In vitro Antigen-In vitro Antigen-Antibody-ReactionsAntibody-Reactions

I- Agglutination Reactions:I- Agglutination Reactions:

Requirements:

1-The antigen(Ag) has to be in particulate form.

2-The presence of electrolytes (saline).

Agglutination can be performed on slides, in tubes, or even in plastic plates with wells (micro-titration plates).

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Agglutinins

Agglutination

TypesTypes: : 1-Slide agglutination: i- Identification and typing of isolated bacteria

(Serotyping):It is a quick reliable test, if the proper antibody (Ab ) is available.

Two drops of saline are placed on a clean slide. A suspension of the isolated organism is prepared in both drops. To, one drop, a drop of known serum (Ab) is added and mixed, the other is left as a control.

Clumping occurs if the serum is specific to the organism.

Slide agglutinationSlide agglutination

ii) Blood grouping: Two drops of blood are placed on a clean slide. To one drop, anti-A is added, and to the other drop, anti-B is added and mixed. Group A red cells will agglutinate with anti-A serum. Group B will agglutinate with anti-B serum. Group AB will agglutinate with anti-A and anti-B sera. Group O will not agglutinate with any of the antisera. This is an example of active haemagglutination.

2-Tube agglutination : It is a semiquantitative test, used mainly to detect Abs in patients' sera.

To serial dilutions of patient's serum, a constant amount of known bacterial suspension is added, then incubated for the required time at the desired temperature. The tubes are examined for the presence of agglutination. The last tube showing agglutination is the end point.

• The reciprocal of the dilution of this end point is the titre, for example, if the dilution is 1/400 then the titre is 400. e.g.Widal test for the diagnosis of enteric fever (typhoid and paratyphoid) and Weil - Felix test for the diagnosis of typhus fever are examples of such tube agglutination tests .

Tube agglutinationTube agglutination

• 3-Haemagglutination(Passive&Active): i) Passive haemagglutination: Red cells

sensitized with the Ag can be used to detect antibodies directed to these antigens that are prepared in a special way and used to coat the RBCs. If antibodies are present, haemagglutination will occur. Example TPHA for detection of treponemal antibodies.

HemagglutinationHemagglutination

Hemagglutination(tube method)Hemagglutination(tube method)

• ii) Active haemagglutination: Some viruses for example influenza virus have haemagglutinin spikes on their surface that are capable of haemagglutinating RBCs. This character can be used in a haemagglutination inhibition test to detect viral antibodies.

• 4-Latex agglutination:

Particles such as latex beads could be conjugated to antibodies and used for agglutination of antigens in solutions. These latex agglutination tests are the basis of a number of kits for the rapid identification of pathogenic bacteria and fungi such as N.meningitidis and Cryptococcus neoformans.

It is also possible to conjugate latex particles with the Ags and used for detecting antibodies in patients' sera or other body fluids.

Latex agglutinationLatex agglutination

Latex agglutinationLatex agglutination

Antigen & AntibodiesAntigen & Antibodies

• 5-Coagglutination test:

This assay uses Staphylococcus aureus as an antibody – carrying particle. It takes the advantage of the ability of protein A on the surface of this bacterium to bind the Fc portion of IgG antibodies. In coagglutination assays, the binding of the antigen to the antibody immobilized via protein A to the bacterial surface causes the S.aureus cells to agglutinate.

CoagglutinationCoagglutination

• 6-Coomb' s test:

i) Direct Coomb' s test: It detects Abs already present on red cells. In erythroblastosis foetalis (E.F.), baby' s red cells are coated with anti- Rh Abs. The cells are washed, then anti-human globulin is added resulting in agglutination.

Coomb’s testCoomb’s test

Incomplete or blocking AbsIncomplete or blocking AbsCoombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests

• Direct Coombs Test– Detects antibodies on erythrocytes

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Patient’s RBCs Coombs Reagent(Antiglobulin)

ii) Indirect Coomb' s test: It detects anti-Rh Abs in mother's serum.

Mother 's serum suspected to have anti-Rh Abs is incubated with Rh positive red cells. Abs will bind to the RBCs but produce no agglutination (similar to E.F.). The cells are then washed , and anti-human globulin prepared in rabbit is added. If the serum contains Abs, clumping of the cells will occur.

Coombs (Antiglobulin)TestsCoombs (Antiglobulin)Tests • Indirect Coombs Test

– Detects anti-erythrocyte antibodies in serum

Patient’s Serum

TargetRBCs

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Coombs Reagent(Antiglobulin)

Step 2

II -Precipitation Reactions:II -Precipitation Reactions:

• Requirements:

1-The Ag has to be soluble(in solution).

2-The Ag and the Ab have to be in optimal concentration.

• Types:

• i) Slide precipitation: as RPR and VDRL for diagnosis of syphilis.

• ii)Tube precipitation :as Lancefield test for grouping streptococci ( Ring test).

iii)Agar ( Gel) Diffusion tests:

A) Double immunodiffusion :

a) Agar gels are poured in plates or on slides. Wells are punched in the gel. The Ag is placed in one well, and the Ab in another well. Both the Ag and Ab will diffuse, and at the site of optimum proportion a precipitation band will occur.

Double ImmunodiffusionDouble Immunodiffusion

• b) Toxin-antitoxin reaction as in Elek's test to diagnose toxigenic strains of C.diphtheria. Strains isolated from carriers are not considered pathogenic unless proved to produce the toxin.

Elek’s testElek’s test

• Elek's test: Strip of filter paper is immersed in diphtheria antitoxin, then placed on the surface of serum agar plate. A heavy inoculum of diphtheria bacilli isolated from case or carrier is inoculated at right angles to the strip. The plate is examined after two days incubation at 37ºC. If the organism is toxigenic, the toxin will diffuse and will precipitate with the antitoxin diffusing from the filter paper and a line of precipitation will be formed at the optimum concentration. Positive and negative strains should also be included in the test as controls.

Double immunodiffusion could be electrically accelerated to give a rapid result in half an hour.

This is termed

counter current immunoelectrophoresis.

B) Single (Radial) immunodiffusion:

The Ab is incorporated in the agarose gel plate. The Ag is placed in a well punched in the agarose. The Ag diffuses in all directions and a precipitation ring will form around the well. The diameter of the ring is proportional to the Ag concentration. This is a quantitative method to measure the concentration of the unknown Ag.

Single radial immunodiffusionSingle radial immunodiffusion

Single radial immunodiffusionSingle radial immunodiffusion

• This technique is used for estimating the amount of different Ig classes in the serum for example IgG. An agarose plate containing anti-IgG is used.

• IgG preparations of known concentration, e.g.(2.5, 5.0 and 10.0 µg/ml) are placed in the first three wells, the unknown sera are placed in the other wells. The plate is left for two days, then the squares of the diameter of the rings containing the known concentrations are plotted against their concentration .

• By measuring the diameters of the unknown sera, one can get out the concentration of the unknown Ag.

• Single immunodiffusion could be electrically accelerated to get rapid results in half an hour. This is termed rocket electrophoresis.

C) Immunoelectrophoresis: It is electrophoretic separation of the serum into its

components followed by immunoprecipitation by specific antisera.

It is done by separating different Ags in an agarose gel poured on a glass slide by placing an electric charge across it. The pH is chosen so that positively charged proteins move to the negative electrode, and the negatively charged proteins to the positive electrode. Then an antiserum(Ab) is allowed to diffuses from a trough cut into the agar along the line of protein migration . As each Ag meets its specific Ab, a curve of precipitin forms in the agarose ( the Ags and Abs form precipitin arcs). This method allows the study of complicated mixtures of Ags as found in serum.

ImmunoelectrophoresisImmunoelectrophoresis

III-Complement Fixation Test III-Complement Fixation Test (C.F.T):(C.F.T):

• This is an antigen antibody reaction that occurs in the presence of a third component known as the complement. The antigen reacts with its specific antibody and the resulting complex fixes the complement.

• The complement is a complex protein component of normal serum and body fluids (except urine and C.S.F.) of man and other animals. It is heat labile and is destroyed by heating at 56ºC for 30 minutes. The guinea pig serum is a common laboratory source of complement.

• The complement can be fixed by Ab after it binds with its specific Ag giving Ag-Ab complex. If the Ag-Ab complex is red cells coated with their antibodies , the fixed complement causes haemolysis of red cells.

The C.F.T. is used in the diagnosis of many diseases by detecting C.F.antibodies in the serum of patients, as in syphilis, whooping cough, chronic gonorrhoea, typhus and other diseases as well as many viral infections.

• Principle of C.F.T: The heated serum of a patient (unknown

antibody) is added to the known antigen, then the standardized complement is added. If the serum contains antibodies specific to the added antigen, they will unite together and fix the complement. If the serum is negative for antibodies, no Ag-Ab complex is formed and the complement is not fixedi.e.left free in solution.

• Up to this stage of the test there is no visible phenomenon to show if the complement is fixed or not(no visible reaction).

• A second step should be done in which an “indicator system” is added made of sheep red cells coated with their antibody. If the complement is free (as with negative sera), it will cause haemolysis of red cells.

• Thus, haemolysis means negative serum. If the complement is fixed (as with positive sera); no haemolysis occurs. Thus no haemolysis means positive serum.

IV- Antibody (Ab) –labeled IV- Antibody (Ab) –labeled assays:assays:

1-Fluorescent Antibody techniques:

• A)Direct immunofluorescence(D I F):

The Ag to be detected is present in a smear or frozen tissue section.

The fluorescein labeled specific Ab is added, and then the slide is washed to remove the unbound Ab. The slide is then examined by an ultraviolet microscope(U.V.). The background is dark and areas with bound fluoresceinated Ab fluoresce green.

• B) Indirect immunofluorescence(I I F): i-For detection of antibodies: This test can be used for detecting

specific antibody in patient's sera. It is done by placing the Ag on a clean slide, the patient' serum is added, then the slide is washed. Antihuman gamma globulin labeled with fluorescein is added, then the slide is washed and examined by the ultraviolet microscope.If green fluorescence is seen, it means that the patient has specific Ab to the Ag.

ii-For detection of antigens:

Specimen suspected to contain the antigen is fixed on a slide, specific antibody is added, then the slide is washed. Antispecies antibody conjugated with fluorescent dye is added, then washed and examined by U.V microscope.

The indirect IF is more sensitive than the direct method, and is more economic and it gives more magnification to the reaction.

Indirect ImmunoflourescenceIndirect Immunoflourescence

• C-Flow cytometer for the detection of leukocyte antigens ( markers or cell surface molecules ).

-It is an apparatus capable of analyzing single cells using monoclonal antibodies that are conjugated with different fluorochromes (e.g. fluorescein isothiocyanate for green light emission, rhodamine for red /orange light emission) as they pass through an orifice at high velocity.

• -This method is termed leukocyte phenotyping based on their size and intracellular granules.The data are analysed by computer.

• -It allows us quantify B-cells, T-cells, monocytes and granulocytes within different sites in the body ( blood, bone marrow, spleen, lymph nodes ) in tissue sections or in fresh cell suspensions.

• -In addition , we can enumerate subsets of these cell types .For example, T-lymphocyte subsets that differ in their functional properties can be distinguished phenotypically.

• -The surface phenotype can also provide clues as to the differentiation state of the cell.

• Precise quantitation of T and B cells in human peripheral blood has made important contributions to our understanding of immunodeficiency disorders, autoimmune diseases, tumour immunity, and immunity to infections.

2- Enzyme Linked 2- Enzyme Linked Immunosorbent Assay(ELISA):Immunosorbent Assay(ELISA):

• Principle of the test: It is based on two assumptions that

antigen or antibody can be attached to a solid phase (plastic surface), but still it retains its immunological activity and that either antigen or antibody can be linked to an enzyme such as alkaline phosphatase or peroxidases and the complex retain both immunological and enzymatic activity.

• Types:

A-Sandwich method ELISA( Double antibody) for detection and measurement of unknown Ag.

1.The wells in polystyrene plastic plates are coated with specific antibody to the antigen.Plates are washed.

2. The test solutions thought to contain antigen are incubated in the sensitized wells. Washing removes unreacted materials, and any antigen remains attached to the immobilized antibody on the plastic surface.

3.The conjugate consisting of enzyme- labelled specific antibody is then incubated in each well. This will react with any antigen already “captured” by the antibody on the well surface. A further washing removes excess conjugate.

4.Finally, the enzyme substrate is added. Its rate of degradation depends on the amount of enzyme- labelled antibody present and that, in turn, depends on the amount of antigen in the test sample. The enzyme substrate is chosen to give a colour change upon degradation, and this can be assessed visually or measured in a spectrophotometer (ELISA reader).

B-Indirect ELISA for detection and measurement of unknown antibody.

1.Wells of polystyrene microplates are sensitized by passive adsorption with the relevant antigen; the plates are then washed.

2.The test samples are incubated in the sensitized wells and the plates

are again washed; antibody present reacts with the immobilized antigen on the well surfaces.

3.Enzyme-labelled anti-human Ig conjugate is incubated in the wells, this reacts with any “captured” antibody in step 2. Enzyme substrate

is added and the plates are incubated, the rate of degradation of the substrate is indicated by a colour change, which is proportional to the

antibody concentration in the test samples in step 2.

4.The reaction is stopped, and the colour changes is assessed visually or in a spectrophotometer.

C-Competitive ELISA; both labelled (known) and unlabelled (unknown) Ag compete for the active site of the enzyme.

3-Radioimmuno assay (RIA):

*Principle:It is based on labelling either the Ag or the Ab with radioactive substance such as iodine or tritiated thymidine. One of them should be known (radioactivelabelled) and the other is unknown.

The amount of radioactivity is measured by gamma or beta counter.

RIARIA

Types:

i) Liquid phase RIA:The test is done in solution.

ii) Solid phase RIA:Ag is coated onto a plastic plate (or tube), then the plate is washed, and the test Ab is added. The plate is washed , then radioactive labeled anti-species Ab is added, then the radioactivity is measured.

iii) Competitive RIA.

4-Chemiluminescence:

Principle: It depends on labelling the Ab with acetylaminofluorene ;a luminescent substance detected by luminometer.

Intradermal skin testsIntradermal skin tests

I-Neutralization tests:

These are in- vivo manifestations of

Ag-Ab reactions.

A-Schick test: To test the susceptibility of a person to diphtheria. A small amount of the diluted diphtheria toxin, is injected intradermally in one forearm and in the other a heated toxin. The development of a localized erythema indicates that, there is no antitoxin to neutralize the toxin, so the person is susceptible to suffer from diphtheria.

B-Dick test:To test the susceptibility of a person to get scarlet fever.The test is done exactly as the above, but using the erythrogenic toxin of Streptococcus pyogenes.

C-Schultz-Charlton reaction:Used for the diagnosis of scarlet fever. A small amount of the antitoxin (anti-erythrogenic toxin) is injected in the red rash. If blanching occurs, it means that the rash is caused by the erythrogenic toxin, and that the person has scarlet fever.

II-Hypersensitivity tests:II-Hypersensitivity tests:

a) Immediate type skin test:

As testing for the sensitivity to penicillin. A small amount of penicillin is injected in the forearm. An immediate wheal and flare indicates that the person is hypersensitive to penicillin.

b)Delayed type skin test: The Ag is injected intradermally, and the

test is read after 48-72 hours. Development of induration and redness at the site of injection indicates a positive test. Delayed type hypersensitivity tests are induced by sensitized lymphoid cells(cellular reaction). Examples: tuberculin, lepromin, brucellin and Ducrey tests.