26 Difference Between Neutralization Assay and Hemagglutination Inhibition Assay

A neutralization assay is a laboratory procedure used to assess an antibody’s or antiviral agent’s capacity to reduce a virus’s or other infectious agent’s infectivity. In order to determine if the virus has been neutralized or inhibited from infecting target cells, the assay requires combining the virus with the test material (antibody or antiviral agent).

The ability of antibodies (typically against influenza viruses) to prevent the agglutination (clumping) of red blood cells by viral hemagglutinins is commonly assessed using the Hemagglutination Inhibition (HI) assay. Hemagglutinins, which are proteins on the virus’s surface, are in charge of the virus’s ability to attach to host cells.

An HI assay follows identical procedures to a general neutralization assay, but with a focus on preventing red blood cell agglutination. The test entails:

The virus is made, and serum from a subject who has been exposed to the virus or received a vaccination against it is obtained.

To produce a variety of concentrations, the serum is diluted in steps.

A predetermined virus concentration is combined with diluted serum. Antibodies to the virus will bind to the viral hemagglutinins and stop them from agglutinating red blood cells if the serum has these antibodies present.

Red blood cells are introduced following incubation. Red blood cells will aggregate if the virus is successful in doing so, creating observable clumps.

The level of agglutination is monitored and noted in the agglutination readout. In comparison to controls without the serum, there will be reduced or no agglutination if the serum’s antibodies are successful in neutralizing the virus.

The hemagglutination inhibition titer is the highest dilution of the serum that inhibits hemagglutination. It gives an indication of the antibody’s capacity to prevent the virus from agglutinating red blood cells.

In virology and immunology, neutralization tests and HI assays are crucial techniques for determining the effectiveness of antibodies, vaccinations, and antiviral medicines. They support the development of treatments and preventive measures by assisting researchers in understanding how well these drugs work to stop viral infections.

S.No.	Aspects	Neutralization Assay	Hemagglutination Inhibition Assay
1	Definition	Measures the ability of antibodies to neutralize a virus or toxin	Measures the ability of antibodies to inhibit the agglutination of red blood cells
2	Purpose	Determines the presence and potency of neutralizing antibodies	Measures the quantity of specific antibodies in a sample
3	Target	Typically used for viral pathogens or toxins	Commonly used for detecting antibodies against certain antigens, including viruses
4	Endpoint	Involves assessing the reduction or prevention of viral infection	Focuses on the prevention of red blood cell agglutination
5	Virus or Antigen	Requires a live virus or toxin to perform the assay	Uses red blood cells as the antigen
6	Viral Growth	Requires a cell culture system for viral growth	Does not require viral growth or a cell culture system
7	Agglutination	Does not involve agglutination of cells	Focuses on inhibiting red blood cell agglutination
8	Mechanism	Determines if antibodies can block viral entry, replication, or other functions	Checks if antibodies can prevent red blood cell clumping
9	Readout	Typically, the readout is based on a reduction in virus-induced cytopathic effects	Readout is based on the absence of visible agglutination
10	Assay Duration	Can take several days to perform	Can be completed relatively quickly
11	Viral Titer Estimation	Not used for estimating viral titer	Can be used to estimate antibody titer
12	Antibody Detection	Not primarily used for detecting antibodies	Mainly used to detect antibodies in a sample
13	Viral Strain Specificity	Can be used to determine the specificity of neutralizing antibodies	May not provide information about antibody specificity
14	Suitable for All Viruses	May not work for all viruses due to the need for specific cell lines	Can be adapted for various viruses and antigens
15	Clinical Applications	Commonly used in vaccine development and serology testing	Primarily used in serology testing and blood typing
16	Sensitivity	Generally highly sensitive to neutralizing antibodies	Sensitive to the presence of specific antibodies
17	Viral Load Estimation	Does not estimate viral load	Does not estimate viral load
18	Viral Variants Detection	Can be used to assess the efficacy against viral variants	Not typically used to detect viral variants
19	Requirement for Red Blood Cells	Doesn’t require the use of red blood cells	Depends on the presence of red blood cells
20	Viral Species Specificity	May be specific to certain viral species or strains	Not specific to viral species or strains
21	Vaccine Testing	Used to evaluate the efficacy of vaccines	Not used to evaluate vaccine efficacy
22	Antigen-Antibody Interaction	Assesses the blocking of virus-antibody interactions	Measures the inhibition of antigen-antibody reactions
23	Commonly Used in	Virology and immunology research	Blood banks and serology laboratories
24	Examples of Use	Used for assessing immunity to influenza, HIV, and other viral infections	Used for ABO and Rh blood typing and measles antibody detection
25	Risk of Contamination	Requires strict biosafety measures due to live virus use	Less risk of contamination as it doesn’t involve live viruses
26	Result Interpretation	Focuses on the reduction in viral infection or cytopathic effects	Concentrates on the prevention of red blood cell agglutination

Frequently Asked Questions (FAQ’S)

Q1. What purposes do neutralization assays serve?

Neutralization tests are used to assess the efficacy of vaccines, check the strength of antiviral medications, research the immune response to infections, and comprehend the neutralization properties of antibodies made in response to vaccination or naturally occurring illness.

Q2. What are the essential procedures for carrying out a neutralization assay?

Preparing viral samples, incubating the virus with the test material, introducing the mixture to target cells, enabling infection to take place, and calculating the decline in infection rates relative to control samples are the essential processes.

Q3. Neutralization tests can be used to research what kinds of viruses?

Numerous viruses, including influenza, HIV, hepatitis viruses, coronaviruses (such as SARS-CoV-2), and more can be studied using neutralization assays.

Q4. What role does the HI assay play?

The HI test is frequently used in influenza research and vaccine development to gauge the amount of antibodies that neutralize various viral strains. It aids in determining how well a vaccine works to produce defense-enhancing antibodies.

Q5. Do HI assays target influenza viruses specifically?

The HI assay can be modified for various hemagglutinating agents, even though influenza viruses are the ones most frequently associated with it. However, depending on the drug being examined, different methods and reagents may be used.

Q6. What procedures are followed when doing an HI assay?

Preparing red blood cells, dilution and incubation of test serum or antibodies with the virus, addition of red blood cells, and watching for agglutination are the main processes. The level of hemagglutination inhibition is determined by the maximum dilution that does not cause agglutination.