A monoclonal antibody-based immunoassay for measuring the potency of 2009 pandemic influenza H1N1 vaccines

Abstract

Background: The potency of inactivated influenza vaccines is determined using a single radial immunodiffusion (SRID) assay. This assay is relatively easy to standardize, it is not technically demanding, and it is capable of measuring the potency of several vaccine strain subtypes in a multivalent vaccine. Nevertheless, alternative methods that retain the major advantages of the SRID, but with a greater dynamic range of measurement and with reduced reagent requirements, are needed.

Objectives: The feasibility of an ELISA-based assay format was explored as an alternative potency assay for inactivated influenza vaccines.

Methods: Several murine monoclonal antibodies (mAbs), specific for the 2009 pandemic H1N1 influenza virus hemagglutinin (HA), were evaluated for their potential to capture and quantify HA antigen. Vaccine samples, obtained from four licensed influenza vaccine manufacturers, included monovalent bulk vaccine, monovalent vaccine, and trivalent vaccine. Traditional SRID potency assays were run in parallel with the mAb-ELISA potency assay using the reference antigen standard appropriate for the vaccine samples being tested.

Results: The results indicated that the ELISA potency assay can quantify HA over a wide range of concentrations, including vaccine at subpotent doses, and the ELISA and SRID potency values correlated well for most vaccine samples. Importantly, the assay was capable of quantifying A/California HA in a trivalent formulation.

Conclusions: This study demonstrates the general feasibility of the mAb approach and strongly suggests that such ELISAs have potential for continued development as an alternative method to assay the potency of inactivated influenza vaccines.

Keywords: Influenza virus vaccine; monoclonal antibody; potency assay.

Figure 1

Binding and specificity of mAbs used to capture A/California hemagglutinin (HA). Purified murine mAbs (A–E) and rabbit polyclonal antibody (F) were used to coat ELISA plates at 2 μg/ml prior to binding dilutions of inactivated pandemic H1N1 (A/California), seasonal H1N1 (A/New Caledonia and A/Solomon Islands) and seasonal H3N2 (A/New York) antigen. (A) 4F8 mAb, (B) 5C12 mAb, (C) 4A10 mAb, (D) 3A7 mAb, (E) 1C5 mAb and (F) rabbit polyclonal antibody.

Figure 2

ELISA and single radial immunodiffusion (SRID) potency of A/California monovalent vaccine bulk samples. Potency and standard error of A/California monovalent vaccine bulks (MV) from manufacturer #2 (A) and manufacturer #3 (B) were determined by traditional SRID analysis and by ELISA using five A/California-specific mAbs.

Figure 3

ELISA and single radial immunodiffusion (SRID) potency of A/California monovalent vaccines. Potency and standard error of A/California monovalent vaccines (MVV) from manufacturer #1 (A), and manufacturer #2 (B), manufacturer #3 (C) and manufacturer #4 (D) were determined by traditional SRID analysis and by ELISA using five A/California-specific mAbs.

Figure 4

ELISA and single radial immunodiffusion (SRID) potency of trivalent vaccines containing A/California. Potency and standard error of trivalent vaccines containing A/California hemagglutinin (HA) from manufacturer #2 (A) and manufacturer #4 (B) were determined by traditional SRID analysis and by ELISA using five A/California-specific mAbs.

Figure 5

ELISA and single radial immunodiffusion (SRID) potency of A/California vaccine samples subjected to temperature stress. A/California vaccine from manufacturer #1 (A) and A/California vaccine bulk from manufacturer #2 (B) were incubated at 43°C for 1 week before analysis by SRID and ELISA using five A/California-specific mAbs. The percentage drop in potency compared to replicate vaccine samples stored at 4°C is indicated.