Immunoassay T tiTestingDr Mir Majid MossalaeieDr. Mir Majid Mossalaeie

(DCLS)(DCLS)Parseh Medical

LaboratoryTehran-Iran (2012)

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Tehran-Iran (2012)

Introduction

• Antibody/Antigen reaction provides theAntibody/Antigen reaction provides the means of generating a measurable result.

• “Immuno” refers to an immune response that causes the body to generate

tib diantibodies.

• “Assay” refers to a test• “Assay” refers to a test.

• An immunoassay is a test that uses

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• An immunoassay is a test that uses immunocomplexing when antibodies and antigens are brought together.

Immunoassay Definitionsy

• An antibody is a protein produced in theAn antibody is a protein produced in the body to a foreign substance.

A ti i th b t th t th b d• An antigen is the substance that the body is trying to eliminate by mounting an immune response.p

• An analyte is anything measured by a laboratory testlaboratory test.

• Immunoassays may measure either the

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Immunoassays may measure either the antigen or antibody.

Summary of Immunoassay Techniq esTechniques

• Immunoassays use one or more select• Immunoassays use one or more select antibodies to detect analytes of interest.interest.

• Analyte may be naturally present.

• Analyte may be those that the body producesproduces.

• Analyte may be those that does not normally occur in the body

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normally occur in the body.

Structure of Antibodies

• Antibodies are a type• Antibodies are a type of protein called immunoglobinsimmunoglobins.

• Most common protein is immunoglobin G.

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Preparation of Monoclonal and Polyclonal AntibodiesPolyclonal Antibodies• Polyclonal antiserum is generated in• Polyclonal antiserum is generated in

animals (sheep, rabbits or goats) with the introduction of antigens into thethe introduction of antigens into the animals bloodstream.

• The antiserum (serum from blood containing the desired antibodies)containing the desired antibodies) contains a mixture of antibodies, each of which may bind to different antigen

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binding sites (epitopes).

Preparation of Monoclonal and Polyclonal AntibodiesPolyclonal Antibodies

• Antiserum contains a mixture of antibodies.

• This mixture of antibodies are called ployclonal antibodies.

• An antigen that has multiple sites for g pantibody binding is called a mutivalent antigen.

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Preparation of Monoclonal and Polyclonal AntibodiesPolyclonal Antibodies

• Monoclonal antibodies are highly specific• Monoclonal antibodies are highly specific for a single epitope on a multivalent antigen.

• They are produced from a single cell lineThey are produced from a single cell line using hybridoma technology and mouse myeloma cell lines.y

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Production of Monoclonal AntibodiesAntibodies

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Categories of Immunoassay Tests• Competitive• Competitive

• Noncompetitive

• Homogeneous

• Heterogeneous

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Categories of Immunoassay TestsTests

• Labels may be applied to either theLabels may be applied to either the antibody…

• or the antigen.

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Competitive and Noncompetitive AssaysAssays

• In a competitive format, unlabeled analyte (usually the antigen) in the test sample is(usually the antigen) in the test sample is measured by its ability to compete with the labeled antigen in the immunoassay.

• In a competitive immunoassay, less label measured in the assay means more of the unlabeled (test sample) antigen is presentunlabeled (test sample) antigen is present.

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Competitive and Noncompetitive AAssays

• There are two• There are two versions of the competitive pformat:

• One step format• One step format

• Two step format

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Competitive and Noncompetitive AssaysAssays

• Noncompetitive assay formats give the• Noncompetitive assay formats give the highest level of sensitivity and specificity.p y

• They are normally used to measure critical analytes such as cardiac andcritical analytes such as cardiac and hepatitis markers.

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Competitive and Noncompetitive AssaysAssays

• Noncompetitive assay formats can• Noncompetitive assay formats can use either one step or two step methodsmethods.

• In the two step assay format, there p y ,are wash steps in which the sandwich binding complex is g pisolated and washed to remove excess unbound labeled reagent.

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g

Competitive and Noncompetitive AssaysAssays

• In noncompetitive• In noncompetitive assays, the measurement of themeasurement of the labeled analyte (usually the(usually the antibody) is directly proportional to the p pamount of antigen present in the

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sample.

Heterogeneous and Homogeneous Immunoassays Methods

• Immunoassays that require• Immunoassays that require separation of the bound Ab-Ag* complex are referred to as beingcomplex are referred to as being heterogeneous immunoassays.

• Those that do not require separation f d t hare referred to as homogeneous

immunoassays.

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gHomogeneous Immunoassays MethodsMethods

• Homogeneous methods have generally• Homogeneous methods have generally been applied to the measurement of small analytes such as abused and therapeutic y pdrugs.

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Immunoassay Detection TechniquesTechniques

• RIARIA

• EIAEIA

• ELISA

• FPIA

• CLIA / ECLIA

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Radioimmunoassayy

• Radioimmunoassay (RIA) techniques• Radioimmunoassay (RIA) techniques were developed in the 1960s and use radioactive isotopes as a labelradioactive isotopes as a label

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Enzyme Immunoassayy y

• In enzyme immunoassays (EIA)• In enzyme immunoassays (EIA), enzyme labels are used instead of radioactive labelsradioactive labels.

• Typical enzyme labels include yp yalkaline phosphatase, horseradish peroxidase and β-galatosidase.p β g

• EIA tests typically use a change in l i i f li h h

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color, emmission of light or other signal.

Enzyme Immunoassayy y

• Enzyme Linked Immunosorbent• Enzyme Linked Immunosorbent Assay (ELISA) is an application of solid phase heterogeneous sandwichsolid phase heterogeneous sandwich immunoassay that combines enzyme antibody label reagent with aenzyme-antibody label reagent with a solid phase bound antibody.

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Fluorescence Polarization ImmunoassayImmunoassay

• Fluorescence Poloarization Immunoassy• Fluorescence Poloarization Immunoassy (FPIA) is a type of homogeneous competitive fluoresence immunoassay.p y

• With competitive binding, antigen from the specimen and antigen fluorescein (AgF)specimen and antigen-fluorescein (AgF) labeled reagent compete for binding sites on the antibody.on the antibody.

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Fluorescence Polarization IImmunoassay

• FPIA is used to provide accurate and• FPIA is used to provide accurate and sensitive measurements of small toxicological analytes such astoxicological analytes such as therapeutic drugs and drugs of abuseabuse.

• The FPIA reagent includes the gantibody reagent, a tracer, and a pretreatment detergent.

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p g

Fluorescence Polarization IImmunoassay

• FPIA uses three concepts to measure• FPIA uses three concepts to measure specific analytes in a homogeneous format:format:

• Fluorescence

• Rotation of molecules in solution

• Polarized light

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Fluorescence

• Fluorescein is a fluorescence label that• Fluorescein is a fluorescence label that absorbs light at 490 nm and releases this energy at 520 nm.gy

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Rotation of Molecules in S l tiSolution• Larger molecules rotate more slowly• Larger molecules rotate more slowly

in solution that smaller molecules.

• Because of this, we can distinguish between the smaller antigen-gfluorescein (AgF) label from antibody bound antigen-fluorescein (Ab-AgF).g ( g )

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Polarized Lightg

• When polarized light is absorbed by• When polarized light is absorbed by AgF, the molecule rotates quickly before the light is emitted asbefore the light is emitted as fluorescence.

• When the larger-sized Ab-AgF complex absorbs the polarized light,complex absorbs the polarized light, it rotates more slowly and the light is emitted in the same plane and the

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detector can measure it.

Polarized Lightg

• FPIA results in an• FPIA results in an inverse response curve such that lower levels of patient analyte result in a higher signal.

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Electrochemiluminescence• This novel

electrochemiluminescence detectionelectrochemiluminescence detection technology can be used for all immunoassay principles.immunoassay principles.

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Principle of ECLIA/ ruthenium (+2)-tris(bipyridyl) [Ru (bpy)3]2+ a Tripropylaminu (TPA)620620 nm

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• The ability of molecules such as ruthenium to yemit light or to luminesce has been studied for more than 20 years. If the light is produced by a chemical reaction, it's called chemiluminescence. If the chemiluminescent reaction is initiated by an electrical stimulation of the molecules, it's then called ,electrochemiluminescence or ECL.

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In ECL technology, this electrochemiluminescent quality is integrated with conventional antigen (Ag) and antibody (Ab) reactions. These reactions take place on the surface of a streptavidin-coated paramagnetic microparticle during two incubations. pa a ag et c c opa t c e du g t o cubat o sThe incubation time is dependent upon the assay.

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consider the thyroid-stimulating hormone (TSH) sandwich test. In the first step, a patient sample i bi d ith t t i iis combined with a reagent containing biotinylated TSH antibody and a second ruthenium conjugated TSH antibody in an assayruthenium conjugated TSH antibody in an assay cup. During a nine-minute incubation, antibodies capture the TSH present in the sample.

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In step two, streptavidin coated paramagnetic microparticles are added. During a second nine-minute incubation, the biotinylated TSH antibody attaches to the streptavidin-coated surface of the microparticles. Adding microparticles during the

d i b ti d th ti i hi hsecond incubation reduces the time in which

nonspecific binding can occur.

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Next, the sample solution is drawn into the ECL measuring cell along with a buffer solution containing tripropylaminecell along with a buffer solution containing tripropylamine (TPA). A magnet located under the electrode captures the microparticles in a thin, even layer on the electrode's surface. Liquid flow rinses away all unbound reagent and sample. This is the bound/free separation process.

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The magnet is removed and voltage is applied to the electrode. Two oxidation reactions are simultaneously initiated, resulting in the oxidation of the ruthenium complex and the TPA in the solution. The two compounds react to produce an excited state of the ruthenium conjugate. Spontaneous decay results in the emission of a co jugate Spo ta eous decay esu ts t e e ss o o aphoton of light at 620 nm.

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This process regenerates the ruthenium complex, which can perform multiple cycles during thewhich can perform multiple cycles during the measurement. The result is amplification of the signal, which adds to the technology's sensitivity.

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Multiple readings are taken by the photomultiplier tube (PMT) during a 0.4 second interval. Measurements are taken at peak ECL intensity. The readings are integrated totaken at peak ECL intensity. The readings are integrated to produce a single value, which is compared to the calibration curve to calculate the sample result. The paramagnetic microparticles are released from theparamagnetic microparticles are released from the electrode surface and thoroughly washed away. Regeneration of the electrode surface is accomplished by controlled variation of the electrode potential. The ECL measuring cell is then ready for another measurement.

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Principle of ECLIAp

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Principle of ECLIA IIp

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Factors Impacting ImmunoassaysImmunoassays• Accuracy and Precision• Accuracy and Precision

• Calibration and Controls

• Assay Interferences

• Human Anti-Mouse Antibodies (HAMA)(HAMA)

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Accuracy and Precisiony

• Accuracy means the assay is• Accuracy means the assay is determining the correct concentrationconcentration.

• Precision is the reproducibility of an p yassay.

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Accuracy and Precisiony

• Sensitivity and specificity are• Sensitivity and specificity are subsets of accuracy and precision.

• An assay that has the ability to produce accurate and precise results and does not produce false positivesand does not produce false positives is considered specific.

• An assay that has the ability to produce accurate and precise results

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and does not produce false negatives is said to be sensitive.

Accuracy and Precisiony

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Calibrators and Controls

• Calibrators are solutions with known• Calibrators are solutions with known concentration values that establish the relationship between the signal response p g pprodused during the assay and the analyte concentration.

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Calibrators and Controls

• It is important that the user follow the• It is important that the user follow the manufacturer’s treatment criteria for the calibrators to ensure that thethe calibrators to ensure that the calibration is accurate.

Th f t l h t h• The manufacturer also has to chose the correct matrix for the calibrators has a signal response that mimicshas a signal response that mimics the signal from patient samples

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Calibrators and Controls

• Controls are samples that contain• Controls are samples that contain known concentrations of analyte and are used to monitor the accuracy andare used to monitor the accuracy and precision of the assay and analyzer.

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Calibrators and Controls

• If the control’s concentration is• If the control s concentration is within 2 SDs of the QC average, then the assay is said to be in control andthe assay is said to be in control and that the results collected are valid

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Assay Interferencesy

• One step assays may be prone to• One step assays may be prone to interferences that affect both sensitivity and specificity.sensitivity and specificity.

• In general sequential assays are lik l t i ld t ltmore likely to yield accurate results

by elimination the adverse contribution of binding proteinscontribution of binding proteins, endogenous interfering substances and general matrix effects due to the

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and general matrix effects due to the extra wash step.

Advantages of Non Radio Isotopic Immunoassays• Automation (Rapid less staff low space )Automation (Rapid, less staff, low space,..)• Stable Calibration• Low Carry OverLow Carry Over• Repeatability (Low Imprecision) CV< 1%• Accuracy (High Reproducibility) Inaccuracy y ( g p y) y

= +/- 0.1%• Wide Linearity Range (Rare Hook Effect)• No dangerous waste (No Radioactivity)• High Sensitivity (Low detection Limit)

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• Long Shelflife

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