Paper-based electrochemical immunosensor for highly sensitive detection of chicken anemia virus.

Chicken anemia virus (CAV) is one of the primary causes of morbidity and mortality in young chickens. Given the importance of timely detection for maintaining livestock quality, there is a pressing need for rapid and field-deployable diagnostic tools. This study introduces a highly sensitive paper-based electrochemical immunosensor (PEI) for the detection of the 60 amino acid N-terminally truncated viral protein 1 (Δ60VP1), a derivative of the CAV capsid (VP1). A custom antibody was produced for precise immunoassay detection, with results obtainable within 30 min using Square Wave Voltammetry (SWV). The underlying mechanism involves an immunocomplex in the sample zone that hinders the electron transfer of redox species, thereby reducing the current signal in proportion to the Δ60VP1 concentration. Under optimal conditions, the detection linearity for Δ60VP1 ranged from 80 to 2500 ng/mL, with a limit of detection (LoD) of 25 ng/mL. This device was then successfully applied to detect VP1 in 29 chicken serum samples, achieving 91.6% sensitivity and 94.1% selectivity. In conclusion, the PEI device presents a promising solution for rapid, sensitive, and disposable detection of chicken pathogens, potentially revolutionizing productivity and quality assurance in chicken farming.

Development of a lateral flow assay for rapid and accurate detection of chicken anemia virus.

Significant challenges to poultry health are posed by chicken anemia virus (CAV), which induces immunosuppression and causes increased susceptibility to secondary infections. The effective management and containment of CAV within poultry stocks require precise and prompt diagnosis. However, a deficiency persists in the availability of low-cost, rapid, and portable CAV detection devices. In this study, an immunochromatographic lateral-flow test strip-based assay was developed for CAV detection using in-house generated monoclonal antibodies (MABs) against CAV viral protein 1 (VP1). The recombinant truncated VP1 protein (Δ60VP1), with amino acid residues 1 to 60 of the native protein deleted, was produced via a prokaryotic expression system and utilized for immunizing BALB/c mice. Subsequently, high-affinity MABs against Δ60VP1 were generated and screened using conventional hybridoma technology combined with serial dilution assays. Two MABs, MAB1, and MAB3, both binding to distinct epitopes of Δ60VP1, were selected for the development of a lateral-flow assay. Sensitivity analysis demonstrated that the Δ60VP1 antigen could be detected by our homemade lateral-flow assay at concentrations as low as 625 ng/mL, and this sensitivity was maintained for at least 6 mo. The assay exhibited high specificity, as evidenced by its lack of reactivity with surrogate recombinant proteins and the absence of cross-reactivity with other chicken viruses and viral antigens. Comparative analysis with quantitative PCR data demonstrated substantial agreement, with a Kappa coefficient of 0.66, utilizing a sample set comprising 305 clinical chicken serum samples. In conclusion, the first lateral-flow assay for CAV detection was developed in this study, utilizing 2 specific anti-VP1 MABs. It is characterized by simplicity, rapidity, sensitivity, and specificity.

Effective production of recombinant Δ60VP1 chicken anemia virus protein in Escherichia coli and its application to a serodiagnostic indirect ELISA.

Chicken anemia virus (CAV) causes severe anemia and immunosuppression in chickens. VP1 is the main capsid protein, and is suitable for diagnostic kit development, however, it has 24 arginine residues in the first forty N-terminal amino acids of the protein causing toxicity to bacteria leading to reduced prokaryotic expression. In this study, a 60 amino acid N-terminally truncated VP1 (Δ60VP1) which removes the toxic region was expressed in Escherichia coli and the resultant insoluble recombinant protein was purified by Ni-NTA affinity chromatography with anionic denaturing detergents. The high amounts of purified Δ60VP1 produced (150 mg/L) retained appropriate antigenicity and the antigen was used to develop an indirect enzyme-linked immunosorbent assay (ELISA) for serological diagnosis of CAV. One hundred fifty-two chicken serum samples (n = 152) were evaluated using the newly developed Δ60VP1 indirect ELISA (cutoff value = 7.58 % S/P). The sensitivity and specificity of the Δ60VP1 indirect ELISA were 87.50 % and 95.31 %, respectively, while the agreement between the Δ60VP1 indirect ELISA and the commercial IDEXX CAV ELISA was 90.79 % (kappa = 0.814). In this study, we have developed an alternative VP1 production platform in E. coli by truncating the N-terminal 60 amino acids (Δ60VP1) and using anionic denaturing detergents during the purification to successfully solubilize the insoluble Δ60VP1. The antigen was purified with high yield and good immunoreactivity, and an indirect ELISA was developed. The assay could potentially be applied to large-scale CAV serosurveillance.