Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Summary

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.

Abstract

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37°C for 60 minutes. After this time, the samples can then be easily scored for ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Protocol

Preparation of 5x Veronal Buffered Saline (VBS)

1. To prepare the Veronal Buffered Saline (VBS), three separate solutions need to be prepared.
2. Prepare solution 1 by dissolving 21.25gm of NaCl and 0.94gm of Sodium Barbitone in 350ml of distilled water. The final concentrations of the NaCl and Sodium Barbitone are 1.02M and 13mM respectively.
3. Prepare solution 2 by dissolving 1.44gm of Barbitone in 125ml of Hot distilled water. The final concentration of Barbitone is 62.5mM.
4. Prepare solution 3 by dissolving 20.33gm of MgCl₂ and 4.41gm of CaCl₂ in 100ml of distilled water. The final concentrati....

Discussion

The haemagglutination assay is a very simple technique that allows for a large number of serum samples to be assessed within a short period of time. It also has the advantage of being read without the need for specialised equipment, with the eye being suitable. This technique can also be modified for the detection of human chorionic gonadotrophin hormone present in urine, as part of a haemagglutination-inhibition test. In relation to a standard haemagglutination test, there are a few areas where care needs to be taken in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Revercells	CSL	02490201	Use at 1% final
Anti-A serum	CSL	02611305
96 well U bottom plate	TRP	92097	Must be U-bottom
Veronal buffered saline			Use at 1x final
37°C room/incubator
Disposable gloves