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CLINICAL CHEMISTRY CHAPTER 6 IMMUNOASSAYS 1 • Introduction – In the last chapter, we discussed a variety of analytical techniques – In this chapter we’ll add some new techniques … They all involve the use of antibody – antigen reactions – Antibody – Antigen reactions have the advantage of being very specific 2 Key Terms • • • • • • • • • • • • • Antibody Antigen Affinity Avidity Competitive Immunoassay Heterogeneous Immunoassay Homogeneous Immunoassay IEP IFE Nephelometry Non-competitive Immunoassay Postzone Prozone • • • • • • • • Tracer ( Tag ) Turbidimetry Haptene Crossreactivity Polyclonal Monoclonal Prozone Postzone 3 • Objectives – Discuss the basic principles of the following immunoassays • • • • • • Immunoelectrophoresis ( IEP ) Immunofixation Electrophoresis ( IFE ) Nephelometry and Turbidimetry Competitive Immunoassays ( RIA, EIA, FIA ) Non-competitive Immunoassays Fluorescence Polarization – Discuss different types of tags or labels used in immunoassays – Classify homogenous, heterogeneous, competitive and noncompetitive immunoassay techniques – Discuss methods of separation of free and bound tagged reagents 4 • Immunoelectrophoresis ( IEP ) – Electrophoresis of antigens is followed by the addition of various antibodies to a parallel trough along the separated proteins – The antibodies diffuse through the agar and form lines of precipitation with their respective antigens – The visible precipitant arcs can be compared to known standards to identify specific protein bands – Or to detect missing bands 5 Immunoelectrophoresis ( IEP ) Step 1. Patient plasma is placed in a well and undergoes electrophoresis. Precipitant lines against 3 proteins = Step 2 + Known anti-sera against one or more proteins is placed in a parallel trough after electrophoresis and diffuses through the agar. Visible lines of precipitation form if antibody antigen reaction occurs. 6 • IMMUNOFIXATION ELECTROPHORESIS ( IFE ) – Antibody is poured over a completed electrophoresis procedure ( performed on an agar surface ) to produce visible precipitation lines 7 • ROCKET ( LAURELL TECHNIQUE ) – Modification of IEP technique – Antigen ( proteins ) undergo electrophoresis in a supporting agarose gel with specific antibody previously mixed into the gel – As antigen moves thru the gel , antigen-antibody complexes form creating visible precipitation lines in the shape of long arches or “rockets” – The length of these “rockets” is proportional to the concentration of antigen 8 • Turbidimetry and Nephelometry – Light is obstructed by insoluble complexes ( usually antibody – antigen ) – Light is obstructed by these insoluble complexes – Turbidimetry measures transmitted light • Photo-detector is placed at 180 degrees from the light source – Nephelometry measures scattered light • Photo-detector is placed at 90 degrees from the light source 9 • Labeled Immunoassays – Antigen or antibody is labeled ( tagged ) with a substance that can be detected later on and allows for the detection of an antibody – antigen reaction – Different binding agents are allowed to attach to substances we want to measure. – The type of binding agent defines what type of assay it is • Antibody • Transport Protein • Hormone receptor Immunoassay Competitive Assay Receptor Assay – Types of tags • Radioactive isotopes • Enzymes • Fluorescent molecules 10 • Competitive Immunoassays – Competition between tagged and un-tagged antigen for limited antibody – Tagged antigen – Untagged antigen – Specific antibody Reagent Patient antigen we want to measure Reagent – Let the competition begin !!! • • • • Mix the three components together Allow the antigens to compete for the limited antibody Antibody will bind with tagged or un-tagged antigen ( it doesn’t care ) Separation Step : Antibody-Antigen complexes are separated from free antigen • Tagged antibody-antigen complex is measured 11 – The tagged antigen and antibody from the reagent kit are constant. – The only variable is the concentration of the patient antigen ( the thing we want to measure ) – A standard curve can be constructed with known antigen concentrations giving the following general results • High concentrations of patient antigen means that more of the antibody-antigen complexes are untagged • Low concentrations of patient antigen means that more of the antibody-antigen complexes are tagged • There is an inverse relationship between patient antigen concentration and tag activity after the separation process 12 Competitive Labeled Immunoassays ( RIA, FIA, EIA ) A competition between tagged antigens ( reagent ) and untagged antigens ( patient )for a limited amount of antibody ( reagent ) 13 Calculation of RIA / FIA / EIA The activity of the tag is measured twice : Before separation step = Total tag activity After separation step = Bound tag activity ( antibody – antigen complex ) Note that the separation process removes all unbound ( free ) tag from the testing % B = B / T x 100 The ratio of the Bound activity to the Total activity ( B / T ) decreases as the concentration of the patient’s ( untagged ) antigen increases. Using Standard solutions of known antigen concentrations, the % B is plotted against the concentrations of the Standards 14 Example of an Competitive Assay Standard Curve B/T 0 Concentration 15 ELISA ( Enzyme Linked Immunosorbent Assay ) - Antibody is adsorbed onto a solid surface Tagged and untagged antigens compete for limited antibody Separation is achieved by pouring off excess unbound ( free ) antigen Enzymatic activity is inversely proportional to patient antigen concentration 16 EMIT ( Enzyme Multiplied Immunoassay Technique ) Enzyme tag Homogenous technique - no separation step of antibody - bound and free antigen Steric hindrance : Antibody binding to the enzyme–tagged–antigen inhibits enzymatic activity Patient antigen concentration is inversely proportional to enzyme activity 17 Immunometric Technique - Immunometric techniques utilize a tagged antibody - Patient antigen concentration is proportional to measured tag activity 18 Fluorescence Polarization Immunoassay • Competitive Immunoassay • Homogenous assay – No separation step required • Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient ) compete for specific antibody in a curvet • The curvet is exposed to polarized fluorescent light • Large molecules ( tagged - antigen – antibody complexes ) emit polarized light, where as smaller molecules ( free tagged antigens ) do not • The amount of polarized light emitted is inversely proportional to the concentration of patient ( untagged ) antigen • Fluorescence Polarization is used by the ABBOTT TDX analyzer, commonly used for Therapeutic Drug Monitoring ( TDM ) 19 Example of Polarized and “Normal” Light - Normal light has wavelengths that occur in all planes - A polarizing filter blocks all planes except one, but the wavelength is unchanged 20 Fluorescence Polarization Tagged antigen Untagged antigen Antibody ( Low concentration of patient antigen ) Most of the limited antibody will bind with fluorescent tagged antigen. Large bound molecules will increase emission of polarized light. 21 Tagged antigen Fluorescence Polarization Untagged antigen Antibody ( High concentration of patient antigen ) Most of the limited antibody will bind with untagged patient antigen. Free unbound fluorescent antigen will decrease emission of polarized light. 22 TOP 10 23