• Hemagglutination Assay

Hemagglutination Assay

HA

A. Reagents and Preparation

Reagents

Prepare all solutions using analytical grade reagents and store at 4 °C.

• Fresh erythrocytes
• Phosphate-buffered saline (PBS): 150 mM NaCl, 20 mM Na-phosphate (pH 7.2), or an appropriate buffer (e.g., Tris– HCl containing 5 mM CaCl 2 for Ca 2+-requiring lectins, or MEPBS: 20 mM PBS (pH 7.2) with 4 mM β-mercaptoethanol, 2 mM EDTA for lectins such as galectins that require a reducing agent)
• Trypsin (Sigma): store at −20 °C
• Glutaraldehyde
• Glycine: store at room temperature
• Neuraminidase (e.g. from Vibrio cholerae)
• Incubation buffer for neuraminidase: 0.1 M acetate buffer containing 1 mM CaCl 2 (pH 5.5) 
• Carbohydrate solution(s) for inhibition assay: dissolve at 10 mg/mL in the same buffer used for the hemagglutination assay
• A 96-well microtiter plate, V shape or U shape

Isolation of Erythrocytes

1. Centrifuge 10 mL of rabbit blood at 500 × g for 5 min at 4 °C. Note: The procedure can be scaled down, depending on the blood volume. 
   
2. Remove the plasma and white cell ghost layer at the top of the pellet, and re-suspend the cells in 50 mL of cold PBS by gently aspirating and expelling them with a pipette, then centrifuge at 500 × g for 5 min at 4 °C. 
   
3. Remove the supernatant and repeat step 2 three times. Store the pelleted erythrocytes at 4 °C until use. Use within 24 h.

Preparation of Trypsinized Glutaraldehyde-Fixed Erythrocytes

1. Suspend the isolated erythrocytes (ca. 5 mL) in 100 mL of 0.1 % (w/v) trypsin solution in PBS. Note: The procedure can be scaled down, depending on the quantity of erythrocytes.
2. Incubate at 37 °C for 1 h, with occasional shaking.
3. Re-suspend in 50 mL of cold PBS and wash by centrifugation, as described in Isolation of Erythrocytes.
4. Suspend trypsinized erythrocytes in 25 mL of 1 % glutaraldehyde in PBS
5. Incubate at room temperature for 1 h with continuous gentle shaking.
6. Centrifuge at 500 × g for 5 min at 4 °C, remove the supernatant, and wash twice with 25 mL of 0.1 M glycine in PBS.
7. Wash with PBS and store as 10 % (v/v) erythrocyte suspension at 4 °C.

Preparation of Sialidase-Treated Erythrocytes

1. Mix the suspension of untreated erythrocytes (10 %, v/v) with an equal volume of incubation buffer (pH 5.5) containing neuraminidase (1 unit/mL)
2. Incubate at 37 °C for 1 h with occasional shaking.
3. Wash with cold PBS and centrifuge at 4 °C as described in Isolation of erythrocytes. Store as a 10 % suspension at 4 °C.

B. Procedure

(HA) Measurement of Hemagglutination

1. Add 25 μL of lectin-containing solutions at physiological salt concentration in the left well of a horizontal row of a microtiter plate.
2. Make a series of twofold dilutions (2 −1, 2 −2, 2 −3 …) of the sample in PBS in the horizontal row. Make a control well using PBS without the lectin.
3. Add 25 μL of PBS to each well.
4. Mix the plate by swirling gently on the top of the bench in a circular or figure “8” motion for 10 s. (For this step, the mix function of the microplate reader can be used.)
5. Add 4 % (v/v) rabbit erythrocyte suspension prepared as above.
6. Mix the plate by gently swirling on the top of the bench in a circular or figure “8” motion for 10 s. Do not use the mix function of the microplate reader.
7. Allow the hemagglutination reaction to proceed for 30–60 min at room temperature.
8. Determine whether agglutination has occurred using the naked eye or a microscope. Hemagglutination activity is expressed by a titer defined as the reciprocal of the maximum dilution giving positive hemagglutination. If a lectin solution causes hemagglutination at a maximum dilution of 2 −9 (=1/512), the titer of the original solution is 512.

(HIA) Measurement of Hemagglutination Inhibition by Carbohydrates

1. Add 25 μL of the carbohydrate solution to the left well of a horizontal row of microtiter plate.
2. Make a series of twofold dilution (2 −1, 2 −2, 2 −3 …) of the carbohydrate solution in PBS in the horizontal line. Make a control well with PBS.
3. Add 25 μL of a fixed titer of lectin solution (titer 2 is most often used) to each well.
4. Mix the contents of the wells and incubate for 30 min at room temperature.
5. Add 4 % (v/v) rabbit erythrocyte suspension prepared above.
6. Mix the plate by gently swirling on the top of the bench in a circular or figure “8” motion. Do not use the mix function of the microplate reader.
7. Allow the hemagglutination reaction to proceed for 1–2 h at room temperature.
8. Determine whether agglutination has occurred by naked eye microscope. The hemagglutination inhibition activity is expressed as the lowest concentration of the sugar solution that completely inhibits hemagglutination.

Notes

• A plate with U-shaped wells is sometimes used instead of one with V-shaped wells for assays using untreated erythrocytes. Choose the type of plate that clearly distinguishes positive and negative agglutination, which may differ depending on the type of erythrocytes.
• Trypsinized glutaraldehyde-fixed rabbit erythrocytes are often used because the trypsin treatment renders cells more sensitive to agglutination and glutaraldehyde fixation allows cells to be stored for a long period (>1 month).
• Hemolysis may occur if the centrifuge speed is too high. Centrifugation is usually operated at lower than 2,000 × g.
• Erythrocytes from various species (chicken, guinea pig, goose, horse, cow, sheep, rabbit, and human types A, B, AB, and O) are widely used.
• Use gloves of the appropriate type and length, eye protection, and a face mask when measuring glutaraldehyde.
• Neuraminidase is most active at pH 5.0–5.5 (e.g., 0.1 M acetate buffer); it retains about 30–50 % activity at pH 7.0 in a phosphate buffer.
• Neuraminidase from Arthrobacter ureafaciens or Clostridium perfringens can also be used.