A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

A novel double antibody sandwich quantitative ELISA for detecting porcine epidemic diarrhea virus infection.

Publication date: Oct 08, 2024

Porcine epidemic diarrhea (PED), a contagious intestinal disease caused by the porcine epidemic diarrhea virus (PEDV), has caused significant economic losses to the global pig farming industry due to its rapid course and spread and its high mortality among piglets. In this study, we prepared rabbit polyclonal antibody and monoclonal antibody 6C12 against the PEDV nucleocapsid (N) protein using the conserved and antigenic PEDV N protein as an immunogen. A double-antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-qELISA) was established to detect PEDV using rabbit polyclonal antibodies as capture antibodies and horseradish peroxidase (HRP)-labeled 6C12 as the detection antibody. Using DAS-qELISA, recombinant PEDV N protein, and virus titer detection limits were approximately 0. 05 ng/mL and 10 50% tissue culture infective dose per mL (TCID/mL), respectively. There was no cross-reactivity with porcine reproductive and respiratory syndrome virus (PRRSV), porcine rotavirus (PoRV), porcine pseudorabies virus (PRV), porcine deltacoronavirus (PDCoV), or porcine circovirus (PCV). The reproducibility of DAS-qELISA was verified, and the coefficient of variation (CV) for intra- and inter-batch replicates was less than 10%, indicating good reproducibility. When testing anal swab samples from PEDV-infected piglets using DAS-qELISA, the coincidence rate was 92. 55% with a kappa value of 0. 85 when using reverse transcription-polymerase chain reaction (RT-PCR) and 94. 29% with a kappa value of 0. 88 when using PEDV antigen detection test strips, demonstrating the reliability of the method. These findings provide fundamental material support for both fundamental and practical studies on PEDV and offer a crucial diagnostic tool for clinical applications. KEY POINTS: • A new anti-PEDV N protein monoclonal antibody strain was prepared • Establishment of a more sensitive double antibody sandwich quantitative ELISA • DAS-qELISA was found to be useful for controlling the PEDV spread.

Concepts Keywords
Biotechnol Animals
Deltacoronavirus Antibodies, Monoclonal
Diarrhea Antibodies, Monoclonal
Horseradish Antibodies, Viral
Sandwich Antibodies, Viral
Antigen detection
Coronavirus Infections
DAS-qELISA
Enzyme-Linked Immunosorbent Assay
Monoclonal antibodies
Nucleocapsid protein
Nucleocapsid Proteins
Nucleocapsid Proteins
PEDV
Rabbits
Reproducibility of Results
Sensitivity and Specificity
Swine
Swine Diseases

Semantics

Type Source Name
disease MESH intestinal disease
disease IDO protein
disease IDO assay
disease MESH virus titer
disease MESH porcine reproductive and respiratory syndrome
drug DRUGBANK Penciclovir
disease MESH Coronavirus Infections
disease MESH Swine Diseases

Original Article

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