ANTIGEN- ANTIBODY INTERACTIONS II COMPLEMENT FIXATION

ANTIGEN- ANTIBODY INTERACTIONS – II

COMPLEMENT FIXATION TEST § Complement, a heat labile factor in normal serum is absorbed during combination of Ag with Abs, lyses RBC’s, kills bacteria, immobilise motile organisms, promote phagocytosis, immune adherence and tissue damage in HS reactions § CFT : - Ability of Ag –Ab complex to fix the complement - Consist of 2 steps an 5 reagents – Ag, Ab, C, Sheep RBC’s, Amboceptor (Rabbit antibody to sheep RBC’s) § MHD (min haemolytic dose) of C & Amboceptor standardised § Step 1 : Ag + Test Serum (Ab) Complement Fixed + Complement § Step 2 : + Haemolytic system No Haemolysis Positive CFT § Eg : Wassermann reaction for Syphilis

INDIRECT COMPLEMENT FIXATION TEST § Done for certain avian and mammalian sera which do not § § fix guinea pig complement (duck, turkey, parrot, horse, cat) Test sera are set up in duplicate and after first step, a standard antiserum known to fix the complement is added to one set If the test serum contained specific antibody, antigen would be used up in the first step and standard antiserum added subsequently would not be able to fix the complement Result : Haemolysis : Positive CFT No Haemolysis : Negative CFT Conglutinating Complement absorption test used for such systems

Conglutinating Complement absorption test § Uses Horse complement which is nonhaemolytic § Indicator system is sensitised sheep RBC’s mixed with bovine serum § Bovine serum contains a beta globulin, conglutinin which acts as antibody to complement and causes agglutination of sheep RBC’s – Conglutination § If the Horse Complement is used up by Ag-Ab interaction in first step, agglutination of sensitised cells will not occur § Result : Conglutination (Haemolysis) : Negative CFT No Conglutination : Positive CFT

Other Complement dependent serological 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 TEST § Neutralisation of virus – animals, eggs & tissue culture § Nutralisation of bacteriophage – Plaque inhibition test § Toxin neutralisation : Bacterial exotoxins and antitoxins § In vivo tests : in animals – Schick test by diphtheria toxin § In vitro test : Antistreptolysin O test § OPSONISATION : - Heat labile substance present in sera which facilitated phagocytosis - Bacteriotropin : Heat stable serum factor with same activity - Opsonic index : Ratio of the phagocytic activity of the patient’s blood for a given bacterium to the phagocytic activity of blood from a normal individual

IMMUNOFLUORESCENCE § Fluorescence – Fluorescent dyes – Fluorescent antibody § Direct Immunofluorescence : Specific antisera labeled with § § fluorescent dyes used for identification of bacteria, virus, and other antigens Eg: Detection of Rabies virus antigen in brain smears Indirect Immunofluorescence : Antiglobulin fluorescent conjugate is used eg : Fluorescent treponemal antibody test A single antihuman globulin fluorescent conjugate is used for detecting human antibody to any antigen Fluorescent dyes : Fluorescein isothiocyanate- blue-green, Lissamine rhodamine- orange-red florescence Used for localising antigen-antibody reactions in situ – Immuno histochemical technique

Fluorochromes § 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 fluoresceintagged 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 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.

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RADIO IMMUNO ASSAY (RIA) § § Berson & Yallow 1959, analyse upto picograms (10 -12 g) Quantitation of hormones, drugs, tumor markers, Ig. E etc. Binder- Ligand Assay Competitive assay in which fixed amounts of antibody and radioisotope labeled antigen react in presence of unlabelled antigen. § Labeled and unlabelled antigen competes for limited binding sites on the antibody § After binding antigen is separated into free and bound fractions and radioactive counts measured, concentration of test antigen calculated from the ratio of bound and total antigen labels, using standard dose response or caliberating curve

ENZYME IMMUNO ASSAYS (EIA) § Versatile, sensitive, simple, economic and free from radiation hazards, facility for automation § Enzyme labeled antigen and antibodies § Homogenous EIA : No need to separate free and bound fractions, eg: Enzyme multiplied Immunoassay technique (EMIT) - Single step, reagents added simultaneously § Heterogenous EIA : Separation of free and bound fractions - Multistep, reagents added sequentially

ELISA (Enzyme Linked Immuno Sorbent Assay) § Immunosorbent – Cellulose, Agarose, Polystyrene or Polyvinyl § § § tubes or Microwells, or Membrane or discs of Polyacrylamide Detection of Antigen by ELISA – Rota virus Ag Detection of Antibody by ELISA – HIV Ab Different types : 1. Direct ELISA 2. Indirect ELISA 3. Competitive ELISA 4. Card & Dipstick methods 5. Casette ELISA Chemiluminescence Immunoassay (CLIA) : Chemiluminescent compounds like Luminol, Acridium esters labelled Ag or Ab Fully automated method

IMMUNO ELECTROBLOT § More sensitive and specific § Combination of 3 separate procedures : 1. Separation of Antigen components by PAGE 2. Blotting of electrophoresed Antigen fraction on Nitrocellulose membrane strips 3. Enzyme Immuno Assay to detect antibody in test sera § Western Blot test for HIV

PAGE IMMUNO BLOTTING

ENZYME IMMUNO ASSAY

Immunochromatographic tests § § § One step Qualitative assay Simple, Economic and reliable method Eg : HBs. Ag detection : - Small casette with membrane impregnated with anti HBs. Ag antibody-colloidal gold dye conjugate - Reacts with the HBs. Ag and gives a coloured band - Controls set up to avoid false negative results

Immuno electron microscopic tests § Immuno electron microscopy : When viral partiles mixed with specific antisera are observed under EM, seen as clumped § Immuno ferritin test : Ferritin (Electron dense substance) conjugated with antibody and reacted with antigen are visualized under EM § Immuno enzyme test : Stable enzymes like Peroxidase, Glucose oxidase, Phosphatases and Tyrosinase can be conjugated with antibodies and when bound with antigen are visualized under EM