Serological detection of avian influenza A(H7N9) virus infections by modified horse red blood cells haemagglutination-inhibition assay (WHO, December 23 2013, edited)

[Giuseppe]

[Source: World Health Organization, full PDF document: (LINK). Edited.]


Laboratory Procedures

Serological detection of avian influenza A(H7N9) virus infections by modified horse red blood cells haemagglutination-inhibition assay

20 December 2013


The WHO Collaborating Center for Reference and Research on Influenza at the Chinese National Influenza Center, Beijing, China, has made available attached laboratory procedures for serological detection of avian influenza A(H7N9) virus infections by modified horse red blood cells haemagglutination-inhibition assay.

This assay supersedes the assay using turkey red blood cells posted on 23 May 2013. The modified assay has been shown to be more sensitive.

For further information please contact us at: gisrs-whohq@who.int


SEROLOGIC DETECTION OF ANTIBODIES TO AVIAN INFLUENZA A(H7N9) VIRUS BY MODIFIED HORSE RED BLOOD CELLS (RBCS) HAEMAGGLUTINATION INHIBITION (HI) ASSAY IN HUMAN SERA

INTRODUCTION

The haemagglutination-inhibition (HI) assay is a traditional method for assessing immune responses to influenza virus haemagglutinin (HA). The HA protein on the surface of influenza virus agglutinates erythrocytes.

Specific attachment of antibody to the antigenic sites on the HA molecule interferes with the binding between the viral HA and receptors on the erythrocytes. This effect inhibits haemagglutination and is the basis for the HI assay.

The presence of specific anti-HA antibodies will inhibit the agglutination, which would otherwise occur between the virus and the RBCs. The choice of the erythrocytes to use in the HI assay is dependent on the virus being tested.

Human influenza viruses bind preferentially to SA receptors containing α2,6-Gal when avian influenza viruses preferentially bind to those containing α2,3-Gal. Horse RBCs express a high proportion of SA α2,3-Gal linkages compared with turkey RBCs.

With H7N9 influenza virus, a significantly improved sensitivity has been observed using horse RBCs for detecting HI antibodies in confirmed H7N9 cases sera than using turkey RBCs.

However, the below factors need to be considered when conducting HI assay using horse RBCs:

1. Higher levels of non-specific agglutinins can be detected in sera samples using horse RBCs.
   • When nonspecific agglutinins are present in serum, agglutination occurs due to the interaction of the nonspecific agglutinins with the RBC.
   • This is independent from the haemagglutination that occurs through the interaction of the influenza virus and the RBC.
   • Specific antibodies to the virus can inhibit the virus-RBC interaction, but may have no impact on the nonspecific agglutinin-RBC interaction resulting in a false negative result in the HI assay.
   • The presence of nonspecific agglutinins can be observed when diluted serum and a solution of RBC are combined and haemagglutination occurs.
   • The serum must be adsorbed with RBC before testing in the HI assay.
   
   
2. During adsorption with horse RBCs, non-specific virus inhibitors may be introduced into serum, which will cause a false positive result in HI assay with horse RBC.
   • We have observed that these non-specific inhibitors can be eliminated by RDE treatment.
   • Sera need to be RDE treated after horse RBC haemadsorption prior to the horse HI assay.
   • Here we describe an improved horse HI assay with modified order of sera treatment procedures (haemadsorption and then RDE treatment) that are necessary to remove non-specific agglutinins to horse RBC as well as nonspecific inhibitors after haemadsorption.
   
   
3. Non-specific false positive HI antibodies were observed in plasma (with anticoagulant of heparin) samples from H7N9 confirmed cases. Plasma is not proper for case diagnosis and seroepidemiology survey when using HI assay.

MATERIALS, SUPPLIES AND EQUIPMENTS

A: MATERIALS and SUPPLIES

1. Influenza virus
   • Live virus
   • BPL inactivated virus can be used in place of the live virus if:
     • 1). It is demonstrated that BPL inactivated virus will yield equivalent HI results compared with live virus.
     • 2). BPL inactivation of the virus is confirmed prior to lowering the biocontainment level from BSL-3 to BSL-2 for laboratory testing.
   
   
2. Serum samples.
   • Note: Serum samples should not be repeatedly freeze-thawed. Ideally, aliquot and store sera at -20 to -70?C.
   
   
3. Horse RBCs in Alsever?s Solution
   • Note:
     • 1). Horse RBCs in Alsever?s solution can be used at a concentration of 1.0% in PBS + 0.5% BSA.
     • 2). It is preferred that the horse blood cells in Alsever?s solution should be as fresh as possible.
   
   
4. Phosphate buffered saline (PBS) (HyClone Cat #SH30256.01) or equivalent
5. Bovine serum albumin, fraction V, protease-free, Roche, cat. # 03116964001 or equivalent
6. 70% EtOH
7. 0.85% saline, sterile
8. Receptor destroying enzyme, RDE (II) ?Seiken?. (Denka Seiken Co., Ltd, cat # 370013 or equivalent) RDE is supplied as a filter-sterilized, lyophilized culture supernatant of Vibrio cholerae Ogawa type 558.
   • Note: Reconstitute each vial of RDE with 20 ml of 0.85% saline or PBS. Use immediately or freeze in single use aliquots at -20?C or colder.
   
   
9. Sterile cotton gauze pads (Fisher, cat. # 22-415-469 or equivalent)
10. Conical centrifuge tubes
11. Assorted pipettes and tips for appropriate volumes.
12. Disposable reservoirs for multi-channel pipettes.
13. Titertube Micro test tubes (Bio-Rad, cat. # 223-9390 or equivalent) and Titertube Plugs (Bio-Rad, cat. # 223-9393 or equivalent)
14. 96-well PCR plate and cover for large-scale screening of human sera
15. 96-well, V-bottom, polystyrene, microtiter plates (Costar, cat. # 3897)

B. Equipment

1. Class II Biological Safety Cabinet (BSC)
2. Haemocytometer
3. Low speed, bench-top centrifuge
4. Standard microscope with 10X cular and 10X or 20X objectives lenses
5. 37?C and 56?C water bath
6. Automation workstation (Beckman Biomek FX,if do large-scale screening of human sera)

METHODS

Horse HI assay can be performed with below steps:

• I. Preparation of 1.0% horse RBCs
• II. Adsorption of Serum with Horse RBCs to Remove Nonspecific Agglutinins
• III. Testing for Nonspecific Agglutinins
• IV. RDE-treatment of Serum to Remove Nonspecific Virus Inhibitors
• V. Determination of HA titer of influenza virus
• VI. HI assay with horse RBCs

• I. Preparation of 1.0% horse RBCs
  • Notes:
    • 1). Horse RBC is prepared at 1.0% (v/v). To standardize the RBC concentrations, cells can be counted by haemocytometer. In our laboratory, the target concentration of a 1.0% (v/v) horse RBC solution has been empirically determined to be approximately 1.5 x 108 cells / ml.
    • 2). The 1.0% RBCs are prepared the day that sera are tested for nonspecific agglutinins and may be stored at 4? C for use on the second day in the HI assay. Discard at the end of the second day.
    
    
  • 1. To start preparation of packed RBCs, carefully collect, using a 10 ml pipette, 5-7 ml of horse RBCs from the bottom of the tube. Remove horse RBCs from the bottom of the container to minimize contamination with cell fragments. Filter through a sterile cotton gauze pad into a 50 ml conical centrifuge tube.
  • 2. Gently fill the conical tube with cold PBS +0.5% BSA and mix gently by inversion.
  • 3. Centrifuge at 2000rpm (for example: 2000 rpm in Thermo 75006445 rotor, Thermo Scientific? Sorvall? Legend? T Plus centrifuge) for 5 minutes at 4?C.
  • 4. Aspirate the supernatant using a 10 ml pipette. Be careful to not disturb the pellet of RBCs.
  • 5. Gently fill the conical tube with cold PBS +0.5% BSA and mix gently by inversion.
  • 6. Centrifuge at 2000rpm for 5 minutes at 4?C.
  • 7. Aspirate the supernatant using a 10 ml pipette. Be careful to not disturb the pellet of RBCs.
  • 8. Carefully repeat the cold PBS +0.5% BSA wash (steps 5-7) one more time for a total of three PBS washes to prevent hemolysis, always handle the RBCs gently, keep the PBS on ice or at 4?C, and do not wash more than 3 times.
  • 9. Aspirate the remaining supernatant with a P1000 microliter pipette for final packed RBCs. Keep packed RBCs on ice.
  • 10. Prepare a 1.0% v/v suspension of RBCs.
    • For example, add 2.5 ml of the packed RBCs from step 9 to 247.5 ml cold PBS +0.5% BSA in a 500 ml glass bottle (rinse with PBS before use). Mix gently by swirling.
    
    
  • 11. To count the RBCs, prepare a 1:200 dilution of the RBC suspension by adding 50 μl of the suspended RBCs (from step 10) to 10 ml cold PBS in a 15 ml conical, polystyrene tube. Mix gently by inversion.
  • 12. Clean the haemocytometer thoroughly with 70% ethanol and dry with lens tissue or a soft lint-free cloth. Clean and dry the cover slip in the same way and place it gently onto the haemocytometer so that it covers the counting area.
  • 13. Transfer 10 μl of the 1:200 diluted RBC (from step 11) onto the haemocytometer loading channel and allow the cells to spread throughout the unit, being careful not to overfill the channel.
  • 14. Count the RBCs in each of the 4 large corner squares of the haemocytometer as shown in Figure 1
  • (...)
  • 15. After counting the RBCs, rinse the chamber and cover slip with 70% ethanol and dry.
  • 16. Based on the RBC count, add cold PBS +0.5% BSA, or more packed RBC to obtain final concentration of 1.5 X 108 cells / ml (1.0% horse RBC).
  
  
• II. Adsorption of Serum with Horse RBCs to Remove Nonspecific Agglutinins
  • 1. Heat-inactivate aliquoted serum samples at 56?C for 30 minutes, then cool down at room temperature.
  • 2. Add 4 part of cold PBS to pre-dilute each serum sample in 1:5.
  • 3. Combine 1 part packed horse RBCs (from step I-9) with 9 parts 1:5 pre-diluted serum in tube (1:10 v/v).
  • 4. Gently invert to mix and incubate for 15 minutes at 4?C for adsorption.
  • 5. Gently invert again and incubate an additional 15 minutes at 4?C for adsorption.
  • 6. Centrifuge at 2000rpm for 5 minutes at 4?C to pellet down RBCs.
  • 7. Carefully remove the adsorbed serum without disturbing the packed cells. Usually, if we do a large number of sera screening by using automation workstation, we prefer to transfer 85% of pre-diluted serum. If we do case serum detection, the total amount of diluted serum recovered manually will be similar to the volume of diluted serum added. The final serum dilution after adsorption is still approximately 1:5.
    • For example (automation workstation): if 20 μl of original serum was pre-diluted into 1:5 to make a total volume of 100 μl, usually 85 μl diluted serum could be recovered after adsorption without disturbing the packed cells.
  
  
• III. Testing for Nonspecific Agglutinins
  • 1. Add 25 μl of PBS to V bottom plate in rows A through H (A1-A11 to H1-H11). Row A (A1-A11) will serve as the sera loading wells.
  • 2. Add 25 μl of each serum after adsorption to the wells A1-A11.
  • 3. Prepare serial 2-fold dilutions by transferring 25 μl of serum from the first row A to H. Discard 25μl after row H.
  • 4. Add 25 μl of PBS to wells containing sera, columns 1-11 for a total volume of 50μl.
  • 5. Add 50 μl of PBS to column 12 on the plate as a RBC control.
  • 6. Gently tap the plates to make sure the sera mix with PBS.
  • 7. Add 50 μl of 1.0% of horse RBCs to all wells.
  • 8. Gently tap the plates to mix. Stack plates and cover with an empty plate.
  • 9. Incubate at room temperature, 22?C to 25?C, for 60 minutes to allow the RBCs to settle.
  • 10. Tilt the plate at 45?to 60? angle. The settled RBCs in column 12 should start ?running? and forming a tear-shape due to gravity. Wait until these RBCs finish ?running? and then record samples where agglutination of RBCs occurred. Agglutinated RBCs do not ?run? or cause a tear-shaped spot to form.
  • 11. Serum samples with RBC agglutination in wells with a serum dilution of 1:20 or higher need to be adsorbed with horse RBCs prior to testing in HI assay. If agglutination is only observed in the first well, the 1:10 serum dilution, then adsorption is not necessary.
  
  
• IV. RDE-treatment of Serum to Remove Nonspecific Virus Inhibitors
  • Notes:
    • Adsorption of sera with horse RBCs can potentially introduce non-specific virus inhibitors to cause false positive HI result. Horse RBC pre-adsorbed sera must be RDE treated prior to the HI assay.
    
    
  • 1. Add RDE that is equal to 3 parts of the original sera volume in the serum recovered from haemadsorption.
    • For example, if 20 μl of original serum was pre-diluted into 1:5 to make a total volume of 100 μl, in step II above, 85μl of serum recovered from haemadsorption. Therefore, there were 17 μl of original serum in the remaining 85 μl of 1:5 sera recovered from haemadosorption.5μl of original vol (25 μl of 1:5) is used for testing of non-specific agglutinin. Finally, there were 12 μl (17 μl - 5 μl) of original serum in the remaining 60μl (85 μl ? 25 μl) of treated serum. Add 36μl RDE (equals to 3 parts of original vol in the sera after haemadsorption).
    
    
  • 2. Incubate RDE-serum mixture at 37?C for 18-20 hrs.
  • 3. Heat serum samples at 56?C for 30 minutes to inactivate RDE.
  • 4. Add appropriate volume of PBS to bring the final dilution to 1:10. Volume of PBS is calculated based on the total volume from step 1.
    • In the example above, adding 3 parts of RDE brings the total volume to 96μl (60 μl + 36 μl). The target volume to make 1:10 dilution of 12 μl original serum equals to 120 μl. So, the volume of PBS needed after RDE-treatment will be 24 μl (120 μl - 96 μl).
    
    
  • 5. The removal of nonspecific agglutinins needs to be confirmed by combining the treated serum with 1.0% horse RBCs as above and observing for the absence of agglutination.
  • 6. Treated sera may store at 4?C overnight. If longer storage is needed, freeze at -20?C or colder.
  
  
• V. Determination of HA Titer of Influenza Virus (Work in BSC)
  • 1. Mark the V bottom plates with the names of the viruses to be tested. Viruses are tested in duplicate. See figure 2.
  • 2. Add 50 μl of PBS to wells 2 through 12 in rows A and B. If more than 1 virus, use the rest of rows as needed.
  • 3. Add 50μl of PBS to the entire H row. This row will serve as the RBC control.
  • 4. Immediately prior to removing virus from vial, gently vortex the vial of virus using three quick pulses. Then add 100 μl of the virus to be tested to wells A1 and B1.
  • 5. Make serial 2-fold dilutions by transferring 50μl from well 1 successively through well 12.Discard 50 μl from well 12.
  • 6. Add 50 μl of 1.0% horse RBC suspension to all wells in rows A, B (or other rows if more than 1 virus), and H on the plate.
  • 7. Gently tap the plates to mix. Stack plates and cover with an empty plate.
  • 8. Incubate at room temperature for 60 minutes.
  • 9. Read the viruses HA titers by tilting the plate at a 45 o to 60o angle. The settled RBCs in row H should start ?running? and form a teardrop-shape due to gravity. Wait until these RBCs finish ?running? and then note the RBC buttons in the virus titrations that ?run?. These RBCs do not exhibit haemagglutination. The highest dilution of virus that causes complete haemagglutination is considered the HA titration end-point. The HA titer is the reciprocal of the dilution of virus in the last well with complete haemagglutination.
  • 10. Dilute virus in PBS to make a working solution containing 8 HAU per 50 μl.
  • 11. Verify that the diluted virus contains 8 HAU per 50 μl by performing a second HA test as described above. The titer of the virus should be 8. If not 8, then adjust the virus concentration by adding virus if < 8 HAU or PBS if > 8 HAU. Do not use until the virus concentration is 8 HAU per 50 μl.
  • 12. Store the working dilution of virus in 4 ?C and use within the same day.
    • Note:
      • The standardized working dilution of virus must have an HA titer of 8 HAU per 50μl. This is the same as 4 HAU per 25 μl. This titer will haemagglutinate the first four wells in the virus back titration in the HA assay. If the working solution does not have an HA titer of 8 in 50μl, it must be adjusted accordingly by adding more virus to increase the number of HAU or by diluting the virus with PBS to decrease the number of HAU.
  
  
• VI. HI Assay with Horse RBCs
  • A. Quality Control
    • 1. Serum controls - Prepare multiple, single use aliquots of RDE-treated control sera and store at -20 ?C to -70?C. Include animal and, if possible, human positive and negative serum controls.
      • For each virus tested, include at least one positive control serum, and one negative control serum.
      
      
    • 2. RDE treatment control ? Ideally, each assay should include a normal animal serum known to contain non-specific inhibitors of haemagglutination that is RDE treated at the same time as the human serum samples being tested.
    • 3. Virus back titration - In each assay, include a virus back titration of the working dilution of the virus.
    • 4. Horse RBC control - The RBC control contains 50 μl PBS and 50 μl of 1.0% horse RBC suspension. A horse RBC control must be added to every plate.
    
    
  • B. HI Assay with Horse RBCs (Work in BSC if handle live virus.)
    • 1. Thaw at room temperature if treated sera are frozen, then keep in 4?C during use.
    • 2. Mark the V bottom plates with the plate number and the names of the viruses accordingly based on experiment design.
    • 3. Column 12 of all plates can be reserved for the RBC control. Positive and negative control sera, and back titration can be run in a separate plate or incorporated in available columns of plates.
    • 4. If Titertubes are used, for duplicate test with one virus, make a serial 2-fold dilution of treated sera by adding 110 μl of treated sera (1:10) to Titertubes in rows A, columns 1-11.
    • 5. Add 55 μl of cold PBS to Titertubes in rows B-H, columns 1-11.
    • 6. Transfer 55 μl of RDE-treated sera from row to row (A -> B -> C?..H) using a P200 multichannel pipette to make serial 2-fold dilutions.
    • 7. Discard 55 μl from row H after mixing.
    • 8. Positive and negative control with appropriate initial dilution should be serially diluted following the same procedure above.
    • 9. Transfer 25 μl of each diluted serum sample from Titertubes into V-bottom plates starting with row H and going to row A. No need to change tips if transferring from the highest dilution (row H) to the lowest dilution (row A). It is critical that the tips must be changed before beginning to pipet the next set of serum samples.
    • 10. If Titertubes are not available, serial dilution of sera samples can be done directly on plates (Figure 3). For each replicate test with one virus, first, add 25 μl of PBS to V-bottom plate in rows B-H, columns 1-11. Second, add 50 μl of RDE-treated sera to row A, columns 1-11. Then, transfer 25 μl RDE-treated sera from row to row (A -> B -> C?..H) to make serial 2-fold dilutions. Discard 25 μl from row H after mixing.
    • 11. Add 25 μl of standardized virus containing 4 HAU to wells containing sera. Note this is the same as 50 μl containing 8 HAU.
    • 12. Gently tap the plates to mix. Stack plates and cover with an empty plate.
    • 13. Incubate virus and sera at room temperature (22? to 25?C) for 30 minutes.
    • 14. Add 50 μl of PBS to column 12. This will serve as the RBC control.
    • 15. Add 50 μl of 1.0% horse RBC suspension to each well.
    • 16. Gently tap the plates to mix. Stack plates and cover with an empty plate.
    • 17. Incubate at room temperature for 60 minutes.
    • 18. Record the HI titers of sera after the 60 minute incubation by tilting the plates at a 45 o to 60o angle. The settled RBCs in column 12 should start ?pulling? or ?running? and form a ?teardrop-shape? due to gravity. Wait until these RBC?s finish ?pulling? and then read the RBC buttons that ?run? or ?stream? in the same way. A well with complete haemagglutination inhibition will look the same as the RBC controls. The serum HI titer is the reciprocal of the serum dilution in the last well with complete haemagglutination inhibition. See figure 3.
    • 19. The dilution of serum if starting from 1:10, for the purpose of determining serum titer, in well A is 1:10, well B 1:20, well C 1:40, well D 1:80, well E 1:160, well F 1:320, well G 1:640, well H 1:1280(Figure 3).
      • Notes:
        • 1- All sera tested with horse HI must be tested for non-specific agglutinin, pre-adsorb as needed, and then treated with RDE prior to HI assay.
        • 2 - In order to ensure optimum results and assay reproducibility, it is essential that test procedures be followed exactly. Occasionally, the HI assay may be difficult to interpret, in such cases, consider the factors presented below:
          • The virus dilutions must be freshly prepared, the HA titer of the virus determined for each HI assay, and the concentration of virus in the working virus solution confirmed in each HI assay.
          • Select virus isolated from same outbreak for optimal antigenic match or use an antigenically equivalent strain.
          • Incubation times must be strictly observed. Plates must be read promptly when the RBC control has completely settled.
          • The quality of RBC may vary. Lot to lot variation may occur.
          • RBC suspension must be standardized in a consistent manner each time as described in step I.
          • Virus stocks must be stored at -70?C. Virus, for use in the HA and HI assays must be kept 4?C until added to the micro titer plates.
      
      
    • 20. Criteria
      • Paired sera (acute and convalescent sera) with a 4-fold rise in HI titer or single sera collected in convalescent phase with HI titer of ≥160 could be considered as H7N9 HI antibody positive.
        • Sera with HI titer of 20-80 should be confirmed by MN or WB assay.

Acknowledgement

This protocol is adapted from the modified horse HAI assay from the United States Centers for Disease Control and Prevention (USCDC). We thank the USCDC and CONSISE (Consortium for the Standardization of Influenza Seroepidemiology) for the technical support and discussion.


Reference

1. Stephenson I et al., Sialic acid receptor specificity on erythrocytes affects detection of antibody to avian influenza haemagglutinin. J Medical Virology 70:391-398 (2003)
2. 2. Modified haemagglutination-inhibition assay using horse RBCs for serologic detection of antibodies to H7 subtype avian influenza virus in human sera. USCDC,http://consise.tghn.org/site_media/media/articles/160713_Modified_Haemagglutination_Inhibition_Assay _Using_Horse_RBCs.pdf

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