ANTIGEN- ANTIBODY ANTIGEN- ANTIBODY

INTERACTIONS – II INTERACTIONS – II

COMPLEMENT FIXATION TESTCOMPLEMENT FIXATION TEST

Complement, a heat labile factor in normal serum is absorbed Complement, a heat labile factor in normal serum is absorbed during combination of Ag with Abs, lyses RBC’s, kills bacteria, during combination of Ag with Abs, lyses RBC’s, kills bacteria, immobilise motile organisms, promote phagocytosis, immune immobilise motile organisms, promote phagocytosis, immune adherence and tissue damage in HS reactionsadherence and tissue damage in HS reactions CFT :- Ability of Ag –Ab complex to fix the complementCFT :- Ability of Ag –Ab complex to fix the complement

- Consist of 2 steps an 5 reagents – Ag, Ab, C, Sheep - Consist of 2 steps an 5 reagents – Ag, Ab, C, Sheep RBC’s, Amboceptor (Rabbit antibody to sheep RBC’s)RBC’s, Amboceptor (Rabbit antibody to sheep RBC’s) MHD (min haemolytic dose) of C & Amboceptor standardisedMHD (min haemolytic dose) of C & Amboceptor standardised Step 1 : Ag + Test Serum (Ab) Complement FixedStep 1 : Ag + Test Serum (Ab) Complement Fixed

+ Complement+ Complement Step 2 : + Haemolytic system No HaemolysisStep 2 : + Haemolytic system No Haemolysis

Positive CFTPositive CFT Eg : Wassermann reaction for SyphilisEg : Wassermann reaction for Syphilis

INDIRECT COMPLEMENT FIXATION TESTINDIRECT COMPLEMENT FIXATION TEST

Done for certain avian and mammalian sera which do Done for certain avian and mammalian sera which do not fix guinea pig complement (duck, turkey, parrot, not fix guinea pig complement (duck, turkey, parrot, horse, cat)horse, cat)

Test sera are set up in duplicate and after first step, a Test sera are set up in duplicate and after first step, a standard antiserum known to fix the complement is standard antiserum known to fix the complement is added to one setadded to one set

If the test serum contain contained specific antibody, If the test serum contain contained specific antibody, antigen would be used up in the first step and standard antigen would be used up in the first step and standard antiserum added subsequently would not be able to fix antiserum added subsequently would not be able to fix the complementthe complement

Result : Haemolysis : Positive CFTResult : Haemolysis : Positive CFT No Haemolysis : Negative CFTNo Haemolysis : Negative CFT

Conglutinating Complement absorption test used for Conglutinating Complement absorption test used for such systemssuch systems

Conglutinating Complement absorption testConglutinating Complement absorption test

Uses Horse complement which is nonhaemolyticUses Horse complement which is nonhaemolytic Indicator system is sensitised sheep RBC’s mixed with Indicator system is sensitised sheep RBC’s mixed with

bovine serumbovine serum Bovine serum contains a beta globulin, conglutinin which Bovine serum contains a beta globulin, conglutinin which

acts as antibody to complement and causes acts as antibody to complement and causes agglutination of sheep RBC’s – Conglutinationagglutination of sheep RBC’s – Conglutination

If the Horse Complement is used up by Ag-Ab interaction If the Horse Complement is used up by Ag-Ab interaction in first step, agglutination of sensitised cells will not occurin first step, agglutination of sensitised cells will not occur

Result : Conglutination (Haemolysis) : Negative CFTResult : Conglutination (Haemolysis) : Negative CFT

No Conglutination : Positive CFTNo Conglutination : Positive CFT

Other Complement dependent Other Complement dependent serological testsserological tests

Immune adherence : Some bacteria react with specific antibodies in the presence of complement and particulate materials such as RBC’s or platelets and aggregate and adhere to cells

Immobilisation tests : Treponema pallidum immobilisation test – gold standard in diagnosis of syphilis

Cytolytic or Cytocidal tests : Vibriocidal antibody test for measurement of anticholera antibodies

NEUTRALISATION TESTNEUTRALISATION TEST Neutralisation of virus – animals, eggs & tissue cultureNeutralisation of virus – animals, eggs & tissue culture Nutralisation of bacteriophage – Plaque inhibition testNutralisation of bacteriophage – Plaque inhibition test Toxin neutralisation : Bacterial exotoxins and antitoxinsToxin neutralisation : Bacterial exotoxins and antitoxins In vivo tests : in animals – Schick test by diphtheria toxinIn vivo tests : in animals – Schick test by diphtheria toxin In vitro test : Antistreptolysin O testIn vitro test : Antistreptolysin O test OPSONISATION OPSONISATION :-:-

- Heat labile substance present in sera which - Heat labile substance present in sera which facilitated phagocytosisfacilitated phagocytosis

- Bacteriotropin : Heat stable serum factor with same activity- Bacteriotropin : Heat stable serum factor with same activity

- Opsonic index : Ratio of the phagocytic activity of the Opsonic index : Ratio of the phagocytic activity of the patient’s blood for a given bacterium to the patient’s blood for a given bacterium to the phagocytic phagocytic activity of blood from a normal individualactivity of blood from a normal individual

IMMUNOFLUORESCENCEIMMUNOFLUORESCENCE

Fluorescence – Fluorescent dyes – Fluorescent antibodyFluorescence – Fluorescent dyes – Fluorescent antibody Direct ImmunofluorescenceDirect Immunofluorescence : Specific antisera labeled with : Specific antisera labeled with

fluorescent dyes used for identification of bacteria, virus, and fluorescent dyes used for identification of bacteria, virus, and other antigens other antigens

Eg: Detection of Rabies virus antigen in brain smearsEg: Detection of Rabies virus antigen in brain smears Indirect ImmunofluorescenceIndirect Immunofluorescence : Antiglobulin fluorescent : Antiglobulin fluorescent

conjugate is used eg : Fluorescent treponemal antibody testconjugate is used eg : Fluorescent treponemal antibody test A single antihuman globulin fluorescent conjugate is used for A single antihuman globulin fluorescent conjugate is used for

detecting human antibody to any antigendetecting human antibody to any antigen Fluorescent dyesFluorescent dyes : Fluorescein isothiocyanate- blue-green, : Fluorescein isothiocyanate- blue-green,

Lissamine rhodamine- orange-red florescenceLissamine rhodamine- orange-red florescence Used for localising antigen-antibody reactions in situ – Used for localising antigen-antibody reactions in situ –

Immuno histochemical techniqueImmuno histochemical technique

FluorochromesFluorochromes Fluorescein, an organic dye that is the most widely used label for

immunofluorescence procedures, absorbs blue light (490 nm) and emits an intense yellow-green fluorescence (517 nm).

Rhodamine, another organic dye, absorbs in the yellow-green range (515 nm) and emits a deep red fluorescence (546 nm). Because it emits fluorescence at a longer wavelength than fluorescein, it can be used in two-color immunofluorescence assays. An antibody specific to one determinant is labeled with fluorescein, and an antibody recognizing a different antigen is labeled with rhodamine. The location of the fluorescein-tagged antibody will be visible by its yellow-green color, easy to distinguish from the red color emitted where the rhodamine-tagged antibody has bound. By conjugating fluorescein to one antibody and rhodamine to another antibody, one can, for example, visualize simultaneously two different cell-membrane antigens on the same cell.

Phycoerythrin is an efficient absorber of light (~30-fold greater than fluorescein) and a brilliant emitter of red fluorescence, stimulating its wide use as a label for immunofluorescence.

Flow Cytometry and FluorescenceFlow Cytometry and Fluorescence The flow cytometer uses a laser beam and light detector to

count single intact cells in suspension. Every time a cell passes the laser beam, light is deflected from the detector, and this interruption of the laser signal is recorded. Those cells having a fluorescently tagged antibody bound to their cell surface antigens are excited by the laser and emit light that is recorded by a second detector system located at a right angle to the laser beam.

The simplest form of the instrument counts each cell as it passes the laser beam and records the level of fluorescence the cell emits; an attached computer generates plots of the number of cells as the ordinate and their fluorescence intensity as the abscissa.

More sophisticated versions of the instrument are capable of sorting populations of cells into different containers according to their fluorescence profile. Use of the instrument to determine which and how many members of a cell population bind fluorescently labeled antibodies is called analysis; use of the instrument to place cells having different patterns of reactivity into different containers is called cell sorting.

++

RADIO IMMUNO ASSAY (RIA)RADIO IMMUNO ASSAY (RIA)

Berson & Berson & Yallow Yallow 1959, analyse upto picograms (10 1959, analyse upto picograms (10 -12-12 g) g) Quantitation of hormones, drugs, tumor markers, IgE etc.Quantitation of hormones, drugs, tumor markers, IgE etc. Binder- Ligand AssayBinder- Ligand Assay Competitive assayCompetitive assay in which fixed amounts of antibody and in which fixed amounts of antibody and

radioisotope labeled antigen react in presence of unlabelled radioisotope labeled antigen react in presence of unlabelled antigen. antigen.

Labeled and unlabelled antigenLabeled and unlabelled antigen competes for limited competes for limited binding sites on the antibodybinding sites on the antibody

After binding antigen is separated into free and bound After binding antigen is separated into free and bound fractions and radioactive counts measured, concentration of fractions and radioactive counts measured, concentration of test antigen calculated from the test antigen calculated from the ratio of bound and total ratio of bound and total antigen labelsantigen labels, using , using standard dose responsestandard dose response or or caliberating curvecaliberating curve

ENZYME IMMUNO ASSAYS (EIA)ENZYME IMMUNO ASSAYS (EIA)

Versatile, sensitive, simple, economic and Versatile, sensitive, simple, economic and free from radiation hazards, facility for free from radiation hazards, facility for automationautomation

Enzyme labeled antigen and antibodiesEnzyme labeled antigen and antibodies Homogenous EIAHomogenous EIA : No need to separate free : No need to separate free

and bound fractions, eg: Enzyme multiplied and bound fractions, eg: Enzyme multiplied Immunoassay technique (EMIT)Immunoassay technique (EMIT)- Single step, reagents added simultaneously- Single step, reagents added simultaneously

Heterogenous EIAHeterogenous EIA : Separation of free and : Separation of free and bound fractions bound fractions - Multistep, reagents added sequentially- Multistep, reagents added sequentially

ELISA (Enzyme Linked Immuno ELISA (Enzyme Linked Immuno Sorbent Assay)Sorbent Assay)

ImmunosorbentImmunosorbent – Cellulose, Agarose, Polystyrene or Polyvinyl – Cellulose, Agarose, Polystyrene or Polyvinyl tubes or Microwells, or Membrane or discs of Polyacrylamidetubes or Microwells, or Membrane or discs of Polyacrylamide

Detection of Antigen by ELISADetection of Antigen by ELISA – Rota virus Ag – Rota virus Ag Detection of Antibody by ELISADetection of Antibody by ELISA – HIV Ab – HIV Ab Different types : Different types :

1. Direct ELISA1. Direct ELISA2. Indirect ELISA2. Indirect ELISA3. Competitive ELISA3. Competitive ELISA4. Card & Dipstick methods4. Card & Dipstick methods5. Casette ELISA5. Casette ELISA

Chemiluminescence Immunoassay (CLIA)Chemiluminescence Immunoassay (CLIA) : : Chemiluminescent compounds like Luminol, Acridium esters Chemiluminescent compounds like Luminol, Acridium esters labelled Ag or Ablabelled Ag or Ab

Fully automated methodFully automated method

IMMUNO ELECTROBLOTIMMUNO ELECTROBLOT

More sensitive and specificMore sensitive and specific Combination of 3 separate procedures :Combination of 3 separate procedures :

1. Separation of Antigen components by 1. Separation of Antigen components by PAGEPAGE

2. Blotting of electrophoresed Antigen fraction 2. Blotting of electrophoresed Antigen fraction on on Nitrocellulose membrane stripsNitrocellulose membrane strips

3. Enzyme Immuno Assay to detect antibody 3. Enzyme Immuno Assay to detect antibody in in test seratest sera

Western Blot test for HIVWestern Blot test for HIV

PAGEPAGE

IMMUNO BLOTTING

ENZYME IMMUNO ASSAY

Immunochromatographic testsImmunochromatographic tests

One step Qualitative assayOne step Qualitative assay Simple, Economic and reliable methodSimple, Economic and reliable method Eg : HBsAg detection :-Eg : HBsAg detection :-

- Small casette with membrane - Small casette with membrane impregnated impregnated with anti HBsAg with anti HBsAg antibody-colloidal gold antibody-colloidal gold dye conjugatedye conjugate

- Reacts with the HBsAg and gives a - Reacts with the HBsAg and gives a coloured bandcoloured band

- Controls set up to avoid false negative - Controls set up to avoid false negative resultsresults

Immuno electron microscopic testsImmuno electron microscopic tests

Immuno electron microscopyImmuno electron microscopy : When viral : When viral partiles mixed with specific antisera are observed under partiles mixed with specific antisera are observed under EM, seen as clumpedEM, seen as clumped

Immuno ferritin testImmuno ferritin test : Ferritin (Electron dense : Ferritin (Electron dense substance) conjugated with antibody and reacted with substance) conjugated with antibody and reacted with antigen are visualized under EMantigen are visualized under EM

Immuno enzyme testImmuno enzyme test : Stable enzymes like : Stable enzymes like Peroxidase, Glucose oxidase, Phosphatases and Peroxidase, Glucose oxidase, Phosphatases and Tyrosinase can be conjugated with antibodies and when Tyrosinase can be conjugated with antibodies and when bound with antigen are visualized under EMbound with antigen are visualized under EM