Immunoassay

HistoryIntroductionPrincipleClassificationApplicationDesigning StepsQuality controlInstrumentTraining video

Contents

History of immunoassayHistory

40 years ago Radioimmunoassay developed for human Insulin1971 Engvall and Perlmann, VanWeeman and Schuurs introducing ELISAIn the 1972, the development of EMIT Enzyme Multiplied Immunoassay (Homogenous Immunoassay)

IntroductionPrincipleDefinitions Antibodies (also known as immunoglobulins abbreviated Ig) are gamma globulin proteins that are found in blood and are used by the immune system to identify and neutralize foreign objects, such as bacteria and viruses.

Definitions- contAntigensA substance that when introduced into the body stimulates the production of an antibody

ImmunoassayA laboratory technique that makes use of the binding between an antigen and its homologous antibody in order to identify and quantify the specific antigen or antibody in a sample

Definitions- contAnalyteThe sample being analyzed and in immunoasssays the analyte is either Antibody or Antigen

Antibody ProductionSpecific antibodies are produced by injecting an antigen into a mammal, such as a mouse, rat or rabbit for small quantities of antibody, or goat, sheep, or horse for large quantities of antibody. Blood isolated from these animals contains polyclonal antibodiesmultiple antibodies that bind to the same antigenin the serum, which can now be called antiserum.

Properties of the antibody-antigen bondClassification of immunochemical detection methodsPrecipitation of antibody/antigen complexesFactors affecting solubilityZone of equivalenceThe precipitin reactionPrecipitateAntibody/Antigenetc.Single radial immunodiffusionAgSingle radial immunodiffusion

rElectroimmunodiffusion+-ImmunoelectrophoresisImmunoelectrophoresisSpecimen-human serum+-ImmunoelectrophoresisPCPCPC+-Immunofixation electrophoresisSPEIgGIgAIgMImmunochemical Methods (handout addendum)Bertholf and Bowman23AgglutinationAgglutininsAntibodies 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 particles5/30/2014Dr.T.V.Rao MD25Agglutination 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 precipitation5/30/2014Dr.T.V.Rao MD26ExamplesSlide Agglutination TestPlate Agglutination TestTube Agglutination TestPassive Agglutination TestMicroscopic Agglutination TestHaemagglutination test (HAT)5/30/2014Dr.T.V.Rao MD27Steps 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)

5/30/2014Dr.T.V.Rao MD28Secondary phenomenon:LATTICE FORMATIONAb + multivalent Ag stable network (visible reaction)conc. of Ag and AbGoverned by physiochemical factors:Ionic strength of milieupHtemperature

5/30/2014Dr.T.V.Rao MD29Secondary 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.5/30/2014Dr.T.V.Rao MD31DIRECT 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 groups5/30/2014Dr.T.V.Rao MD32

5/30/2014Dr.T.V.Rao MD33DIRECT AGGLUTINATIONThese reactions can be performed on slides (rapid tests) or on microliter plates or tubes for Antibody titration if required.5/30/2014Dr.T.V.Rao MD34

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 spp35Pictures 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 agglutination5/30/2014Dr.T.V.Rao MD37Slide Agglutination Test5/30/2014Dr.T.V.Rao MD38

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 Testing5/30/2014Dr.T.V.Rao MD395/30/2014Dr.T.V.Rao MD40

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

5/30/2014Dr.T.V.Rao MD43Passive 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.

5/30/2014Dr.T.V.Rao MD44REVERSE 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 albicans5/30/2014Dr.T.V.Rao MD45

Quantitative Micro Hemagglutination Test (HA)5/30/2014Dr.T.V.Rao MD46

Haemagglutination Test (HA)465/30/2014Dr.T.V.Rao MD47HaemagglutinationRBC47Viral HaemagglutinationSome viruses and microbes contain proteins which bind to erythrocytes (red blood cells) causing them to clump together

NDVAdenovirus IIIAIVIBVMycoplasma

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48 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 5/30/2014Dr.T.V.Rao MD49

49Titer = 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 RBCs50In the absence of anti-virus antibodies

5/30/2014Dr.T.V.Rao MD51ErythrocytesVirusVirus agglutination of erythrocytes51In the presence of anti-virus antibodies

5/30/2014Dr.T.V.Rao MD52ErythrocytesVirusAnti-virus antibodies

Viruses unable to bind tothe erythrocytes525/30/2014Dr.T.V.Rao MD53

53What is Antibody TiterIs the lowest concentration of antibodies against a particular antigen.5/30/2014Dr.T.V.Rao MD54Figure 18.6

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55Readings 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 ) 5/30/2014Dr.T.V.Rao MD5656Coombs 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).5/30/2014Dr.T.V.Rao MD57

Coombs (Antiglobulin)Tests 5/30/2014Dr.T.V.Rao MD58 Incomplete Ab Direct Coombs Test Detects antibodies on erythrocytes+

YYYYYYYYYYYYYYYYYYYPatients RBCsCoombs Reagent(Antiglobulin)Coombs Test Direct ant globulin test (also called the Coombs test, 5/30/2014Dr.T.V.Rao MD59

Coombs (Antiglobulin)Tests Indirect Coombs TestDetects anti-erythrocyte antibodies in serum5/30/2014Dr.T.V.Rao MD60YYYYYPatients SerumTargetRBCs+

Step 1+

YYYYYYYYYYYYYYYYYYYCoombs Reagent(Ant globulin)Step 2Application of Coombs (Antiglobulin)Tests ApplicationsDetection of anti-Rh AbAutoimmune hemolytic anemia5/30/2014Dr.T.V.Rao MD61

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 proteins5/30/2014Dr.T.V.Rao MD62Agglutination InhibitionTestsPregnancy Testing-classic example of agglutination inhibition Human chorionic gonadotropin (hCG)Appears in serum and urine early in pregnancy5/30/2014Dr.T.V.Rao MD63

Agglutination Inhibition5/30/2014Dr.T.V.Rao MD64Urine 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 reactions5/30/2014Dr.T.V.Rao MD65

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.

5/30/2014Dr.T.V.Rao MD68Complement 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

Light reflection

Distribution of scattered radiationNephelometry vs. Turbidimetry0-90TermsTransmittance (T) - The ratio of the radiant flux transmitted by the test substance to that of the incident radiant flux. Terms formerly used include transmittancy and transmission.Turbidance (S) - A measure of the light-scattering effect of suspended particles.Turbidity () - In light-scattering measurements, the turbidity is the measure of the decrease in incident beam intensity per unit length of a given suspension.

Colorimetric, Radiometric, Chemiluminescent, FluorescentImmobilized antibody methodsCoated tubeCoated beadSolid phase antibody methodsCoated tube methodsSpecimenLabeled antigenWashCoated bead methodsMicroparticle enzyme immunoassay (MEIA)Labeled antibodyEEESPGlass fiber matrixMagnetic separation methodsFeFeFeFeFeFeFeFeFeMagnetic separation methodsFeFeFeFeFeAspirate/Wash

ColorimetricELISAImmunoblottingLateral Flow Diffusion (handheld assay)Contact ZoneDetectionZoneInternal Control ZoneFluorescent LatexAb-coated Immobilized AbSamplePadWicking PadAgAgAgAgAgAg

Radioisotope labelsRadiometricEnzyme labels93Enzyme Immunoassay (EIA)94

Fluorescent labelsFluorescent ImmunoassaysTwo ApproachesPlanar microarraysEncoded micoparticle assay (Suspension) xMAP technology by Luminex, the CBA technology by Becton Dickinson BioSciences, and the VeraCode technology by Illumina, as

Chemiluminescent labelsChemiluminescent labels

Chemiluminescent labels

Electrochemiluminescence (ECL)similar to ELISA except that the secondary antibody is labeled with a chemiluminescent label ruthenium (Ru)Electron transfer between the Ru atom and the substrate tripropylamine (TPA) results in photon productionIntroduction to Heterogeneous versus Homogenous ImmunoassayIntroduction to Homogeneous ImmunoassayHomogeneous immunoassaysTypical design of a homogeneous immunoassayNo signalSignalEnzyme-multiplied immunoassay technique (EMIT)EMIT methodEnzymeSSPNo signalSignalEnzymeSImmunoassay Different FormatImmunoassay

Indirect Ag Sandwich Ab Competition Antibody DetectionAntigen Detection

Ab Sandwich

Ag CompetitionApplication

ELISA Manufacturing1. Development2. Optimization3. ValidationBasic Steps for Developing and Running an ImmunoassayIMMUNOASSAY PARAMETERS

1. Analyte (hapten or antigen) to be measured.2 .Sample matrices in which measurements will be made (serum, plasma, cell lysates, culture media etc.)3 .Source of antibody, analyte standards and detection reagents (labeled antibody, enzyme substrates etc). Availability of these reagents is a critical requirement.4 . Detection mode (colorimetric, fluorescence or chemiluminescence) and appropriate plate readers.5 .Type of immunoassay to develop (sandwich, competitive). 6 .Expected analyte concentration ranges to be measured (pg/ml, ng/ml or g/ml) in the sample matrix of choice. IMMUNOASSAY PARAMETERS

7. Data analysis models and format for reporting results. 8. Validation and optimization criteria using statistical experimental design tools. 9. Recovery, accuracy and precision expected at the limits of quantification and the measurable range. 10. Sample throughput, frequency of use, automation and the number of laboratories that would run the assay. 11 Control samples that would be used for optimization, validation and quality control runs.Design Optimization

ELISA Kit Results

117Repeat FLASH animation to solidify what they just did.OIE principles and Methods of Diagnostic Test ValidationCompany Logo

www.themegallery.comManufacturer Standards Harmonized Standard Directive 98/79/ECLaboratory Standards ISO 15189, ISO guide 25 ISO 13485Quantitative QC - Module 7120The Quality Management SystemOrganizationPersonnelEquipmentPurchasing & InventoryProcess ControlInformation ManagementDocuments& RecordsOccurrence ManagementAssessmentProcess ImprovementCustomer ServiceFacilities & Safety Five M of Quality Man Machine FACILITY (SIZE, CONSTRUCTION, LOCATION)

Qualifications, Organization, Job description, Training, etc.Qualification, Calibration ManualMethodologyMaterial/SampleStorageLabel MotivationSOP, Mfr Bruchurewww.themegallery.comMETHOD VALIDATIONRuggednessAccuracyInterferenceSpecificityPrecisionRobustnessSensitivityMfr claimed Performance Characteristics(Quantitative, Qualitative)

Reference IntervalStabilityKnown Panelswww.themegallery.comQuantitative QC - Module 7123Accuracy and PrecisionAccurate = Precise but not BiasedAccurate and Precise

Precisebut Biased Imprecise123Company LogoMeasurement of PrecisionWithin RunIntra-assayBetween-RunInter-assayYour TextYour TextYour TextBetween-lotWithin methodWithin lab

1234

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Validation methods 126AccuracyCloseness of determined value to the true value.Represent Systemic Error. or Bias (X-m)

The acceptance criteria is mean value 15% deviation from true value.

At LOQ, 20% deviation is acceptable.

Accuracy (%) = 100 x Found value - Theoretical valueTheoretical value

Poor PrecisionGood AccuracyGood PrecisionPoor AccuracyPoor PrecisionPoor AccuracyGold StandardSilver StandardOff-Base ModelHit or Miss ModelGood PrecisionGood AccuracyGraphwww.themegallery.comCompany Logo1. Recovery test: Adding a known amount of analyte to a base and measuring the concentration 2. Specificity (cross-reactivity) 3. Interferences 4. Parallelism (Linearity)Correlation Comparing the results to a reference values obtained from a definitive method, How Accuracy DeterminedDirectIndirect

www.themegallery.comCorrelation3. Parameters like m, Y intercept, r, Bias, etc2. Samples: At least 40 samples (~200-300 serum samples)Reference method

Regression Statistics Review:Correlation Coefficient (r) - characterizes the dispersion of results around the line of best fit.Slope - The lean of the line of best fit (proportional bias)Y-Intercept - the point at which the line of best fit intersects the Y axis. (constant bias)Acceptability Criteria:Correlation Coefficient (r) - the closer to 1.0 the betterSlope - The closer to 1.0 the betterY-Intercept - the closer to zero the better

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Indirect Accuracy DetectionRecovery (C-B/A)x100Matrix EffectPrecision

Linearity Measurement123Requires a minimum of 4-5 concent. levels

0, 25%, 50%, 75%, 100% solution3 replicates of each solution tested

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Sensitivity

Definition: Smallest amount of analyte that can be detected under the conditions of the assay1. Lower limit of detection, ie., The least or minimum detection dose (LDD) 2. Minimum distinguishable difference in concentration, Resolution (MDDC) The sensitivity of an analytical method is itsability to give response to small changes inthe absolute amount of analyte present123High sensitivityConcentration (X)added quantityResponse (Y)measuredquantity

Three analytical areas123Xbnot detectedArea of detectionArea of quantificationor CV