocking ELISA to specifically detect anti-RABV neutralizing antibody was developed using the purified RABV glycoprotein (G) expressed in HEK293T cells as coating antigen, and a labeled anti-G neutralizing monoclonal antibody as the blocking antibody. The overall agreement between the blocking ELISA and fluorescent antibody virus neutralization test in detection of clinical serum samples (dogs = 658; cats = 508) was 97.43% (1,136/1,166), with a diagnostic specificity of 95.63% (219/229) and a diagnostic sensitivity of 97.87% (917/937). Further comparison with the commercial ELISA kits and inter-laboratory validation showed that the blocking ELISA had excellent specificity, sensitivity, and reproducibility. In conclusion, the developed method is a potential tool as an alternative to the virus neutralization test for the detection of canine and feline rabies neutralization antibodies following vaccination.IMPORTANCEThis study establishes a blocking enzyme-linked immunosorbent assay (ELISA) for detecting rabies neutralizing antibodies in dogs and cats, demonstrating high sensitivity, specificity, and no cross-reactivity. This method provides a reliable alternative to conventional neutralization assays, facilitating efficient large-scale rabies vaccination assessment, and thereby strengthening global rabies control efforts.

Keywords: blocking ELISA; monoclonal antibody; neutralizing antibody detection; rabies virus glycoprotein; validation.

Fig 1

The experimental procedure for blocking ELISA.

Fig 2

(A) Western blotting analysis of glycoprotein with mAb 25-6C. M: Marker, Lane 1: negative control, Lane 2: purified RABV-G lysate. (B) The optimal concentrations of antigen RABV-G and HRP-25–6C. The x-axis represents the dilution of HRP-25=6C, and the initial concentration of HRP-25-6C is 1 mg/mL; the y-axis represents PI; the circular legend represents the RABV-G coated concentration of 0.5 µg/mL; the square legend represents the RABV-G coated concentration of 0.25 µg/mL.

Fig 3

ROC analysis (A) The ROC curve was performed to determine the cut-off value of the developed blocking ELISA. (B) Different cut-off values correspond to specificity and sensitivity of the blocking ELISA, which were tested by ROC curve analysis.

Fig 4

(A) Analytic sensitivity of the developed RABV-G based blocking ELISA. Twofold serially diluted PC serum ranging from 1:4 to 1:256 were detected via the ELISA. The PI cut-off value of 67.23% was marked with a dashed line. (B) Analytic specificity of the RABV-G based blocking ELISA. Sample ID 1–8 represents 8 CDV and CPV simultaneously immunized dog sera.

Fig 5

Relationship between RVNA titers and corresponding PI in 1,166 dog and cat serum samples.

Fig 6

(A) Comparison of the developed blocking ELISA and three commercial ELISA kits with FAVN for the detection of RVNA on 100 canine sera. (B) Comparison of the developed blocking ELISA and two commercial ELISA kits with FAVN for the detection of RVNA on 100 feline sera. The C kit can only detect canine sera, so it is not tested with cat sera.