Serological / Immunological Methods

Let's continue with the alternative methods, which are serological/ immunological methods. These methods consist of :

• Haemagglutination (HA)
  
• Haemagglutination Inhibition (HI)
  
• Virus neutralisation
  
• Complement fixation
  
• Immunostaining
  
• Immunoprecipitation/ Immunoblot
  
• ELISA

Unfortunately yes, there are many methods to know about. Let's go through one by one slowly..

1. Haemagglutination

Haemagglutination is visible macroscopically and is the basis of haemagglutination tests to detect the presence of viral particles. The test does not discriminate between viral particles that are infectious and particles that are degraded and no longer able to infect cells. Both can cause the agglutination of red blood cells.

-Influenza and other viruses
-Two spike proteins: NEURAMINIDASE , HAEMAGGLUTININ ( Binds specifically to red blood cells)

Steps to haemagglutination:
1. Dispense diluent.
2. Add red blood cells and mix by gently shaking.
3. Allow the red blood cells to settle and observe the pattern.
4. Observe if the cells have a normal settling pattern and there is no auto-agglutination. This will be a distinct button of cells in the micro test and an even suspension with no signs of clumping in the rapid test.

2. Haemagglutinin Inhibition

Serologic tests in which a known quantity of antigen is added to the serum prior to the addition of a red cell suspension. Reaction result is expressed as the smallest amount of antigen which causes complete inhibition of haemagglutination.
It is possible to carry out rapid haemagglutination tests and haemagglutination-inhibition tests on a plate, just as for the bacterial agglutination tests described above. However HA and HI are generally only used in this way to confirm the presence and identity of a haemagglutinating antigen.

• HA conducted in the presence of antibody
  
• Neutralisation of virus inhibits agglutination
  
  

3. Virus Neutralisation

Antibody neutralisation prevents/ lowers virus infectivity.

4. Complement fixation

The complement fixation test is an immunological medical test that can be used to detect the presence of either specific antibody or specific antigen in a patient's serum. It was widely used to diagnose infections, particularly with microbes that are not easily detected by culture methods, and in rheumatic diseases. However, in clinical diagnostics labs it has been largely superseded by other serological methods such as ELISA and by DNA-based methods of pathogen detection, particularly PCR.

• Meditated by antibody
  
• Antibody binds to antigen


• Complement cascade of molecules in blood serum initiated, causing lysis of infected cell or pathogen
  

5. Immunostaining

- Immunoflourescence

-Immuno-gold EM

Immunoflourscence: A laboratory technique to identify specific antibodies or antigens. Antibody identification is usually performed on blood (serum).

• Antibody tagged with flourescent dye
• Antibody attached specifically to antigen
• View specimen under exciting light
• Flourscence microscope

Immunogold Electron Microscopy :

• Same principle as immunoflourscence
  
• Gold particles attached to antibodies (nanometer size particles)


• Viewed under EM to localise specific proteins or antigens
  
  
  

6. Immunoprecipitation/Immunoblot

Immunoprecipitation:

- Antigen radioactivity labelled
- Reaction with anitibody
- Isolate anitibody –anitigen complex
- Run through SDS-PAGE
- Detect through x ray film

Immunoblot:

- Western blot analysis
- Whole protein sample run through SDS-PAGE
- Transfer to nitrocellulose membrane
- Antibody attaches specifically to one protein
- Antibody labelled with sensitive indicator
- Colour reaction with streptavidin

7. (LASTLY.) ELISA

ELISA is a widely-used method for measuring the concentration of a particular molecule (e.g., a hormone or drug) in a fluid such as serum or urine. It is also known as enzyme immunoassay or EIA.
The molecule is detected by antibodies that have been made against it; that is, for which it is the antigen. Monoclonal antibodies are often used.

The test requires:
- the antibodies fixed to a solid surface, such as the inner surface of a test tube;
- a preparation of the same antibodies coupled to an enzyme. This is one (e.g., β-galactosidase) that produces a colored product from a colorless substrate.

Performing the Test:

1. The tubes are filled with the antigen solution (e.g., urine) to be assayed. Any antigen molecules present bind to the immobilized antibody molecules.

2. The antibody-enzyme conjugate is added to the reaction mixture. The antibody part of the conjugate binds to any antigen molecules that were bound previously, creating an antibody-antigen-antibody "sandwich".

3. After washing away any unbound conjugate, the substrate solution is added.

4. After a set interval, the reaction is stopped (e.g., by adding 1 N NaOH) and the concentration of colored product formed is measured in a spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.

ELISA can also be adapted to measure the concentration of antibodies. In this case,

1. The wells are coated with the appropriate antigen.

2. The solution (e.g., serum) containing antibodies is added.

3. After they have had time to bind to the immobilized antigen,

4. an enzyme-conjugated anti-immunoglobulin is added, consisting of

- an antibody against the antibodies being tested for. For example, if human anti-HIV antibodies are being assayed, then antibodies (raised in a goat or rabbit against human immunoglobulins) are conjugated to
- the enzyme.

5. After washing away unreacted reagent, the substrate is added.

6. The intensity of the color produced is proportional to the amount of enzyme-labeled antibodies bound (and thus to the concentration of the antibodies being assayed).

Literally hundreds of ELISA kits are manufactured for
- research
- human and veterinary diagnosis

Some examples:
-screening donated blood for evidence of viral contamination
-measuring hormone levels
-detecting infections

That's all for the serological/immunological methods! It's a very long list but we hope you all understand the methods better with our explanations and diagrams.

The very last method used for detection will be up in the next post!

stay tuned.

:DD