Development of a Broad-Spectrum Antigen-Capture ELISA Using Combined Anti-p26 Polyclonal and Monoclonal Antibodies for Detection of Equine Infectious Anemia Virus

Abstract

Equine Infectious Anemia Virus (EIAV) poses significant diagnostic challenges due to its genetic variability and the limitations of conventional nucleic acid detection methods. This study developed an antigen-capture, enzyme-linked immunosorbent assay (AC-ELISA) for the detection and quantification of the EIAV capsid protein p26. The assay utilized a monoclonal antibody (1G11) specific to the p26 protein as the capture antibody and a polyclonal antibody as the detection antibody, forming a highly specific and sensitive detection system. Under optimized conditions, the detection limit of the AC-ELISA was 1.95 ng/mL, with a good linear relationship observed between 1.95 ng/mL and 60.5 ng/mL of p26 protein. Additionally, the AC-ELISA effectively distinguished EIAV from other equine viruses, including equine herpesvirus 1 (EHV-1), equine arteritis virus (EAV), and equine influenza virus (EIV), without cross-reactivity. Importantly, the AC-ELISA demonstrated the ability to detect multiple EIAV strains, including virulent strains, attenuated strains, and strains from other countries, highlighting its broad applicability across diverse EIAV isolates. Compared to western blot and reverse transcriptase assays, the AC-ELISA exhibited higher sensitivity and strong correlation in quantifying the EIAV p26 protein. The assay is simple, rapid, and cost-effective, making it suitable for both laboratory research and clinical applications. It provides a powerful tool for EIAV detection and quantification, supporting future vaccine development and clinical trials.

Keywords: AC-ELISA; EIAV; p26.

Figure 1

Genome structure of EIAV. The genome of EIAV consists of three genes encoding structural proteins (gag, pol, env) and three genes encoding accessory proteins (rev, tat, S2), with a total length of approximately 8.2 kb. The precursor protein of the structural protein gag, Pr55gag (p55), has a molecular mass of about 55 kDa. As the viral particle matures, Pr55gag is cleaved by the protease encoded by the pol gene, producing four major structural proteins: capsid protein (CA, p26), matrix protein (MA, p15), nucleocapsid protein (NC, p11), and core protein (p9).

Figure 2

Comparison of amino acid sequences of the p26 protein among different EIAV strains. Darker red colors indicate lower consistency.

Figure 2

Comparison of amino acid sequences of the p26 protein among different EIAV strains. Darker red colors indicate lower consistency.

Figure 3

Preparation of antibodies and validation of their specificity. (A) Purified p26 protein of EIAV with His tag M: protein maker; 1: uninduced; 2: induced with IPTG; 3–4: conducted with wash buffer; 5–7: purified His-p26 protein. (B) The samples of purified p26-His protein were diluted to various concentrations for the WB and detected by pAb and 1G11 mAb. (C) The vCMV3-8 was diluted to various concentrations for the WB and detected by pAb and 1G11 mAb.

Figure 4

Establishment of the AC-ELISA. (A) Optimization of the coating concentration of the capture antibody. (B) Optimization of the dilution of pAb. (C) Optimization of the blocking buffer. Error bars indicate the SD from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Figure 5

Specificity of the ELISA for p26. (A) Major viruses that infect equine were selected (EAV, EIV, EHV, and EIAV) to assess the specificity of AC-ELISA across different viruses. EIAV-luc as positive control, PC and dilution buffer as negative control, NC. (B) Different EIAV strains were selected to evaluate the broad-spectrum of p26 AC-ELISA.

Figure 6

Sensitivity of the ELISA for p26. (A) Different concentrations of purified p26 protein with a serial two-fold dilution from 1 μg/mL to 0.002 μg/mL were tested. (B) The linear range of determination for EIAV p26 detection was 0.002–0.06 μg/mL. (C) Serially two-fold-diluted EIAV-luc was quantified by AC-ELISA. The amount of EIAV-luc in the minimum virus dilution (8×) with the OD value in the dynamic range was calculated using the linear equation of p26. There was also a good linear relationship from 1:8 to 1:4096 dilutions of EIAV-luc. The error bar shows the means of triplicate. Blue data points: Raw experimental measurements used to generate the calibration curve. Red regression line: Quantification of the relationship between Y-axis and X-axis.

Figure 7

Comparison of the novel AC-ELISA and other methods. (A) A serially diluted EIAV-luc was quantified by AC-ELISA. (B) The EIAV-luc was serially diluted and used for reverse transcriptase activity. (C) The EIAV-luc was serially diluted and used for western blot detection of the p26 protein. (D) Correlation analysis between AC-ELISA and real-time qPCR detection of EIAV-luc. (E) Correlation analysis between AC-ELISA and real-time qPCR detection of EIAV-luc. Blue data points: Raw experimental measurements used to generate the calibration curve. Red regression line: Quantification of the relationship between Y-axis and X-axis.