The Hemagglutination Assay (HA) is a simple and easy method to obtain semi-quantitative data on the sugar binding and specificity of a lectin. An active lectin binds erythrocytes (RBCs) by recognising a cell-surface carbohydrate, forming a cross-linked network in suspension. This process is known as hemagglutination, and it is the basis of this assay. By serially diluting the lectin in a 96-well microtiter plate, and adding a constant quantity of erythrocytes, the lectin activity can be estimated - the erythrocytes that are bound to lectin will form a lattice that coats the well, whereas unbound erythrocytes will 'sink'.
HA can be easily modified into a Hemagglutination Inhibition Assay (HIA). The basis of the HIA is that carbohydrates that bind lectin will prevent attachment of lectin onto RBC cell-surface carbohydrates; hemagglutination can be inhibited when carbohydrates (sugar solution) are present.
See below for a general protocol, with recommendations, for using HA, and HIA, to determine lectin activity.
A. Reagents and Preparation
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
- 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
- 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.
- 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.
- 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
- 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.
- Incubate at 37 °C for 1 h, with occasional shaking.
- Re-suspend in 50 mL of cold PBS and wash by centrifugation, as described in Isolation of Erythrocytes.
- Suspend trypsinized erythrocytes in 25 mL of 1 % glutaraldehyde in PBS
- Incubate at room temperature for 1 h with continuous gentle shaking.
- 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.
- Wash with PBS and store as 10 % (v/v) erythrocyte suspension at 4 °C.
Preparation of Sialidase-Treated Erythrocytes
- Mix the suspension of untreated erythrocytes (10 %, v/v) with an equal volume of incubation buffer (pH 5.5) containing neuraminidase (1 unit/mL)
- Incubate at 37 °C for 1 h with occasional shaking.
- Wash with cold PBS and centrifuge at 4 °C as described in Isolation of erythrocytes. Store as a 10 % suspension at 4 °C.
(HA) Measurement of Hemagglutination
- Add 25 μL of lectin-containing solutions at physiological salt concentration in the left well of a horizontal row of a microtiter plate.
- 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.
- Add 25 μL of PBS to each well.
- 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.)
- Add 4 % (v/v) rabbit erythrocyte suspension prepared as above.
- 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.
- Allow the hemagglutination reaction to proceed for 30–60 min at room temperature.
- 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
- Add 25 μL of the carbohydrate solution to the left well of a horizontal row of microtiter plate.
- 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.
- Add 25 μL of a fixed titer of lectin solution (titer 2 is most often used) to each well.
- Mix the contents of the wells and incubate for 30 min at room temperature.
- Add 4 % (v/v) rabbit erythrocyte suspension prepared above.
- 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.
- Allow the hemagglutination reaction to proceed for 1–2 h at room temperature.
- 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.
- 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.