المحاضرة 3 - فايروسات عملي

Serological and Immunological Methods in Virus Detection

Immunological techniques are used to diagnose virus diseases. This is done so by demonstrating an antigen-antibody reaction to reveal evidence of virus infection. 

Virus infection can be shown by:

• *Serology; where development of antibody to the virus at the time of or just after symptoms of disease.
• Virus being present in the blood of the patients or other tissues.
• Antibody: proteins produced by the immune system which help defend against antigens. The variable regions are thought to be the place for recognition and binding with the antigen
• Antigen: Any molecule that induces production of antibodies when introduced in the body is called antigen. Or any "thing", foreign to the immune system e.g. bacteria, viruses, (or their parts), pollen, etc 

1. يا نجاتي انفخ ( لقح ) البللين - pollen

There are several serological and immunological methods and these methods include:

1. Haemagglutination Assay (HA) 

2. Haemagglutination Inhibition (HI)

3. Virus neutralization

4. Complement fixation

5. Immunostaining

6. Immunofluorescence

7. RIA

8. ELISA

Haemagglutination Assay (HA)

A quantitative of viruses by haemagglutination is known as haemagglutination assay (HA). Haemagglutination is a particular form an agglutination which involves the participation of red blood cells (RBC). Some viruses and other microbes contain proteins which bind to erythrocytes ( red blood cells) causing them to clump together.

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This test is not antigen-antibody test.{ Heamagglutination: is a biological phenomenon present in some viruses which have heamagglutinin antigen on their surface like paramyxoviruses and orthomyxoviruses}, the other antigen which present on the surface of these viruses is neuraminidase which responsible for Elution. The titer of heamagglutination is found out before carrying out the heamagglutination inhibition test 

Purposes of use:

1-To diagnose and characterize viruses which contain heamagglutinin in their surface.

2-To found out titer of virus.

3-To perform another test (heamagglutination inhibition test). 

Factors affecting on heamagglutination test:

1-Temperature

2-Type of washed red blood cells:

3-PH:

Haemagglutination Inhibition (HI)

The agglutination of red blood cells by the haemagglutinating viruses are blocked by the antiviral antibody. 

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اختصار لما ورد في اعلاه

1. RBC + RBC = NO REACTION
2. RBC + VIRUS = HAEMAGGLUTINATION
3. RBC + ANTIBODY + VIRUS = HAEMAGGLUTINATION INHIBATION

Virus Neutralisation (VNT)

In the case of cell cultures and laboratories animals, antibody prevents or lowers the viral infectivity. This technique is difficult and slow as it requires long and hard work. This method work to detect the presence of viral neutralizing, the antibodies bind to the viral particles.

VNT procedure: mixing dilutions of antibodies with standardized amount of virus, incubation them and cultured into cells, egg or animal to have a clear cytopathic effect (CPE) observation

Steps of NVT

1- Serial dilutions of serum (virus-neutralizing Ab) with known virus incubated for 1-2h at 37°C

2- Add the mixture in step 1 with cell culture then incubated at 37°C

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3- Examine for CPT under light microscope (The CPE could be, rounding of cells, change in texture (granular, hyaline, glassy or formation of syncytium)

Results that expected for this test is

No Ab in serum + virus, there is no neutralization, there is CPF Ab in serum + virus, the is a neutralization thus, there is no CPE

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