Hemagglutination assay

Authors：

Category

Sugar binding proteins

Protocol Name

Authors

St-Pierre, Guillaume
Glycobiology and Bioimaging laboratory, Research Centre for Infectious Diseases, CHUQ, Laval University, Quebec

Milot, Valérie
Glycobiology and Bioimaging laboratory, Research Centre for Infectious Diseases, CHUQ, Laval University, Quebec

Sato, Sachiko *
Glycobiology and Bioimaging laboratory, Research Centre for Infectious Diseases, CHUQ, Laval University, Quebec

*To whom correspondence should be addressed.

Reagents

●	Heparined tubes (BD, Franklin Lakes, NJ: Cat. #362753)
●	PBS(-) 1x
●	Glutaraldehyde (Thermo Fisher Scientific Inc., Waltham, MA: Cat. #O2957-1)
●	Sodium Azide (NaN₃ ) (Thermo Fisher Scientific Inc.: Cat. #S227^I-25)
●	96 wells round bottom plates (Corning, Corning, NY: #3799)
●	α-Lactose (Sigma-Aldrich, St. Louis, MO: Cat. #L3635)
●	15 mL and 50 mL tubes (Corning: #430729 and 430829)

Instruments

●	Centrifuge (for 50 mL tubes; ours is Beckman Allegra 6R centrifuge)
●	37°C incubator
●	Tube rotator (we use a Thermo Scientific Labquake Tube Rotator)

Methods

Preparation of red blood cells (RBC)

1)	Collect ~10 mL of blood in heparined tubes.	Comment 0

2)	Spin at 3,500 rpm 5 min.	Comment 0

3)	Remove the buffy coat as far as possible.	Comment 0

4)	Wash the red blood cells 3 times in PBS (spin between each wash).	Comment 0

5)	Dilute RBC at 8% in PBS-3% glutaraldehyde and rotate 1 h at RT.	Comment 0

6)	Wash 5 times in PBS-0,0025% NaN₃ (spin between each wash).	Comment 0

7)	Resuspend at 3 to 4% in PBS-0,0025% NaN_3.	Comment 0

Store at 4ºC (good for ~3 months).

Comment 0

Calibration of red blood cells

1)	Put PBS : 95 to 60 μL by well.	Comment 0

2)	Add RBC : 5 to 40 μL by well to the PBS.	Comment 0

3)	Incubate at 37ºC for 30 min.	Comment 0

Incubate at 4ºC for 2 h.

Possible results: Tight button: Negative

Spread out RBC: Positive

(see Fig. 1)

Comment 0

Assay

1)	Calculate your galectin concentration by Bradford assay.	Comment 0

2)	Put 10 μM of your galectin in 190 μL total volume (PBS) in well A.	Comment 0

3)	Put 95 μL PBS in wells B to H (line H is 95 μL PBS 1x only).	Comment 0

4)	Do a serial dilution (1:2) from well A to G. (you now have 95 μL of diluted galectin per well; line H is 95 μL PBS 1x only).	Comment 0

5)	Add 5 μL RBC per well and gently suspend RBC by using the pipette.	Comment 0

Incubate at 37ºC for 30 min.

Comment 0

Notes

Calibration of red blood cells

Objective: To know how much red blood cells to put in each well; avoiding false positives

Note 1: Fig. 1B shows that the most optimal volume of this RBC preparation is 5 μL in this preparation as we obtained a clear tight button with this amount of RBC and expected galectin-induced hemagglutination activity, which is inhibited by the antagonist, lactose.
Note 2: Results should be clear after 30 min, but it can be clearer after 2 h at 4°C.

Assay

We usually use 5 μL of RBC in each well. The volumes in the next section are calculated for this amount of RBC. If you need to use more or less than 5 μL of RBC, adjust the others volumes to obtain 100 μL final in each well.

Note 1: Normally galectin-3 induces hemagglutination above 1 μM.
Note 2: The lactose is an inhibitor of the lectin activity of the galectins. It competes with others possible ligands. At the concentration of 150μM, lactose inhibit completely hemaglutination induced by the galectin-3 (Fig. 1B, column 3).

Figure & Legends

Copyrights

Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses

Date of registration：2014-07-31 09:34:38