Antigen and Antibody Reactions

Agglutination Tests
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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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Slide Agglutination Test

Used for serotyping (e.g. Salmonella) Antigen: isolated Salmonella in suspension Antibody: specific antisera against Salmonella Place test Salmonella in a drop of saline on a slide Add a drop of antiserum, mix and rock slide for approx 1 minute Examine for agglutination
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Slide Agglutination Test

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Agglutination Test

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OTHER DIRECT AGGLUTINATION TESTS

The particle antigen may be a bacterium. e.g.: Serotyping of E. coli, Salmonella using a specific antiserum The particle antigen may be a parasite. e.g.: Serodiagnosis of Toxoplasmosis The particle antigen may be a red blood cell. e.g.: Determination of blood groups
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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.

Complement fixation (CF)

Antibody and antigen allowed to combine in presence of complement If complement is fixed by specific antigen-antibody reaction, it will be unable to combine with indicator system Precautions Serum must be heat-activated Stored serum becomes anti-complementary Extensive QC/standardization required Only use for IgM antibodies

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

Antigen + Antibody absent + Complement

Step 2:
At 37C Haemolysis (Test Negative)

Free Complement + Haemolytic system 1 Hour

Results and Interpretations:

No haemolysis is considered as a positive test. haemolysis of erythrocytes indicative of a negative test. 1 2 3 4 A

Microtiter plate showing Haemolysis (Well A3, A3 and B4) and No Haemolysis (Well

Quantitative Micro Hemagglutination Test (HA)

Haemagglutination Tests
(HA)
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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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HEMAGGLUTINATION INHIBITION TEST (HI)

VIRUSE
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SERUM
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In the absence of anti-virus antibodies

Erythrocytes

Virus

Virus agglutination of erythrocytes

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In the presence of anti-virus antibodies

Erythrocytes

Virus

Anti-virus antibodies Viruses unable to bind to the erythrocytes

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Heterophile Agglutination Tests

Weil Felix Test or Reaction in Serodiagnosis of typhus fevers is heterophile agglutination test and sharing of common antigen between typhus Rickettsiae and some strains of Proteus bacilli
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Other tests
Red cells as antigens 1 Paul Bunnell test 2 Cold agglutination test

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Paul Bunnell test

Based on the principle of presence of sheep agglutinins in the sera of infectious mononucleosis patents who are absorbed by OX red cells but not by guinea pig kidney extract
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Cold Agglutination test

Positive in Mycoplasma ( Primary Atypical ) Pneumonia The patients sera agglutinated human O group erythrocytes at 4 o c the agglutination being reversible at 37 0 c

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Coombs (Antiglobulin)Tests Applications

Detection of anti-Rh Ab

Autoimmune hemolytic anemia

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Coombs (Antiglobulin)Tests
Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes

+
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

+
Target RBCs

Coombs Reagent (Antiglobulin) Dr.T.V.Rao MD

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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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Reverse Passive Agglutination

Antibody rather than antigen is attached to a carrier particle
For the detection of microbial antigens such as: Group A and B streptococcus Staphylococcus aureus Neisseria meningitidis Haemophilus influenzae Rotavirus Cryptococcus neoformans Mycoplasma pneumoniae Candida albicans
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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

Antigens on Cells or on Tissue Sections

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Fluorescence

Double layer Sandwich

UV Light

Antigens
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Enzyme Immunoassay ( EIA )

Introduced in 1966 alternative to fluorescent methods Versatile, simple economical Absence of radiation. EIA means measuring enzymes labelled antigen, hapten, antibody
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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

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
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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Enzyme Linked Immuno Assay

The Technique involves use of Immuno-Sorbant and absorbing material specific for one of the components of reaction, the antigen or antibody
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Components in ELISA testing

Conjugate Horseradish peroxidase Substrate - O-phenyle diamine dihydrochloride The test is conducted in solid phase Polystyrene, Polyvinyl or polycarbonate tubes or in plastics
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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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Different methods of ELISA

Direct and Indirect ELISA Sandwich Non competitive Sandwich
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ELISA most popularly used method

The ELISA is probably the most commonly used immunological assay because of its versatility, sensitivity (ability to detect small amounts of antigen or antibody), specificity (ability to discriminate between closely related but antigenically different molecules), and ease of automation. Although some of the substrates are carcinogenic, they are generally considered safer than radioisotopes used in RIA (radioimmunoassay).
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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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Radio Immuno Assay Berson and Yallow

Besides fluorescent dyes other labels can be used Uses with Radio isotopes Variety of tests are done for detection of antigen or antibody The term binder ligand assay has been used The minute amounts of substances can be detected Used in Biology and Medicine
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Rosalyn S. Yalow and Sol Berson

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RIA ( Radio Immuno Assay )

The technique was introduced in 1960 by Berson and Yalow as an assay for the concentration of insulin in plasma. It represented the first time that hormone levels in the blood could be detected by an in vitro assay.
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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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Direct and Indirect 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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Western Blot Test

The method originated from the laboratory of George Stark at Stanford. The name western blot was given to the technique by W. Neal Burnette and is a play on the name Southern blot, a technique for DNA detection developed earlier by Edwin Southern. Detection of RNA is termed northern blotting.
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Appearance of test readings

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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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Uses for flow cytometry

Percentage of a total population of cells Measuring antigen density within a population of cells Multiple antibodies can be used to assess several cell surface antigens simultaneously Clinical analysis (tumor characterization)
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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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Chemiluminescence's Immuno Assay

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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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Also called as Dot Methods

The tests can be done by paramedical staff, as they are simple to read

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
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