Lab.6 viral serological tests

By Assist. Lect. Mays Jassim

Serology is used widely to diagnose viral infections as many of the viruses cannot be easily cultured.

Serology means the study of serum and can be used to detect both antibody and antigen (e.g. hepatitis B surface antigen).

Several techniques have been developed, but the fundamental principles are similar for all.

Antibodies are produced as a host response to viral infection.

1. IgA is produced at the local site of infection and provides local immunity, for example in the gut or respiratory tract.

2. IgM is the first antibody to appear after humoral immunity response (produced in blood) which can be detected as early as a couple of days after an acute infection, then it declines and is normally undetectable by about 3 months after infection

3. IgG antibody, which appears from 7–15 days after onset of infection, it persists for life and is responsible for providing lifelong immunity to the particular virus.

Acute or recent infection can therefore be diagnosed by: demonstrating the presence of virus specific IgM (IgG may or may not be present)

Past infection or immunity is diagnosed by:

demonstration of virus specific IgG alone (and absence of IgM).

Principle of serological techniques

Assays may be • qualitative (e.g. give only a yes or no answer). e.g. immune-chromatographic assay (ICA)

• or be quantitative (e.g. measure the antibody level). e.g. ELIZA, Neutralization (NI), Heamagglutination Inhibition (HI) tests.

All serological techniques that detect antibody are based on the principle of adding:

specific viral antigen + patient serum (in the kit) (ABs)

An indicator system is used to detect whether such a complex has been formed (e.g. enzyme or florescent stain or RBCs/ depending on the technique).

These techniques can be reversed to detect the presence of viral antigen instead, such as hepatitis B surface antigen (HBsAg), in the patient’s serum.

techniques

indicator system antigen/antibody complex.

Principle of viral hemagglutination:

Many viruses have hemagglutinin (hemagglutinate RBCs) that are able to adhere to the surface of erythrocytes by reaction of agglutinin protein (this specific protein project from envelop of orthomyxoviruses such as: (Influenza A, B and C), mumps, Newcastle, rubella and respiratory syncytial virus) and receptor sites on RBCs membrane (mostly glycoprotein) thus reaction occurs as visible agglutination. The linking together of the red blood cells by the viral particles results in clumping. This clumping is known as haemagglutination.

1. haemagglutination inhibition tests (HAI)

The screen test is very simple and gives indication to the presence of virus and viral replication in clinical samples and experimental tests.

Procedure of viral hemagglutination 1-Prepare serial 2-fold dilution of allantoic fluid in saline (1:2)-(1:1024). 2-Add 50µl of 1% RBCs suspension suspension (guinea pig RBCs or human group RBCs) of the species to 50µl of each viral dilution of allantoic fluid. Mix and incubate at R.T. (25Ċ°) for 1hour. 3-Examine for hemagglutination, the concentration of the viral agent with highest dilution of embryonic fluid (allantoic fluid) at which complete visible agglutinate occur in the end point and 50µl is dilution contains 1HAU/50µl.

Titer = 32 HA units/ml

1:8

1:2 1:2 1:2 1:2 1:2

8 16 32 64 128 256

virus

serial dilution

mix with

red blood

cells side view

top view

Note: To obtain 4HAU/50µl must be diluents the original allantoic fluid according to the following formula: Reciprocal dilution containing 1 HAU/50 µl Dilution of fluid containing 4HAU/50µl = -------------------------------------------------------------- 4 = 32 /4 = 8

haemagglutination inhibition tests (HAI)

These tests detect antibodies to viruses (rubella, influenza) that possess a haemagglutinin antigen. These are also relatively insensitive and can give non-specific reactions, and have mostly been replaced by more sensitive and specific techniques.

Procedure of haemagglutination inhibition tests (HAI)

1. Mix 50µl of allantoic fluid (virus) containing (4HAU/50µl) with 50µl (1:10) dilution of hyper immune type specific antisera (antibodies).

inactivate the complement in antiserum at 56Ċ water bath for 30 min prior to use).

2. Add 50µl of 1% RBCs suspension for each tube.

Set up control: • Virus control containing 50µl allantioc fluid and 50µl 1% RBCs

suspension.

• RBCs control containing 50µl RBCs suspension and 50µl normal saline.

3. Incubate all tubes at R.T. for 1hour.

4. Identify of the virus is indicated by the type of antiserum that inhabits the hemagglutination reaction.

Haemagglutination inhibition test (HIA)

You can identify the type of the viral isolate (influenza virus/ H1NI, H5N1, H3N2) from embryonic fluid or tissue culture or clinical samples.

Thank you