Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses^1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)³. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity⁴, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method⁵. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.

Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses^6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide⁶, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)⁷. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera^7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used.

Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

1. Preparation of reagents and equipment

1. Before starting the slot blot procedure, prepare 20_mls of 4M urea solution in Tris-buffered saline (20 mM Tris, 137 mM NaCl, pH 7.6) (TBS) for the hemagglutinin, or HA, slot blot, or 20_mls of a 0.01% Zwittergent solution in TBS for the neuraminidase, or NA, slot blot. While 4M urea should be prepared fresh every time, a 10% Zwittergent stock solution in dH₂O is stable for at least 6 months at room temperature and can therefore be diluted to 0.01% in TBS b.......

Quantitative determination of influenza viral HA and NA are critical for vaccine research and development since these two surface proteins are most important viral components inducing immune responses^6-11. Previously reported immunological methods for the detection of these proteins require strain specific antibodies. The simple, reproducible and rapid slot blot method to quantify the HA and NA antigens described here are suitable for all influenza A viral HA and NA proteins since the antibodies recognize thei.......

The authors would like to thank Mrs. Monika Tocchi for editorial review of the manuscript. AMH is supported by a Scholarship from King Abdulaziz University, through the Saudi Arabian Cultural Bureau in Canada.