Antigen and Antibody Reactions Agglutination Tests
Antigen and Antibody Reactions Agglutination Tests
Antigen and Antibody Reactions Agglutination Tests
Agglutination Tests
Dr.T.V.Rao MD
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DIRECT AGGLUTINATION
-Test patient serum against large,
cellular antigens to screen for the presence of antibodies. Antigen is naturally present on the surface of the cells. In this case, the Ag-Ab reaction forms an agglutination, which is directly visible.
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Agglutination Test
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Introduction:
The complement fixation test (CFT) was extensively used in syphilis serology after being introduced by Wasserman in 1909. Complement is a protein (globulin) present in normal serum. Whole complement system is made up of nine components: C1 to C9 Complement proteins are heat labile and are destroyed by heating at 56C for 20 30 minutes. Complement binds to Ag-Ab complex When the Ag is an RBC it causes lysis of RBCs.
Complement takes part in many of the immunological reactions. It gets absorbed during the combination of antigens and antibody.
Principle
This property of antigenantibody complex to fix the complement is used in complement fixation test for the identification of specific antibodies.
The haemolytic system containing sheep erythrocytes (RBC) and its corresponding antibody (amboceptor) is used as an indicator which shows the utilization or availability of the complement. If the complement is fixed then there will be no lysis of sheep erythrocytes, thus denoting a positive test.
If the complement is available then there will be haemolysis which is a property of complement, denoting a negative test.
Components of CFT
Test System Antigen: It may be soluble or particulate.
Antibody: Human serum (May or may not contain Antibody towards specific Antigen)
Complement: It is pooled serum obtained from 4 to 5 guinea pigs. It should be fresh or specially preserved as the complement activity is heat labile (stored at -30 C in small fractions). The complement activity should be initially standardized before using in the test. Indicator System (Haemolytic system) Erythrocytes: Sheep RBC Amboceptor (Hemolysin): Rabbit antibody to sheep red cells prepared by inoculating sheep erythrocytes into rabbit under standard immunization protocol.
Positive Test
Step 1:
At 37C Antigen + Antibody + Complement (from serum) Complement gets fixed 1 Hour
Step 2:
Fixed Complement complex + Haemolytic system At 37C No Haemolysis 1 Hour (Test Positive)
Negative Test
Step 1:
At 37C Complement not fixed 1 Hour
Step 2:
At 37C Haemolysis (Test Negative)
Microtiter plate showing Haemolysis (Well A3, A3 and B4) and No Haemolysis (Well
HEMAGGLUTINATION
Detects antibody to erythrocyte antigens sufficient concentration of antibody present-> antibody cross-link= agglutination non-reactive/insufficient antibody present= no agglutination
Binding different antigens on the RBC surface = detect antibodies to antigen other than those present in the cells
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Haemagglutination
RBC
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Viral Haemagglutination
Some viruses and microbes contain proteins which bind to erythrocytes (red blood cells) causing them to clump together
NDV Adenovirus III AIV IBV Mycoplasma
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VIRUSE
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SERUM
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Erythrocytes
Virus
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Erythrocytes
Virus
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Other tests
Red cells as antigens 1 Paul Bunnell test 2 Cold agglutination test
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Coombs (Antiglobulin)Tests
Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes
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Patients RBCs Coombs Reagent (Antiglobulin)
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Coombs (Antiglobulin)Tests
Indirect Coombs Test
Detects anti-erythrocyte antibodies in serum
Step 1 Patients Serum Step 2
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Target RBCs
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Passive Agglutination
An agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfaces Particle carriers include:
Red blood cells Polystyrene latex Bentonite charcoal
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Passive Agglutination
Passive agglutination has been used in the detection of :
Rheumatoid factor Antinuclear antibody in LE Ab to group A streptococcus antigens Ab to Trichinella spiralis
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Coagglutination
Name given to systems using inert bacteria as the inert particles to which the antibody is attached S.aureus: most frequently used because it has protein A in its outer surface that naturally adsorbs the Fc portion of the antibody
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Highly specific but not very sensitive in detecting small quantities of antigen
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False-Positive Result
If injected with hCG to trigger ovulation or to lengthen luteal phase of menstrual cycle. Chorioepithelioma, hydatidiform mole or ingestion of aspirin To detect the presence of a testicular tumor in men False Negative Testing before reaching detectable levels of hCG.
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Immunofluorescence
Antibodies can be labeled with fluorescent dye Can localize binding sites on cell Dyes: Fluorescein, rhodamine, phycoerythrin can be conjugated to Fc region of Ab (so antigen binding is unaffected Absorb at one wavelength and emit at another
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Fluorescence
UV Light
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Fluorescence
UV Light
Antigens
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ELISA
Enzyme Linked Immuno-Sorbant Assay
Peroxidase Enzyme is permanently attached to Antibody Probe
Substrate that turns from clear to green
Ag
Ag
Microtiter ELISA
Antigens are immobilized to the plastic surface of a Dr.T.V.Rao MD Microtiter Plate
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ELISA
Enzyme Linked Immuno-Sorbant Assay
Peroxidase Enzyme is permanently attached to the Antibody Probe
Microtiter ELISA
Antigens are immobilized to the plastic surface of a Dr.T.V.Rao MD Microtiter Plate
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ELISA
The ELISA (Enzyme-Linked ImmunoSorbant Assay) can be used both qualitatively and quantitatively to measure antigen-antibody binding. Depending on what variation you use, it will detect antigen (hormones, enzymes, microbial antigens, illicit drugs) or antibody (anti-HIV in the screening test for HIV infection) in body fluids or tissue culture supernatants.
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ELISA
Enzyme-Linked Immuno-Sorbant Assay, also called ELISA, Enzyme
ImmunoAssay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.
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ELISA plate
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ELISA methodology
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA).
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ELISA methodology
After the antigen is immobilized the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bioconjugation
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Sandwich ELISA
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Uses of ELISA
Helps detection of Antigens, Antibodies, hormones and Enzymes Eg in Microbiology Antigens HbS Ag Antibodies HIV, HCV, CMV, several other disease
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Uses of RIA
Used for detection of Hormones, enzymes,tumour markers IgE and viral antigens RIA is a competitive binding assay fixed amount of antibody and radiolabelled antigen react in the presence of unlabelled antigen Detection is done for free and bound fractions, ratios calculated
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Immunofluorescence
The purpose of immunofluorescence is to detect the location and relative abundance of any protein for which you have an antibody. Once you have antibodies to your favourite protein, you can use them to indicate where the protein is located.
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Immunofluorescence
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Fluorescent Methods
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Western Blot
Western blot analysis can detect your protein of interest from a mixture of a great number of proteins. Western blotting can give you information about the size of your protein (with comparison to a size marker or ladder in kDa), and also give you information on protein expression (with comparison to a control such as untreated sample or another cell type or tissue).
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Flowcytometry
FACS- fluorescence-activated cell sorter Analyze cell populations Sort cells with different features into different containers (e.g., T and B cells; cells that are producing a cellsurface marker from those that are not)
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Chemiluminescence's
Chemiluminescence's Chemical reaction emitting energy in the form of light Chemilumiscence - Luminol or acridinium esters causes signal in the process of antigen antibody reaction Signal can be amplified, measured, and the concentration of the analyses sample Uses the automated methods. Increasingly used where the volume of work is large
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Immunochromatographic Tests
One step in diagnosing Simple Economical. Reliable Eg HbsAg A small cassette system containing a membrane impregnated with antiHbsAg antibody colloidal gold dye conjugate The membrane is exposed at three windows on the cassette
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Immunochromatographic Tests
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Immunochromatographic Tests
A colored band appears at the second window Control also can be recorded
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Helps in emergency rooms. The results are available within few minutes The HIV and HBV infections can be done at the earliest
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Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing world
Email doctortvrao@gmail.com
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