The hemagglutination test was developed in the 1940s as a method to quantify the relative concentration of viruses, bacteria or antibodies. Active lectins bind red blood cells (RBCs) by recognizing cell surface carbohydrates, forming a cross-linked network in suspension. This process is known as hemagglutination. The hemagglutination test is a well-established, simple, short and inexpensive method that is widely used for virus detection and antibody titer measurement.
Antibodies can be detected and measured by the hemagglutination method. This relies on the ability of the antibody to cross-link RBCs by interacting with antigens on the RBC surface. Creative Biolabs offers hemagglutination testing services for antibody detection. We use standard protocols to perform the assay.
For general protocols and recommendations for determining antibody activity using hemagglutination assays, please see below.
|Solutions and Reagents
|Phosphate buffer (PBS), fresh erythrocytes, trypsin, dilution buffer, incubation buffer
|Antigen, erythrocyte suspension, incubation buffer
Prepare the blood of the animal you need and perform centrifugation. Remove the supernatant, gently aspirate the precipitate and resuspend. Repeat several times. The isolated red blood cells can be fixed using trypsin and sialidase. After completion, wash and resuspend for storage.
Set up antibody sample group and control group. Place equal amounts of sample and control into each well of different rows in a microtitration plate. Add equal amounts of dilution buffer to the first well, mix and perform serial dilutions. The same amount of reagent is added from one well to another, and so on.
Add a fixed amount of antigen of known titer to each well of the 96-well plate. Exclude control wells that do not require the addition of antigen. Allow the plate to react at room temperature for a time set according to specific requirements. Then add the erythrocyte suspension prepared as above to each well. Mix the plate by gently rotating it to allow adequate mixing of the reaction. Allow the hemagglutination reaction to proceed at room temperature for 30-60 minutes.
Use the naked eye or microscope to determine if agglutination is occurring. Antibody activity is expressed as a titer, defined as the reciprocal of the maximum dilution at which hemagglutination occurs.
No precipitation in pure RBC control wells
Positive control is negative
Bad hemagglutination results
These are our common questions about hemagglutination experiments and their solutions. We are committed to understanding your current relevant test problems and facilitating the process of hemagglutination experiments. We hope our information will be of practical reference to you.
For research use only. Not intended for any clinical use.