Quantitative determination of influenza virus hemagglutinin in a primary liquid reference standard by polyacrylamide gel electrophoresis followed by densitometry: Method optimization

INTRODUCTION. In the absence of an international reference standard for the antigen and the corresponding antiserum for quantifying influenza virus hemagglutinin (HA) content by single radial immunodiffusion (SRID), as well as during pandemics or other emergencies, validated alternative methods for HA quantification are required.

AIM. Optimization of the method for certifying a primary reference standard for quantification of influenza virus hemagglutinin using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by densitometric analysis of the gels.

MATERIALS AND METHODS. The HA content (%) in the reference standard was determined by SDS-PAGE under reproducibility conditions (two laboratories). HA concentration (µg/mL) was calculated from the total protein content determined by the Lowry method. The SRID method was used to evaluate the accuracy of HA quantification. The CombiStats software was used for plate image processing, calculation of the squared diameters of precipitation rings, and determination of HA content in the samples. Sucrose and glycerol in the virus concentrate and the diluted virus concentrate (DVC) were quantified by high-performance liquid chromatography.

RESULTS. When performing SDS-PAGE with deglycosylation, the addition of 0.072 mM 2-mercaptoethanol for sample processing eliminates the need for urea. The HA concentrations in DVC determined by SDS-PAGE using 4–12% Bis-Tris gels in Laboratory 1 (135.8 µg/mL) and 4–20% Tris-glycine gels in Laboratory 2 (135.5 µg/mL) were virtually identical; the coefficient of variation did not exceed 4% in either laboratory. The HA concentration determined by SDS-PAGE in Laboratory 1 was comparable to the SRID data (123.6 µg/mL). The content of excipients (glycerol and sucrose) in DVC did not exceed 55 µg/mL and did not affect the results of protein determination.

CONCLUSIONS. Optimal SDS-PAGE conditions were established for certifying a primary liquid reference standard for quantification of influenza virus HA, yielding results comparable to those of the SRID method. The next phase of the study will involve testing the sample preparation conditions and quantification methods for all HA types (H1N1, H3N2, and B) relevant to the 2025–2026 season.

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