Agglutination tests

SerologyAGGLUTINATION TESTS and IMMUNOASSYS BASICSDr.T.V.Rao MD

Methods for Ag-Ab detectionPrecipitationAgglutinationHemagglutination and Hemagglutination inhibitionViral neutralization testRadio-immunoassaysELISAImmunofluorescenceImmmunoblottingImmunochromatography1/12/2013Dr.T.V.Rao MD2

2AgglutinationAgglutininsAntibodies that produce such reactionsInvolves two-step process:Sensitization or initial bindingLattice formation or formation of large aggregates

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AgglutinationTypes of particles that participate in such reactions:ErythrocytesBacterial cellsInert carriers such as latex particles1/12/2013Dr.T.V.Rao MD4Agglutination testsAntibodies can agglutinate multivalent particulate antigens, such as Red Blood Cells (RBCs) or bacteriaSome 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 precipitation1/12/2013Dr.T.V.Rao MD5ExamplesSlide Agglutination TestPlate Agglutination TestTube Agglutination TestPassive Agglutination TestMicroscopic Agglutination TestHaemagglutination test (HAT)1/12/2013Dr.T.V.Rao MD6Steps in AgglutinationPrimary phenomenon (SENSITIZATION)First reaction involving Ag-Ab combination Single antigenic determinant on the surface particleInitial reaction: rapid and reversibleCross link formation visible aggregates (stabilization)

1/12/2013Dr.T.V.Rao MD7Secondary phenomenon:LATTICE FORMATIONAb + multivalent Ag stable network (visible reaction)conc. of Ag and AbGoverned by physiochemical factors:Ionic strength of milieupHtemperature

1/12/2013Dr.T.V.Rao MD8Secondary Phenomenon Lattice FormationThe Fab portion of the Ig molecule attaches to antigens on 2 adjacent cells-visible results in agglutinationIf both antigen and antibody are SOLUBLE reaction will become visible over time, ie, precipitation

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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.1/12/2013Dr.T.V.Rao MD10DIRECT AGGLUTINATIONThe particle antigen may be a bacterium. e.g.: Serotyping of E. coli, Salmonella using a specific antiserumThe particle antigen may be a parasite. e.g.: Serodiagnosis of ToxoplasmosisThe particle antigen may be a red blood cell. e.g.: Determination of blood groups1/12/2013Dr.T.V.Rao MD11

1/12/2013Dr.T.V.Rao MD12DIRECT AGGLUTINATIONThese reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required.1/12/2013Dr.T.V.Rao MD13

Positive NegativeAg-Ab complexDirect agglutination Principlecombination of an insoluble particulate antigen with its soluble antibody forms antigen-antibody complex particles clump/agglutinate used for antigen detectionExamplesbacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp14Pictures public domain

Slide Agglutination TestUsed for serotyping (e.g. Salmonella)Antigen: isolated Salmonella in suspensionAntibody: specific antisera against SalmonellaPlace test Salmonella in a drop of saline on a slide Add a drop of antiserum, mix and rock slide for approx. 1 minuteExamine for agglutination1/12/2013Dr.T.V.Rao MD16

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Positive NegativeAg-Ab complexDirect agglutination Principlecombination of an insoluble particulate antigen with its soluble antibody forms antigen-antibody complex particles clump/agglutinate used for antigen detectionExamplesbacterial agglutination tests for sero-typing and sero-grouping e.g., Vibrio cholerae, Salmonella spp18Pictures public domainSlide Agglutination Test1/12/2013Dr.T.V.Rao MD19

Tube Agglutination TestAlso 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 @37CParticular antigen clumps at the bottom of the test tubeTest is read at 50% agglutinationQuantitativeConfirmatory test for ELISA reactorsExample: Brucellosis screening , Widal Testing1/12/2013Dr.T.V.Rao MD201/12/2013Dr.T.V.Rao MD21

Tube Agglutination TestNo agglutinationAgglutination1/101/201/401/801/1601/320Neg. ctrlIn this case, the titre is 1/40Tube Agglutination TestPassive AgglutinationAn agglutination reaction that employs particles that are coated with antigens not normally found in the cell surfacesParticle carriers include:Red blood cellsPolystyrene latexBentonitecharcoal

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Passive AgglutinationPassive agglutination has been used in the detection of :Rheumatoid factorAntinuclear antibody in LEAb to group A streptococcus antigensAb to Trichinella spiralis

1/12/2013Dr.T.V.Rao MD24Passive Agglutination TestConverting a precipitating test to an agglutinating testChemically link soluble antigen to inert particles such as LATEX or RBCAddition of specific antibody will cause the particles to agglutinateReverse PAT: antibody linked to LATEXe.g. Lancefield grouping in Streptococci.

1/12/2013Dr.T.V.Rao MD25Reverse passive agglutinationPrincipleantigen binds to soluble antibody coated on carrier particles and results in agglutination detects antigensExampledetecting cholera toxin1/12/2013Dr.T.V.Rao MD26

26REVERSE PASSIVEAgglutination TestsAntibody rather than antigen is attached to a carrier particleFor the detection of microbial antigens such as:Group A and B streptococcusStaphylococcus aureusNeisseria meningitidesHaemophilus influenzaRotavirusCryptococcus neoformansMycoplasma pneumoniaeCandida albicans1/12/2013Dr.T.V.Rao MD27

1/12/2013Dr.T.V.Rao MD28The latex particles are coated with IgG and mixed with the patient's serum

Quantitative Micro Hemagglutination Test (HA)1/12/2013Dr.T.V.Rao MD29

Haemagglutination Test (HA)291/12/2013Dr.T.V.Rao MD30HaemagglutinationRBC30Viral HaemagglutinationSome viruses and microbes contain proteins which bind to erythrocytes (red blood cells) causing them to clump together

NDVAdenovirus IIIAIVIBVMycoplasma

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31 Comparison of Mycoplasma synoviae hemagglutinating antigens by the hemagglutination-inhibition testAuthors: T H Vardaman, J H DrottImpact factor: 1.07, Cited half life: >10.0, Immediacy index: 0.29Journal: Avian DiseasesMycoplasma synoviae (MS) strains were isolated from the trachea of hens from MS-positive broiler breeder flocks having progeny condemnations due to airsacculitis. Hemagglutinating (HA) antigens were made from several strains. The HA antigen made from the 95th medium passage of MS FMT strain was compared with that of the standard MS WVU 1853 strain by the hemagglutination-inhibition (HI) test. Sixty-six sera from 10 MS-positive flocks, 4 Mycoplasma gallisepticum (MG)-positive sera, and 7 normal sera were used in the HI test. The geometric mean (GM) HI titer with MS WVU 1853 antigen was 98.69 on 66 sera from 10 MS-positive flocks, whereas the GM HI titer with MS FMT antigen was 226.21.Viral Hemagglutinationthe 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 1/12/2013Dr.T.V.Rao MD32

32Readings 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 28(1:256 dilution contains (1 HA unit) (one haemagglutinating unit) 1/12/2013Dr.T.V.Rao MD3333Titer = 32 HA units/mlHemagglutination test: method1:81:21:21:21:21:28163264128256virusserial dilutionmix with red blood cellsside viewtop viewOne HA unit :minimum amount of virus that causes complete agglutination of RBCs34In the absence of anti-virus antibodies

1/12/2013Dr.T.V.Rao MD35ErythrocytesVirusVirus agglutination of erythrocytes35In the presence of anti-virus antibodies

1/12/2013Dr.T.V.Rao MD36ErythrocytesVirusAnti-virus antibodies

Viruses unable to bind tothe erythrocytes361/12/2013Dr.T.V.Rao MD37

37What is Antibody TiterIs the lowest concentration of antibodies against a particular antigen.1/12/2013Dr.T.V.Rao MD38Figure 18.6

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39Readings 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 (29).(the lowest concentration of Abs which inhibit HA caused by the virus ) 1/12/2013Dr.T.V.Rao MD4040Coombs Test an Agglutination TestThe Coombs test is actually two separate tests: the "direct" and "indirect" Coombs tests. Both aim to identify autoimmune haemolysis of red blood cells (erythrocytes).1/12/2013Dr.T.V.Rao MD41

Coombs (Antiglobulin)Tests 1/12/2013Dr.T.V.Rao MD42 Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes+

YYYYYYYYYYYYYYYYYYYPatients RBCsCoombs Reagent(Antiglobulin)Coombs Test Direct ant globulin test (also called the Coombs test, 1/12/2013Dr.T.V.Rao MD43

Coombs (Antiglobulin)Tests Indirect Coombs TestDetects anti-erythrocyte antibodies in serum1/12/2013Dr.T.V.Rao MD44YYYYYPatients SerumTargetRBCs+

Step 1+

YYYYYYYYYYYYYYYYYYYCoombs Reagent(Ant globulin)Step 2Application of Coombs (Antiglobulin)Tests ApplicationsDetection of anti-Rh AbAutoimmune hemolytic anemia1/12/2013Dr.T.V.Rao MD45

Agglutination InhibitionBased on the competition between particulate and soluble antigens for limited antibody combining siteLack of agglutination is indicator of a positive reactionUsually involves haptens complexed with proteins1/12/2013Dr.T.V.Rao MD46Agglutination InhibitionTestsPregnancy Testing-classic example of agglutination inhibition Human chorionic gonadotropin (hCG)Appears in serum and urine early in pregnancy1/12/2013Dr.T.V.Rao MD47

Agglutination Inhibition1/12/2013Dr.T.V.Rao MD48Urine AntiserumNo hCG in urine:Anti-hCG free hCG in urine:Anti-hCG neutralizedCarriers coated with hCG addedCarriers coated with hCG addedAGGLUTINATION of carriers:Negative test for hCGNOT PREGNANT NO AGGLUTINATION of carriers:Positive test for hCGPREGNANT Co-agglutinationCo 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 reactions1/12/2013Dr.T.V.Rao MD49

CoagglutinationName given to systems using inert bacteria as the inert particles to which the antibody is attachedS.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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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.

1/12/2013Dr.T.V.Rao MD52Rickettsia and SerologyRickettsia is a genus of motile, Gram-negative, non-spore forming, highly pleomorphic bacteria that can present as Cocci (0.1 m in diameter), rods (14 m long) or thread-like (10 m 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 test1/12/2013Dr.T.V.Rao MD53

Weil and Felix contribute for testingIn 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. 1/12/2013Dr.T.V.Rao MD54

54Edmund Weil attended the German University of Prague. He obtained his doctorate in 1903 and then worked as 2nd assistant under the pathological anatomist Hans Chiara (1851-1916). In 1905 he entered the hygienically institute, at which he assumed leadership of the serological department in 1918. He was habilitated for hygiene in 1909, becoming ausserordentlicher professor in 1914. He died in Prague in 1922 from an infection of spotted fever he had attracted in a laboratory in Poland . n 1911, with the neuropsychologist Viktor Kafka, Weil described a previously unknown bacteriological reaction, now known as Weil-Kafka reaction. At the time, Kafka was an assistant in the psychiatric clinic of the German faculty in Prague. Weil-Felix reaction A Heterophile agglutination TestA 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. 1/12/2013Dr.T.V.Rao MD55

Weil-Felix a Heterophile agglutination testThe agglutination reactions, based on antigens common to both organisms, determine the presence and type of Rickettsial infectionBecause 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.

1/12/2013Dr.T.V.Rao MD56Reading/Grading Agglutination Reactions

Done by gently shaking the tubes containing the serum and cells, and observing the cell button as it is dispersedHard shaking must be avoided because this may yield to false resultAttention should also be given to whether discoloration of the supernatant is present (Hemolysis).

1/12/2013Dr.T.V.Rao MD57Interpretations 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 infectionsTest may be negative in 50 percent scrub typhus 1/12/2013Dr.T.V.Rao MD58

Weil-Felix testindicated in when patients present with rashesTest 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)..

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Latex Agglutination

Antibody molecules can be bound to each latex beadsIt 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)

1/12/2013Dr.T.V.Rao MD60Complement fixation TestThe 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 complexWhen the Ag is an RBC it causes lysis of RBCs.PrincipleComplement takes part in many of the immunological reactions. It gets absorbed during the combination of antigens and antibody.

This property of antigenantibody 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 CFTTest SystemAntigen: 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 TestStep 1: At 37CAntigen + Antibody + Complement Complement gets fixed (from serum) 1 Hour Step 2: At 37CFixed Complement complex + Hemolytic system No Hemolysis 1 Hour (Test Positive)Negative TestStep 1: At 37CAntigen + Antibody absent + Complement Complement not fixed 1 Hour Step 2: At 37CFree Complement + Hemolytic system Hemolysis 1 Hour (Test Negative)Results and Interpretations:No hemolysis is considered as a positive test. hemolysis of erythrocytes indicative of a negative test. 1 2 3 4AB

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

Radio-immunoassaysPrincipleRadioactively labelled-antibody (or antigen) competes with the patients 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 boundLimited use due to the problems with handling radioisotopeExampleHBsAgThyroid function test1/12/2013Dr.T.V.Rao MD68ResponseAntibody68Radio Immuno Assay

Radio-immunoassays:Performance, applicationsAdvantageshighly sensitivecan be used for detection of small quantitiesquantification possible Limitationsexpensive requires isotopesTime taken1 day1/12/2013Dr.T.V.Rao MD70

70Labeling techniqueEnzyme-linked Immuno-Sorbant assay (ELISA)Principleuse of enzyme-labeled immunoglobulin to detect antigens or antibodies signals are developed by the action of hydrolyzing enzyme on chromogenic substrateoptical density measured by micro-plate readerExamplesHepatitis A (Anti-HAV-IgM, anti-HAV Iggy)71ELISA

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Micro-plate reader96-well micro-platePositive result72Picture public domainLabeling techniqueTypes 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 targetHydrolysis signal from Ag-Ab complex (enzyme-labeled) is measuredAntigen or antibody in serum is then calculatedNo need to remove the excess/unbound Ag or Ab from the reaction plate or tubes)

73Graphic self preparedLabeling techniqueTypes of ELISA used in the detection of antigens and antibodiesNon-competitive must remove excess/unbound Agor Ab before every step of reactionsDirect ELISA Indirect ELISASandwich ELISA Ab Capture ELISA (similar to sandwich ELISA but in 1st step, anti-Ig (M or G) is coated on the plateThen antibodies in patient serum are allowed to capture in next step

74Graphic self prepared

ELISA:Performance, applicationsAdvantagesAutomated, inexpensiveObjectiveSmall quantities requiredClass specific antibodies measurable LimitationsExpensive initial investment Variable sensitivity / specificity of variable testsCross contamination Time taken - 1 day1/12/2013Dr.T.V.Rao MD7575Labeling technique

Cell infected with Dengue virusV. CholeraeImmuno-fluorescencePrincipleUse fluorescein isothiocyanate labeled-immunoglobulin to detect antigens or antibodies according to test systemsRequires a fluorescent microscopeExamplesHerpes virus IgMDengue virusRabies virusScrub and murine typhus

76Photos public domainImmunofluorescence Helps in Diagnosis of Various Diseases

Immuno-fluorescence:Performance, applicationsAdvantagesSensitive and specificCan be used for discrepant analysis LimitationsExpensive (Reagents and equipment)SubjectiveCross reactivityNon-specific immuno-fluorescence Time taken few minutes to few hours.1/12/2013Dr.T.V.Rao MD7878Labeling techniqueTypes of immuno-fluorescence

Direct immuno-fluorescence Used to detect antigenIndirect and sandwich immuno-fluorescenceAntigen detectionAntibody detection1/12/2013Dr.T.V.Rao MD7979Graphic self preparedWestern-blot analysisPrincipleAntigens are separated by Poly Acrylomide Gel Electrophoresis (PAGE) and trans-blotted onto nitrocellulose/nylon membranesAntibodies in serum react with specific antigens Signals are detected according to the principles of test systemsAntibodies against microbes with numerous cross-reacting antibodies identified more specificallyExamplesT. pallidum, B.burgdorferi, Herpes simplex virus types 1 and 21/12/2013Dr.T.V.Rao MD80

Anti HIV-180Photo public domainWestern-blot analysis Serum, saliva, urine can be testedKits are commercially availableRecombinant immuno-blotting assays (RIBA) uses recombinant proteins

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) lineEither antibody specific for the labelled antibody, or antigen, is bound at the control line1/12/2013Dr.T.V.Rao MD82Lysing agendLabled AB.Test band(bound AB)Control band(bound AB)Nitrocellulose stripBoundABFree labled AB82Graphics redone with reference to http://www.wpro.who.int/sites/rdt/what_is_rdt.htmImmuno-chromatography: Principle If antigen is present, some labelled antibody will be trapped on the test lineExcess-labelled antibody is trapped on the control line 1/12/2013Dr.T.V.Rao MD83Captured Ag-labelled Ab-complexCaptured labelled AbLabelled AB-AG-complexCaptured by bound AB of test bandLabelled AB-AG-complexCaptured by bound AB ofcontrol band83Graphics redone with reference to http://www.wpro.who.int/sites/rdt/what_is_rdt.htmImmuno-chromatography: Performance, applicationsAdvantagesCommercially availableSingle use, rapid testEasy to performCan detect antigen or antibodyCan be used in the fieldLimitationsCostConcern validated dataTime taken - 1 hour1/12/2013Dr.T.V.Rao MD84

84Chemiluminescent Immunoenzymatic AssayProcess 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. 1/12/2013Dr.T.V.Rao MD85

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