Variability in a commercially available enzyme-linked immunosorbent assay system. I. Assay variability

Area of Science:

• Veterinary Immunology
• Diagnostic Assays
• Virology

Background:

• Enzyme-linked immunosorbent assays (ELISA) are crucial for detecting antibodies against avian pathogens like infectious bronchitis virus (IBV) and infectious bursal disease virus (IBDV).
• Variability in ELISA kit performance can compromise diagnostic accuracy and disease surveillance.

Purpose of the Study:

• To characterize and quantify the variation in ELISA kits used for IBV and IBDV detection.
• To evaluate the impact of specific assay parameters on the reliability of ELISA results.

Main Methods:

• Experiment 1 assessed inter-lot, intra-lot, and inter-day/technician variability using serum pools of varying titers.
• Experiment 2 investigated the effect of ortho-phenylene-diamine (OPD) substrate incubation time on assay outcomes.
• Experiment 3 examined the influence of different serum dilution methods on observed titers.

Main Results:

• Significant variations were observed among ELISA kit lots and test plates for both IBV and IBDV.
• OPD incubation time and serum dilution methods significantly affected mean titers, except for low-titered IBV samples.
• High-titered IBDV samples often reached maximum absorbance, limiting detection capabilities.

Conclusions:

• ELISA kits for IBV and IBDV exhibit considerable variability, potentially impacting diagnostic sensitivity and specificity.
• Standardization of assay parameters, including incubation times and dilution protocols, is critical for reliable avian disease diagnostics.