HomeMicrobiologyHemagglutination Assay- Precept, Varieties, Methodology, Makes use of

Hemagglutination Assay- Precept, Varieties, Methodology, Makes use of

The antigen and antibody response wherein the antigen-antibody advanced fashioned is seen within the type of clumps is named agglutination.

It happens on the floor of the cells or parts concerned as antigens are expressed on their floor. It happens between insoluble antigens and soluble antibodies.

Agglutination response is of two varieties: 

  • Direct agglutination: It contains slide agglutination, tube agglutination, coombs’ check, and heterophile agglutination check.
  • Passive agglutination: It contains latex agglutination, hemagglutination check and, cooaglutination check.

Hemagglutination is a kind of passive agglutination response.

Hemagglutination assay (HA) is a kind of immunoassay wherein erythrocytes are used as service particles and are generally most popular for serological prognosis of varied infections.

George Hirst was the individual to find hemagglutination assessments. He was an American virologist.


  • RBC suspension: It’s coated with antigens particular to the antibody to be detected or antibodies particular to antigens to be detected; therefore known as a service particle. RBCs could be of people, sheep, chicks, and so forth.
  • Serum or blood pattern
  • Microtitre plates resembling 96 well-microtitre (V-bottomed) plate
  • Diluent: Phosphate Buffered Saline (PBS)
  • Destructive and Constructive Management Samples
  • RDT kits (usually with slides, reagents, and management) can be found in case of Speedy Hemagglutination assay

Precept of Hemagglutination Assay

The first theme of the hemagglutination check is that when any antigens current on the floor of Purple Blood Cells are available in contact with any complementary antibody and vice-versa, they mix to agglutinate and kind noticeable clumps, which could be noticed clearly distinguishing the constructive check from the unfavorable one.

Sorts of Hemagglutination Assay

It’s of primarily two varieties primarily based on the methodology:

A. Speedy Hemagglutination Assay

Because the identify suggests, in round one minute, this check can decide the presence of a haemagglutinating agent. Therefore, it will also be known as Speedy Analysis Take a look at (RDT). The unfavorable and constructive management samples have to be examined solely as soon as when testing a number of samples. 

Each time a haemagglutination check is carried out, the settling sample of the pink blood cell suspension have to be examined. That is carried out by combining diluent with pink blood cells and permitting them to settle.

i. The diluent needs to be disbursed.

ii. Add pink blood cells and gently shake them to mix.

iii. Permit the pink blood cells to settle earlier than inspecting the sample

iv. Study the cells to see if they’re setting in a standard sample and haven’t any auto-agglutination. Within the micro-agglutination assay, there might be a definite button of cells and a good suspension with no indicators of clumping within the speedy assay.

Process of Speedy Hemagglutination Assay

  1. Place 4 separate drops of 10% hen pink blood cells on a glass slide or the offered one within the equipment.
  2. Add one drop of every management and check pattern to every drop of blood together with PBS. To dispense every pattern, use separate suggestions, pipettes, or a flamed loop.
  3. At first, PBS is dropped, adopted by management and unidentified samples.
  4. It needs to be blended for one minute by rotating the slide or tile.
  5. Observe the consequence and examine it with the constructive and unfavorable management offered within the equipment to research the consequence.

B. Micro-hemagglutination Assay

This methodology is helpful for testing the presence or absence of haemagglutinin in allantoic fluid from many embryonated eggs. It’s a extra time-consuming methodology than RDT. Purple blood cells are dissolved in a 1% answer. Cells settle sooner in V-bottom plates, and the slight distinction between constructive and unfavorable outcomes is bigger than in U-bottom plates.

Process of Micro-hemagglutination Assay

  1. Fill out a recording sheet with details about the samples being examined. The samples and controls might be positioned within the wells indicated on this sheet.
  2. Take the pattern of about 50 ml with a micropipette and dispense it right into a effectively of the microwell plate. Use a special tip for every pattern to stop contamination of samples.
  3. Place unfavorable and constructive controls on one of many plates.
  4. Pour 50 mL of PBS into every effectively. These wells will function auto-agglutination controls for pink blood cells.
  5. Fill every effectively with 25 mL of 1% pink blood cells.
  6. Gently faucet the plate’s sides to combine. Cowl the plate with a plate cowl.
  7. Let the plate stand for about 40 minutes, and observe/file the info.

Consequence Interpretation of Hemagglutination Assay

The looks of clumps within the case of agglutinated suspension can point out a constructive check in all assessments. It may be in contrast with the constructive management set to research correctly. A constructive check means that the respective pattern is contaminated with antibodies or antigens associated to a pathogen.

Observe: The clumps could be noticed on the backside of the effectively within the case of Micro-well and on the floor within the case of RDT.

Hemagglutination Assay

Purposes of Hemagglutination Assay

  • It may be used to detect the humoral immune response of the physique in opposition to any illness or infective brokers.
  • It may be used for figuring out completely different blood cell varieties or teams.
  • It might detect and quantify viral infections resembling paramyxovirus, influenza, and so forth.
  • Completely different speedy prognosis check kits primarily based on hemagglutination are designed. For e.g. an RDT equipment for detecting HbsAg in case of Hepatitis B an infection.
  • It might additionally detect varied bacterial infections, resembling syphilis.

Benefits of Hemagglutination Assay

  • Easy to carry out
  • Not as costly as many different assessments
  • Sorts of gear used are usually simply accessible.
  • Quick interpretation of outcomes (as within the case of RDT)

Limitations of Hemagglutination Assay

  • Faults in incubation time and focus of RBC could result in fallacious outcomes.
  • Various factors thought-about within the response have to be particular. Their non-specificity could lead to an incorrect consequence.
  • Dedication of quantitative values and consequence interpretation require certified people.
  • Consequence interpretation is made manually with none digital information, so the completely different observers might need errors or fluctuations in evaluation.


  1. S. Miyaishi, F. Moriya, (2005) Encyclopedia of Analytical Science (Second Version)
    Parija S.C., (2009), Textbook of Microbiology and Immunology, 2nd version, Elsevier, a division of Reed Elsevier India Personal Restricted, pg. 108
  2. Sukhadeo B. Barbuddhe and Deepak B. Rawool, Strategies in Microbiology, 2020
  3. Townsend, A., Rijal, P., Xiao, J. et al. A haemagglutination check for speedy detection of antibodies to SARS-CoV-2. Nat Commun 12, 1951 (2021). https://doi.org/10.1038/s41467-021-22045-y
  4. Thangavelu, C. P., & Koshi, G. (1980). Micro-indirect hemagglutination check for detection of antibodies to the Ibc protein of group B Streptococcus. Journal of medical microbiology, 12(1), 1–6. https://doi.org/10.1128/jcm.12.1.1-6.1980


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