1. 1 CLINICAL CHEMISTRY CHAPTER 6 IMMUNOASSAYS

2. 2 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

3. 3 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

4. 4 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

5. 5 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

6. 6 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. Step 1. Patient plasma is placed in a well and undergoes electrophoresis. + = Precipitant lines against 3 proteins Immunoelectrophoresis ( IEP )

7. 7 IMMUNOFIXATION ELECTROPHORESIS ( IFE ) –Antibody is poured over a completed electrophoresis procedure ( performed on an agar surface ) to produce visible precipitation lines

8. 8 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

9. 9 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

10. 10 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 Immunoassay Transport Protein Competitive Assay Hormone receptor Receptor Assay –Types of tags Radioactive isotopes Enzymes Fluorescent molecules

11. 11 Competitive Immunoassays –Competition between tagged and un-tagged antigen for limited antibody –Tagged antigenReagent –Untagged antigenPatient antigen we want to measure –Specific antibodyReagent –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

12. 12 –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

13. 13 Competitive Labeled Immunoassays ( RIA, FIA, EIA ) A competition between tagged antigens ( reagent ) and untagged antigens ( patient )for a limited amount of antibody ( reagent )

14. 14 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

15. 15 Example of an Competitive Assay Standard Curve B / T Concentration 0

16. 16 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

17. 17 EMIT ( Enzyme Multiplied Immunoassay Technique ) 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 Enzyme tag

18. 18 Immunometric Technique - Immunometric techniques utilize a tagged antibody - Patient antigen concentration is proportional to measured tag activity

19. 19 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 )

20. 20 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

21. 21 Tagged antigen Untagged antigen Most of the limited antibody will bind with fluorescent tagged antigen. Large bound molecules will increase emission of polarized light. Antibody ( Low concentration of patient antigen ) Fluorescence Polarization

22. 22 ( 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. Fluorescence Polarization Tagged antigen Untagged antigen Antibody

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