Optimization of Antigen Preparation for Hemagglutination Inhibition Assay of Newcastle Disease Virus Serology

Summary

This article presents a simplified method for preparing 4 hemagglutination units antigen for Newcastle disease virus serological testing. By accurately determining the hemagglutination titer of the antigen, combined with a more rigorous back-titration method, and a well-defined adjustment process, it enhances test efficiency, reduces false positives, and improves disease surveillance in poultry.

Abstract

Accurate assessment of Newcastle disease virus (NDV) antibody titers is crucial for effective poultry disease control and surveillance. This article introduces an optimized method for preparing 4 hemagglutination units (4-HAU) antigen solution, a key component of the hemagglutination inhibition assay (HI) used in NDV serological detection. Unlike conventional methods, which involve time-consuming and undefined back-titration and adjustment steps, this approach streamlines this process by accurately measuring the HA titer using an initial series of dilutions (1:3, 1:5, 1:7, and 1:9). We also provide a specific method for adjusting or reformulating based on the back-titration results, reducing the need for repeated back-titrations. In addition, we evaluated the effect of the accuracy of the 4-HAU antigen solution on serum HI titer and found that when the titer of the 4-HAU antigen was lower than 3, it resulted in the appearance of false-positive HI samples. By providing an accurate method and minimizing computational tasks, this approach increases test efficiency and reliability, contributing to improved disease surveillance and control in poultry populations.

Introduction

Newcastle Disease (ND) is a widespread and severe poultry affliction recognized globally^1,2,3. It manifests through various symptoms such as high fever, respiratory distress, dysentery, nervous disorders, and mucosal hemorrhage⁴. The causative agent, Newcastle Disease Virus (NDV), has endured for almost a century, afflicting more than 200 avian species, including chickens, ducks, geese, and pigeons⁵. Transmission primarily occurs through direct or indirect contact with infected birds, with poultry, pigeons, and free-ranging bird....

Protocol

Approval for the protocol was granted by the local institutional animal care and use committee. All procedures involving live virus antigens and clinical serum samples were performed in a Biosafety Level 2 laboratory, in compliance with established safety protocols.

1. Preparation of 1% chicken RBC suspension

1.  Add 3 mL of Alserver's solution (anticoagulant) into a sterile 15 mL conical centrifuge tube.
2. Collect 1 mL of blood from the wing vein of .......

Discussion

The optimized method proposed in this article presents an approach for accurately preparing the 4-HAU antigen solution. Although the OIE and EU guidelines suggest initial dilutions for HA titration, they do not provide precise details regarding the dilution ratios to be used or offer specific methodologies^11,12. Furthermore, although the FAO and OIE recommend back-titration as a means of enhancing the accuracy of 4-HAU, they lack a clear protocol^11,<.......

Materials

Name	Company	Catalog Number	Comments
Sterile Centrifuge Tube (15 mL)	Labshark	130201030
Air-cooled Low-speed Tabletop Centrifuge	Titan	LDC-5
Alserve's Solution	Sangon Biotech	E607058
Centrifuge	DLAB	9032002121
Disposable Pasteur Pipette	Titan	SWXG-004
Disposable Sterile Syringe(1 mL)	Beyotime	FS801-30PCS
Disposable V-bottom Microtiter Plate(96-well)	Labshark	130207001
Microplate Shaker	Jiangsu Xinkang Medical Equipment Co., Ltd	XK96-3
NDV HI Negative Sera	Qingdao Regen Diagnostics Development Center
NDV HI Positive Sera	Qingdao Regen Diagnostics Development Center
NDV HI Test Antigen	Qingdao Regen Diagnostics Development Center
PBS Solution (1x)	Adamas Life	C8020
Sterile Centrifuge Tube (1.5 mL)	Labshark	130201012