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Heamagglutination is inhibited when antibodies are present because antibodies to influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to inhibit heamagglutination about virus A/California/07/2009 (H1N1) and A/Brisbane/10/2007 (H3N2).

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Haemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies

Immunology > Antibody analysis > Antibody function
Authors: Ying Wu*
Ying WuAffiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3175
MyungSam Cho*
MyungSam ChoAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3176
David Shore*
David ShoreAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3177
Manki Song
Manki SongAffiliation: International Vaccine Institute, Seoul, Korea
Bio-protocol author page: a3178
JungAh Choi
JungAh ChoiAffiliation: International Vaccine Institute, Seoul, Korea
Bio-protocol author page: a3179
Tao Jiang
Tao JiangAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
Bio-protocol author page: a3180
Yong-Qiang Deng
Yong-Qiang DengAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
Bio-protocol author page: a3181
Melissa Bourgeois
Melissa BourgeoisAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3182
Lynn Almli
Lynn AlmliAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3183
Hua Yang
Hua YangAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3184
Li-Mei Chen
Li-Mei ChenAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3185
Yi Shi
Yi ShiAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3186
Jianxu Qi
Jianxu QiAffiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Bio-protocol author page: a3187
An Li
An LiAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: College of Veterinary Medicine, Guangxi University, Nanning, China
Bio-protocol author page: a3188
Kye Sook Yi
Kye Sook YiAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3189
MinSeok Chang
MinSeok ChangAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3190
Jin Soo Bae
Jin Soo BaeAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3191
HyunJoo Lee
HyunJoo LeeAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3192
JiYoung Shin
JiYoung ShinAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3194
James Stevens
James StevensAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Bio-protocol author page: a3195
SeoungSuh Hong
SeoungSuh HongAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
Bio-protocol author page: a3196
Cheng-Feng Qin
Cheng-Feng QinAffiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China
For correspondence: qincf@bmi.ac.cn
Bio-protocol author page: a3197
George F. Gao
George F. GaoAffiliation 1: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China
Affiliation 2: Research Network of Immunity and Health, Beijing Institutes of Life Science, Chinese Academy of Sciences, Beijing, China
Affiliation 3: Office of Director-General, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China
For correspondence: gaof@im.ac.cn
Bio-protocol author page: a3198
Shin Jae Chang
Shin Jae ChangAffiliation: Biotechnology Research Institute, Celltrion, Inc., Incheon, South Korea
For correspondence: ShinJae.Chang@celltrion.com
Bio-protocol author page: a3199
 and Ruben O. Donis
Ruben O. DonisAffiliation: Influenza Division, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
For correspondence: rvd6@cdc.gov
Bio-protocol author page: a3200
 (*contributed equally to this work)
Vol 6, Iss 11, 6/5/2016, 1116 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1828

[Abstract] Heamagglutination is inhibited when antibodies are present because antibodies to influenza virus will prevent attachment of the virus to red blood cells. The highest dilution of antibody that prevents hemagglutination is called the HI titer. The human monoclonal antibodies generated from single human B cells were tested to characterize their ability to inhibit heamagglutination about virus A/California/07/2009 (H1N1) and A/Brisbane/10/2007 (H3N2).
Keywords: Haemagglutination inhibition, Influenza, Antibody

Materials and Reagents

  1. Materials
    1. 96 well microtiter plates (V bottom) (Corning, catalog number: 3897 )
    2. Tips for multichannel pipette (Gilson, model: D10 , D200 and D1000 )
    3. Centrifuge tubes (SARSTEDT AG & Co, catalog number: 72.69 0)

  2. Reagents
    1. Viral antigen
      1. a. H1N1
        A/California/07/2009
      2. H3N2
        A/Brisbane/10/2007
        Note: Viruses were amplified in embryonated eggs. Refer to Manual for the laboratory diagnosis and virological surveillance of influenza, WHO.
    2. Erythrocytes
      Turkey red blood cells (RBCs)
      Note: Both in-house RBCs and commercial RBCs are usable.
    3. Antibody
      Human monoclonal antibodies CT146, CT147, CT149, CT164 and CD166
      Notes:
      1. Maximum concentration of antibody is 20 μg/ml.
      2. Expressed in CHO cell
      3. Purified according to manufacturer’s instruction [HiTrap™ MabSelect SuRe (GE Healthcare, catalog number: 11-0034-93 )]
    4. Other reagents
      1. Receptor destroying enzyme (RDE) (DENKA SEIKEN CO., catalog number: 370013 )
      2. Phosphate buffered saline (1x PBS) (pH 7.2) (Sigma-Aldrich, catalog number P4417 )
      3. Physiological saline (0.85% NaCl) (Sigma-Aldrich, catalog number: S9888 )

Equipment

  1. Haemacytometer (Hausser Scientific, catalog number: 1492 )
  2. Multichannel pipette (Gilson, model: PIPETMAN Neo® Multichannel )
  3. Centrifuge (Beckman Coulter, model: Allegra X-15R )
  4. Water bath (JULABO GmbH, model: MB13 )
  5. Inverted routine microscopy (Nikon Instruments Inc., model: TS-100 )

Procedure

  1. Preparation of erythrocytes
    1. Prepare the diluted RBC for washing step: 5 ml of RBC 45 ml of 1x PBS (room temperature).
    2. Washing step (just one time): Centrifuge the diluted RBC, 2,000 x g, 10 min, 25 °C.
    3. Discard supernatant and then suspend the RBS pellet with 20 ml (initial volume) of 1x PBS.
    4. 1:100 dilution (0.5 ml of RBC suspension from step A3 49.5 ml of 1x PBS).
    5. Transfer to 10 μl of RBC of step d onto hemacytometer and count the RBC in each of the 4 squares to calculate the final volume of RBC.
    6. Calculate the final volume of RBC by formula below (Formula is referred to Manual for the laboratory diagnosis and virological surveillance of influenza, WHO).
      1. Formula
        Final volume = total number of cells counted X initial volume /160
      2. Example of calculation
        Initial volume =20
        Total number of RBC = 320
        Final volume = 320 x 20/160 (160 is used as constant when use avian red blood cells; Chicken or turkey) = 40
        Then, add 20 ml of 1x PBS to 20 ml of RBC to make 40 ml and use it for HI assay.

  2. Heamagglutination (HAU) titration of viral antigen (Serial dilution is described on Figure 1)
    1. Add 50 μl of PBS to the V bottom 96 wells in column 2 to 12.
    2. Add 100 μl of each antigen to the first wells of rows.
    3. Make serial 2-fold dilutions by transferring 50 μl from the first to successive wells of each row using multi-channel pipette.
    4. Discard the final 50 μl.
    5. Add 50 μl of prepared RBCs to each well.
    6. Incubate the plate at room temperature for 30 min to 1 h.
    7. Record the HAU results refer to Example of HAU titers below Figure 2.


      Figure 1. Serial dilution of test sample for HAU titration. Add 50 μl of PBS to the all wells in column 2 to 12 of a v-bottom 96-well plate and then add 100 μl of test sample to the first well of each row. Transfer 50 μl of sample to the next wells using multi -channel pipette to make 2-fold dilution. Dilution factor is 1 to 2,048.


      Figure 2. Example of HAU titers. Hemagglutinin on the surface of influenza virus binds to the sialic acid receptors of red blood cells and then creates lattice structure. The agglutinated lattice maintains the red blood cells in a suspended and shows as a reddish solution. Heamagglutination titer (HAU) is the dilution factor of the last well showing reddish solution. HAU titer of sample 1 to sample 8 is 16, 8, 32, 32, 32, 16, 32 and 64.

  3. Preparation of standardized antigen for HI test
    1. Prepare 4 heamagglutination units (HAU) of antigen by dilution of antigen.
      Example of dilution: If antigen HAU is 16, 4 fold dilution with 1x PBS for 4 HAU.

  4. Heamagglutination inhibition (HI) test (Serial dilution is described on Figure 3)
    1. Add 25 μl of PBS to the V bottom 96 wells in column 2 to 12.
    2. Add 50 μl of the antibody (diluted with PBS, 1:10) to the well in row A1 to H1.
    3. Transfer 25 μl of the antibody from the column1 to 12 for 2 fold serial dilution.
    4. Add 25 μl of standardized antigen to all wells of plates.
    5. Mix the plates using a laboratory shaker for 10 sec or by manually agitating the plates thoroughly.
    6. Cover the plates and incubate at room temperature for 30 min.
    7. Add 50 μl of prepared RBC to the all wells and incubate at room temperature for 30 min.
    8. Observe the haemagglutination inhibition. If sample inhibits haemagglutination, RBCs appear as a dot by sink to bottom of well. Record the results refer to Example of HI titers below (Figure 4).


      Figure 3. Serial dilution of test sample for HI assay. Add 25 μl of PBS to all wells in column 2 to 12 of a V-bottom 96-well plate and then add 50 μl of test sample (pre diluted 1:10) to the first well of each row. Transfer 25 μl of sample to the next wells using multi -channel pipette to make 2-fold dilution. Dilution factor is 10 to 20,480.


      Figure 4. Example of HI titers. If antibodies bind to the viral particles, influenza virus are effectively blocked from causing hemagglutination. The HI titer value is the last dilution factor of antibody showing completely inhibited hemagglutination. HI titer of sample 1 to sample 8 is 160, 80, 80, 320, 80, 160, 80 and 80.

Notes

  1. Inactivation of nonspecific inhibitors (in case of using serum)
    1. Reconstitute the RDE with the volume of physiological saline (0.85% NaCl).
    2. Add 3 volumes of RDE to 1 volume of antibody.
    3. Incubate overnight in 37 °C water bath.
    4. Heat in a 56 °C water bath for 30 min to inactivate any remaining RDE.
    5. Cool at room temperature and add 6 volumes of physiological saline. The final dilution of antibody is 1:10.

Acknowledgements

We thank Dr. Jun Myung Kim and Dr. Sang Hoon Han for providing blood samples from Severance Hospital, and Dr. Yasuo Watanabe of SC World, Inc. for assistance in ISAAC assays for antibody screening. This study was supported by a grant of the Korea Healthcare technology R&D Project‚ Ministry of Health&Welfare‚ Republic of Korea. (Grant No. A103001), the National Natural Science Foundation of China (NSFC, Grant No. 81401671), the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDB08020100), the China National Grand S&T Special Project (Grant No. 2015ZX09304005) and. G. F. G. is a leading principal investigator of the NSFC Innovative Research Group (Grant No. 81321063). The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention or the Agency for Toxic Substances and Disease Registry.

References

  1. Manual for the laboratory diagnosis and virological surveillance of influenza. WHO global influenza surveillance network...


How to cite: Wu, Y., Cho, M., Shore, D., Song, M., Choi, J., Jiang, T., Deng, Y., Bourgeois, M., Almli, L., Yang, H., Chen, L., Shi, Y., Qi, J., Li, A., Yi, K. S., Chang, M., Bae, J. S., Lee, H., Shin, J., Stevens, J., Hong, S., Qin, C., Gao, G. F., Chang, S. J. and Donis, R. O. (2016). Haemagglutination Inhibition (HI) Assay of Influenza Viruses with Monoclonal Antibodies. Bio-protocol 6(11): e1828. DOI: 10.21769/BioProtoc.1828; Full Text



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