serology

. The branch of medical immunology concerned with antigen Antibody reactions in vitro is serology. The usefulness of serological test is dependent on its sensitivity and specificity

Terms used in evaluating test methodology

SensitivityAnalytical Sensitivity – ability of a test to detect very small amounts of a substance Clinical Sensitivity – ability of test to give positive result if patient has the disease (no false negative results)

Specificity

Analytical Specificity – ability of test to detect substance without interference from cross-reacting substances Clinical Specificity – ability of test to give negative result if patient does not have disease (no false positive results)

Affinity

Affinity refers to the strength of binding between a single antigenic determinant and an individual antibody combining site.

Any foreign substances which when introduced into an animal, can stimulate a specific immune response, in the form of production of antibodies and specific reactive T-lymphycytes. Antigens have the ability to combine specifically with the antibodies.


Are immunoglobulin that react specifically with antigen that stimulated their production

Features of Ag-Ab reactions:

The reaction is highly specific There is no denaturation of Ag or Ab during the reaction The strength of binding (the affinity) is proportional to the fit of the antigen with its antibodies combining site
Ab
Ag
Ab
Ag
Ab
Ag
Ab
Ag



Diagnosis of infectious diseases: known antigen preparations are used to detect circulating antibodies in patient's serum as evidence of a current or previous infection with that agent OR known antibodies are used to detect antigens associated with an infectious agent directly in body fluids.

A\ Unlabelled tests Precipitation reactions (Ag is soluble) precipitation. Agglutination reactions (Ag is particles) clumping. Complement fixation reactions. B\ Labelled tests 1-Immuno-fluorescence reactions 2 - ELISA.

This is an Ag-Ab reaction in which the Ag is soluble (eg: Protein ; Bacterial toxin). When antigens and antibody mixed in the proper proportion, they form large macromolecular complexes called precipitates in the presence of electrolytes at the suitable temperature and pH.

Precipitation Curve

Precipitation Reactions
Involve soluble antigens with antibodies


Agar Gel diffusion method: 1- Double diffusion: a- Elek’s Toxigenicity Test: b- Ouchterlony method: 2- Single radial immunodiffusion.

Elek’s Toxigenicity Test: Principle:

To determined the toxigenic strain of C. diphtheriae
Toxin production by C. diphtheriae can be demonstrated by a precipitation reaction between exotoxin and diphtheria antitoxin.
Procedure:
1. Place a strip of filter paper saturated with diphtheria antitoxin on a serum agar plate before it set.
3. Incubate the plate at 35oC for 24 hrs.
2. Streak the test organism across the plate at right angle to the filter paper.

Results:

Positive test: formation of four radiating lines resulting from the precipitation reaction between exotoxin and diphtheria antitoxin.

Ouchterlony method:

Wells are punched in the agar. The antigen in one well and the antibody is placed in another. Both antigen and antibody diffuse radially from wells toward each other, as equivalence is reached, avisible line of precipitation, aprecipitin line forms

Ouchterlony method:

2
1
C
3
Ag
2
1
C
3
Ag


Single radial immunodiffusion ( Mancini): The antibody is mixed with the agar before pouring it in the plate, while the antigen is placed in a well punched in the agar. the Ag diffuses in all directions, and where its concentration is optimal in relation to the antibody, a precipitation ring will form around the well. The diameter of the well depends on the Ag concentration.

The diameter of the precipitin ring is directly proportional to the amount of antigen loaded into the well.
Ab in gel
Diameter
Ag concentration
Ag
Ag
Ag


The interaction between antibody and a particulate antigen results in visible clumping called agglutination

Examples of agglutination reactions

Slide agglutination Tube agglutination

Example of Slide agglutination: Blood grouping (ABO grouping) There are 4 blood groups depending on the presence or absence of either or both two types of antigens A and B on the surface of RBCs.

Anti-A

Anti-B
Drop of blood
Agglutination in 1 only gp A Agglutination in 2 only gp B Agglutination in 1 and 2 gp AB No Agglutination in 1 or 2 gp O
1
2

Example of tube agglutination (Widal test)

A tube test used to determining of agglutinating antibodies in the serum of patient with typhoid fever.

0.5ml transfer from tube to tube

Discard0.5ml
0.5ml saline per tube + 0.5 Salmonella Ag
1:20
1:40
1:80
1:160
1:320
1:640
1:1280
1:2560
1ml serum
control

Complement fixation test

The complement fixation test is an immunological medical test looking for evidence of infection. It tests for the presence of either specific antibody or specific antigen in a patient's serum.
Two systems are used in CFT:1- Test system:The serum sample (heated to 56°)Measured amount of Ag.Complement (Guinea pig serum). if the serum contains the specific Ab→ Ag-Ab complexes→ will fix all the complement. Ag + Ab + C fixation Ag + C No fixation (free complement)


2- Indicator system e.g; sensitized sheep RBCs. (Sheep RBCs coated with their specific Abs).
It uses sheep red blood cells (sRBC), anti-sRBC antibody and complement, plus specific antigen (if looking for antibody in serum) or specific antibody (if looking for antigen in serum( If either the antibody or antigen is present in the patient's serum, then the complement is completely utilized, so the sRBCs are not lysed. But if the antibody (or antigen) is not present, then the complement is not used up, so it binds anti-sRBC antibody, and the sRBCs are lysed.

Result: +ve reaction No Lysis -ve reaction Haemolysis

These are Ag-Ab reactions in which Ab is labelled with fluorescein. Fluorescein is a dye which emits greenish fluorescence under UV light. There are two ways for this test; Direct immunofluorescence, Indirect immunofluorescence.


In this test a fluorescein-labelled Ab is added to detect the presence of Ag in tissue section fixed on a microscopic slide. A drop of the labelled Ab is placed on the section and left to react for some mint. the excess unattached Ab is washed Examine under UV rays. If Ag is present fluorescence If Not No fluorescence Disadvantage: expensive method (for each Ag we need specific labelled Ab)

The test is used to detect Ab in patients serum. Fluorescein labelled anti-human Ig is used . Known Ag is fixed on a slide Add the patient's serum & allow to react for some time the excess is washed, add Fluorescein labelled anti-human Ig (attach to the Fc portion of the human Ig if present). examine under UV.

positive test for rabies

ELISA (Enzyme- Linked Immuno Sorbent Assay)
The ELISA (Enzyme- Linked Immuno Sorbent Assay) has become one of the most widely used serological test for antibody or antigen detection. the test involve the linking of various “label” enzymes to either antigens or antibodies. Enzymes used in ELISA include: Alkaline phosphatasePeroxidase β galactosidase

Direct ELISA (double Ab technique): used for detection of Ags. Known specific Ab is found by adsorption onto a plastic surface. Clinical sample is added (if Ag present it will bind to the Ab) enzyme-labelled specific Ab is addad (attach to the fixed Ag if present) wash the excess add the substrate


If Ab specific to Ag change the color If Not specific No color change Dark yellow highly +ve Yellow moderate +ve Color less --ve Disadvantages: expensive method (for each Ag we need specific Ab labelled)

Antigens

+
Antibody labelled with enzyme
wash
+
substrate


Indirect ELISA: In this test an enzyme- labelled anti-human Ig is used to detect the presence of specific Abs in patients’ sera. Known Ag is fixed by adsorption onto a plastic surface.The serum sample is added ( if specific Ab is present, it will bind the fixed Ag).WashAdd the enzyme-labelled antihuman Ig wash the excess add the substrate, then quantitatively measure for the degree of color change.

Antigens

+
Antibody
=
wash
+
Ig enzyme labelled
=
+
substrate
=