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Methods for Ag-Ab detection:

Methods for Ag-Ab detection Precipitation Agglutination Hemagglutination and Hemagglutination inhibition Viral neutralization test Radio-immunoassays ELISA Immunofluorescence Immmunoblotting Immunochromatography 1/11/2013 Dr.T.V.Rao MD 2


Agglutination Agglutinins Antibodies that produce such reactions Involves two-step process: Sensitization or initial binding Lattice formation or formation of large aggregates 1/11/2013 Dr.T.V.Rao MD 3


Agglutination Types of particles that participate in such reactions: Erythrocytes Bacterial cells Inert carriers such as latex particles 1/11/2013 Dr.T.V.Rao MD 4

Agglutination tests:

Agglutination tests Antibodies can agglutinate multivalent particulate antigens, such as Red Blood Cells (RBCs) or bacteria Some viruses also have the ability to agglutinate with RBCs. This behavior is called agglutination. Serological tests based on agglutination are usually more sensitive than those based on precipitation 1/11/2013 Dr.T.V.Rao MD 5


Examples Slide Agglutination Test Plate Agglutination Test Tube Agglutination Test Passive Agglutination Test Microscopic Agglutination Test Haemagglutination test (HAT) 1/11/2013 Dr.T.V.Rao MD 6

Steps in Agglutination:

Steps in Agglutination Primary phenomenon (SENSITIZATION) First reaction involving Ag-Ab combination Single antigenic determinant on the surface particle Initial reaction: rapid and reversible Cross link formation  visible aggregates (stabilization) 1/11/2013 Dr.T.V.Rao MD 7

PowerPoint Presentation:

Secondary phenomenon: LATTICE FORMATION Ab + multivalent Ag  stable network (visible reaction) conc. of Ag and Ab Governed by physiochemical factors: Ionic strength of milieu pH temperature 1/11/2013 Dr.T.V.Rao MD 8

Secondary Phenomenon :

Secondary Phenomenon Lattice Formation The Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutination If both antigen and antibody are SOLUBLE reaction will become visible over time, ie, p recipitation 1/11/2013 Dr.T.V.Rao MD 9


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. 1/11/2013 Dr.T.V.Rao MD 10


DIRECT AGGLUTINATION 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 1/11/2013 Dr.T.V.Rao MD 11

PowerPoint Presentation:

1/11/2013 Dr.T.V.Rao MD 12


DIRECT AGGLUTINATION These reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required. 1/11/2013 Dr.T.V.Rao MD 13

Direct agglutination :

Positive Negative Ag-Ab complex Direct agglutination Principle combination of an insoluble particulate antigen with its soluble antibody forms antigen-antibody complex particles clump/agglutinate used for antigen detection Examples bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp

Slide Agglutination Test:

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 1/11/2013 Dr.T.V.Rao MD 16

PowerPoint Presentation:

1/11/2013 Dr.T.V.Rao MD 17

Direct agglutination :

Positive Negative Ag-Ab complex Direct agglutination Principle combination of an insoluble particulate antigen with its soluble antibody forms antigen-antibody complex particles clump/agglutinate used for antigen detection Examples bacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp

Slide Agglutination Test:

Slide Agglutination Test 1/11/2013 Dr.T.V.Rao MD 19

Tube Agglutination Test:

Tube Agglutination Test Also known as the standard agglutination test or serum agglutination test (SAT) Test serum is diluted in a series of tubes (doubling dilutions) Constant defined amount of antigen is then added to each tube and tubes incubated for ~20h @37 ° C Particular antigen clumps at the bottom of the test tube Test is read at 50% agglutination Quantitative Confirmatory test for ELISA reactors Example: Brucellosis screening , Widal Testing 1/11/2013 Dr.T.V.Rao MD 20

PowerPoint Presentation:

1/11/2013 Dr.T.V.Rao MD 21 Tube Agglutination Test

Tube Agglutination Test:

No agglutination Agglutination 1/10 1/20 1/40 1/80 1/160 1/320 Neg. ctrl In this case, the titre is 1/40 Tube Agglutination Test

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 1/11/2013 Dr.T.V.Rao MD 23

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 1/11/2013 Dr.T.V.Rao MD 24

Passive Agglutination Test:

Passive Agglutination Test Converting a precipitating test to an agglutinating test Chemically link soluble antigen to inert particles such as LATEX or RBC Addition of specific antibody will cause the particles to agglutinate Reverse PAT: antibody linked to LATEX e.g. Lancefield grouping in Streptococci. 1/11/2013 Dr.T.V.Rao MD 25

Reverse passive agglutination:

Reverse passive agglutination Principle antigen binds to soluble antibody coated on carrier particles and results in agglutination detects antigens Example detecting cholera toxin 1/11/2013 Dr.T.V.Rao MD 26

REVERSE PASSIVE Agglutination Tests:

REVERSE PASSIVE Agglutination Tests 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 meningitides Haemophilus influenza Rotavirus Cryptococcus neoformans Mycoplasma pneumoniae Candida albicans 1/11/2013 Dr.T.V.Rao MD 27

PowerPoint Presentation:

1/11/2013 Dr.T.V.Rao MD 28 The latex particles are coated with IgG and mixed with the patient's serum

Quantitative Micro Hemagglutination Test (HA):

Quantitative Micro Hemagglutination Test (HA) 1/11/2013 Dr.T.V.Rao MD 29 Haemagglutination Test (HA)


1/11/2013 Dr.T.V.Rao MD 30 Haemagglutination RBC

Viral Haemagglutination:

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 1/11/2013 Dr.T.V.Rao MD 31

Viral Hemagglutination:

Viral Hemagglutination the attachment of viral particles by their receptor sites to more than 1 cell. As more and more cells become attached in this manner agglutination becomes visible 1/11/2013 Dr.T.V.Rao MD 32

Readings The results :

Readings The results Titer: The maximum dilution that gives visible agglutination. The end point: is the well with the lowest concentration of the virus where there is haemagglutination 2 4 8 16 32 64 128 256 512 1024 2048 4096 The HA titer of this virus in this row is 256 or 2 8 (1:256 dilution contains (1 HA unit) (one haemagglutinating unit) 1/11/2013 Dr.T.V.Rao MD 33

PowerPoint Presentation:

Titer = 32 HA units/ml Hemagglutination test: method 1:8 1:2 1:2 1:2 1:2 1:2 8 16 32 64 128 256 virus serial dilution mix with red blood cells side view top view One HA unit :minimum amount of virus that causes complete agglutination of RBCs

In the absence of anti-virus antibodies:

In the absence of anti-virus antibodies 1/11/2013 Dr.T.V.Rao MD 35 Erythrocytes Virus Virus agglutination of erythrocytes

In the presence of anti-virus antibodies:

In the presence of anti-virus antibodies 1/11/2013 Dr.T.V.Rao MD 36 Erythrocytes Virus Anti-virus antibodies Viruses unable to bind to the erythrocytes

PowerPoint Presentation:

1/11/2013 Dr.T.V.Rao MD 37

What is Antibody Titer:

What is Antibody Titer Is the lowest concentration of antibodies against a particular antigen. 1/11/2013 Dr.T.V.Rao MD 38 Figure 18.6

PowerPoint Presentation:

1/11/2013 Dr.T.V.Rao MD 39

Readings :

Readings The end point is the well with the lowest concentration of the serum where a clear button is seen. 2 4 8 16 32 64 128 256 512 1024 2048 4096 The antibody titer in this row will be 512 (2 9 ). (the lowest concentration of Abs which inhibit HA caused by the virus ) 1/11/2013 Dr.T.V.Rao MD 40

Coombs Test an Agglutination Test:

Coombs Test an Agglutination Test The Coombs test is actually two separate tests: the "direct" and "indirect" Coombs tests. Both aim to identify autoimmune haemolysis of red blood cells (erythrocytes). 1/11/2013 Dr.T.V.Rao MD 41

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests 1/11/2013 Dr.T.V.Rao MD 42 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)

Coombs Test Direct ant globulin test (also called the Coombs’ test, :

Coombs Test Direct ant globulin test (also called the Coombs’ test, 1/11/2013 Dr.T.V.Rao MD 43

Coombs (Antiglobulin)Tests :

Coombs (Antiglobulin)Tests Indirect Coombs Test Detects anti-erythrocyte antibodies in serum 1/11/2013 Dr.T.V.Rao MD 44 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 (Ant globulin) Step 2

Application of Coombs (Antiglobulin)Tests :

Application of Coombs (Antiglobulin)Tests Applications Detection of anti-Rh Ab Autoimmune hemolytic anemia 1/11/2013 Dr.T.V.Rao MD 45

Agglutination Inhibition:

Agglutination Inhibition Based on the competition between particulate and soluble antigens for limited antibody combining site Lack of agglutination is indicator of a positive reaction Usually involves haptens complexed with proteins 1/11/2013 Dr.T.V.Rao MD 46

Agglutination Inhibition Tests:

Agglutination Inhibition Tests Pregnancy Testing - classic example of agglutination inhibition Human chorionic gonadotropin (hCG) Appears in serum and urine early in pregnancy 1/11/2013 Dr.T.V.Rao MD 47

Agglutination Inhibition:

Agglutination Inhibition 1/11/2013 Dr.T.V.Rao MD 48 Urine Antiserum No hCG in urine: Anti-hCG free hCG in urine: Anti-hCG neutralized Carriers coated with hCG added Carriers coated with hCG added AGGLUTINATION of carriers: Negative test for hCG NOT PREGNANT NO AGGLUTINATION of carriers: Positive test for hCG PREGNANT


Co-agglutination Co agglutination is similar to the latex agglutination technique for detecting antigen (described above). Protein A, a uniformly distributed cell wall component of Staphylococcus aureus, is able to bind to the Fc region of most IgG isotype antibodies leaving the Fab region free to interact with antigens present in the applied specimens. The visible agglutination of the S. Aureus particles indicates the antigen-antibody reactions 1/11/2013 Dr.T.V.Rao MD 49


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 1/11/2013 Dr.T.V.Rao MD 50

Highly specific but not very sensitive in detecting small quantities of antigen :

Highly specific but not very sensitive in detecting small quantities of antigen 1/11/2013 Dr.T.V.Rao MD 51

Co agglutination Test:

Co agglutination Test Agglutination test in which inert particles (latex beads or heat-killed S aureus Cowan 1 strain with protein A) are coated with antibody to any of a variety of antigens and then used to detect the antigen in specimens or in isolated bacteria. 1/11/2013 Dr.T.V.Rao MD 52

Rickettsia and Serology:

Rickettsia and Serology Rickettsia is a genus of motile, Gram-negative, non-spore forming, highly pleomorphic bacteria that can present as Cocci (0.1 μm in diameter), rods (1–4 μm long) or thread-like (10 μm long). Obligate intracellular parasites Because of this, Rickettsia cannot live in artificial nutrient environments and are grown either in tissue or embryo cultures (typically, chicken embryos are used). Still we have to dependent on Weil Felix test 1/11/2013 Dr.T.V.Rao MD 53

Weil and Felix contribute for testing:

Weil and Felix contribute for testing In 1915, Weil and Felix showed that serum of patients infected with any member of the typhus group of diseases contains agglutinins for one or more strains of O X Proteus. In cases of typhus fever the reaction usually appears before the sixth day and reaches its height in the second week. 1/11/2013 Dr.T.V.Rao MD 54

Weil-Felix reaction – A Heterophile agglutination Test:

Weil-Felix reaction – A Heterophile agglutination Test A Weil-Felix reaction is a type of agglutination test in which patients serum is tested for agglutinins to O antigen of certain non-motile Proteus and Rickettsial strains(OX19, OX2, OXk) OX19, OX2 are strains of Proteus vulgaris. OXk is the strain of Proteus mirabilis. 1/11/2013 Dr.T.V.Rao MD 55

Weil-Felix a Heterophile agglutination test:

Weil-Felix a Heterophile agglutination test The agglutination reactions, based on antigens common to both organisms, determine the presence and type of Rickettsial infection Because Rickettsia are both fastidious and hazardous, few laboratories undertake their isolation and diagnostic identification Weil-Felix test that is based on the cross-reactive antigens of OX-19 and OX-2 strains of Proteus vulgaris . 1/11/2013 Dr.T.V.Rao MD 56

Reading/Grading Agglutination Reactions :

Reading/Grading Agglutination Reactions Done by gently shaking the tubes containing the serum and cells, and observing the cell button as it is dispersed Hard shaking must be avoided because this may yield to false result Attention should also be given to whether discoloration of the supernatant is present (Hemolysis). 1/11/2013 Dr.T.V.Rao MD 57

Interpretations in Weil-Felix reaction :

Interpretations in Weil-Felix reaction Sera from endemic typhus agglutinate OX19, OX2. Tick borne spotted fever agglutinate OX19, OX2. Scrub Typhus agglutinate OXk strain Test is negative in rickettsia pox, trench fever and Q-fever. False positive reaction may occur in urinary or other Proteus infections Test may be negative in 50 percent scrub typhus 1/11/2013 Dr.T.V.Rao MD 58

Weil-Felix test indicated in when patients present with rashes:

Weil-Felix test indicated in when patients present with rashes Test for diagnosis of typhus and certain other Rickettsial diseases. The blood serum of a patient with suspected Rickettsial disease is tested against certain strains of (OX-2, OX-19, OX-K).. 1/11/2013 Dr.T.V.Rao MD 59

Latex Agglutination :

Latex Agglutination Antibody molecules can be bound to each latex beads It will increase the potential number of exposed antigen-binding sites . When an antigen is present in test specimen, it may bind to the latex bead thus forming visible cross-linked aggregates . Latex particles can be coated with antigen (pregnancy testing, rubella antibody testing) 1/11/2013 Dr.T.V.Rao MD 60

Complement fixation Test:

Complement fixation Test 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 hemolytic 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 hemolysis which is a property of complement, denoting a negative test .

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 (Hemolytic system) Erythrocytes: Sheep RBC Amboceptor (Hemolysins): 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 + Hemolytic system No Hemolysis 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 + Hemolytic system Hemolysis 1 Hour ( Test Negative)

Results and Interpretations::

Results and Interpretations: No hemolysis is considered as a positive test . hemolysis of erythrocytes indicative of a negative test . 1 2 3 4 A B Microtiter plate showing Hemolysis (Well A3, A3 and B4) and No Hemolysis (Well


Radio-immunoassays Principle Radioactively labelled-antibody (or antigen) competes with the patient’s unlabeled antibody (or antigen) for binding sites on a known amount of antigen (or antibody) Reduction in radioactivity of the antigen-patient antibody complex compared with control test is used to quantify the amount of patient antibody / antibody bound Limited use due to the problems with handling radioisotope Example HBsAg Thyroid function test 1/11/2013 Dr.T.V.Rao MD 68 Response Antibody

Radio Immuno Assay:

Radio Immuno Assay

Radio-immunoassays: Performance, applications:

Radio-immunoassays: Performance, applications Advantages highly sensitive can be used for detection of small quantities quantification possible Limitations expensive requires isotopes Time taken 1 day 1/11/2013 Dr.T.V.Rao MD 70

Enzyme-linked Immuno-Sorbant assay (ELISA):

Labeling technique Enzyme-linked Immuno-Sorbant assay (ELISA) Principle use of enzyme-labeled immunoglobulin to detect antigens or antibodies signals are developed by the action of hydrolyzing enzyme on chromogenic substrate optical density measured by micro-plate reader Examples Hepatitis A (Anti-HAV-IgM, anti-HAV Iggy)


ELISA 1/11/2013 Dr.T.V.Rao MD 72 Antibody Response Micro-plate reader 96-well micro-plate Positive result

Types of ELISA (Ag Abs tests):

Labeling technique Types of ELISA (Ag Abs tests) Competitive Antigen or antibody are labeled with enzyme and allowed to compete with unlabeled ones (in patient serum) for binding to the same target Hydrolysis signal from Ag-Ab complex (enzyme-labeled) is measured Antigen or antibody in serum is then calculated No need to remove the excess/unbound Ag or Ab from the reaction plate or tubes)

Types of ELISA used in the detection of antigens and antibodies:

Labeling technique Types of ELISA used in the detection of antigens and antibodies Non-competitive must remove excess/unbound Ag or Ab before every step of reactions Direct ELISA Indirect ELISA Sandwich ELISA Ab Capture ELISA (similar to sandwich ELISA but in 1 st step, anti-Ig (M or G) is coated on the plate Then antibodies in patient serum are allowed to capture in next step

ELISA: Performance, applications:

ELISA: Performance, applications Advantages Automated, inexpensive Objective Small quantities required Class specific antibodies measurable Limitations Expensive initial investment Variable sensitivity / specificity of variable tests Cross contamination Time taken - 1 day 1/11/2013 Dr.T.V.Rao MD 75


Labeling technique Cell infected with Dengue virus V. Cholerae Immuno-fluorescence Principle Use fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systems Requires a fluorescent microscope Examples Herpes virus IgM Dengue virus Rabies virus Scrub and murine typhus

Immunofluorescence Helps in Diagnosis of Various Diseases :

Immunofluorescence Helps in Diagnosis of Various Diseases

Immuno-fluorescence: Performance, applications:

Immuno-fluorescence: Performance, applications Advantages Sensitive and specific Can be used for discrepant analysis Limitations Expensive (Reagents and equipment) Subjective Cross reactivity Non-specific immuno-fluorescence Time taken few minutes to few hours. 1/11/2013 Dr.T.V.Rao MD 78

Types of immuno-fluorescence:

Labeling technique Types of immuno-fluorescence Direct immuno-fluorescence Used to detect antigen Indirect and sandwich immuno-fluorescence Antigen detection Antibody detection 1/11/2013 Dr.T.V.Rao MD 79

Western-blot analysis:

Western-blot analysis Principle Antigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranes Antibodies in serum react with specific antigens Signals are detected according to the principles of test systems Antibodies against microbes with numerous cross-reacting antibodies identified more specifically Examples T. pallidum, B.burgdorferi, Herpes simplex virus types 1 and 2 1/11/2013 Dr.T.V.Rao MD 80 Anti HIV-1

Western-blot analysis :

Western-blot analysis Serum, saliva, urine can be tested Kits are commercially available Recombinant immuno-blotting assays (RIBA) uses recombinant proteins

Immuno-chromatography: Principle :

Immuno-chromatography: Principle Dye-labelled antibody, specific for target antigen, is present on the lower end of nitrocellulose strip or in a plastic well provided with the strip. Antibody, also specific for the target antigen, is bound to the strip in a thin (test) line Either antibody specific for the labelled antibody, or antigen, is bound at the control line 1/11/2013 Dr.T.V.Rao MD 82 Lysing agend Labled AB. Test band (bound AB) Control band (bound AB) Nitrocellulose strip Bound AB Free labled AB

Immuno-chromatography: Principle :

Immuno-chromatography: Principle If antigen is present, some labelled antibody will be trapped on the test line Excess-labelled antibody is trapped on the control line 1/11/2013 Dr.T.V.Rao MD 83 Captured Ag-labelled Ab-complex Captured labelled Ab Labelled AB-AG-complex Captured by bound AB of test band Labelled AB-AG-complex Captured by bound AB of control band

Immuno-chromatography: Performance, applications:

Immuno-chromatography: Performance, applications Advantages Commercially available Single use, rapid test Easy to perform Can detect antigen or antibody Can be used in the field Limitations Cost Concern validated data Time taken - 1 hour 1/11/2013 Dr.T.V.Rao MD 84

Chemiluminescent Immunoenzymatic Assay:

Chemiluminescent Immunoenzymatic Assay Process for the quantitative and qualitative determination of antigens, antibodies and their complexes by means of a chemiluminescing labelling substance activated or excited to Chemiluminescence's by an analytical reagent. By means of a serological reaction, initially an antigen/antibody complex is formed which is treated with a chemiluminescing conjugate containing chemiluminescing triphenylmethane dyes and the chemiluminescence of the chemiluminescing complex formed is measured. 1/11/2013 Dr.T.V.Rao MD 85

PowerPoint Presentation:

Programme Created by Dr.T.V.Rao MD for Medical Students in the Developing World Email 1/11/2013 Dr.T.V.Rao MD 86

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