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Antigen Antibody
Reactions and clinical utility
Mohammad Mukhit Kazi
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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.
Mohammad Mukhit Kazi, Lecturer SDCH Pune
• 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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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)
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antigens x Antibody
Bacteria Virus
Ag
Ag
Antigen
binding site
Antigen binding siteVariable
constant
Light chain
Heavy chain
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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.
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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.
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Serological tests
• Precipitation reaction
• Agglutination reaction
• Complement fixation test
• Radioimmunoassay
• ELISA
• Immunofluorescence
• Western BlotMohammad Mukhit Kazi, Lecturer
SDCH Pune
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.
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antibody excess Zone of equivalence
Antigen excessAntigen
Antibody
Zone Phenomenon
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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.
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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.
Mohammad Mukhit Kazi, Lecturer SDCH Pune
TYPES OF IMMUNODIFFUSION TESTS
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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)
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antigen in well
Ring of precipitation
Antibody in agar gel
A single diffusion in two dimensions
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antiserum in well
Line of precipitate
Antigen 2
Agar gel on a slide
Antigen 1
Double diffusion in two dimensions
(Lines of nonidentity)
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antiserum in well
Line of precipitate
Antigen 2
Agar gel on a slide
Antigen 1
Double diffusion in two dimensions
(Partial identity)
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antiserum in well
Antigen 2
Agar gel on a slide
Antigen 1
Double diffusion in two dimensions
(Lines of complete identity)
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Trough cut in agar
Agar covered microscopic slide
Well cut in agar
Antigen placed in well
Antigen components separated by electrophoresis
ImmunoelectrophoresisMohammad Mukhit Kazi, Lecturer
SDCH Pune
Trough filled with antibody
Immunoelectrophoresis
Antibody diffuses towards separated antigen components
Precipitin band form where antibody and antigen meet at optimal proportions
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Antiserum (Ab) in agarose gel
Precipitin arcs (rockets)
Antigen wells
Rocket electrophoresis (One dimensional single electroimmuno diffusion)
Increasing concentration of antigen
+
_
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Ag AbAgarose
Wells containing antigen and antibody
Precipitin line
Slide
Electric current
Counterimmunoelectrophoresis
(One dimensional double electroimmunodiffusion)
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
Cowan strain of S. aureusImmunoglobulin
Cowan strain coated with immunoglobulin
Antigen
Agglutination
Coagglutination
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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 Mohammad Mukhit Kazi, Lecturer SDCH Pune
Unknown antigen
Slide
Fluorescein labeled specific Ab
Fluorescence under UV light
(positive test)
Direct immunofluorescence testMohammad Mukhit Kazi, Lecturer
SDCH Pune
Indirect immunofluorescence test
Known Ag
Slide
Patient serum containing Ab
Ag + Ab
Fluorescein labeled Antiglobulin
Fluorescence under UV light
(Positive test)Mohammad Mukhit Kazi, Lecturer
SDCH Pune
Radioimmunoassay
Unlabeled Ag (Test sample)
Known radio labeled Ag
Specific Ab against Ag
Mix
Incubate
Free fraction
Bound fraction
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune
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
Mohammad Mukhit Kazi, Lecturer SDCH Pune