Neutralization Test and Western Blotting

Serological test/Diagnosis

The antigen-antibody test is used in diagnosis and control of a microbial disease.

Application of serological test

• To diagnose a microbial disease when the pathogen is not present in a routine specimen or if present is not easily isolated and identified by other available techniques. Example like Syphillis, rickettsial disease, enteric fever, viral hepatitis.
• To provide an early diagnosis or presumptive diagnosis of disease. Example Meningitis and Cholera.
• To identify serotype of a pathogen that has been isolated by culture. Example Vibrio cholera
• To screen for rises in antistreptolysins ‘o’ in the diagnosis of Rheumatic fever, acute glomerulonephritis.

There is various method of serological test such as:

1. Neutralization test
2. Precipitation
3. Agglutination
4. Complement fixation
5. ELISA
6. Radioimmunoassay

Neutralization test

This test is based on the principle that specific antibodies are able to neutralize biological effects of viruses, toxins, and enzymes. They used for can be used for detection of either antigen or antibody. Two types of neutralizing test are:

• Virus neutralization test
• Toxin neutralizing test

Virus neutralization test

When grown in various culture systems, viruses produce biological effects. Neutralization of these effects by a specific antibody is used in the identification of viruses. Two examples of virus neutralization test are described as follows:

• Neutralization of cytopathic effect, CPE
• Hemagglutination inhibition (HAI) test

• Neutralization of cytopathic effect, CPE

Neutralizing antibodies react with their homologous viruses by attaching to certain critical antigenic determinants on the virus particle. Due to this virus becomes incapable of initiating infection in the susceptible host cells.

For example, Viruses such as poliovirus produced a characteristic cytopathic effect when grown in appropriate tissue culture system. Patient’s serum to be tested for antibodies to poliovirus is mixed with the known suspension of poliovirus. After appropriate incubation, the mixture is inoculated into a susceptible cell line. After incubation, absence of cytopathic effect in the cell line shows that the patient’s serum contains neutralizing antibodies to the polio virus. The cytopathic effect will be observed in the absence of antibody in the patient’s serum.

• Hemagglutination inhibition (HAI) test

Tests using RBC agglutination are called hemagglutination test. Tryponema palladium hemagglutination (TPHA) test is one of the examples of hemagglutination test which uses chicken erythrocytes/RBC coated with an extract of Tryponema palladium. These red cells are agglutinated by antibody in the serum of a patient with syphilis.

Some viruses have the ability to induced hemagglutination when to combine in vitro with erythrocytes from specific animal sources. For example, Rubella virus can agglutinate red cells from newborn chicken. This activity can be neutralized by neutralizing antibodies to the virus.

Antibody to a hemagglutinating virus can be detected first by reacting patient’s serum with the virus. After appropriate incubation, the red cell suspension of susceptible species is added to the mixture. If an inhibiting antibody is present in the patient's serum hemagglutination will not occur.

Character of HAI test

• This reaction involves agglutination using RBC.
• Here, an antigen is attached to a surface of RBC and then patient’s serum is added.
• Antibody binds with antigen coated with RBC then appear visible clumping.
• It may take several hours to perform a test.
• The specificity and sensitivity depend on antigen used and how cells are prepared.
• This test can be used for detection of antibody.
• Example: Tryponema palladium Hemagglutination for the diagnosis of syphilis (TPHA)

Toxin neutralizing test

Exotoxin produced by some bacteria such as Corynebacterium diphtheria or Clostridium species are strongly antigenic and induced the formation of neutralizing antibodies (antitoxins) in their host. Effects of the toxins can be neutralizing by antitoxins and can be demonstrated in vitro.

An example of toxin neutralizing test in vitro is Nagler’s reaction which is used for identification of toxin-producing Clostridium perfringens from the clinical specimen. The alfa-toxin (lecithinase) of the pathogen produces opalescence in the medium containing egg yolk. For the test, half of the Petri dish containing egg yolk agar is smeared with antitoxin. The isolate is incubated in a streak across the plate, starting from antitoxin free area to the antitoxin area. After anaerobic incubation at 37C, the colonies of Clostridium perfringens in the antitoxin free shows a zone of opacity while those on the antitoxin area show no opacity while those on the antitoxin area show no opacity due to neutralization of the toxin by the antitoxin.

Western blotting

Western blotting is used to identify a specific protein in a complex mixture of protein. Specific proteins from the mixture are separated by SQS PAGE & the separated protein is transferred to a nitrocellulose membrane by electrotransfer. Protein antigens are detected by flooding the nitro cellular with radiolabeled or enzyme-linked polyclonal or monoclonal antibodies specific for the protein of interest.

The antigen-antibody complex formed in the band is visualized by following ways:

• If the protein of interest is bound by a radiolabeled and antibodies, its position on the blot is determined by autoradiography.
• If the enzyme-labeled antibody is used to link the protein in the band, a chromogenic substance is added so that the color product are developed.

Steps followed in western blotting technique for antibody detection:

• Separation of known antigen of well-defined molecular weight by SQS-PAGE
• Blotting into nitrocellulose
• Probing of separated bands of known antigens with sample suspected of containing ab specific for one or more of these antibodies
• Detection of Ag-Ab complex in bond by using either radiolabeled or enzyme linked anti-human globulin.

Application

Western blotting is a confirmatory test for HIV where antibodies that react with one or more viral proteins are detected.

REFERENCE

Cheesbrough, M. Medical Laboratory Manual for Tropical Countries. Vol. Vol 2. ELBS London, 2007.

Tille, P. Diagnostic Microbiology. 13th. Elsevier, 2014.

D, Grenwood, Slack RCB and Peutherer J. Medical Microbiology. Dunclude Livingstone: ELBS, 2001.