2013-05-22-世界卫生组织-Serological_detection_of_avian_influenza_A_H7N9_virus_infections_by_turkey_haemagglutination_inhibition_assay_12页_162kb

Summary of Laboratory Procedures for Serological Detection of Avian Influenza A(H7N9) Virus Infections by Turkey Haemagglutination-Inhibition Assay

Core Content

This document outlines the laboratory procedures for the turkey haemagglutination-inhibition (HAI) assay, a serological method used to detect antibody responses to avian influenza A(H7N9) virus in human and animal sera. The HAI assay is a traditional and reliable technique for evaluating immune responses to the virus's haemagglutinin (HA) protein. It has been updated to use horse red blood cells (RBC) as a more sensitive alternative, effective from 20 December 2013. The updated protocol is available at the provided link.

Main Points

• Purpose: Detect antibody responses to A(H7N9) virus in human and animal sera using the HAI assay.
• Key Assay Components:
  • Virus strains: Must be inactivated with beta-propiolactone (BPL) and stored at -70°C.
  • Serum samples: Should be treated with receptor-destroying enzyme (RDE) to remove nonspecific inhibitors.
  • Turkey RBC: Used for the HAI assay due to their mixed expression of α2,3-Gal and α2,6-Gal sialic acid receptors, which allows for detection of both avian and human-type antibodies.
• Procedure Overview:
  1. Stock virus preparation: Inoculate 9–10-day-old embryonated hens' eggs and harvest allantoic fluid after incubation.
  2. Serum treatment: RDE is used to inactivate nonspecific inhibitors in serum, followed by a 1:10 dilution.
  3. RBC preparation: Turkey blood is collected in ACD solution, washed, and standardized to a 1% suspension.
  4. HA titration: Virus is serially diluted to determine the HA titer, which is used to prepare a standardized working solution (8 HAU/50μl).
  5. HAI assay: Serum is serially diluted, and standardized virus is added to each well. The HAI titer is determined based on the last dilution that inhibits haemagglutination.

Key Information

Materials Required

• Virus strains: Live or BPL-inactivated viruses in allantoic fluid.
• Serum samples: Human and animal sera must be treated with RDE.
• Buffers and reagents: ACD solution for turkey RBC, PBS, RDE.
• Equipment: Water baths, centrifuges, BSC, refrigerator, freezer.
• Supplies: Centrifuge tubes, microtiter plates, pipettes, etc.

Quality Control

• Serum controls: Include both negative and positive controls for animal and human sera.
• Virus back titration: Ensures the virus working solution contains the correct HA units (8 HAU/50μl).
• RBC control: Helps determine the appropriate incubation time and ensures consistent results.

Procedure Steps

• Virus stock preparation: Requires BSL3 conditions for handling infectious H7N9 virus.
• Serum treatment: Involves RDE reconstitution, incubation, and heat inactivation.
• RBC preparation: Blood is collected, washed, and standardized to 1% TRBC.
• HA titration: Virus is diluted and tested for haemagglutination to determine the HA titer.
• HAI assay: Serum is diluted, and virus is added to wells. RBCs are then added, and haemagglutination is observed.
• Interpretation: Results are recorded using symbols (+, +/-, -) based on the degree of haemagglutination inhibition.

Considerations for Accuracy

• The HAI titer is the reciprocal of the last dilution that inhibits haemagglutination.
• Standardization of the virus working solution and RBC suspension is essential for reliable results.
• Nonspecific agglutinins must be removed by adsorption with RBCs if present in the serum.
• Control sera and RBC controls are necessary to ensure the specificity and validity of the assay.
• The procedure must be followed exactly to ensure optimal results and avoid false interpretations.

Conclusion

The turkey HAI assay is a sensitive and specific method for detecting antibody responses to A(H7N9) virus. It is important to maintain strict quality control, standardize reagents, and follow the procedure precisely. The assay is particularly useful when virus isolation is not feasible, and it is recommended to use RDE-treated sera and standardized RBC suspensions to ensure accurate and reliable results.