Pathogenic pig coronaviruses found in the intestine (enteric) produce similar symptoms, namely diarrhea with a reduced ability to digest food leading to dehydration and starvation. Transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV) are three common pathogens of this type. They are typically diagnosed by laboratory tests targeting the viral spike (S) protein, however these tests have yet to be multiplexed. Multiplexing can reduce sample consumption, reagent volumes, and time till detection.
Luis Giménez-Lirola of Iowa State University, Ames, USA, and colleagues recently detailed the research and development of a parallel dot ELISA-like multiplex immunoassay called AgroDiag PorCoV. It simultaneously detects TGEV, PEDV and PDCoV, if present, using antibodies. The study was published in the Journal of Immunological Methods.
Seven week old pigs were experimentally inoculated with two strains of TGEV, one PEDV, one PDCoV, and mock control. From these pigs 480 serum samples were collected and banked.
A dot based immunoassay chip was fabricated in a clean room environment. The surface of 96-well plates were functionalized for protein binding by coating with Innobiochips’ dextran polysaccharide backbone.
Affinity purified recombinant S1 proteins (TGEV, PEDV, PDCoV) and pig IgG were arrayed in triplicate in the wells by 130nm dot printing using Scenion’s sciFLEXARRAYER SX®. For detection, test serum was applied, incubated and washed followed by application of the colourimetric detection antibody and imaging. A unique dot pattern was evident for the three viruses.
TGEV, PEDV, and PDCoV could be detected in a specific dose sensitive manner, with viral load in serum samples from each pig increasing over time.
“The colorimetric detection is sensitive, as for a given concentration of antibody in the sample, the quantity of antibody captured on the spot per unit volume is generally higher than if distributed throughout a larger surface. The diagnostic performance of this assay was evaluated on known status samples. Altogether, the results of this study demonstrate that the AgroDiag PEC multiplex immunoassay is an efficient and reliable test for differential detection of PECs,” concluded the authors.