Determining the Reactivity and Titre of Serum using a Haemagglutination Assay
January 29th, 2010
Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.
Determining the reactivity and titer for serum using a hemagglutinin assay. A gluten nation is a reaction that takes place between soluble antibodies and particulate antigens. These can include cells or carry molecules.
Epitopes on the surface of the carrier allow an antibody to bind and cross-link the carrier together leading to the formation of a complex lattice called an antigen antibody complex. Hem agglutination is a specific form of agglutination where antibodies bind to red blood cells, which act as the particulate antigen. Red blood cells are useful targets as they're both readily available and the results can easily be seen by the naked eye.
Serum samples are serially diluted and then placed into a 96 well ubo plate. A suspension of red blood cells Riva cells is then added. When antibodies present at a high concentration binding to the red blood cells takes place forming a large lattice.
As this lattice settles by gravity, it conforms to the shape of the ubo plate. When viewed from above, a broad sheet of red blood cells is seen. This is a positive hemagglutinin reaction.
When antibody levels are low, no lattice can form and the red blood cells conform to the shape of the UBO plate and concentrate at the base forming a small pellet. This is a negative hem agglutination reaction. This diagram shows a positive reaction.
The cells form a lattice as they settle, they conform to the shape of the U bottom plate. On the left and to the right, we see the top view where a broad sheet of red blood cells is seen. If an intermediate amount of antibodies present, some letters formation will be observed.
Hem agglutination also allows for the reactivity of different serum samples to be tested against the known antigen. The titer of the serum sample can also be determined. The titer being the last dilution where a positive reaction was seen.
Normally, the higher the antibody concentration in the serum sample, the higher the titer. This diagram shows a negative hem agglutination reaction. Insufficient antibody present to cross-link the red blood cells.
As the cells settle by gravity, they slide down the side of the well forming a tight pellet at the bottom. The diagram to the right shows the red blood cells when viewed from above. For this experiment, you'll need 1 96 well U bottom plate.
It is essential that a U bottom plate is used since flat bottom plates will not allow the cells to sediment into a pellet making interpretation impossible. A pet able to deliver 50 microliters tests serum a 1%suspension of Riva cells and a one-time solution of verone buffer saline to each well of row. A one to a 12, deliver 50 microliters of roal buffer saline.
This is used as your diluent. Now place 50 microliters of serum into well, A one. This is your first one in two dilution.
Using the perpet, ensure the serum sample is thoroughly mixed. Once mixed, remove 50 microliters from well A one and transfer to well A two. This is your second one in two dilution, giving you a dilution of fourfold.
Continue this dilution series using wells three through to 11. Do not place any serum into, well, 12 as this is your roal buffer saline negative Control. Now take your 1%Suspension of Riva cells, which has been thoroughly mixed and transfer 50 microliters into, well, a one through to well A 12.
Once the cells Have been added, cover the plate and gently tap the plate to ensure that the samples and the cells are mixed. Now incubate at 37 degrees for 60 minutes. A humidified incubator is appropriate.
During the 60 minute incubation period, antigen antibody interactions will take place and the cells will begin to settle. The video footage you're viewing is a time lapse photography of the entire 60 minute period. This is a closeup of wells five through to 10.
We have already examined, well, 12, which was your VBS control in this. Well, the cells had formed a tight pellet indicating that the assay had functioned appropriately. Wells five and six are examples of positive hem agglutination.
Well, seven is an example of an intermediate agglutination result while wells eight, nine, and 10 are negative for hem agglutination. Based on this result, the titer would be determined based on the dilution for well, seven as this was the last well, where a positive reaction was observed.
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Overview
This article demonstrates the haemagglutination assay, a technique used to detect and quantify antibodies that bind to red blood cells. The method involves serial dilution of serum samples, mixing with red blood cells in a U-bottom microtitre plate, and visual scoring of agglutination patterns to determine antibody reactivity and titre. The assay is rapid, simple, and allows for direct observation of results.
Key Study Components
Area of Science
- Immunology
- Serology
- Hematology
Background
- Haemagglutination is a specific form of agglutination where antibodies bind to red blood cells, causing them to clump together.
- Red blood cells serve as convenient particulate antigens due to their availability and the ease of observing agglutination visually.
- This assay is commonly used for blood grouping, antibody titre determination, and viral quantification.
- The technique relies on the formation of antigen-antibody complexes that create visible lattices or pellets depending on antibody concentration.
Purpose of Study
- To demonstrate the preparation and execution of a haemagglutination assay.
- To show how to interpret positive, negative, and intermediate agglutination reactions.
- To explain how to determine the titre of a serum sample using this assay.
Methods Used
- Serial dilution of serum samples in a 96-well U-bottom microtitre plate.
- Addition of a 1% suspension of red blood cells (Revercells) to each well.
- Incubation at 37°C for 60 minutes to allow antigen-antibody interactions and cell settling.
- Visual scoring of agglutination patterns to assess antibody reactivity and titre.
Main Results
- Positive haemagglutination is indicated by a broad sheet of red blood cells forming a lattice in the well.
- Negative reactions result in a tight pellet of red blood cells at the bottom of the well.
- Intermediate reactions show partial lattice formation.
- The titre is determined as the last dilution where a positive reaction is observed.
Conclusions
- The haemagglutination assay is a straightforward and effective method for assessing antibody reactivity and titre.
- Results are easily interpreted visually, making the assay accessible and rapid.
- Proper plate selection (U-bottom) and technique are critical for accurate interpretation.
