Hemagglutination Inhibition Assay

Angie Derick Tomas Renee

Hemagglutination Inhibition Principle

Viral Particles bind surface structures of certain species erythrocytes forming hemagglutination reaction. The Hemagglutination Inhibition Assay exploits the Rubella virus tendency to agglutinate Human Group O blood, goose blood, and chicken blood to detect for viral antibodies in patient serum.

Hemagglutination Inhibition:
Patient Serum with Viral Antibodies Present

Patient Serum has been pretreated with kaolin to remove nonspecific agglutination inhibitors like Beta Lipoprotein and nonspecific antibodies to the RBC. In step 1 patient serum containing antibodies is mixed with a known amount of Rubella viral antigen

Hemagglutination Inhibition:
Patient Serum with Viral Antibodies Present

If the patient serum contains antibodies they will have reacted to the viral Rubella viral antigen given that the concentrations are equal during step 1. In step 2 Red blood cells are added, they can be Human type O, chicken or goose when testing for rubella antibodies. Given that equal amounts of Antigen to Antibody were present during t he first step and all the viral antigens were bound by the patient antibody the viral particle should not be able to agglutinate the added RBC.

Hemagglutination Inhibition:
Patient Serum with Viral Antibodies Present

No agglutination should be visible if the concentration of Antigen to antibodies is equal. This is due to the bound viral antigen.

Hemagglutination Inhibition:
Patient Serum without Viral Antibodies Present

Patient Serum has been pretreated with kaolin to remove nonspecific agglutination inhibitors like Beta Lipoprotein and nonspecific antibodies to the RBC.
Step 1 patient Serum suspected of containing antibodies is mixed with a known amount of Rubella viral antigen

Hemagglutination Inhibition:
Patient Serum without Viral Antibodies Present

The patient serum does not contain antibodies. In step 2 Red blood cells are added, they can be Human type O, chicken or goose when testing for rubella antibodies. Lack of antibodies to rubella virus means the virus will not be bound in the serum and will be able to agglutinate the added red blood cells.

Hemagglutination Inhibition:
Patient Serum without Viral Antibodies Present

The patient serum does not contain antibodies to prevent agglutination of red blood cells by the virus particles.

Hemagglutination Inhibition Assay Recap

antibodies are trying to inhibit hemagglutination

by the viral particles in serum positive for antibodies against the virus being tested By using a measured amount of virus to test the sample for agglutination you can preform a titer on the serum. The highest titer will determine the antibody titer of the serum

HIA
Red Blood Cells used for HIA

testing procedure include Turkey, Horse and Human erythrocytes. Chicken Red Blood cells are the most commonly used for HIA testing. The preference for chicken blood is due to the fact that they are nucleated erythrocytes. They skink faster because of their heavier weight. Thus allowing to speed up the testing

http://www.dreamstime.com/chicken-blood-thumb6166734.jpg

IgG and IgM

The test is nonspecific for Ig

classes To make the test specific you can separate the Immunoglobins through precipitation and chromatographic methods
Protein absorption Sucrose density

fractionalization Non-affinity size exclusion chromatography Affinity chromatography Immunodiffusion Ouchterlony IgM is thermally active at 4-

HIA Disadvantages
Time consuming due to pretreating serum

Technique dependent for maintaining accuracy of

results Results must be visually interpreted (meaning no automation) Results are subjective

False Negatives
Low titer / prozone reaction

Accidental removal of Antibody being tested by

pretreatment process

http://webmedia.unmc.edu/alliedhealth/trudell/immuno%20review/05%20immuno%20review_slide0112_image

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False Positives
Subjective interpretation of results can give a

falsely high titer Not removing all nonspecific inhibitors in pretreatment process
Lack of procedure control means that there is no

way to verify if nonspecific inhibitors were removed or not

Hemagglutination Inhibition Assay : Testing for Rubella Antibodies

Hemagglutination occurs when rubella virus

antigens binds to red blood cell membrane when introduced into the serum. Hemagglutination Inhibition (HAI) assay is a reference method of quantifying the presence of rubella antibodies by lack of agglutination in the patient serum. If antibodies for rubella are present, then there is no agglutination. If antibodies are not present or in low amount , then there is agglutination.

Hemagglutination Inhibition Assay : Testing for Rubella Antibodies

In a serum sample, rubella antigens are added

along with RBCs and presence of clumping is observed. If there is a lack of agglutination, then there antibodies present. If there is slight amount of agglutination, the amount of antibodies present are verified with a titer. If the titer is greater than 1:10, the patient is said to be immunized against rubella.

Patient Immunity and Rubella Virus

Initial exposure results in IgG and IgM production

against rubella antigens. Immunity against rubella is measured by IgG and IgM levels where IgG persist for a lifetime duration and IgM is present for up to six months. Testing for rubella can confirm immunity or recent infection by serum levels.

Other Applications of Hemagglutination Principle: Methods in

Influenza detection
HIA is easily adapted for detection of different

viral infections. One specific application of HIA is used for seasonal influenza It is the Gold Standard of testing in Korea It IDs the presence of antibodies to hemagglutinin protein subtype, produced by specific Avian Influenza virus isolate Currently 16 hemagglutining (HA) and 9 neuraminidase (NA) subtypes of AIV isolated.

Other Applications of Hemagglutination Principle: Methods in

Influenza detection
Influenza virus particles have an envelope

protein called the hemagglutinin,HA. HA binds to sialic acid receptors on cells. The virus envelope protein is capable of binding to erythrocytes causing lattice formation Hemagglutin Inhibition is tested on chicken sera for HA protein Patient antibodies to influenza virus prevents lattice formation Serum without antibodies will demonstrate hemagglutination in all wells

Other Applications of Hemagglutination Principle: Methods in

Influenza detection

Avian Influenza virus specific antibodies may be

detected as early as 7 days after infection Titer is dependent of the antigenic relatedness of the isolate and the specific serum being used. Interpretation can be challenging
False Positives by non-specific inhibitors and

naturally occurring agglutins on RBCs.

Other Applications of Hemagglutination Principle: Methods in

Influenza detection and epidemiology
South Korea uses intensive documentation of Avian

Influenza test results to survey and control the spread of Highly pathogenic AIV (HPAIV) and notifiable AIVs (NAIVs) This has been in operation since the first H5N1 subtype HPAIV outbreak in 2003. Calculate the susceptibility of a population to Avian Influenza infection Compare HI titers of HA protein to Avian Influenza attack rates in populations When used in this manner, the HI assay is a powerful epidemiological tool.

Test Modifications : Latex

Agglutination Inhibition and hCG testing
A modified Hemagglutination Inhibition test can also be used in the lab for hCG detection
This variant test uses latex beads rather than erythrocytes for agglutination detection, but still applies the inhibition principle of the

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Test Modifications : Latex

Agglutination Inhibition and hCG testing
Testing is done for hCG secreted in urine 2. Urine specimen is mixed with anti-hCG 3. A coupled hCG latex reagent added to the mixture
1.

Results : Non-Pregnant Patient: Coupled hCG reagent reacts with anti-hCG resulting in agglutination Pregnant Patient: anti-hCG will be neutralized by hCG in the urine resulting in no agglutination

No hCG in patient urine

Latex carriers coated with hCG added

Negative Latex Inhibition Agglutination Test for hCG Non-Pregnant Individual

Carriers demonstrate agglutination

AntihCG free floating

hCG in patient urine

Positive Latex Inhibition Agglutination Latex Test for hCG carriers

coated with hCG added

Pregnant Individual

Anti-hCG neutralize d

Carriers do not demonstrate agglutinatio n

Foretel Slide Test for Pregnancy

Reagents
anti-hCG (mouse

Limitations and Notes

Sensitivity of 0.3 IU/ml

monoclonal) Polystyrene latex particles chemically coupled to hCG

Qualitative Method

of hCG Laboratory use only Storage of reagent at 28C

Semi-Quantitative Method
24 hour urine sample Progressive dilution series No agglutination in highest dilution hCG 24 hours = S x D x V

First morning urine sample

False Readings
Choriocarcinoma present in patient

Values of hCG > 250 IU/ml

Testing serum rather than urine

References
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3194935/ http://medical-dictionary.thefreedictionary.com/Rubella+Test

http://www.ftb.hr/files/journals/1/articles/456/public/456-448-1-PB.pdf
http://www.bioorganica.org.ua/UBAdenovo/pubs_3_2_05/Nikolayenko.p

df Data on file: Tulip Diagnostics (P) Ltd. Turgon. Immunology and Serology in Laboratory Medicine, 4th ed. St.Louis, Missouri: Elsevier Science, 2009. Print. ISBN-9780323043823 Racaniella, Vincent. Influenza hemagglutination inhibition assay. Virology Blog: About Viruses and Viral Disease. 27 May 2009. Pedersen, JC. Hemagglutination Inhibition Test for Avian Influenza Virus Subtype Indentification and the Detection and Quantitation of Serum Antibodies to the Avian Influenza Virus. Methods in Molecular Biology, 2008, Volume 436, 53-66. Kim, Hye-Ryoung, Lee Kyoung-Ki, et al. Comparison of serum treatments to remove nonspecific inhibitiors from chicken sera for the hemagglutination inhibition test with inactivated H5N1 and H9N2 avian Influenza A virus subtypes. Journal of Veterinary Diagnostic Ivestigation 24(5) 954-958. 2012.