Antigen Antibody

Reactions and clinical utility

Mohammad Mukhit Kazi

Objectives

• To know the principles of serological reactions / tests

• To know the technical aspects of serological reactions

• To know the application or clinical use of serological reactions / tests

• To know various serological tests for clinical diagnosis

Classification of antigen-antibody interactions

1. Primary serological tests: (Marker techniques)

e.g. – Enzyme linked immuonosorbent assay (ELISA)

– Immunoflorescent antibody technique (IFAT)

– Radio immunoassay (RIA)

2. Secondary serological tests:

e.g. – Agglutination tests

– Complement fixation tests (CFT)

– Precipitation tests

– Serum neutralization tests (SNT)

–Toxin-antitoxin test

3.Tertiary serological test:

e.g. – Determination of the protective value of an anti serum in an animal.

• Definition: Observable combination of antigen and antibody

• Importance:

A) In vivo – form basis of antibody mediated immunity in infectious disease, or of tissue injury in hypersensitivity and autoimmune diseases

B) In vitro – i) Help in diagnosis of infections ii) Help in epidemiological surveys iii) Identification of infectious agents iv) Identification of non infectious agents like

enzymes

Antigen antibody reactions in vitro are called serological reactions

General features of Antigen – Antibody Reactions

1. Reactions are specific2. Entire molecules react and not fragment3. No denaturation of antigen of Ag/Ab during reaction4. Combination occurs at the surface5. Combination is firm but reversible

6. Both the Ag and Ab combine in varying proportion

Affinity – Intensity of attraction between Ag and Ab molecules

Avidity – Strength of bond after the Ag – Ab complexes are formed.

Sensitivity:

ability of a test to detect very small amounts of a substance

Specificity:

ability of test to give positive result if patient has the disease (no false negative results)

Antigens x Antibody

Bacteria Virus

Ag

Ag

Antigen

binding site

Antigen binding siteVariable

constant

Light chain

Heavy chain

Antibodies (depicted as Y-shaped structures) form a heterogeneous population of molecules with different specificities. A cross-reaction of an antibody population (an anti-serum) with a foreign antigen (in the middle) occurs only, if the homologous and the foreign antigen are at least partially equipped with the same determinants. Every antibody has two identical binding sites for antigen determinants.

Primary stage

• Initial interaction between Ag and Ab

• No visible effect

• Rapid

• Occurs even at low temperature

• Obeys general laws of physical chemistry & thermodynamics

• Reversible reaction

Secondary Stage

Demonstrable effect produces e.g. precipitation, aggluitnation, lysis of cells, killing of live antigens, neutralization of toxins, complement fixation, immobilization of motile organisms, enhancement of phagocytosis

Tertiary stage

Some Ag – Ab reactions in vivo lead to

• Neutralization

• Destruction of injurious agent

• Tissue damage

• Includes humoral immunity, clinical allergy, and other immunological diseases

Measurement of Ag – Ab reaction

Measured in terms of Mass (mg of nitrogen) Units or titer

Titer: Highest dilution of serum (Ab) which gives an observable reaction with the Ag in a particular test.Antigen can also be titrated against sera (Ab).

Sensitivity – Ability of a test to identify correctly all those who have the disease i.e. true positive

Specificity – Ability of a test to identify correctly all those who do not have the disease i.e. true negative

In general, sensitivity and specificity of a test are in reverse proportion.

Serological tests

• Precipitation reaction

• Agglutination reaction

• Complement fixation test

• Radioimmunoassay

• ELISA

• Immunofluorescence

• Western Blot

Precipitation Reaction

When a soluble antigen combines with its antibody in the presence of electrolytes at a suitable temp and pH the Ag - Ab complex forms an insoluble precipitate. If instead of sedimenting, the precipitate remains suspended, the reaction is called as flocculation.

Medium used :

Liquid or gels like agar, agarose and polyacrylamide.

Antibody excess Zone of equivalence

Antigen excessAntigen

Antibody

Zone Phenomenon

Mechanism of precipitation

• Marrack (1934) – Lattice hypothesis

Application of precipitation:

- Can be used as qualitative or quantitative test

- Very sensitive test for antigen detection, can detect as little as 1µg of protein

- Important for forensic application like identification of blood, seminal stains.

APPLICATION OF PRECIPITATION REACTIONS

• Carried out as qualitative and quantitative for detection of antigen and antibody

• Very sensitive test for detecting antigen (Relatively less sensitive for detection of antibody)

• Capable of detecting little quantity of antigen proteins as 1ug

• Used for identification of blood and seminal stains and food adulterants

Application in diagnostic bacteriology

• Ring test– Typing of streptococci and pneumococci– C- reactive protein– Ascoli’s thermoprecipitin test for diagnosis of antrax– Used in detection of adulteration of food stuffs

• Slide test– VDRL for diagnosis of syphilis

• Tube test– Kahn test for syphilis and standardization of toxins and

toxoids

IMMUNODIFFUSION (precipitation in gel)

When an Ab and its Ag are placed in an agar gel they diffuse towards each other and form an opaque band of precipitation at the junction of their diffusion front.

TYPES OF IMMUNODIFFUSION TESTS

Antigen

Precipitin band

Antibody in agar gel

Plain agar

Single and double diffusion in one dimension

Single diffusion (Oudin procedure)Double diffusion (Okley - Fulthrope procedure)

Antigen in well

Ring of precipitation

Antibody in agar gel

A single diffusion in two dimensions

Antiserum in well

Line of precipitate

Antigen 2

Agar gel on a slide

Antigen 1

Double diffusion in two dimensions

(Lines of nonidentity)

Antiserum in well

Line of precipitate

Antigen 2

Agar gel on a slide

Antigen 1

Double diffusion in two dimensions

(Partial identity)

Antiserum in well

Antigen 2

Agar gel on a slide

Antigen 1

Double diffusion in two dimensions

(Lines of complete identity)

Trough cut in agar

Agar covered microscopic slide

Well cut in agar

Antigen placed in well

Antigen components separated by electrophoresis

Immunoelectrophoresis

Trough filled with antibody

Immunoelectrophoresis

Antibody diffuses towards separated antigen components

Precipitin band form where antibody and antigen meet at optimal proportions

Antiserum (Ab) in agarose gel

Precipitin arcs (rockets)

Antigen wells

Rocket electrophoresis (One dimensional single electroimmuno diffusion)

Increasing concentration of antigen

+

_

Ag AbAgarose

Wells containing antigen and antibody

Precipitin line

Slide

Electric current

Counterimmunoelectrophoresis

(One dimensional double electroimmunodiffusion)

AGGLUTINATION REACTION

When a particulate antigen or an antigen present on the surface of cell (red cell or bacterium) or an inorganic particle ( e.g. polystyrene latex coated with antigen) is mixed with its antibody in the presence of electrolytes at suitable temperature and pH, the particles are clumped or agglutinated

Application of agglutination reaction

• Slide agglutination

– Identification of cultures e.g. Salmonella, Bordetella pertussis– Typing of pneumococci and streptococci– Blood grouping

• Tube agglutination

– Serological diagnosis of • enteric fever (Widal)• brucellosis • typhus fever (Weil – Felix)

– Blood grouping– Coomb’s test

Latex particles

Antibody

Antigen

Agglutination

Antibody coated latex particles

Latex agglutination test

ASO, CRP, RA

Passive agglutination test

e.g. Rose Waaler test in Rheumatoid arthritis

Passive agglutination

Precipitation reaction can be converted to agglutination reaction by coating soluble antigen on the surface of carrier particles such as RBCs, latex, bentonite, gelatin

More sensitive for detection antibodies

Examples

1. RA factor

2. Latex tests

3. TPHA

Negative CFT

Complement Sheep erythrocytes coated with amboceptor (Indicator system)

Lysis

Positive CFT

Ag Ab Complement

Complement is fixed in Ag – Ab reaction

Indicator system

No lysis

As complement is not free to act on indicator system

Complement fixation test

Cowan strain of S. aureusImmunoglobulin

Cowan strain coated with immunoglobulin

Antigen

Agglutination

Coagglutination

Sandwich ELISA Indirect ELISA Competitive EILSA

Conjugate is washed out as Ag is not free to bind the conjugate

Substrate

Enzyme (tagged to conjugate is not there to act on substrate

Color product

Antigen

Antibody

Conjugate (Ab to Ab)

Conjugate (Ab to Ag)

ELISA

Unknown antigen

Slide

Fluorescein labeled specific Ab

Fluorescence under UV light

(positive test)

Direct immunofluorescence test

Indirect immunofluorescence test

Known Ag

Slide

Patient serum containing Ab

Ag + Ab

Fluorescein labeled Antiglobulin

Fluorescence under UV light

(Positive test)

Radioimmunoassay

Unlabeled Ag (Test sample)

Known radio labeled Ag

Specific Ab against Ag

Mix

Incubate

Free fraction

Bound fraction

MCQsQ1. A large lattice is formed when

a. Antigen is in excessb. Antibody is in excessc. Antigens and antibodies are in optimal proportiond. Non of above

Q.2. Precipitation reaction is relatively less sensitive for detection ofa. Antibodiesb. Antigensc. Antigen antibody complexesd. Complement

Q3. Ring test is used for a. Typing of streptococci and pneumococcib. C-reactive protein testc. Ascoli’s thermoprecipitation testd. All of the above

Q4. Widal test is used to diagnosea. Syphilisb. Typhoidc. AIDSd. Typhus fever

Q.5. VDRL test is an example ofa. ring testb. Slide testc. tube testd. None of above

Q6. Agglutination reaction is more sensitive than precipitation for detection ofa. Antigenb. Antibodyc. Antigen antibody complexesd. Complement

Q7. Amount of various immunoglobulin classes can be measured by a. Single diffusion in one dimensionb. Double diffusion in one dimensionc. Single diffusion in one dimensiond. Double diffusion in two dimension

Q8.Anti- Rh antibodies area. IgG typeb. IgD typec. IgA typed. IgE type

LAQ

Q.1. Define agglutination reaction and discuss the principle, application of agglutination reactions giving suitable examples

Q.2 Define precipitation reaction and discuss the principle, application precipitation reactions giving suitable examples

Q.3 Discuss the principle, various types and clinical applications of ELISA technique

Q.4 Short notes

1. Counterimmunoelctrophoresis

2. ELISA

3. Complement fixation test