Reproducibility of antigen-immobilized enzyme-linked immunosorbent assay (ELISA) and sandwich ELISA for quantitative detection of NNV particles

• Published in: Journal of virological methods Vol. 275; p. 113754
• Main Authors: Gye, Hyun Jung, Nishizawa, Toyohiko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01-01-2020
• Subjects: Aggregation state; Animals; Antibodies, Viral; Antigens, Viral - immunology; Cell Line; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Enzymes, Immobilized; Fish Diseases - diagnosis; Fish Diseases - virology; Fishes - virology; Nervous necrosis virus; Nodaviridae - isolation & purification; Quantitative detection; Reproducibility; Reproducibility of Results; Ultrafiltration; Ultrafiltration; Nervous necrosis virus; Reproducibility; Aggregation state; Quantitative detection; ELISA
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2019.113754

•NNV particles passing through a ultrafilter at 3×105 MWCO could be monometric.•Reproducibility for detecting NNV particles by antigen-immobilized ELISA was low.•Sandwich ELISA had better reproducibility and available range of quantitativeness.
Nervous necrosis virus (NNV) is a fish virus belonging to family Nodaviridae. In this study, we prepared partially aggregated and monometric NNV particles to determine reproducibility of two different enzyme-linked immunosorbent assays (ELISAs): antigen-immobilized ELISA and sandwich ELISA. Passing ratios of purified NNV particles through ultrafilters with molecular weight cut off (MWCO) of 105, 3 × 105 and 106 were 0%, 35.2% and 80.3%, respectively, suggesting that purified NNV particles were partially aggregated whereas those in filtrates with MWCO of 3 × 105 could be monometric. Both NNV particles were subjected to ELISAs. Reduction ratios of ELISA values by 2-fold dilution of antigens were 50% in sandwich ELISA regardless of aggregation state of NNV particles. In contrast, those in antigen-immobilized ELISA were 42% (partially aggregated NNV) to 43% (monometric NNV), which were lower than the theoretical value (50%). This could be due to changes in aggregation state of NNV particles during dry-immobilization. Sandwich ELISA has excellent reproducibility from five times of experiments, in comparison with antigen-immobilized ELISA. Furthermore, available range of regression lines (R2 > 0.99) in sandwich ELISA was wider than that in antigen-immobilized ELISA. These results revealed that sandwich ELISA had better quantitativeness, reproducibility and available range of ELISA values than antigen-immobilized ELISA.