Abstract
To formulate inactivated influenza vaccines, the concentration of hemagglutinin (HA) must be accurately determined. The standard test currently used to measure HA in influenza vaccines is the Single Radial Immunodiffusion (SRID) assay.We developed a very rapid, simple and sensitive alternative quantitative HA assay, namely the Latex Agglutination Assay (LAA). The LAA uses the Spherotest® technology, which is based on the agglutination of HA-specific immunoglobulin-coated latex beads. The amount of HA in a sample is calculated from the level of bead agglutination by a simple absorbance measurement at 405nm against a standard curve generated using a monovalent vaccine standard.In less than 2hours, tens of samples could be quantified using the LAA as opposed to 2days for the SRID assay. Ten steps are required to complete an SRID assay as compared to 6 steps for the LAA, from sample preparation through spectrophotometric analysis. Furthermore, the limit of detection of the LAA was found to be approximately 15ng HA/mL, similar to an ELISA, with the quantification of less than 1.8μg HA/mL. The quantification limit of the SRID is usually considered to be approximately 5μg HA/mL.The development of the assay and a comparison of the titers obtained by SRID and LAA for several monovalent vaccines corresponding to various strains were performed. For A/H5N1 and A/H1N1 monovalent vaccines, the LAA was found to be linear and accurate as compared to the SRID. The precision of the LAA was close to that of the standard test, and good reproducibility from one laboratory to another was observed. Moreover, the LAA enabled HA quantification in AlOOH-adjuvanted and in emulsion-adjuvanted low-dose vaccines as well as unadjuvanted vaccines.In conclusion, LAA may be useful to rapidly and accurately measure influenza HA protein in monovalent vaccines, especially in those containing less than 5μg/mL of HA in the presence of an adjuvant.
Highlights
Influenza viruses are negative stranded RNA viruses of the Orthomyxoviridae family
The present study was conducted to develop a simple, sensitive and rapid alternative assay method that could be used on a routine basis to quantify influenza vaccine samples within two hours
Unlike other latex bead agglutination assays previously described for detecting influenza viruses (Chen et al, 2007), the Latex Agglutination Assay (LAA) presented here is based on the Spherotest® technology, which produced accurate HA quantification of seasonal and pandemic monovalent vaccines
Summary
Influenza viruses are negative stranded RNA viruses of the Orthomyxoviridae family. Three types of influenza viruses, influenza A, B and C, are capable of infecting humans, with influenza A and B being the most common circulating types. Influenza A viruses are classified into subtypes based on the antigenic identity of the two major surface glycoproteins on the virion, hemagglutinin (HA) and neuraminidase (NA). Both proteins lead to an antibody responses upon infection and antibodies against HA confer protective immunity (Gomez Lorenzo and Fenton, 2013), while antibodies against NA reduce severity of disease by restricting viral replication (Johansson and Cox, 2011). Global influenza epidemics emerge seasonally and typically occur during the winter seasons of the northern and southern hemispheres. Seasonal influenza epidemics result annually in 3–5 million cases of severe illness and 250,000–500,000 deaths worldwide (WHO, 2016). Vaccination is considered the most effective strategy to reduce the large morbidity and mortality caused by influenza infection (Poland et al, 2001; Zambon, 1999)
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