BASIC PRINCIPLEBASIC PRINCIPLE

The binding of The binding of antibodies (Ab) and antibodies (Ab) and their complementary their complementary Antigens (Ag)Antigens (Ag) forms forms the basis of all the basis of all immunochemical immunochemical techniques.techniques.

Kricka LJ, 2001

BASIC PRINCIPLEBASIC PRINCIPLE

The exquisite specificity and theThe exquisite specificity and the high affinity of high affinity of antibodies for specific antigensantibodies for specific antigens, , coupled with coupled with thethe unique ability of antibodies to cross link unique ability of antibodies to cross link antigensantigens, , allow the identification and allow the identification and quantitation of specific substances by a variety quantitation of specific substances by a variety of methods.of methods.

[pathmicro.med.sc.edu/mayer/ab-ag-rx.htm]

ANTIBODIES AND ANTIGENSANTIBODIES AND ANTIGENS

Antibodies (Ab) are immunoglobulins that can Antibodies (Ab) are immunoglobulins that can bind specificallybind specifically to a wide array of natural and to a wide array of natural and synthetic antigens.synthetic antigens.

Antigens (Ag) are materials capable of Antigens (Ag) are materials capable of reacting reacting with an antibodywith an antibody, , without necessarily being without necessarily being capable of inducing antibody formation.capable of inducing antibody formation.

Kricka LJ, 2001

HETEROGENEITYHETEROGENEITY

Immunization with a single antigenic Immunization with a single antigenic determinant determinant produces a variety of Abproduces a variety of Ab with with different antibody-combining sites and with a different antibody-combining sites and with a range of Ab affinity.range of Ab affinity.

This is termed the This is termed the heterogeneity of the heterogeneity of the immune response.immune response.

Feldkamp CS. 2003

SPECIFICITYSPECIFICITY

For every reagent Ab, the specificity of its For every reagent Ab, the specificity of its immunochemical reactivity is theimmunochemical reactivity is the single single most important factor in the success or most important factor in the success or failure of any immunological technique used failure of any immunological technique used in the clinical laboratory.in the clinical laboratory.

Specificity refers toSpecificity refers to the ability of the Ab to the ability of the Ab to restrict its reaction to a defined group of restrict its reaction to a defined group of molecules.molecules.

Feldkamp CS. 2003

CROSS REACTIVITYCROSS REACTIVITY

Cross reactivity of Ag and Ab isCross reactivity of Ag and Ab is a by product of a by product of the heterogeneity of the immun responsethe heterogeneity of the immun response..

Thus cross reactivity may result from Thus cross reactivity may result from similar or similar or identical antigenic determinantidentical antigenic determinant in different Ag. in different Ag.

This reaction between Ab and the undesired This reaction between Ab and the undesired Ag Ag is termed is termed Cross reactivity.Cross reactivity.

Feldkamp CS. 2003

Rheumatic FeverComplication arising from infection with Streptococcus pyogenesantibodies to bacterial proteins (antigen or streptolysin) cross-react with myocardial andmuscle proteins.

ANTIBODY AFFINITYANTIBODY AFFINITY

The strength of the binding of a single The strength of the binding of a single antigenic determinant to an antibodyantigenic determinant to an antibody is a is a function of the closeness of fit and is function of the closeness of fit and is called antibody affinity. called antibody affinity.

Antibody affinity is Antibody affinity is an expression of the an expression of the attraction between molecules of Ab and attraction between molecules of Ab and AgAg. .

Feldkamp CS. 2003

ANTIBODY AFFINITYANTIBODY AFFINITY

Reagent Ab generally fall into two categories, Reagent Ab generally fall into two categories, those of those of high affinityhigh affinity and those of and those of low affinitylow affinity..

Polyclonal Ab in the reagent maybe a mixture Polyclonal Ab in the reagent maybe a mixture of both, but reagents in whichof both, but reagents in which high affinity are high affinity are predominant should be usedpredominant should be used..

This results inThis results in a strong union with the Aga strong union with the Ag that that isis not readily reversiblenot readily reversible and that willand that will not be not be influenced greatly by alteration of the reaction influenced greatly by alteration of the reaction conditions.conditions.

Feldkamp CS. 2003

ANTIBODY AVIDITYANTIBODY AVIDITY

Avidity isAvidity is a measure of stability of Ab-Ag a measure of stability of Ab-Ag complexcomplex and is partially dependent on and is partially dependent on the affinity of each Ab for its the affinity of each Ab for its complementary antigenic determinant.complementary antigenic determinant.

Feldkamp CS. 2003

ANTIBODY AS REAGENTSANTIBODY AS REAGENTS

Immunochemical reactions form the basis of a Immunochemical reactions form the basis of a diverse range of sensitive and specific clinical diverse range of sensitive and specific clinical assays. In a typical immunochemical analysis, assays. In a typical immunochemical analysis, an an antibody is used as a reagent to detect an antibody is used as a reagent to detect an antigen of interest.antigen of interest.

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ANTIGEN AS ANALYTESANTIGEN AS ANALYTES

Numerous naturally occurring molecules or Ag Numerous naturally occurring molecules or Ag that are proteins, glycoproteins, or lipoproteins that are proteins, glycoproteins, or lipoproteins can be detected and measured easily in can be detected and measured easily in biological fluids biological fluids if specific reagent Ab are if specific reagent Ab are available.available.

In addition, many small molecules such as In addition, many small molecules such as drugs and hormones can be measured.drugs and hormones can be measured.

Feldkamp CS. 2003

Classes of ImmunoassayClasses of Immunoassay

Photon countingPhoton countingChemiluminescent Chemiluminescent compoundscompounds

Chemiluminescent Chemiluminescent immunoassayimmunoassay

Photon countingPhoton countingFluorophoresFluorophoresFluorescent immunoassayFluorescent immunoassay

Spectrophotometry, Spectrophotometry, fluorometry, photon fluorometry, photon countingcounting

EnzymesEnzymesEnzyme immunoassayEnzyme immunoassay

Photon countingPhoton countingRadioisotopes (Radioisotopes (125125I, I, 33H)H)RadioimmunoassayRadioimmunoassay

Naked eye, pattern analyzer, Naked eye, pattern analyzer, spectrophotometry, particle spectrophotometry, particle countingcounting

Blood cells, artificial Blood cells, artificial particlesparticlesParticle immunoassayParticle immunoassay

Naked eye, turbidity, Naked eye, turbidity, nephelometrynephelometryNot requiredNot requiredPrecipitation immunoassayPrecipitation immunoassay

Signal DetectionSignal DetectionLABELSLABELS

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Precipitin and Nephelometric Precipitin and Nephelometric ImmunoassayImmunoassay

Precipitin ReactionPrecipitin Reaction PrinciplesPrinciplesThe precipitate that forms when large complexes of antigen and The precipitate that forms when large complexes of antigen and

antibody combine to form an insoluble lattice.antibody combine to form an insoluble lattice.

LimitationLimitation Low limit of sensitivity ( 0.1 Low limit of sensitivity ( 0.1 –– 0.5 mg/dl) 0.5 mg/dl) Condition of temperature, pH and ionic strength of the medium, and Condition of temperature, pH and ionic strength of the medium, and

the antibody characteristics of avidity and affinity were affect the the antibody characteristics of avidity and affinity were affect the formation of the immune precipitateformation of the immune precipitate

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Qualitative Precipitant Assay Methods Single immunodiffusion (Williams, 1970)

Double immunodiffusion (Garvey, 1977)

Double immunodiffusion in two dimension (Williams, 1970)

Electroimmunodiffusion reaction (Ritzmann, 1975)

Immunoelectrophoresis (Rose, 1973)

Semiquantitative Precipitant Assay Methods Single radial immunodiffusion (Mancini, 1965; Fahey,

1965)

Single dimension electroimmunodiffusion (Axelsen, 1975) “rocket” electrophoresis

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Immunonephelometry

PrinciplesPrinciples Small aggregates that can scatter light quickly Small aggregates that can scatter light quickly

form, when an antibody and an antigen combine form, when an antibody and an antigen combine in solution, and giving a turbid appearance to in solution, and giving a turbid appearance to the solutionthe solution

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Particle Immunoassay Particle Immunoassay (Agglutination assay)(Agglutination assay)

Clumping and sedimentation of an antigen after reaction Clumping and sedimentation of an antigen after reaction with antwith antiibodybody

PrinciplesPrinciplesPassive particles aglutination (indirect aglutination)Passive particles aglutination (indirect aglutination)Active particles aglutination (direct aglutination)Active particles aglutination (direct aglutination)Aglutination inhibitionAglutination inhibition

LimitationLimitationOnly semiquantitativeOnly semiquantitative

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Particle Immunoassay Particle Immunoassay (Agglutination assay)(Agglutination assay)

HemagglutinationHemagglutination Gelatin Particle AgglutinationGelatin Particle Agglutination Latex AgglutinationLatex Agglutination Latex Turbidimetric AssayLatex Turbidimetric Assay Particle-Counting ImmunoassayParticle-Counting Immunoassay

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Aglutination AssayAglutination Assay

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Passive particles aglutination Passive particles aglutination (indirect aglutination)(indirect aglutination)

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hCG agglutination inhibitionhCG agglutination inhibition

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hCG agglutination inhibitionhCG agglutination inhibition

RadioimmunoassayRadioimmunoassay

PrinciplesPrinciplesCompetitive binding reaction to antibodies Competitive binding reaction to antibodies between labeled antigens and non labeled between labeled antigens and non labeled antigensantigens

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AdvantagesAdvantages1.1. Precision and high sensitivityPrecision and high sensitivity2.2. Ease of isotope conjugationEase of isotope conjugation3.3. Stability against interference from the assay among Stability against interference from the assay among

otherother

DisadvantagesDisadvantages1.1. Short shelf life of the reagentsShort shelf life of the reagents2.2. the need against hazardous radioactivitythe need against hazardous radioactivity

RadioimmunoassayRadioimmunoassay

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RadioimmunoassayRadioimmunoassay

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Sandwich RIASandwich RIA

(1)cpm=counts per minute

Enzyme ImmunoassayEnzyme ImmunoassaySensitive technique that uses an enzyme-Sensitive technique that uses an enzyme-antibody-antigen combination absorbed onto antibody-antigen combination absorbed onto the sides of a test wallthe sides of a test wall

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Advantages Advantages 1.1. Sensitive assay can be developed by the amplification effect of Sensitive assay can be developed by the amplification effect of

enzymesenzymes2.2. Reagents are relatively cheap and can have a long shelf lifeReagents are relatively cheap and can have a long shelf life3.3. A wide variety of assay configurations can be developedA wide variety of assay configurations can be developed4.4. Equipment can be inexpensive and is widely availableEquipment can be inexpensive and is widely available

DisadvantagesDisadvantages1.1. Measurement of enzyme activity can be more complex than Measurement of enzyme activity can be more complex than

measurement of the activity of some types of radioisotopesmeasurement of the activity of some types of radioisotopes2.2. Enzyme activity can be affected by plasma constituensEnzyme activity can be affected by plasma constituens3.3. NNot as sensitive as radioimmunoassayot as sensitive as radioimmunoassay

Enzyme ImmunoassayEnzyme Immunoassay

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Heterogeneous EnzymeHeterogeneous Enzyme ImmunoassayImmunoassay (need (need separation of B/F fraction)separation of B/F fraction)

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Competitive assaay

Non Competitive assaay

Heterogeneous Enzyme Heterogeneous Enzyme ImmunoassayImmunoassay

Colorimetric Enzyme ImmunoassayColorimetric Enzyme ImmunoassayEnzyme reaction is performed by using chromogenic Enzyme reaction is performed by using chromogenic substrates to develop a color by prime catalitic reactionsubstrates to develop a color by prime catalitic reaction

Fluorescent Enzyme ImmunoassayFluorescent Enzyme ImmunoassayIdentical to other EIAs except that they use fluorescent Identical to other EIAs except that they use fluorescent substrate. This assay generate a signal intensity that is substrate. This assay generate a signal intensity that is at least one order of magnitude greater than colometric at least one order of magnitude greater than colometric EIAsEIAs

Chemiluminescent Enzyme ImmunoassayChemiluminescent Enzyme ImmunoassayUse chemiluminescent substrate Use chemiluminescent substrate (HRP, AP, etc) (HRP, AP, etc) that that react with various enzymes employed as labels. react with various enzymes employed as labels.

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Enzyme-Linked Immunosorbent Assay (ELISA) - Multi-Lingual Captions.mp4

Do not require washing stepsDo not require washing steps Less sensitive than heterogeneous EIAsLess sensitive than heterogeneous EIAs Classified as competitive and non Classified as competitive and non

competitive binding assaycompetitive binding assay

Homogeneous Enzyme Homogeneous Enzyme ImmunoassayImmunoassay

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Homogeneous Enzyme Homogeneous Enzyme ImmunoassayImmunoassay

Enzyme-Multiplied Immunoassay TechniqueEnzyme-Multiplied Immunoassay Technique

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Substrate-Labeled Fluorescent ImmunoassaySubstrate-Labeled Fluorescent Immunoassay

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Enzyme Inhibitory Homogeneous ImmunoassayEnzyme Inhibitory Homogeneous Immunoassay

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Cloned Enzyme Donor ImmunoassayCloned Enzyme Donor Immunoassay

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Fluorescent ImmunoassayFluorescent Immunoassay

Fluorescent compounds as immunochemical labels to Fluorescent compounds as immunochemical labels to detect antigens in tissue sectiondetect antigens in tissue section

ClasificationClasificationHeterogeneous Fluorescent ImmunoassayHeterogeneous Fluorescent Immunoassay

- Fluoroimmunometric- Fluoroimmunometric- Radial Partition Immunofluorometric Assay- Radial Partition Immunofluorometric Assay- Time-Resolved Fluoroimmunoassay- Time-Resolved Fluoroimmunoassay

Homogeneous Fluorescent ImmunoassayHomogeneous Fluorescent Immunoassay- Fluorescence Polarization Assay- Fluorescence Polarization Assay- Fluorescence Excitation Transfer Immunoassay- Fluorescence Excitation Transfer Immunoassay- Fluorescent Protection Immunoassay- Fluorescent Protection Immunoassay

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Fluorescent ImmunoassayFluorescent Immunoassay

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Fluorescent Polarization ImmunoassayFluorescent Polarization Immunoassay

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Chemiluminescent Chemiluminescent ImmunoassayImmunoassay

Use chemiluminescence molecules as Use chemiluminescence molecules as labelslabels

Labels of chemiluminescent compounds Labels of chemiluminescent compounds produce light electrochemically on the produce light electrochemically on the surface of electrodes and can be applied surface of electrodes and can be applied to homogeneous assay formatsto homogeneous assay formats

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Chemiluminescent ImmunoassayChemiluminescent Immunoassay

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Rapid and Simple Test Devices for Rapid and Simple Test Devices for Point of Care TestingPoint of Care Testing

Immunofiltration Assay DevicesImmunofiltration Assay Devices Immunochromatographic DevicesImmunochromatographic Devices

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Rapid and Simple Test Devices for Rapid and Simple Test Devices for Point of Care TestingPoint of Care Testing

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Rapid and Simple Test Devices for Rapid and Simple Test Devices for Point of Care TestingPoint of Care Testing

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Rapid and Simple Test Devices for Rapid and Simple Test Devices for Point of Care TestingPoint of Care Testing

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ReferenceReference1.1. Ashihara, Y.; Kasahara, Y.; Nakamura, RM; Immunoassay and Ashihara, Y.; Kasahara, Y.; Nakamura, RM; Immunoassay and

Immunochemistry; Clinical Diagnostic and Management by Immunochemistry; Clinical Diagnostic and Management by Laboratory Methods; 20Laboratory Methods; 20thth ed; 821-849; WB. Saunders Company; ed; 821-849; WB. Saunders Company; 2001.2001.

2.2. Carpenter, AB; Antibody-Based Methods; Clinical Laboratory Carpenter, AB; Antibody-Based Methods; Clinical Laboratory Immunology; 6-48; ASM Press; 2002.Immunology; 6-48; ASM Press; 2002.

3.3. Stepp, AC; Laboratory Procedures; 95-97; 238-293; The CU Stepp, AC; Laboratory Procedures; 95-97; 238-293; The CU Mosby Company; 1989.Mosby Company; 1989.

4.4. Bassion, S; Immunological Reactions; Clinical Chemistry; 153-Bassion, S; Immunological Reactions; Clinical Chemistry; 153-164; WB Saunders Company; 1998.164; WB Saunders Company; 1998.

5.5. Hurtubise, PE et.al; Immunochemical Techniques; Clinical Hurtubise, PE et.al; Immunochemical Techniques; Clinical Chemistry; 165-206; WB Saunders Company; 1998.Chemistry; 165-206; WB Saunders Company; 1998.