Antigen and Antibody Reactions basics : Antigen and Antibody Reactions basics Dr.T.V.Rao MD Dr.T.V.Rao MD 1 Classification of antigen-antibody interactions : Classification of antigen-antibody interactions Primary serological tests: (Marker techniques) e.g.
Enzyme linked immuono sorbent assay (ELISA)
Immuno florescent antibody technique (IFAT)
Radio immuno assay (RIA)
Secondary serological tests: e.g.
Complement fixation tests (CFT)
Serum neutralization tests (SNT)
Tertiary serological test: e.g.
Determination of the protective value of an anti serum in an animal. Dr.T.V.Rao MD 2 Types of Antigen and Antibody reactions : Types of Antigen and Antibody reactions Agglutination tests
Double diffusion precipitation tests
Western blot tests
Complement fixation tests
Immunoassays Dr.T.V.Rao MD 3 Serological Tests : Serological Tests Antigen and antibody reactions in vitro are known as serological tests
What Happens can be studied in 3 stages
1st Antigen and antibody react with visible effects, obeys the laws of physical dynamics. But reversible
The reaction is effected by weaker intermolecular forces – Vander Waal-s forces
Ionic bonds, Hydrogen bonding not by covalent bonding
Can be detected by Radioactive isotopes, fluorescent dyes Dr.T.V.Rao MD 4 What happens in Antigen and Antibody reactions : What happens in Antigen and Antibody reactions React with each other in a observable manner.
1 Helps antibody mediated immunity in infection, and tissue injury
2 Helps diagnosis of Infections.
3 In epidemiological surveys
4 Detections and quantization of antigens and antibodies Dr.T.V.Rao MD 5 Detectable reactions 2nd Stage : Detectable reactions 2nd Stage Reaction can occur as
3 Lysis and killing of live antigens
4Neutralizatiobn of toxins
5 Fixation of complement
6 Immobilization of motile microbes
7 Enhancement of Phagocytosis. Dr.T.V.Rao MD 6 General features of Antigen and antibody reactions. : General features of Antigen and antibody reactions. Specific reaction – combines with specific antigen
Entire molecule reacts not fragments
No denaturation of antigen or antibody
Combination occurs as surface antigens to surface of antibodies
Commination is firm but reversible depends on affinity and avidity
Both antigens and antibodies participate
Combine in varying proportions Bivalent and multivalent Dr.T.V.Rao MD 7 Terms used in evaluating test Methodology : Terms used in evaluating test Methodology Sensitivity
Analytical 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) Dr.T.V.Rao MD 8 Specificity : 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) Dr.T.V.Rao MD 9 Affinity : Affinity Antibody affinity is the strength of the reaction between a single antigenic determinant and a single combining site on the antibody.
It is the sum of the attractive and repulsive forces operating between the antigenic determinant and the combining site . Dr.T.V.Rao MD 10 Avidity : Avidity Avidity is a measure of the overall strength of binding of an antigen with many antigenic determinants and multivalent antibodies
Avidity is influenced by both the valence of the antibody and the valence of the antigen.
Avidity is more than the sum of the individual affinities. Dr.T.V.Rao MD 11 Slide 12: Dr.T.V.Rao MD 12 Dilution : Dilution Estimating the antibody by determining the greatest degree to which the serum may be diluted without losing the power to given an observable effect in a mixture with specific antigen Dr.T.V.Rao MD 13 Measurement of Antigen and Antibody reactions : Measurement of Antigen and Antibody reactions Measured as Mass Nitrogen (microgram)
As titer Dr.T.V.Rao MD 14 Titer : Titer Different dilutions of serum are tested in mixture with a constant amount of antigen and greatest reacting dilution is taken as the measure or Titer Dr.T.V.Rao MD 15 Common methods in creating dilutions : Common methods in creating dilutions Dr.T.V.Rao MD 16 Expression of Titers : Expression of Titers Expressed in term of the was in which they are made
Dilution 1 in 8 is a dilution made by mixing one volume of serum with seven volumes of diluents (Normal Saline )
Incorrect to express dilution as 1/8 Dr.T.V.Rao MD 17 Majority Diagnostic tests are Serological tests : Majority Diagnostic tests are Serological tests There are several serology techniques that can be used depending on the antibodies being studied. These include: ELISA, agglutination, precipitation, complement-fixation, and fluorescent antibodies. Dr.T.V.Rao MD Antigens x Antibody : Antigens x Antibody Dr.T.V.Rao MD 19 How Antigen – Antibody reaction occurs : How Antigen – Antibody reaction occurs Primary stage – Interaction between antigen and antibody occurs without any visible effects
The reaction is rapid even at low temperature but the reaction is reversible
Weaker _ intermolecular forces Van der wall’s force and ionic bonds
Can be measured with radioactive isotopes and Florescent dyes. Dr.T.V.Rao MD 20 Ag-Ab interactions : Ag-Ab interactions Bonds:
Van der Waals forces
Each bond is weak; many are strong
To “hold” they must be close requiring high amts of
complementarity! Dr.T.V.Rao MD 21 Slide 22: Nature of Ag/Ab Reactions Dr.T.V.Rao MD 22 What happen futher ? : What happen futher ? 1 Precipitation
2 Lysis of cells.
3 Killing of live antigen
4 Neutralization of toxins
5 Complement fixation
6 Immobilization of motile microbes
7 Enhancement of Phagocytosis
8 Entire molecule react no fragmented
9 No denaturation
10 Combination occurs on surface
11 Firm but reversible Dr.T.V.Rao MD 23 Karl Landsteinar (1868-1943) : Karl Landsteinar (1868-1943) An Austrian physician by training, Landsteiner played an integral part in the identification of blood groups. He demonstrated the catastrophic effect of transfusing with the wrong type of blood, Dr.T.V.Rao MD 24 Serology : Serology The branch of laboratory medicine that studies blood serum for evidence of infection and other parameters by evaluating antigen-antibody reactions in vitro Dr.T.V.Rao MD 25 Serology : Serology Serology is the scientific study of blood serum. In practice, the term usually refers to the diagnostic identification of antibodies in the serum
We can detect antigens too Dr.T.V.Rao MD 26 Types of serological tests : Types of serological tests Agglutination tests
Double diffusion precipitation tests
Western blot tests
Complement fixation tests
Immunoassays Dr.T.V.Rao MD 27 Immunology/ Serology? Precipitation Reactions : Immunology/ Serology? Precipitation Reactions Capillary tube precipitation (Ring Test)
Ouchterlony Double Diffusion (Immunodiffusion)
Rocket Electroimmunodiffusion (EID)
Counterimmunoelectrophoresis (CIEP) Dr.T.V.Rao MD Antigen – Antibody reactions presenting with precipitation : Antigen – Antibody reactions presenting with precipitation Dr.T.V.Rao MD 29 Precipitation - Reaction : Precipitation - Reaction In precipitation antigen combines with its antibody in the presence of electrolytes ( Nacl ) at a suitable temperature and Ph the antigen and antibody complexes form a insoluble precipitate suspended as floccules.
Reaction can take place in liquid medium, gels, agar, agarose, polyacrylamide Dr.T.V.Rao MD 30 Precipitation : Precipitation Principle
Soluble antigen + antibody (in proper proportions) –> visible precipitate
Lattice formation (antigen binds with Fab sites of 2 antibodies)
Double diffusion (Ouchterlony)
Single diffusion (radial Immunodiffusion)
Immunofixation Dr.T.V.Rao MD 31 How precipitation occurs : How precipitation occurs Precipitation occurs with most antigens because the antigen is multivalent (i.e. has several antigenic determinants per molecule to which antibodies can bind). Antibodies have at least two antigen binding sites (and in the case of IgM there is a multimeric complex with up to 10 antigen binding sites), thus large aggregates or gel-like lattices of antigen and antibody are formed
Specific reaction Antigen combines with homologusantibody - vice versa Dr.T.V.Rao MD 32 Mechanism of Precepitation : Mechanism of Precepitation Lattice hypothesis – Multivalent antigens combine with bivalent antibodies in varying proportions depending on the antigen and antibody ratio in the reacting mixture Dr.T.V.Rao MD 33 Slide 34: Precipitation Reactions Dr.T.V.Rao MD 34 Applications of precipitation reactions : Applications of precipitation reactions Qualitative and quantitative
More useful for antigen detection to as least as 1 microgram
Blood, seminal stains, Food adulteration Dr.T.V.Rao MD 35 Slide test -Flocculation test – VDRL test : Slide test -Flocculation test – VDRL test VDRL Test – a drop of VDRL antigen to a drop of patients serum,
The reaction observed under microscope
Observe for flocculation reaction
A Khan test is done in a test tube Dr.T.V.Rao MD 36 Non reactive and Reactive VDRL Tests : Non reactive and Reactive VDRL Tests Dr.T.V.Rao MD 37 Agglutination+: ve RPR Agglutination : - : Agglutination+: ve RPR Agglutination : - Dr.T.V.Rao MD 38 Tube test - Precipitation : Tube test - Precipitation Kahn test for Syphilis – a tube flocculation test.
Quantitative tube flocculation test used in standardarisation of toxin/toxoid
Serum dilution of toxin or toxoid is added to tubes containing a fixed quantity of antitoxin
The amount of toxin that flocculates optimally with one unit of antitoxin is defined as Lf dose Dr.T.V.Rao MD 39 Single diffusion in one DirectionO : Single diffusion in one DirectionO Oudin Procedure Dr.T.V.Rao MD 40 Ring Test : Ring Test The reaction is demonstrated by layering antigen solution over the column of antigen in a narrow tube, The precipitate forms at the junction of two liquids.
Eg Ascoli’s thermo precipitate test
Grouping of streptococci
C-reactive protein Dr.T.V.Rao MD 41 Oudin Procedure : Oudin Procedure Antibodies in agar gel
Above antigen is layered
Single diffusion in one direction
Called Qudin procedure
Antibody is incorporated in agar
Antigen diffuses down
Precipitation concentration of antigen at the site increases due to diffusion Dr.T.V.Rao MD 42 Slide 43: Dr.T.V.Rao MD 43 Double diffusion in one dimensionOakley Fulthrope procedure : Double diffusion in one dimensionOakley Fulthrope procedure Antibodies in gel
Antigens and antibodies moves towards each other
Forms precipitate Dr.T.V.Rao MD 44 Single Diffusion In two DimensionsRadial Immunodiffusion : Single Diffusion In two DimensionsRadial Immunodiffusion Radial Immunodiffusion is an Immunodiffusion technique used in immunology to detect quantity of antigen by measuring the radius surrounding samples of the antigen, marking the boundary between it and antibody Dr.T.V.Rao MD 45 Single Diffusion In two DimensionsRadial Immunodiffusion : Single Diffusion In two DimensionsRadial Immunodiffusion Antigen is added to the wells cut on the surface of the gel
It diffuses radially from well and forms a ring shaped band of precipitation
The halo of precipitation diameter gives the estimate of concentration of antigen
Used in estimation of Immunoglobulin's Dr.T.V.Rao MD 46 Precipitation in Agar : Precipitation in Agar These tests are done in agar
Tested for passive immuno diffusion in Agarose
There are several methods in testing this procedure Dr.T.V.Rao MD 47 Gel electrophoresis allows to separate pieces of DNA. : Gel electrophoresis allows to separate pieces of DNA. Dr.T.V.Rao MD 48 Elek’s Immunodiffusion Test : Elek’s Immunodiffusion Test Elek Immunodiffusion test. Sterile filter paper impregnated with diphtheria antitoxin is imbedded in agar culture medium. Isolates of C diphtheria are then streaked across the plate at an angle of 90° to the antitoxin strip. Toxigenic C diphtheria is detected because secreted toxin diffuses from the area of growth and reacts with antitoxin to form lines of precipitin Dr.T.V.Rao MD 49 Immuno Diffusion Test : Immuno Diffusion Test Can demonstrate the immunological identity (or not) of two antigen samples; radial Immunodiffusion Dr.T.V.Rao MD 50 Radial Immunodiffusion : Radial Immunodiffusion Radial Immunodiffusion, a variation of the agar precipitation technique, is used in clinical immunology for the detection and quantitation of all classes of Immunoglobulin's, complement, ceroplastic, transferring, and other serum components Dr.T.V.Rao MD 51 Double Diffusion in one DimensionOakley – Fulthrope procedure : Double Diffusion in one DimensionOakley – Fulthrope procedure In Double diffusion in one direction antibody in agar gel moves through the layer of plain agar to the antigen above
They react to each other
Form a band of precipitation
Virus antigen is placed in the central well and diffuses outwards. Wells A and C contain positive sera, well B contains a negative sample. The black areas show where antibody in the positive sera have bound to virus antigen and formed a precipitate Dr.T.V.Rao MD 52 Double diffusion in two dimensions Ouchterlony procedure : Double diffusion in two dimensions Ouchterlony procedure Both antigen and antibody diffuses independently through agar gel in two dimensions horizontally and vertically
Done in a slide or petridish
In 12 – 48 hours line of precipitates are formed
Useful in serological identification Dr.T.V.Rao MD 53 Immunoelectrophoresis: : Immunoelectrophoresis: In Immunoelectrophoresis, a complex mixture of antigens is placed in a well punched out of an agar gel and the antigens are electrophoresed so that the antigen are separated according to their charge. After electrophoresis, a trough is cut in the gel and antibodies are added. As the antibodies diffuse into the agar, precipitin lines are produced in the equivalence zone when an antigen/antibody reaction occurs. Dr.T.V.Rao MD 54 Immuno-electrophoresis : Immuno-electrophoresis Mixtures containing multiple antigen species which cross react with the same antiserum may be analysed by running them first on an analytical gel, then cutting a strip from that gel and laying it in a slit cut into the immuno-electrophoresis gel to form a large well. The result is a pattern which shows the positions of strongly reacting antigen species. Dr.T.V.Rao MD 55 Immunoelectrophoresis : Immunoelectrophoresis Immunoelectrophoresis--migration of molecules due to electric charge Positive particles travel to cathode Negative particles travel anode Precipitin specificity provides a critical indicator of identity Dr.T.V.Rao MD 56 Immuno Electrophoresis : Immuno Electrophoresis In this procedure the electrophoretic separation of compatible antigen into constituent protein followed by Immunodiffusion against its antiserum resulting in separate precipitation line, indicating relation between each individual protein in the antibody Dr.T.V.Rao MD 57 Electrical gel Immunoelectrophoresis : Electrical gel Immunoelectrophoresis The reaction is electrically driven, different antigens are separated according to their charges under electrical fields
Number of antigens can be identified Dr.T.V.Rao MD 58 Countercurrent electrophoresis : Countercurrent electrophoresis Method
Ag and Ab migrate toward each other by electrophoresis
Used only when Ag and Ab have opposite charges Qualitative
Rapid Dr.T.V.Rao MD 59 Counter current Immunoelectrophoresis : Counter current Immunoelectrophoresis In this procedure movement of antigens towards the anode and antibody towards the cathode through agar under electric field
Useful studies on CSF
Hepatitis B surface antigen detection
Detection of fetoproteins Dr.T.V.Rao MD 60 Imunoelectrphoresis (IEP)Qualitative : Imunoelectrphoresis (IEP)Qualitative A serum sample is electrophoresed through an agar medium.
A trough is cut in the agar and filled with Ab.
A precipitin arc is then formed.
Because Ag diffuses radially and Ab from a trough diffuses, the reactants meet in optimal proportions for precipitation. Dr.T.V.Rao MD 61 Rocket electrophoresisLaurel : Rocket electrophoresisLaurel The reactions appear as rocket
Driven by electric current
Rocket” Immuno-Electrophoresis is used as a rapid way to quantitate antigen in complex samples. Dr.T.V.Rao MD 62 Immuno-electrophoresis gel : Immuno-electrophoresis gel Mixtures containing multiple antigen species which cross react with the same antiserum may be analyzed by running them first on an analytical gel, then cutting a strip from that gel and laying it in a slit cut into the immuno-electrophoresis gel to form a large well. The result is a pattern which shows the positions of strongly reacting antigen species. Dr.T.V.Rao MD 63 Counterimmunoelectrophoresis : Counterimmunoelectrophoresis Dr.T.V.Rao MD 64 Electro Immunodiffusion : Electro Immunodiffusion Dr.T.V.Rao MD 65 Slide 66: Dr.T.V.Rao MD 66 Rocket electrophoresis : Rocket electrophoresis Crossed Immunoelectrophoresis of antigens and antiserum. In the first dimension, proteins are separated by standard electrophoresis. The separated proteins are then run into the second dimension gel at an angle of 90° from the first dimension. Dr.T.V.Rao MD 67 Serology can be done on various specimens : Serology can be done on various specimens Some serological tests are not limited to blood serum, but can also be performed on other bodily fluids such as semen and saliva, which have (roughly) similar properties to serum.
Serological tests may also be used forensically, generally to link a perpetrator to a piece of evidence (e.g., linking a rapist to a semen sample). Dr.T.V.Rao MD 68 Measurement of Precipitation by Light : Measurement of Precipitation by Light Antigen-antibody complexes, when formed at a high rate, will precipitate out of a solution resulting in a turbid or cloudy appearance.
Turbidimetry measures the turbidity or cloudiness of a solution by measuring amount of light directly passing through a solution.
Nephelometry indirect measurement, measures amount of light scattered by the antigen-antibody complexes. Dr.T.V.Rao MD 69 Slide 70: Cross Reactivity The ability of an individual Ab combining site to react with more than one antigenic determinant.
The ability of a population of Ab molecules to react with more than one Ag Dr.T.V.Rao MD 70 For Articles of Interest on microbiology follow me on ….. : Dr.T.V.Rao MD 71 For Articles of Interest on microbiology follow me on ….. Programme created by Dr.T.V.Rao MD for Medical and Paramedical students in the Developing World : Programme created by Dr.T.V.Rao MD for Medical and Paramedical students in the Developing World Email
email@example.com Dr.T.V.Rao MD 72