2013-05-22-世界卫生组织-Serological_detection_of_avian_influenza_A_H7N9_infections_by_microneutralization_assay_16页_679kb

Summary of Laboratory Procedures for Serological Detection of Avian Influenza A(H7N9) Infections by Microneutralization Assay

This summarizes the WHO laboratory procedures for detecting avian influenza A(H7N9) infections using a microneutralization assay, adapted from WHO guidelines. The procedure is highly sensitive and specific, detecting neutralizing antibodies in serum samples without viral detection.

Introduction

Serological methods are used for retrospective diagnosis, epidemiology, and vaccine evaluation. The microneutralization assay detects antibodies against the influenza hemagglutinin (HA) protein by assessing if sera neutralize viral infectivity. A ≥4-fold increase in antibody titers between acute and convalescent-phase sera indicates a recent infection.

Procedure Overview

The assay consists of three parts:

1. Determination of tissue culture infectious dose (TCID50) for virus titration.
2. Virus microneutralization assay to test serum samples.
3. ELISA for detecting viral nucleoprotein (NP) in infected cells.

Key steps include serial dilutions of sera and virus, incubation with MDCK cells, fixation, and antigen-antibody detection using primary and secondary antibodies.

Controls and Quality Assurance

• Positive controls: Known antisera (e.g., acute-convalescent human sera).
• Negative controls: Normal sera not exposed to the virus.
• Cell controls: Monitor cell health and plate-to-plate variation.
• Virus titer should be determined before assay, and factors like virus, cell passage, and serum quality can affect results.

Materials Required

• Equipment: Biosafety cabinet (BSL-2 for seasonal viruses, BSL-3+ for H7N9), water baths, incubator, microscope, ELISA reader, centrifuge.
• Supplies: Cell culture flasks, microtiter plates, pipettes, reagents for cell culture and ELISA.
• Reagents: MDCK cells (<25-30 passages), D-MEM medium, PBS, specific antibodies (anti-Influenza A NP monoclonal mouse antibody, conjugated goat anti-mouse IgG).

Safety Considerations

Handle viruses appropriately: BSL-2 for low pathogenic, BSL-3+ for highly pathogenic strains like H7N9. Serum samples must be heat-inactivated at 56°C for 30 minutes.

Data Interpretation

• Calculate neutralizing antibody titers based on optical density (OD492) readings from ELISA.
• A ≥4-fold rise in titers (or seroconversion) is diagnostic for recent infection.
• For single serum samples, compare against control titers.

Key Cautions

• Avoid repeated freeze-thaw cycles for sera.
• Ensure MDCK cells are low passage and confluent.
• Proper washing steps in ELISA to avoid false positives.
• Include back-titration to confirm virus viability.

This protocol is critical for accurate laboratory diagnosis and surveillance of influenza like H7N9.