Transcript File - SOUTHERN MEDICAL UNIVERSITY

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
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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 contain 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
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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).
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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)
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Berson & Yallow 1959, analyse upto picograms (10 -12 g)
Quantitation of hormones, drugs, tumor markers, IgE 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
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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 : HBsAg detection :- Small casette with membrane impregnated
with anti HBsAg antibody-colloidal gold
dye conjugate
- Reacts with the HBsAg 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