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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.
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.
Newcastle Disease (ND) is a widespread and severe poultry affliction recognized globally1,2,3. 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 birds serving as potential reservoirs6. Despite NDV's single serotype, its genetic diversity poses significant challenges to disease management and control efforts3,7.
Vaccination serves as the primary strategy for controlling ND, complemented by stringent biosecurity measures5,8. Various commercial vaccines are globally available for poultry, provoking robust serum antibody responses following immunization9. These antibodies play a critical role in mitigating symptom severity upon exposure to virulent strains and in curbing interflock transmission10. Standard revaccination protocols, typically involving live attenuated vaccines administered every 6-12 weeks, are standard practice in regions endemic to ND9. Routine monitoring of postvaccination antibody titers in commercial poultry flocks is essential for assessing vaccine effectiveness11,12,13. Low antibody titers post vaccination may indicate vaccine failure, prompting timely corrective measures such as supplementary vaccination or investigation into potential immunosuppressive factors affecting immune response14.
Multiple techniques are utilized for detecting serum antibodies against NDV, including enzyme-linked immunosorbent assay, hemagglutination inhibition (HI), and neutralization assay9,13,15,16. Each method presents unique advantages and limitations concerning sensitivity, specificity, and cost-effectiveness.
This article delineates a step-by-step protocol based on the World Organization for Animal Health (OIE)'s protocol for conducting HI to quantify serum-specific antibody titers against NDV11. Hemagglutination (HA), a phenomenon induced by certain enveloped viruses like NDV, involves the clumping of red blood cells (RBCs)9. The hemagglutinin-neuraminidase (HN) protein on the NDV surface interacts with RBCs, resulting in cell clumping and lattice formation2. HI assay is preferred as a serological method due to its ability to assess serum antibody specificity towards the HN protein of NDV8,9. Furthermore, its cost-effectiveness and independence from specialized instrumentation render it accessible and practical for routine use.
To improve assay efficiency, we refined the workflow of the OIE protocol11, with a focus on attaining more accurate antigen HA titers and offering detailed adjustments for the 4 hemagglutination units (4-HAU) antigen solution. Additionally, through comparative analysis, we assessed the impact of 4-HAU accuracy on HI results, providing valuable insights for field practitioners. This approach is not limited to NDV antibody testing but extends to the detection of viral subtypes and other hemagglutinating viruses, including measles, polyomavirus, mumps, and rubella.
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
2. Reconstitution of lyophilized antigen or serum
3. HA titration of antigens
Figure 1: Schematic diagram illustrating the procedure for diluting of the antigen solution in the hemagglutination assay. The antigen solution is diluted in PBS at ratios of 1:3, 1:5, 1:7, and 1:9, respectively. PBS in wells or centrifuge tubes is represented in blue, while antigens are shown in green. Please click here to view a larger version of this figure.
Figure 2: Schematic layout of a microplate used in the hemagglutination assay. The first column of rows 1-5 contains undiluted and various initial dilutions of the antigen solution, followed by a two-fold serial dilution from left to right until column 11. PBS control is placed in the last column. The gradient from dark to light indicates decreasing antigen concentration. Please click here to view a larger version of this figure.
Antigens | Highest dilution column of complete HA | HA titer of undiluted stock |
Undiluted stock | a | 2a |
3-fold dilution | b | 3 x 2b |
5-fold dilution | c | 5 x 2c |
7-fold dilution | d | 7 x 2d |
9-fold dilution | e | 9 x 2e |
Table 1: HA assay results and calculation of HA titer of stock solution.
4. Preparation of the 4-HAU antigen solution
5. Back-titration of the 4-HAU antigen solution
Figure 3: Schematic diagram of dilution procedure of 4 hemagglutination units' solution in back-titration. The 4-HAU working solution is diluted with PBS at ratios of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7. PBS is depicted in blue, and antigens in green. Please click here to view a larger version of this figure.
Figure 4: Schematic layout of the wells for back-titration and representative result. The diluted 4-HAU working solution is transferred to a new row, with an additional PBS control added. The gradient from dark to light green indicates antigen concentration from high to low. Representative results demonstrate that the 1:4 dilution is the highest dilution showing complete hemagglutination. Please click here to view a larger version of this figure.
HA titer of back-titration | Replenishment of antigen or PBS |
1:2 | Va |
1:3 | Va/3 |
1:5 | Vp/4 |
1:6 | Vp/2 |
1:7 | 3Vp/4 |
Table 2: Reference table for adjusting 4-HAU concentration based on back-titration results. Va and Vp represent the volumes of antigen and PBS used in the formulation of 4-HAU antigen solution, respectively.
HA titer of back-titration | Dilution factor correction |
1:2 | D/2 |
1:3 | 3D/4 |
1:5 | 5D/4 |
1:6 | 3D/2 |
1:7 | 7D/4 |
Table 3: Reference table of dilution factor correction for re-preparation of 4-HAU antigen. D represents the dilution factor previously used in 4-HAU antigen preparation.
6. Preparation of serum
7. HI assay
Figure 5: Schematic diagram of hemagglutination assay workflow. PBS is represented in blue, serum in yellow, the 4-HAU antigen solution in green, and the 1% chicken RBC suspension in red. Arrows indicate the order of liquid dispensation. Please click here to view a larger version of this figure.
Validation of the titer of 4-HAU antigen solution formulated using the optimized method
The study employed various dilutions of the antigen stock solution to accurately determine the HA titer, facilitating the calculation of the dilution factor for preparing the 4-HAU antigen solution. The results reveal that the optimized method is both efficient and precise, reducing the number of repetitive back-titration and adjustment procedures. In the first scenario, an initial HA titer of ~512 yielded a fin...
TheΒ 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 methodologies11,12. Furthermore, although the FAO and OIE recommend back-titration as a means of enhancing the accuracy of 4-HAU, they lack a clear protocol11,<...
The authors have no conflicts of interest to declare.
C.C. was supported by Taicang Technology program (TC2021JC16), and Innovation Team Funds of Suzhou Chien-shiung Institute of Technology (2023JXKYTD01). H.Y. was supported by Taicang Technology program (TC2021JC11). Both of them were supported by Start-up Fund for New Ph.D. Researchers of Suzhou Chien-Shiung Institute of Technology.
Name | Company | Catalog Number | Comments |
Β Sterile Centrifuge Tube (15 mL)Β | Labshark | 130201030 | |
Air-cooled Low-speed Tabletop CentrifugeΒ | 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)Β | Adamas Life | C8020 | |
Sterile Centrifuge Tube (1.5 mL) | Labshark | 130201012 |
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