1. MLAB 2401: Clinical Chemistry Keri Brophy-Martinez Immunoassays 1

2. General Considerations In an immunoassay, an antibody molecule recognizes and binds to an antigen. Binding is related to: – Concentration of each reactant – Specificity of antibody for antigen – Affinity & avidity for pair – Environmental conditions Temperature pH: best in the neutral range of 6.0-7.5 Time: need adequate time for complete binding of antibody & antigen

3. General Considerations Meet the antibody – Protein – IgG important in chemistry – Produced in response to foreign invaders Humoral Acquired 3

4. Antibodies Monoclonal – Arise from one cell line – Provide high specificity Polyclonal – Arise from many cell lines 4

5. General Considerations the antigen – Elicits an antibody response – Has multiple sites (epitopes)to bind antibodies 5

6. General Considerations An antigen-antibody reaction – Requires affinity and avidity – Determined by Law of Mass Action 6 Antigen + Antibody Antigen-Antibody Complex

7. Affinity 7 Attraction between the antibody and antigen  As antigens and antibodies come together, a chemical bond forms  Stability depends on the “fit” of the connection Most antibodies have a high affinity for their antigens Property of the antigen

8. Avidity Overall strength of antigen/antibody bond formed Property of the antibody The greater the avidity- the less likely to have cross- reactivity 8

9. Law of Mass Action Governs the reversibility of the antigen-antibody reaction. Reversible reaction, visible reaction occurs when the rate of binding exceeds the rate of dissociation. As affinity and avidity increases, strengthens reaction.

10. Zone of Equivalence: Antigen-Antibody Complexes Become visible when antibody/antigen concentrations are in the Zone of Equivalence Zone of Equivalence – Optimal ratio of concentration of antibody to concentration of antigen that results in maximal precipitation – 2-3 Antibodies: 1 Antigen 10

11. Prozone – Antibody excess – No precipitation 11

12. Postzone Antigen excess – No precipitation 12

13. General Principles of Immunoassays Immunoassay Labels Competitive Immunoassays Noncompetitive Sandwich Immunoassays 13

14. 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 Types of tags  Radioactive isotopes (RIA)  Enzymes (EMIT, ELISA)  Fluorescent molecules  Luminescent labels 14

15. 15 Fluorescence Polarization Immunoassay Competitive Immunoassay Homogenous assay – No separation step required Fluorescent – tagged antigen ( reagent ) and untagged antigen ( patient ) compete for specific antibody (reagent) in a cuvet The cuvet 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 )

16. Fluorescence Polarization Immunoassay 16

17. 17 Lumininescence/Chemiluminescence The process of exciting molecules by chemical means and measuring the light emitted as the molecules return to their ground/unexcited state. Competitive & heterogeneous Applications include quantitation of drugs, steroid and peptide hormones, etc.

18. Labeled Immunoassay Heterogeneous or homogeneous Heterogeneous assays called separation assays – Require multiple steps – Careful washing of surface to remove unbound reagents and samples. Homogeneous assays do NOT require a separation step. – Mix reagents and patient sample. – Measure the labeled product.

19. Competitive Immunoassays Labeled known and patient unknown are added to reaction and “compete” for the target. – For example, looking for an antibody. – Add labeled reagent antibody of known specificity, patient sample and known antigen. – Patient antibody competes with reagent antibody for the target antigen. – Concentration is inversely proportional to results. High concentrations of patient antigen means that more of the antibody-antigen complexes are unlabeled Low concentrations of patient antigen means that more of the antibody-antigen complexes are labeled 19

20. Competitive Labeled Immunoassays 20

21. Non-Competitive Labeled Immunoassays “Sandwich” A labeled reagent antibody is used to detect an antigen Direct relationship between the concentration of the antigen and the bound antibody. 21

22. References Bishop, M., Fody, E., & Schoeff, l. (2010). Clinical Chemistry: Techniques, principles, Correlations. Baltimore: Wolters Kluwer Lippincott Williams & Wilkins. Sunheimer, R., & Graves, L. (2010). Clinical Laboratory Chemistry. Upper Saddle River: Pearson. 22