rHA1 was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the separated protein bands were visualized by Coomassie blue staining and silver staining. The proteins were transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA, USA) and blocked with 5% skim milk in phosphate buffered saline (PBS) containing tween-20 (PBST, 1× PBS and 0.05% Tween-20) for 1 hour at room temperature (RT). The membrane was incubated with an anti-HA1 mouse monoclonal antibody (1:1,000, ATGen, Seoul, Korea) in 0.2% bovine serum albumin (BSA) in PBST after 3 washes with PBST at RT. The membranes were incubated at 4℃ overnight. Horseradish peroxidase (HRP)-conjugated goat anti-mouse antibody (1:3,000, Bio-Rad) as a secondary antibody in 5% skim milk-PBST was added after PBST washing and incubated for 1 hour at RT. After washing with PBST 3 times, the membranes were developed using a chemiluminiescent substrate (Amersham ECL, GE Healthcare, Buckinghamshire, UK) and exposing the protein side of the membrane to X-ray film.

A total of 147 subjects was enrolled in this study. Forty-seven patients with influenza infection were confirmed by real-time polymerase chain reaction using nasopharyngeal swabs from patients with presenting fever. Blood samples from the patients were obtained at the time of H1N1 diagnosis. The control group of 30 subjects showed no flu-like symptoms or confirmed H1N1 infection. Samples from 70 subjects were collected before vaccination (pre-vaccination) and resampled at 4 weeks after vaccination with H1N1-inactivated influenza vaccine (post-vaccination). This study was approved by the Institutional Review Board Committee of Gangnam Severance Hospital (Seoul, Korea; IRB No. 3-2009-0170). Informed consents were obtained from all participants and all procedures were carried out in accordance with the relevant guidelines and regulations.

The HI assay was performed as described previously [5]. Briefly, sera were treated with receptor destroying enzyme at 30℃ for 20 hours and inactivated at 56℃ for 1 hour. The samples were serially diluted with PBS in the V-bottom 96-well plates then mixed with a standard amount of virus (4 HAU). The plates were incubated for 30 minutes at RT, and 0.5% of chicken red blood cells were loaded into each well and further incubated for 30 minutes at 25℃. The HI endpoint was the highest serum dilution at which agglutination was not observed.

Serum neutralizing antibody titers were determined by conducting a multi-well plate cell culture assay. Briefly, MDCK cells were seeded at 1.5×10⁴ cells/well in a 96-well culture plate and cultured at 37℃, and 5% CO₂ in an incubator to form a monolayer. Serially diluted samples pretreated with receptor destroying enzyme were mixed separately with 100 TCID₅₀ of A/New York/3571/2009 (H1N1) and incubated at 37℃ for 1 hour. The serum and virus mixture was added to the monolayer of MDCK cells. The plates were further incubated for 18 hours at 37℃ and washed with PBS 3 times, and fixed with 80% acetone for 10 minutes. The presence of viral proteins was confirmed by the monoclonal antibody against influenza nucleoprotein (NP), and the end point of the neutralizing titer was determined as the highest serum dilution at which NP was reduced by 50%.

Optimal dilutions of purified H1N1-rHA1 antigen were determined by various conditions and repeated experiments. For antigen coating, the indicated concentration of rHA1 antigens (7.5 ng/mL) was diluted in cold carbonate buffer (pH 9.6) in 96-well microplate and incubated at 4℃, overnight. Antigen-free carbonate the buffer was coated for endpoint assay and the background was subtracted. 3% of BSA in PBST was used for blocking (37℃ for 1 hour). After washing the plate 5 times, serum samples were diluted and pre-incubated with blocking buffer containing supernatant of the High-Five cell culture medium (10 µg/mL of protein concentration) at RT for 10 minutes. Next, 100 µL of diluted serum samples (1:100) was applied in duplicate to each antigen-coated and non-coated well and incubated for 90 minutes at 37℃. After the plates were washed 5 times with PBST, HRP-conjugated rabbit anti-human IgG (1:10,000, Sigma-Aldrich, St. Louis, MO, USA) and anti-mouse IgG Ab (1:5,000, Santa Cruz Biotechnology, Dallas, TX, USA) were added to each well matching the sample origin. Next, the plates were incubated for 90 minutes at 37℃ and washed 7 times with PBST. For color development, 100 µL of 3,3′,5,5′-tetra-methylbezidine in acidic buffer (KPL, Gaithersburg, MD, USA) was added to each well and incubated for 15 minutes at RT. The reaction was stopped by adding an equal volume of 2.5 N H₂SO₄. The optical density (OD) was determined at a wavelength of 450 nm using a microplate reader within 30 minutes. Endpoint titers of human H1N1-HA1-specific Abs were expressed as the OD₄₅₀ value of rHA1 uncoated well subtraction from rHA1-coated wells.

All data were expressed as the mean±standard deviation. Statistical comparisons between experimental groups were performed using the Student's t test. The sensitivity and specificity of ELISA for detecting the anti-HA1 specific antibody were reported by calculating the area under the receiving operator characteristic curve. The correlation between the anti-HA1 antibody and HI titer was determined by Pearson correlation test. GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA) was used for all analyses. p<0.05 was considered statistically significant.

Recombinant H1N1-HA1-18-344 (rHA1) was expressed in the baculovirus insect system (Fig. 1A, B). Proteins were produced by High-Five cells after vector transformation. Purified rHA1 protein was confirmed by Coomassie blue and silver staining (Fig. 1C). H1N1 proteins of the expected size of 46 kDa were isolated.

The ELISA system using rHA1 protein was developed and validated to detect anti-rHA1 antibodies in the serum samples from influenza-infected patients and controls. According to this ELISA, OD values of patients were increased than controls (0.942±0.371 vs. 0.212±0.086, p<0.001) (Fig. 2A). And in vaccinated individuals, anti-rHA1 antibody levels were significantly increased after vaccination compared with levels from pre-vaccination status (Fig. 2B). From these data, this ELISA system using rHA1 could be useful in diagnosis of influenza infection and in discrimination of vaccination status in human.

The ELISA system was compared to standard detection methods for influenza virus infection, such as the HI assay and MNT. As shown in Fig. 2C, this ELISA system could be a sensitive and specific method for detecting the anti-rHA1 antibody in influenza-infected patients. The detection performance of this ELISA system was comparable to that of the HI assay and MNT.

The ELISA system using rHA1 protein was also validated in an animal model. The control (PBS) and vaccinated groups were compared. The vaccinated groups were immunized with influenza virus A/California/04/2009 (CA04) vaccine. The vaccines contained 3 different amounts of antigens, such as 0.2, 0.5, and 0.8 µg in each single dose of vaccine, respectively. All mice were immunized with PBS or vaccines twice in 2 weeks apart and challenged at 2 weeks after the last vaccination (Fig. 3A). Body weight changes and the survival rate of vaccinated and non-vaccinated groups were monitored after influenza virus challenge (Fig. 3B, C). Compared to the control PBS group, the vaccinated groups showed lower body weight changes (Fig. 3B) and death rates (Fig. 3C). The serum levels of anti-rHA1 antibodies were measured in both non-immunized and immunized mice by ELISA. The increased levels of antibodies were detected in all vaccinated mice but not in PBS group. The amount of antigens in vaccines did not affect the production of anti-rHA1 antibodies in this study (Fig. 3D). And the levels of anti-rHA1 antibodies were significantly correlated with the results of HI assay using the same samples (r=0.508, p<0.001) (Fig. 3E).

Taken together, anti-rHA1 antibodies were detected by the ELISA system in vaccinated mice. The positive correlation between anti-rHA1 antibody measured by ELISA and titers in HI assay was also observed in the animal model.