Antigen and Antibody Reactions Agglutination Tests

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Antigen and Antibody Reactions

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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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 AGGLUTINATIONTESTS

• 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

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 56°C for 20 – 30 minutes.

• Complement binds to Ag-Ab complex• When the Ag is an RBC it causes lysis of RBC’s.

Principle• Complement takes part in many of the immunological reactions.

It gets absorbed during the combination of antigens and antibody.

• This property of antigen–antibody 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 CFTTest 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 37°CAntigen + Antibody + Complement Complement gets fixed

(from serum) 1 Hour

• Step 2:

At 37°CFixed Complement complex + Haemolytic system No Haemolysis

1 Hour (Test Positive)

Negative Test Step 1:

At 37°CAntigen + Antibody absent + Complement Complement not fixed

1 Hour

Step 2:

At 37°CFree Complement + Haemolytic system Haemolysis

1 Hour (Test Negative)

Results and Interpretations:• No haemolysis is considered as a positive test. • haemolysis of erythrocytes indicative of a negative test.

1 2 3 4

A

B

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

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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 SERUM

19

In the absence of anti-virus antibodies

Erythrocytes

VirusVirus agglutination of erythrocytes

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In the presence of anti-virus antibodies

Erythrocytes

Virus Anti-virus antibodies

Viruses unable to bind tothe 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

+ ↔

Patient’s RBCs Coombs Reagent(Antiglobulin)

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Coombs (Antiglobulin)Tests • Indirect Coombs Test

– Detects anti-erythrocyte antibodies in serum

Patient’s Serum

TargetRBCs

+ ↔Step 1

+ ↔Coombs Reagent

(Antiglobulin)

Step 2

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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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Antigens on Cells or on Tissue Sections

UV Light

Fluorescence

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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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Ag

Peroxidase Enzyme is permanently attached to Antibody Probe

Microtiter ELISAAntigens are immobilized to the plastic surface of a

Microtiter Plate

Enzyme Linked Immuno-Sorbant Assay

ELISA

Ag

Substrate that turns from clear to green

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Ag

Peroxidase Enzyme is permanently attached to the Antibody Probe

Microtiter ELISAAntigens are immobilized to the plastic surface of a

Microtiter Plate

Enzyme Linked Immuno-Sorbant Assay

ELISA

Ag

Substrate that turns from clear

to green

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ELISA• The ELISA (Enzyme-Linked Immuno-

Sorbant 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 AssayBerson 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 cell-surface 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

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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