Antigen and Antibody Reactions Agglutination Tests

Category: Education

Presentation Description

Antigen and Antibody Reactions Agglutination Tests


Presentation Transcript

Antigen and Antibody Reactions Agglutination Tests Dr.T.V.Rao MD :

Antigen and Antibody Reactions Agglutination Tests Dr.T.V.Rao MD Dr.T.V.Rao MD 1


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. Dr.T.V.Rao MD 2

Slide Agglutination Test:

3 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 Dr.T.V.Rao MD

Slide Agglutination Test:

4 Slide Agglutination Test Dr.T.V.Rao MD

Agglutination Test:

Agglutination Test Dr.T.V.Rao MD 5


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 Dr.T.V.Rao MD 6


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) :

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:

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:

Positive Test Step 1: At 37°C Antigen + Antibody + Complement Complement gets fixed (from serum) 1 Hour Step 2: At 37°C Fixed Complement complex + Haemolytic system No Haemolysis 1 Hour ( Test Positive)

PowerPoint Presentation:

Negative Test Step 1: At 37°C Antigen + Antibody absent + Complement Complement not fixed 1 Hour Step 2: At 37°C Free Complement + Haemolytic system Haemolysis 1 Hour ( Test Negative)

Results and Interpretations::

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

Quantitative Micro Hemagglutination Test (HA):

14 Quantitative Micro Hemagglutination Test (HA) Haemagglutination Tests (HA) Dr.T.V.Rao MD


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 Dr.T.V.Rao MD 15


16 Haemagglutination RBC Dr.T.V.Rao MD

Viral Haemagglutination:

17 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 Dr.T.V.Rao MD



In the absence of anti-virus antibodies:

19 In the absence of anti-virus antibodies Erythrocytes Virus Virus agglutination of erythrocytes Dr.T.V.Rao MD

In the presence of anti-virus antibodies:

20 In the presence of anti-virus antibodies Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes Dr.T.V.Rao MD

PowerPoint Presentation:

21 Dr.T.V.Rao MD

Heterophile Agglutination Tests:

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 Dr.T.V.Rao MD 22

Other tests:

Other tests Red cells as antigens 1 Paul Bunnell test 2 Cold agglutination test Dr.T.V.Rao MD 23

Paul Bunnell test :

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 Dr.T.V.Rao MD 24

Cold Agglutination test:

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 Dr.T.V.Rao MD 25

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests Applications Detection of anti-Rh Ab Autoimmune hemolytic anemia Dr.T.V.Rao MD 26

PowerPoint Presentation:

Dr.T.V.Rao MD 27

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Patient’s RBCs Coombs Reagent (Antiglobulin) Dr.T.V.Rao MD 28

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum Y Y Y Y Y Patient’s Serum Target RBCs + ↔ Step 1 + ↔ Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Coombs Reagent (Antiglobulin) Step 2 Dr.T.V.Rao MD 29

PowerPoint Presentation:

Dr.T.V.Rao MD 30

Passive Agglutination:

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 Dr.T.V.Rao MD 31

Passive Agglutination:

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 Dr.T.V.Rao MD 32

Reverse Passive Agglutination:

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 albican s Dr.T.V.Rao MD 33


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 Dr.T.V.Rao MD 34

PowerPoint Presentation:

Highly specific but not very sensitive in detecting small quantities of antigen Dr.T.V.Rao MD 35

False-Positive Result :

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. Dr.T.V.Rao MD 36

Immunofluorescence :

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 Dr.T.V.Rao MD 37

PowerPoint Presentation:

Antigens on Cells or on Tissue Sections UV Light Fluorescence Dr.T.V.Rao MD 38

PowerPoint Presentation:

Fluorescence Double layer Sandwich UV Light Antigens Dr.T.V.Rao MD 39

Enzyme Immunoassay ( EIA ):

Enzyme Immunoassay ( EIA ) Introduced in 1966 alternative to fluorescent methods Versatile, simple economical Absence of radiation. EIA means measuring enzymes labelled antigen, hapten, antibody Dr.T.V.Rao MD 40

PowerPoint Presentation:

Ag Peroxidase Enzyme is permanently attached to Antibody Probe Microtiter ELISA Antigens are immobilized to the plastic surface of a Microtiter Plate Enzyme Linked Immuno-Sorbant Assay ELISA Ag Substrate that turns from clear to green Dr.T.V.Rao MD 41

PowerPoint Presentation:

Ag Peroxidase Enzyme is permanently attached to the Antibody Probe Microtiter ELISA Antigens are immobilized to the plastic surface of a Microtiter Plate Enzyme Linked Immuno-Sorbant Assay ELISA Ag Substrate that turns from clear to green Dr.T.V.Rao MD 42


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. Dr.T.V.Rao MD 43


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. Dr.T.V.Rao MD 44

Enzyme Linked Immuno Assay:

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 Dr.T.V.Rao MD 45

Components in ELISA testing:

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 Dr.T.V.Rao MD 46

ELISA plate:

ELISA plate Dr.T.V.Rao MD 47

ELISA methodology:

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). Dr.T.V.Rao MD 48

ELISA methodology:

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 Dr.T.V.Rao MD 49

Sandwich ELISA:

Sandwich ELISA Dr.T.V.Rao MD 50

Different methods of ELISA:

Different methods of ELISA Direct and Indirect ELISA Sandwich Non competitive Sandwich Dr.T.V.Rao MD 51

ELISA – most popularly used method:

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). Dr.T.V.Rao MD 52

Uses of ELISA:

Uses of ELISA Helps detection of Antigens, Antibodies, hormones and Enzymes Eg in Microbiology Antigens – HbS Ag Antibodies HIV, HCV, CMV, several other disease Dr.T.V.Rao MD 53

Radio Immuno Assay Berson and Yallow:

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 Dr.T.V.Rao MD 54

Rosalyn S. Yalow and Sol Berson:

Rosalyn S. Yalow and Sol Berson Dr.T.V.Rao MD 55

RIA ( Radio Immuno Assay ):

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 . Dr.T.V.Rao MD 56

Uses of RIA:

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 Dr.T.V.Rao MD 57


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. Dr.T.V.Rao MD 58


Immunofluorescence Dr.T.V.Rao MD 59

Fluorescent Methods:

Fluorescent Methods Dr.T.V.Rao MD 60

Direct and Indirect Methods:

Direct and Indirect Methods Dr.T.V.Rao MD 61

Western Blot:

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).  Dr.T.V.Rao MD 62

Western Blot Test:

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. Dr.T.V.Rao MD 63

Appearance of test readings:

Appearance of test readings Dr.T.V.Rao MD 64

Flowcytometry :

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) Dr.T.V.Rao MD 65

PowerPoint Presentation:

Dr.T.V.Rao MD 66

Uses for flow cytometry :

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) Dr.T.V.Rao MD 67


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 Dr.T.V.Rao MD 68

Chemiluminescence's Immuno Assay:

Chemiluminescence's Immuno Assay Dr.T.V.Rao MD 69

Immunochromatographic Tests:

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 Dr.T.V.Rao MD 70

Immunochromatographic Tests:

Immunochromatographic Tests Dr.T.V.Rao MD 71

Immunochromatographic Tests:

Immunochromatographic Tests A colored band appears at the second window Control also can be recorded Dr.T.V.Rao MD 72

Also called as Dot Methods:

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 Dr.T.V.Rao MD 73

PowerPoint Presentation:

Programme Created by Dr.T.V.Rao MD for Medical and Paramedical Students in the Developing world Email Dr.T.V.Rao MD 74

authorStream Live Help