Herpes simplex virus

Human immunodeficiency virus Coronavirus (covid-19)

Lab. 9 viral serological

techniques

by assist. Lect. Mays jassim

2- Complement fixation test

This test is based on the principle that when an antigen/antibody complex is formed it will ‘fix’ (bind) complement, so free complement is not available to lyse red blood cells (RBCs) that are added as indicator. Complement fixation tests have been extensively used in the past to aid clinical diagnosis; because of : 1- their complexity. 2- relative in-sensitiveness.

So, they are now being replaced by newer tests such as EIA.

3- Immunofluorescence tests (IF or IFT)

These assays use the same principle as ELISA, and like EIA they can be constructed to detect either viral antibody or antigen in the patient specimen. However, instead of the enzyme/substrate detector system of ELISA, fluorescein-labelled anti-human antibody is used to detect a positive reaction, which appears as (apple-green) fluorescence under a light microscope.

To look for viral antigen (RSV, influenza A etc.), tissue from the patient’s secretions (e.g. nasopharyngeal aspirate) are fixed to a spot on the glass slide and fluorescein-labelled monoclonal antibody against the virus is added.

IFT principle

procedure

Immunofluorescence test is also rapid serological tests. but the disadvantage is that they require subjective interpretation and good expertise.

Zika virus- infected cells,diagnosed by IFA test

4- Latex agglutination (LA) and gelatin particle agglutination test (GPAT)

• Latex particles are coated with viral antibody.

• The antibody-coated

particles are specifically agglutinated by virus.

• Then the agglutination is evident macroscopically within minutes.

rabies Latex agglutination test for in saliva specimen virus

Latex agglutination test of fecal supernatant in patients with diarrhea. Agglutination of latex particles were occurred, when group A rotavirus antigens present in fecal sample and this agglutination interpreted with the naked eye in few min

5- Immunochromatographic assay/ ICA (rapid test): are simple cellulose-based devices intended to detect the presence of a target analyte (antigen or antibody) in liquid sample without the need for specialized and costly equipment.

1. sample pad 2. Conjugate pad (site of labeled antibodies) 3. Test line (site of primary antibodies for Antigens detection. or site of specific antigen for antibodies detection. 4. Control line (site of secondary antibodies (anti- labeled antibody)) 5. Absorbent pad (absorb the remains of the reaction elements). 6. Plastic adhesive pad. 7. Nitro-cellulose membrane.

The protocol of ICA for antigen detection

1. mixing the patient’s sample (blood, stool, sputum) with a lysing agent (buffer solution) in a test strip or well. To release the antigen from the cells.

2. on the conjugated pad, 2-3 drops of buffer containing sample , have been added. The antigen then mixed with labeled antibody in the pad and flown up the strip across the test line.

3. In the test line, if antigen is present, the antigen- labeled antibody complex will be captured by the primary- antibody to make antigen-labeled- primary antibody complex. And the positive result appears as a red or purple line.

4. In the control line, the labeled antibody form a complex with the secondary antibody and appears as a line indicate for the validity of the strips.

The protocol of ICA for antibodies detection

1. on the conjugated pad, 2-3 drops of patient serum have been added. The labeled antibody make a complex with the serum antibodies to form patient antibody- labeled antibody complex, then the complex and to the test line.

2. In the test line, the patient serum antibodies with labeled antibodies captured by the antigen specific for serum patient antibodies, to result in a colored line (red or purple).

3. In the control line, the labeled antibodies captured by anti- labeled antibodies (i.e. secondary antibodies). And the result appear as a colored line.

Herpes simplex virus Hepatitis B virus

Human immunodeficiency virus Coronavirus (covid-19)

TORCH rapid test

Rapid test detect the serum antibodies (IgM) against five categories of microorganisms. They checked at a time altogether, because they all responsible for causing abortion for pregnant women and birth defects to newborns. T = mean Toxoplasmosis causing by Toxoplasma gondii R= Rubella C= Cytomegalovirus H= Herpes Simplex disease (HSV-1, HSV-2).

Thank you