Clin Exp Vaccine Res.  2019 Jan;8(1):70-76. 10.7774/cevr.2019.8.1.70.

Development of a HA1-specific enzyme-linked immunosorbent assay against pandemic influenza virus A H1N1

• Shim DH
• Kim MJ
• Cha HR
• Park ES
• Kim AR
• Park JH
• Park HC
• Song D
• Lee JM

• KMID: 2438969
• DOI: http://doi.org/10.7774/cevr.2019.8.1.70

Abstract

PURPOSE
Enzyme-linked immunosorbent assay (ELISA) has been used in the diverse field to evaluate influenza virus infection; for the surveillance, diagnosis, efficacy evaluation, and development of the vaccine. The aim of this study was to establish an ELISA for detecting HA strain-specific antibodies using recombinant pandemic A H1N1 (pH1N1) HA1 (rHA1) protein.
MATERIALS AND METHODS
rHA1 was produced in baculovirus system. The clinical performance of the developed ELISA was validated using human serum samples, by comparison with standard methods for detecting a neutralizing antibody; hemagglutination inhibition (HI) assay and microneutralization test (MNT). The ability of the ELISA system to evaluate the efficacy test of an influenza vaccine was explored by measuring antibody levels in the serum of vaccinated mice.
RESULTS
Our ELISA could detect anti-rHA1 antibody in influenza-infected patients and vaccinated subjects. Compared to HI assay and MNT as reference methods, our method showed good performance in detection of anti-rHA1 antibody. Detection of the anti-rHA1 antibody in vaccinated mice and its correlation with titers in HI assay was also proved in a mice model.
CONCLUSION
An ELISA system using rHA1 of pH1N1 influenza virus was developed, and showed good clinical performance in diagnosis of influenza virus infection and evaluation of the vaccination efficacy in both human and animal models.

Keyword

Influenza A virus; H1N1 subtype; Infection; Enzyme-linked immunosorbent assay; Vaccination

MeSH Terms

Animals
Antibodies
Antibodies, Neutralizing
Baculoviridae
Diagnosis
Enzyme-Linked Immunosorbent Assay*
Hemagglutination
Humans
Influenza A virus
Influenza Vaccines
Influenza, Human*
Methods
Mice
Models, Animal
Orthomyxoviridae*
Pandemics*
Vaccination
Antibodies
Antibodies, Neutralizing
Influenza Vaccines

Figure

• 

  Fig. 1 Cloning and expression of recombinant HA1 proteins of pH1N1. (A) Synthetic HA1 gene sequence including restriction enzyme sites. (B) Sequences of the recombinant protein, HA1 gene of influenza virus A/New York/3571/2009 (H1N1) was cloned into the 6×-His-tagged baculovirus transfer vector pAcGP67A. (C) rHA1 protein using baculovirus system was determined by Coomassie blue staining and silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis. HA1, hemagglutinin 1; rHA1, recombinant H1N1 HA1 protein.

• 

  Fig. 2 Validation of the ELISA system in human. (A, B) rHA1 protein could detect anti-HA1-specific antibody effectively both in patients with influenza virus infection (A) and after vaccination (B). (C) A table of diagnostic performance on the ELISA system, in comparison with HI assay and MNT. ***p<0.001, patients vs. controls (A), pre-vaccination vs. post-vaccination (B). ELISA, enzyme-linked immunosorbent assay; rHA1, recombinant H1N1 HA1 protein; HA1, hemagglutinin 1; HI, hemagglutination inhibition; MNT, microneutralization test; CI, confidence interval.

• 

  Fig. 3 Measurements of anti-rHA1 antibodies in mice. (A) Experimental schedule of the vaccination and challenge in mice. (B, C) Body weight changes (B) and survival rate (C) in control and vaccinated mice after influenza virus infection. (D) The level of anti-rHA1 antibodies were measured by enzyme-linked immunosorbent assay in all groups of mice. (E) Anti-rHA1 antibodies were positively correlated with titers in HI assay (r=0.508, p<0.001). Black dots, PBS; gray dots, 0.2 µg; blue dots, 0.5 µg; red dots, 0.8 µg. rHA1, recombinant H1N1 hemagglutinin 1 protein; HI, hemagglutination inhibition; PBS, phosphate buffered saline; d.p.i., days post infection. (A) *p<0.05, ***p<0.001, compared to PBS group.

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