Indirect Haemagglutination Test | IHA


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Sop's for Indirect Haemagglutination Test (IHA)
Indirect Haemagglutination test /IHA is used for assay of serum Antibodies after vaccination e.g HS vaccine produces antibodies specific against HS pathogen. Causative agent of HS (haemorhagic septicemia is Pasteurella Multocida which is non-hemolytic, i.e can not directly agglutinate RBC's that's why we use Indirect Haemagglutination Test (IHA) for detection of antibodies in serum.
Equipment/Glassware Required:

  1. Centrifuge machine
  2. Centrifuge tube 15ml
  3. Micotitration plates U-shaped 96 wells
  4. Multichannel Pipette,12 channel/8 channel
  5. Single channel pipette
  6. Troughs/Petri plates
  7. Pasteur pipettes
  8. Glass pipette 1ml and 10 ml
  9. Pipetting Aid

Chemicals/Ingredients Required
  1. sheep RBCS/Human Blood Group O
  2. 0.9% normal saline (pH 7.2-7.4) /PBS
  3. Formalin 37% stabilized with 10% ethanol
  4. Antigen (extracted from pasteurella multocida serotype B:2)
  5. Blood Agar
  6. Serum to be tested
  7. EDTA
1. Preparation of Antigen:
  • Pasteurella multocida serotype B:2 is cultured on blood Agar and is incubated at 37C for 24 hours. After 24 hours, purity of culture is checked by slide prepartion, grams staining and observation under microscope.
  • The pure growth is suspended in 12ml of 0.3% (V/V) formal saline solution.
  • The suspension is heated at 56C in water bath for 30 minutes to dissolve surface capsular antigen.
  • The suspension is centrifuged at 5000 rpm for 10 minutes and supernatant is used as antigen and pallet is discarded.

2. Preparation of RBC's

  • 0.2ml of washed RBC's packed cell volume (PCV) is mixed with 2ml of antigen to make 10% solution.
  • This solution is kept at 37C for one hour.
  • After one hour mixture is centrifuged at 1500 rpm for 15 minutes.
  • The supernatant is discarded and RBC's are again washed twice in Normal Saline/PBS and PCV of sensitized RBC's (SRBCS) obtained.

3. Working Solution of SRBCS

The working solution of SRBC's is prepared by mixing 0.2ml of SRBC's with 19.8ml of normal saline/PBS to make 1% solution.

Test Procedure
  • Test is carried out in plastic U-shaped 96 wells titration plates.
  • 50 ul of 0.9% normal saline/PBS is added in each well using multichannel pipette.
  • 50 ul of test serum is added in first well.
  • Two fold serial dilution of test serum is prepared starting from 1:2 in normal saline/PBS in HA plates till 10th well and mixed thoroughly preventing bubble & froth formation.
  • 11th well is kept as positive control (known serum of P.multocida+NS) and 12th well is kept as negative control (RBC's +NS).
  • 50 ul of 1% SRBC's suspension is added in each well.
  • Prepared plates are incubated at 37C for half an hour and results are recorded.
  • The test plates are then shifted at 4C for overnight and results are recorded again.

Note: please use separate tips for each step.
Interpretition of results
Positive : No button formation i.e Agglutination of RBC's
Negative : Button formation with sharp margins
Titer: Highest dilution showing agglutination.
Control : +ve control---serum+ Normal Saline + SRBC, -ve control---RBC's +Normal saline (bead formation)
1:8 >>> Border line protections
1:8 - 1:32 >>> Protective Titer
1:64 and above >>> Showing infection in herd.