Hemagglutination Inhibition Assay

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Viral Particles bind surface structures of certain specie’s

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 Principle

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

I f 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 the 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

HIARed 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 procedure.

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°-

22°C IgG is optimal at 37°C

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

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http://webmedia.unmc.edu/alliedhealth/trudell/immuno%20review/05%20immuno%20review_slide0112_image057.jpg

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.

In a serum sample, rubella antigens are added along with RBC’s 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.

Hemagglutination Inhibition Assay : Testing for Rubella Antibodies

Patient Immunity and Rubella VirusInitial 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 ID’s 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.

 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

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.

Other Applications of Hemagglutination Principle: Methods in Influenza detection and epidemiology

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 testhttp://2.imimg.com/data2/WT/AR/MY-2478375/medox-bio-latex-agglitination-teaching-kit-250x250.jpg

Test Modifications : Latex Agglutination Inhibition and hCG testing

1. Testing is done for hCG secreted in urine2. Urine specimen is mixed with anti-hCG 3. A coupled hCG latex reagent added to the mixture

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

Anti-hCG free

floating

Latex carriers coated with hCG

added

Carriers demonstra

te agglutinati

on

Negative Latex Inhibition

Agglutination Test for hCG

Non-Pregnant Individual

hCG in patient urine

Anti-hCG

neutralized

Latex carriers coated with hCG

addedCarriers do not

demonstrate

agglutination

Positive Latex Inhibition

Agglutination Test for hCG

Pregnant Individual

Foretel® Slide Test for PregnancyReagents

Limitations and Notes

anti-hCG (mouse monoclonal)

Polystyrene latex particles chemically coupled to hCG

Sensitivity of 0.3 IU/ml of hCG

Laboratory use onlyStorage of reagent at 2-8°C

Qualitative Method • First morning urine

sample• 24 hour urine sample• Progressive dilution series• No agglutination in highest

dilution• hCG 24 hours = S x D x V

Semi-Quantitative Method

False ReadingsChoriocarcinoma present in patientValues of hCG > 250 IU/mlTesting 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.pdf 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.