Immunological TechniquesImmunological Techniques

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SUTIRTA YASA IWPSUTIRTA YASA IWP

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Learning Task Laboratories Learning Task Laboratories testtest

1.1. Explain the precipitin reactions, what are antibody Explain the precipitin reactions, what are antibody excess zone, equivalence zone and antigen excess excess zone, equivalence zone and antigen excess zonezone

2.2. Explain the differential of immuno-double diffusion Explain the differential of immuno-double diffusion and immunoelectrophoresis.and immunoelectrophoresis.

3.3. Explain the differential of direct and indirect immuno Explain the differential of direct and indirect immuno techniques method.techniques method.

4.4. Mention the immunoassay using labeled reagents for Mention the immunoassay using labeled reagents for detecting antigens and antibodies.detecting antigens and antibodies.

5.5. Explain the competitive assay and two-site capture Explain the competitive assay and two-site capture assay techniques.assay techniques.

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Self assessmentSelf assessment

1.1. Mention the principle of methods on Mention the principle of methods on immunoassay techniques ?immunoassay techniques ?

2.2. What’s the meaning of equivalence What’s the meaning of equivalence zone ?zone ?

3.3. Mention the reaction marked on Mention the reaction marked on haemagglutination methods haemagglutination methods

4.4. Mention the label used on the ELISA Mention the label used on the ELISA methods?methods?

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What is the laboratory diagnostic tests What is the laboratory diagnostic tests can be used ?can be used ?

What is the performance of laboratoryWhat is the performance of laboratory ??

How many techniques are used How many techniques are used commonlycommonly??

For example, the methods used to For example, the methods used to measured :measured :– antigens and antibodiesantigens and antibodies– Inflammatory factor : ILs,IFNs, CRFInflammatory factor : ILs,IFNs, CRF

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Most of the common used are outlined in this lecture

The Laboratory personal As The Laboratory personal As journalist on a war ??journalist on a war ??

• ..

Sensitivity of test

Ab

Ab

Mikroorganisme / agent, enter/ invasion

As detective What happened?Damage?Many signal?Soluble mediator?Detect byHematology test ?Biochemistry test ?Immunol test ?

Host, defense mechanisms:celluler & humoral

Agent : growing, expandingExcretion toxic substance

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LLaboratory diagnostic tests aboratory diagnostic tests can be usedcan be used : : (Nicoll D, 2004) (Nicoll D, 2004)

– Tests can be helpful for Tests can be helpful for screeningscreening– Tests can be helpful for Tests can be helpful for diagnosisdiagnosis– Finally , tests can be helpful in patient Finally , tests can be helpful in patient

managementmanagement• EvaluateEvaluate the severity of disease the severity of disease

• Estimate Estimate prognosisprognosis

• MonitorMonitor the course of disease ( progression, the course of disease ( progression, stability, or resolution)stability, or resolution)

• DetectDetect disease recurrence disease recurrence

• Select drugs and adjust Select drugs and adjust therapytherapy(Nicoll D(Nicoll D., McPhee, Pignone. ., McPhee, Pignone. Basic Principles of Diagnostic Test Use and InterpretationBasic Principles of Diagnostic Test Use and Interpretation On : On : Pocket guide to Pocket guide to

Diagnostic TestsDiagnostic Tests .Nicoll D eds. Fourth Edition. Lange Medical Books/McGraw-Hill. New York. 2004:1-21) .Nicoll D eds. Fourth Edition. Lange Medical Books/McGraw-Hill. New York. 2004:1-21)

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Performance of diagnostic tests (Nicoll D, Performance of diagnostic tests (Nicoll D, 2004):2004):– Tests preparation : Tests preparation : test selectedtest selected

– Patient Patient preparationpreparation (appropriate specimens) (appropriate specimens)– Specimen collection (identification , labeling, Specimen collection (identification , labeling,

tourniquet time > hemoconcentration, lyses of blood tourniquet time > hemoconcentration, lyses of blood cell, special handling, storage )cell, special handling, storage )

– Accuracy : true value; Precision : reproducibility when Accuracy : true value; Precision : reproducibility when repeated on the sample.repeated on the sample.

– Reference ranges are method and laboratory specific. Reference ranges are method and laboratory specific. Often represent test results found in 95% of small Often represent test results found in 95% of small population presumed to be healthy. population presumed to be healthy.

– InterferingInterfering factors. factors. ExternalExternal factors such as ingestion factors such as ingestion of of drugsdrugs. . InternalInternal factors such as abnormal physiologic factors such as abnormal physiologic states. states.

– SensitivitySensitivity and and specificityspecificity. Test sensitivity is the . Test sensitivity is the likelihood that likelihood that a diseased patient has positive test a diseased patient has positive test (no (no diseased patients have negative tests). Test specificity diseased patients have negative tests). Test specificity is likelihood that is likelihood that a healthy patient has a negative testa healthy patient has a negative test (no healthy patients have positive tests). (no healthy patients have positive tests).

(Nicoll D., McPhee, Pignone. (Nicoll D., McPhee, Pignone. Basic Principles of Diagnostic Test Use and InterpretationBasic Principles of Diagnostic Test Use and Interpretation On : On : Pocket guide to Diagnostic TestsPocket guide to Diagnostic Tests .Nicoll D eds. .Nicoll D eds. Fourth Edition. Lange Medical Books/McGraw-Hill. New York. 2004:1-21)Fourth Edition. Lange Medical Books/McGraw-Hill. New York. 2004:1-21)

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Access is anAccess is anImmunoassay SystemImmunoassay SystemAccess is anAccess is anImmunoassay SystemImmunoassay System

Cardiac Markers

Chemical (Clinical Chemistry)

10-12 10-9 10-6 10-3 10-0

mg/mL g/mLpg/mL ng/mL g/mL

Thyroid Hormones

Cancer Markers

Vitamins

Immunological (Immunoassay)

Therapeutic Drugs

Fertility Hormones

Allergy Markers

Infectious Disease

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Laboratory on immunoassayLaboratory on immunoassayThere are two major type measured :There are two major type measured :– Non Specific measuredNon Specific measured : :

hematology, hematology,

biochemistry, biochemistry,

urinary test urinary test – Specific measuredSpecific measured : : serologyserology testtest may be may be

developed a number of its own techniques, developed a number of its own techniques, particularly those based on the antigen-particularly those based on the antigen-antibody interaction (can be helpful for antibody interaction (can be helpful for diagnosis)diagnosis)

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Divide this talk into:Divide this talk into:

1.1.Basic conceptsBasic concepts

2.2.Direct/indirect methodsDirect/indirect methods

3.3.Competitive / sandwich methodsCompetitive / sandwich methods

4.4.Precipitation methodsPrecipitation methods

5.5.Agglutination methodsAgglutination methods

6.6.Amplification methodsAmplification methods

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1. Basic concepts 1. Basic concepts • Based on : Ab + Ag = Ab:Ag (are not visualized)Based on : Ab + Ag = Ab:Ag (are not visualized)• Ab:Ag (in proportions at or near equivalence, can be Ab:Ag (in proportions at or near equivalence, can be

seen by seen by colourcolour (enzyme, fluorecence, protein stain); (enzyme, fluorecence, protein stain); solid phasesolid phase (ery, bentonite, latex); (ery, bentonite, latex); X rayX ray ( (: detect : detect by machinary counter),by machinary counter), chemiluminescent chemiluminescent as marker.as marker.

• In vivo similar to in vitroIn vivo similar to in vitro– Van der WaalsVan der Waals EnergyEnergy : : specificityspecificity of Ag & Ab of Ag & Ab

– ColumbicColumbic EnergyEnergy : : electricityelectricity– Distance between antigen and antibody Distance between antigen and antibody

– pH, hydrogen, hydrophobic, electrostatic, etcpH, hydrogen, hydrophobic, electrostatic, etc

• The immunoassay using labeled reagents are: Elisa (using The immunoassay using labeled reagents are: Elisa (using enzyme), RIA (using radioisotopes) , IF (using florescent) , enzyme), RIA (using radioisotopes) , IF (using florescent) , chemiluminescent (using electron)chemiluminescent (using electron)

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2. Direct, indirect methods2. Direct, indirect methods

• Direct methodsDirect methods

• Indirect methods.Indirect methods.

Measured: Ag

Measured: Antibody, and its responses

Ab labeled

Ab labeled

Complement

Based on methods

direct indirect

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1st stept

3. Sandwich and competitive 3. Sandwich and competitive assayassay

• Sandwich Assay Sandwich Assay

• Competitive AssayCompetitive Assay

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Serum: present Ab Px

enzyme (color as marker)

enzyme (color as marker)

• Competitive AssayCompetitive Assay

• Sandwich AssaySandwich Assay

Serum: Px Ab

Ab: reagent labeled

PhotoPhoto

metermeter

Ad

sorb

ent

Consentration of test

Ad

sorb

ent Concentration

of test

1414Serum: Px Ab Ab: reagent labeled Color

Color

Immunoassay methodsImmunoassay methods

Divided three major methods:Divided three major methods:• PrecipitationPrecipitation methods methods : high : high

concentration, in solutionconcentration, in solution• AgglutinationAgglutination methods methods : Ag / : Ag /

Immunoglobulin on solid phased Immunoglobulin on solid phased • AmplificationAmplification methods methods : low : low

concentrationconcentration( methods ; ELISA, RIA, IMUNOFLURESENSI) / ( methods ; ELISA, RIA, IMUNOFLURESENSI) /

labeled methodlabeled method

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4. Precipitation4. Precipitation methods methods• We observe the antigen-antibody reactions depend on We observe the antigen-antibody reactions depend on

their ability to their ability to precipitateprecipitate when combined in when combined in proportions at or near proportions at or near equivalenceequivalence..

• By performing these reactions in By performing these reactions in agar gelsagar gels• Example:Example:

– Simple diffusion (Simple diffusion (qualitativequalitative))– Immunoelectrophoresis, single radial immunodiffusion Immunoelectrophoresis, single radial immunodiffusion

((quantitative = gram/L; mg/dlquantitative = gram/L; mg/dl))

• Developed to quantitative methods : Developed to quantitative methods : immunoelectrophoresis & radial immune diffusion immunoelectrophoresis & radial immune diffusion

• On the immunoelectrophoresis methods, antigens On the immunoelectrophoresis methods, antigens separated in agar gel’s by placing an electric charge separated in agar gel’s by placing an electric charge across it before being visualized by precipitation across it before being visualized by precipitation

• These techniques operate in the range of These techniques operate in the range of 20 ug/ml to 20 ug/ml to 2mg/m2mg/ml of antigen or antibodyl of antigen or antibody

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The classical illustration of the Ag-Ab reaction invitro is the precipitin reaction

The Ag-Ab reactions was their ability to precipitate when combined in proportions at or near equivalenceAntibody excess zone : Ag is insufficient to react with Ab

Free Ab in the supernatantEquivalence zone : Ag is sufficient to combine with Ab

neither free Ag and Ab can detected in the supernatantAntigen excess zone: Ag exceeds that required to bind the Ab

Free Ag in the supernatant

Precipitation reactions, the Precipitation reactions, the immuno-double –diffusion immuno-double –diffusion techniquetechnique

The plate are left for at least 24 hour, during which time the antigen and antibody diffuses out to form soluble Ag-Ab complexes, can be visualized by staining the precipitin arcs with a protein stain such as Coomassie blue.

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This technique mayThis technique may

be used to determinebe used to determine

the relationship the relationship

between Ag(blue)between Ag(blue)

and test Ab (yellow)and test Ab (yellow)

IMMUNOELECTROPHORESISIMMUNOELECTROPHORESIS

• ..

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1. Antigen are separated in an agar gel by placing an electric charge across it. The gel’s pH is chosen so that positively charged protein move to the negative electrode and negatively charged protein to the positive2. A trough is then cut between the wells and filled with the antibody, which is left to diffuse. 3. The antigen and antibody form precipitin arcs

IMMUNOELECTROPHORESISIMMUNOELECTROPHORESIS• ..

Glob glob -2-glob -1-glob

AlbuminPre Alb

ANTI SERUM

2020

IMMUNOELECTROPHORESISIMMUNOELECTROPHORESIS

Albumin , 1, 2, , GlobulinCirrhosis hepatic : low albumin , high Globulin

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SDMEry

5. Agglutination5. Agglutination methods methods• Haemagglutination : antibody may Haemagglutination : antibody may

detected and measured by detected and measured by haemahaemagglutination (Ery)gglutination (Ery)

• the ability of antibody to bind the the ability of antibody to bind the antigen on their surfaceantigen on their surface

• As solid phase are : erythrocyteAs solid phase are : erythrocyte, latex, benthonic., latex, benthonic.Example, measure used to widal test Example, measure used to widal test

, CRF, RF, HCG, blood grouping, CRF, RF, HCG, blood grouping• These techniques operate levels of These techniques operate levels of less less

than than 1 ug/ml1 ug/ml

SD

MLA

TE

K SD

MLA

TE

K

SD

MLA

TE

K SD

MLA

TE

K

SDMEry

SDMEry

SDMEry2222

AGGLUTINATIONSAGGLUTINATIONS• Agglutination Agglutination slideslide method method

• Agglutination Agglutination tubetube method method

• Applications: Widal test, bacterial IdentificationApplications: Widal test, bacterial Identification2323

Incomplete AntibodyIncomplete Antibody

• Incomplete antibody on surface red cellsIncomplete antibody on surface red cells

Assays that detect antibodies to red cell antigens.

Application: Coomb’s test (anti –immunoglobulin) : red cells hemolytic

Reagent

Sample agglutination

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6. Amplification6. Amplification methods methods ELISA (Enzyme Linked Immunosorbent ELISA (Enzyme Linked Immunosorbent Assay)Assay)

..

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Flow cytometryFlow cytometry

• ..

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This technique used to detecting Ag in solution or detect surface molecules on cellAb is immobilized onto the sensing surface and the test Ag solution is passed overthe surface. Light is focused onto the gold film which is part of the sensing surface andis reflected. The intensity of light reflected is reduced defend on Ag-Ab reaction

Pulse Editing (Patented)Pulse Editing (Patented)Cell flows through centre of apertureCell flows through centre of aperture

Good Pulse

Diluent stream

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Oscilloscope

Sensing Zone

Neutrophil A White cell passes through WBC A White cell passes through WBC apertureaperture

The Coulter principleThe Coulter principle

Oscilloscope2828

Conclusion Conclusion Clinical Laboratories Clinical Laboratories

– Pre analytic Pre analytic (yours area)(yours area)– Analytic Analytic – Post analyticPost analytic

Divided three major methods:Divided three major methods:• PrecipitationPrecipitation methods methods • AgglutinationAgglutination methods methods• AmplificationAmplification methods methods

Application the theory at yours cases Application the theory at yours cases

• Good luckGood luck

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