Monoclonal Antibody Preparation Against Nucleoprotein of Avian Influenza Virus Subtype H9N2


Maryam Hashemi ORCID 1 , Rasool Madani ORCID 2 , * , Mahmoudreza Aghamaali ORCID 3 , Tara Emami ORCID 2 , Fariba Golchinfar ORCID 2

1 Department of Biology, University Campus 2, University of Guilan, Rasht, Iran

2 Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran

3 Department of Biology, Faculty of Sciences, University of Guilan, Rasht, Iran

How to Cite: Hashemi M, Madani R, Aghamaali M, Emami T, Golchinfar F. Monoclonal Antibody Preparation Against Nucleoprotein of Avian Influenza Virus Subtype H9N2, Iran Red Crescent Med J. Online ahead of Print ; 21(10):e95906. doi: 10.5812/ircmj.95906.


Iranian Red Crescent Medical Journal: 21 (10); e95906
Published Online: November 3, 2019
Article Type: Research Article
Received: July 2, 2019
Revised: October 3, 2019
Accepted: October 8, 2019




Background: In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide. Influenza virus type A, which belongs to the family Orthomyxoviridae, is responsible for a serious infectious disease. H9N2 AVI subtype circulates in the poultry worldwide, causing significant economic losses and infections in humans, also domestic and wild animals.

Objectives: The purpose of the present experimental research was monoclonal antibodies (MAbs) production in contradiction of the NP of avian influenza virus (AIV) H9N2 subtype to detect AIV antigens and antibodies in the Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran. in 2017.

Methods: The conserved protein of NP from H9N2A/Chicken/Iran/259/2014 virus, with 60 kDa molecular weight, was isolated using the electroelution method. The purified protein was applied for Australian BALB/c mice immunization. After evaluating immunization by ELISA assay, the spleen of immunized mice was isolated and hybridized with SP2/0 myeloma cell. Next, the hybridized cell was cultured, and clone soups were collected after 15 days to examine antibodies via ELISA assay. The produced antibodies using Western blotting and antibody isotype kits were characterized.

Results: Thirty antibody-producing clones were examined for reactivity against Nucleoprotein (NP), the antigen, at 1 µg/mL concentration. According to the ELISA assay of antibody titers, two (3/F10 and 2/D7) out of 30 antibodies were bound to the antigen with titer 0.863 and 1.641, respectively. Two hybrid clones, 2D7 and 3F10, which produced anti-NP antibodies, were isolated and cultured. Characterizing of the produced antibodies using Western blotting was performed using H9N2 virus; finally, two clone soups (3/F10 and 2/D7) reacted with the NP virus protein. According to the isotyping of antibodies produced by 3F10 and 2D7 clones, 3F10 clone produced IgG1 with a κ chain, and IgG1 concentration was 1.997, based on ELISA assay. Also, 2D7 clone produced IgG2a with a κ chain, and IgG2a concentration was obtained 1.951.

Conclusions: According to the findings of our study, the produced antibody might be used in the diagnosis of influenza.


Avian Influenza Virus H9N2 Subtype Inbred BALB C Influenza A Virus Influenza in Birds Mice Monoclonal Antibody Nucleoprotein (NP) Poultry Diseases

Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License ( which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited

1. Background

In recent years, various poultry diseases have posed risks to this industry. Respiratory and infectious diseases are the most common diseases, which are caused by microbial factors, such as viruses, bacteria, and fungi. According to previous findings, influenza disease is recognized as a significant life-threatening disease in the industry of poultry worldwide (1).

Avian influenza virus (AIV) H9N2 strain was isolated from turkey in the United States in 1966, firstly (2). Though viruses of H9 are categorized as low-pathogenicity avian influenza viruses (LPAI), a fatality rate above 50% has been reported (3) and making it difficult for poultry veterinarians to diagnose (4).

H9N2 AVI subtype circulates in the poultry worldwide, causing significant economic losses and infections in humans, also domestic and wild animals (3, 5, 6). H9N2 AIV can transmit from birds to humans and other mammals such as pigs (7-10). H9N2 human infections have been incessantly reported, but there were no H9N2 vaccines available in humans (11). Generally, AIV from the family Orthomyxoviridae is polymorphic, with a single-stranded, eight-part RNA genome (12). Type A is an epidemic factor and known as the most virulent and contagious in poultry influenza (13).

At present, the most appropriate strategy for influenza management is early detection and control (14). An important part of influenza control programs is the accurate and rapid detection of viruses in different animal species. Respiratory illnesses have similarities to the symptoms; therefore, diagnosis is important issue in the management of this pandemic (15). Currently, serological diagnostic techniques, such as agar gel propagation (AGP), enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI), are usually applied as important tools for antibodies detection towards specific viral antigens (16). Generally, effective detection and monitor are necessary for the influenza infection management (17).

The results of some studies have shown that internal proteins, such as NPs, are one of the significant influenza A virus determinant; therefore, NP is a specific type of antigen, useful for influenza viruses A, B, and C detection. It is known that NP is a highly conserved antigen among a variety of influenza viruses (18). Accordingly, isolation of conserved NPs from H9N2 viruses and preparation of monoclonal antibodies against NPs can be helpful in identifying the virus in different species, based on serological methods (19-22) and competitive ELISA assay (23).

Use of monoclonal antibodies against conserved proteins, such as NP, can be introduced as an appropriate strategy for immediate detection of influenza virus in a variety of animal species; this method can increase the diagnosis efficiency and accuracy, in comparison with polyclonal antibodies (24). NPs, which are suitable for serological tests given their conservation among different influenza A virus strains (25, 26), can detect antibodies to all AIV subtypes.

2. Objectives

In the current research, monoclonal antibodies (mAbs) were manufactured and characterized against the purified H9N2 NP for use in immunodiagnostic tests.

3. Methods

3.1. Viruses

AIV strain A/flash chicken/Iran/772/1998 (H9N2) was acquired from the Department of Influenza Reference Laboratory, Razi Vaccine and Serum Research Institute (Karaj, Iran) and stored at -70°C 1n 2017.

3.2. Isolation of NP Antigen via Gel Electrophoresis

In this experimental study, that performed in the Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran, Standard bovine serum albumin (BSA) protein (Sigma, St. Louis, MO, USA) was used, based on the Lowry method to determine the amount of proteins in the viral solution (27). For analyzing proteins with biological activity, a non-denaturing system is required. For this purpose, a non-denaturing system, according to the Davis method, was used to purify proteins (28, 29). NP was separated via polyacrylamide gel electrophoresis (PAGE). A Model 422 Electro-Eluter (Bio-Rad) was also used based on the manufacturer’s guidelines to recover NP.

The concentration of purified protein was defined by the Lowry method (27) at an optical density (OD) of 750 nm, and protein quantity was calculated by using BSA (Sigma, USA) as standard. The purified NP protein (60 kDa) was finally analyzed based on Tricine-SDS-PAGE method (30). It should be noted that Tricine-SDS-PAGE gels are usually applied to segregate proteins below 100 kDa.

3.3. Immunization with NPs

Two female BALB/c mice (4 - 6 weeks), acquired from Razi Vaccine and Serum Research Institute (Iran), subcutaneously were immunized with 40 µg of purified NP, mixed in a complete Freund’s adjuvant (Sigma, USA). Also, using the same protein in incomplete Freund’s adjuvant (Sigma, USA), three booster vaccinations were performed within three-week intervals. Blood sampling was performed one week after the third boost (31). The polyclonal antiserum titer was measured after the final immunization via indirect ELISA, using AIV NP as antigen (32).

3.4. Generation and Selection of Hybridoma Cells

The SP2/0-Ag14 myeloma cell line expansion was accomplished in RPMI-1640 medium, supplemented with 20% fetal bovine serum (FBS) one week before fusion. On the day of cell fusion, the myeloma cells total number in the spleen of the mouse was 1 × 108 cells in two flasks. Final immunization was carried out with the same antigen in saline at a dose of 28 µg via intravenous injection three to four days before fusion. One day before fusion, a non-immunized BALB/c mouse spleen cells with Sp2/0 myeloma cells were sowed in 96-well plates of hybridoma cells to support the growth of hybridoma cells.

On the day of fusion, myeloma cell line SP2/0-Ag14 was maintained in the log phase and examined by an inverted microscope to assure sufficient growth of cells for fusion. Next, spleen cells of mouse were applied to fuse with Sp2/0 myeloma cells at a 4:1ratio in the 50% polyethylene glycol (PEG; Sigma) presence. The cell suspension (100 µL) was gently aspirated to every well of 96-well flat-bottom plate and at 37°C incubation in a 5% CO2 incubator.

Afterward incubation during three days, the wells were examined under an inverted microscope. Next, 100 µL of hypoxanthine aminopterin thymidine (HAT; Sigma) and 20% (v/v) FBS in RPMI medium (Gibco Invitrogen Co., Paisley, UK) were added to each well and placed in a 5% CO2 incubator at 37°C. On days 2, 4, 6, 8, and 10, the cells were fed with HAT, and on day 12, the feeding protocol was repeated using hypoxanthine-thymidine (HT) medium. From day 16 onwards, the wells were not fed with HAT or HT. The hybridomas were prepared to screen, as the majority of wells containing the grown cells demonstrated the confluence of 10% - 25%.

3.5. Screening of Secreted Antibodies by ELISA Assay

Hybridoma supernatants containing the grown cells in the wells were screened using an NP antigen via indirect ELISA assay. NP antigens were coated onto a Maxisorp microtiter plate (Nunc, Roskilde, Denmark) at an optimal concentration of 1 µg/mL and overnight incubation at 4°C in a coating buffer (0.1 M carbonate/bicarbonate; pH: 9.6). Next, the antigen-coated plate was blocked with 200 µL/well of blocking buffer (5% non-fat dry milk in PBST) for 90 minutes. Then, 100 µL of hybridoma supernatant was added to every well with 75 minutes incubation. In addition, 100 µL of horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:1000; Sigma, USA) was added to each well, and the plate was incubated for 75 minutes. Finally, the plate was incubated with 100 µL of BM Blue POD substrate (Roche Diagnostics GmbH, USA) for 20 minutes in darkness at room temperature.

By adding 0.1 M sulfuric acid the reaction was terminated, and OD was read at 450 nm by a microplate spectrophotometer (BioTek Instruments Inc., USA). Every step of incubation was conducted at 37°C after that with three times plate washing using a washing buffer (0.01 M PBS; pH: 7.5), comprising 0.05% (v/v) Tween-20 (PBST). Hybridoma supernatants, which tested positive on NP-based ELISA assay, were transferred to a 24-well plate and grown in a CO2 incubator. Then, positive clones were grown to larger volumes and immediately frozen in liquid N2 in FBS (Gibco, USA), containing 10% DMSO.

3.6. Characterization of mAbs Against NP

3.6.1. ELISA Assay

ELISA assay was used to screen clone antibodies by coating NP with the concentration obtained using the checkerboard method. The clone soup was evaluated to determine the antibody titer.

3.6.2. Western Blotting

Western blotting was applied to approve the reactivity of different mAbs using AIV H9N2 subtype. The H9N2 virus protein solution was subjected to 10% SDS-PAGE under reduced conditions. Next, protein electrophoresis was performed and protein bands onto a nitrocellulose membrane, by a Trans-Blot semi-dry transfer cell (BioRad, USA) were transferred. After blocking the blotted membrane in 3% BSA solution, it was incubated for 90 minutes at room temperature. The membrane was washed with PBST three times for 10 minutes. Then, with the hybridoma supernatant, the membrane was incubated overnight at 4°C. With PBST for three minutes, the membrane was washed five times and then incubated again for one hour in a rabbit anti-mouse IgG secondary antibody (HRP-conjugated; 1:1000 in 5% BSA). After washing the membrane, it was immersed in a precipitating substrate solution, containing chloronaphthol for 30 minutes until the protein band appeared.

3.7. Determination of Antibody Isotypes

By a mouse mAb isotyping kit (Amersham Bioscience, UK) and ELISA assay isotyping were performed. ELISA assay was conducted based on the formerly explained method with an NP protein concentration of 1 µg/mL. Then, anti-mouse IgG2a antibodies, and anti-mouse IgG, anti-mouse IgG1were added to the conjugated peroxidase phase, and the rest of the procedure was carried out similar to the indirect ELISA method.

4. Results

4.1. NP Protein Purification

Based on the Lowry method, the protein amount in the solution of purified protein was 0.296 mg/mL. The analysis results of Tricine-SDS-PAGE demonstrated only one protein band with a 60 kDa molecular weight (Figure 1).

Analysis of Tricine-SDS-PAGE of purified NP protein
Figure 1. Analysis of Tricine-SDS-PAGE of purified NP protein

4.2. Hybridoma Cell Production and Checkerboard Method

To obtain the optimal antigen concentration for evaluation of monoclonal antibodies, checkerboard method was applied. For this purpose, serial concentrations of NP protein were coated, and serum samples of immunized mice were evaluated at different dilutions against the positive control serum. All experiments were done triplicated. The optimal antigen concentration was found to be 1 µg/mL (Table 1).

Table 1. Results of Checkerboard Method for Determining the Optimal Antigen Concentration of NP Protein
Dilution of sera ↓Serum of mice ↓0.25 µg/mL of NP antigen0.5 µg/mL of NP antigen1 µg/mL of NP antigen2 µg/mL of NP antigen
1:20Mouse 10.5320.7990.9910.968
Mouse 20.6300.8540.9321
1:40Mouse 10.4980.6800.7360.718
Mouse 20.5740.6360.5910.593
1:80Mouse 10.3580.4070.4700.366
Mouse 20.3780.4950.5880.337

4.3. Screening of Clones for Antibody Production by ELISA Assay

Thirty antibody-producing clones were examined for reactivity against NP (antigen) at 1 µg/mL concentration, which was determined based on the checkerboard method using indirect ELISA assay. All experiments were done triplicated. According to the ELISA assay of antibody titers, two (3/F10 and 2/D7) out of 30 antibodies were bound to the antigen (Table 2).

Table 2. Screening of Clones for Antibody Production by ELISA Assay
Clone Screening/NP-Coated, 1 µg/mL
Positive control2.442
Negative control0.2

4.4. Western Blotting of Hybridized Clones and Polyclonal Sera

Western blotting was performed using H9N2 virus, which was subjected to 10% SDS-PAGE under reduced conditions. As can be seen, two clone soups reacted with the NP virus protein. Also, Western blotting of polyclonal serum was performed with several immune response protein bands (Figure 2).

Western blotting screening of different hybridoma supernatants via the analysis of SDS-PAGE of AIV H9N2 subtype. Lane 1: molecular weight marker; lane 2: serum of an immunized mouse with H9N2; lane 3: 2D7 hybridoma supernatant; lane 4: 3F10 hybridoma supernatant; and lane 5: SDS-PAGE results of AIV H9N2 subtype.
Figure 2. Western blotting screening of different hybridoma supernatants via the analysis of SDS-PAGE of AIV H9N2 subtype. Lane 1: molecular weight marker; lane 2: serum of an immunized mouse with H9N2; lane 3: 2D7 hybridoma supernatant; lane 4: 3F10 hybridoma supernatant; and lane 5: SDS-PAGE results of AIV H9N2 subtype.

4.5. Isotyping of Antibodies Produced by 3F10 and 2D7 Clones

Isotyping of antibodies produced by 3F10 and 2D7 clones was examined quantitatively using isotyping and ELISA assays. According to the qualitative method, 3F10 clone produced IgG1 with a κ chain, and IgG1 concentration was 1.997, based on ELISA assay. Also, 2D7 clone produced IgG2a with a κ chain, and an IgG2a concentration was obtained 1.951 (Figures 3 and 4 and Table 3).

Table 3. Isotyping of Antibodies Produced by 3F10 and 2D7 Clones Based on ELISA Assay
Isotyping IgGTotal IgGIgG1IgG2a
Isotyping of antibodies produced by 3F10 and 2D7 clones based on the qualitative method
Figure 3. Isotyping of antibodies produced by 3F10 and 2D7 clones based on the qualitative method
Isotyping of antibodies produced by 3F10 and 2D7 clones based on the qualitative method
Figure 4. Isotyping of antibodies produced by 3F10 and 2D7 clones based on the qualitative method

5. Discussion

Avian influenza is a significant viral disease worldwide. H9N2 viruses have relatively fewer pathogenesis types, whereas over the past decade, there have been many reports of damage or loss in these strains (3, 14, 33). NP is the most important influenza virus internal protein, which can help detect influenza viruses A, B, and C. Previously research have shown that NP can be a proper target for the antiviral drugs development against influenza virus different types by stimulating immune responses (34, 35).

AIV NP is conserved between different subtypes and strains of the influenza virus (36). In the present study, NP was purified using the electroelution method on the Native PAGE gel. Native gel was used to preserve the biological activity and native structure of the protein. Considering the low amount of viruses, methods such as chromatography were not applicable for purifying the protein. Generally, the technique used in the present study is applicable not only for soluble proteins, but also for membrane proteins. The separation of different proteins by this method has been also reported in previous studies (37).

The protein that purified was evaluated using Tricine-SDS PAGE, and a 60-kDa band was observed. Therefore, the immune cells of spleen were hybridized with myeloma SP2/0 cells. Since the mice were immunized with NP, all clones producing antibodies were directed against NP. In addition, ELISA assay was used to screen the antibodies of clones by coating the NP protein. Clones secreting antibodies were cultured and transferred to new flasks. Frequent screening of 30 clones based on ELISA assay indicated two clones with high antibody titers. Also, clone F10 from plate 3 and clone D7 from plate 2 were cultured in new flasks, and their antibodies were evaluated. In addition, the antibodies were characterized based on Western blotting, and then, antibody isotypes were identified.

HI test is the most common method for evaluating antibody titers against influenza virus. This test evaluates the increase in antibody titers against the virus by controlling blood clots. In this test, antibodies act against HA of viruses and prevent the binding of virus by the red blood cells. However, this process is relatively insensitive for antibody detection (38).

Although NP is a reasonably well-preserved AIV protein with antigenic variations, it occurs much more slowly than HA and NA proteins (39). Analysis of phylogenetic of virus strains was derived from various hosts discovered that the NP gene might vary up to a maximum amino acid difference of 11% (40). Therefore, it is essential to apply NP antigens derived from circulating AIV strains for the mAbs development in order to increase the sensitivity of mAb-based diagnostic assays.

In the study, it is the first time that native NPs antibodies purified from mice were investigated. Recombinant NPs don’t have post-translational modifications and often have some additional amino acids which interrupt the production of native antibodies, but it is a time-consuming process. Generally, mAbs against NP protein is a key part in the diagnosis of AIV infections (41). In the current research, mAbs were developed against type A-specific NPs, derived from the H9N2 subtype, for their probable usage in detection tests of antigen or antibody against all type-A influenza viruses in poultry. The findings showed that mAbs against NP could be useful in the development of the immunochromatographic tests for detection of antigen or competitive ELISA assays for diagnosis of AI antibody in the poultry.



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