Immunoassays

1. Immunoassays Katarzyna, Reyna, Becca, Ismael

2. History • The first immunoassay was described by Berson and Yalow in 1959. Their work resulted in their receipt of the Nobel Prize in Medicine in 1977. Since this introduction, immunoassays have evolved considerably. • The first homogenous immunoassay (no separation step required) was the Enzyme Multiplied Immunoassay Technique (EMIT), which enabled adaptation of this assay onto automated chemistry platforms. • Today, immunoassays play a prominent role in the analysis of many clinical laboratory analytes such as proteins, hormones, drugs, and nucleic acids. • Although unskilled technicians can run immunoassays, the development of these tests requires knowledge in many areas of immunology and protein chemistry. • Syva immunoassays which were commercially produced first were developed to be used in the methadone maintenance treatment programs. When the US military explored methods to curb the drug addiction problem in troops from Vietman, further development in drug testing emerged and expanded. • Major developments in homogenous immunoassays have been due to the development of illicit drug testing and therapeutic drug testing programs.

3. Immunoassay Tests • Immunoassays are chemical tests used to detect or quantify a specific substance, the analyte, in a blood or body fluid sample, using an immunological reaction. Immunoassays are highly sensitive and specific. Their high specificity results from the use of antibodies and purified antigens as reagents.  • An antibody is a protein (immunoglobulin) produced by B-lymphocytes (immune cells) in response to stimulation by an antigen. Immunoassays measure the formation of antibody-antigen complexes and detect them via an indicator reaction.  • Immunoassays may be qualitative (positive or negative) or quantitative (amount measured).  • There are several different methods used in immunoassay tests.

4. Immunoassay Tests Continued • Immunoprecipitation. The simplest immunoassay method measures the quantity of precipitate, which forms after the reagent antibody (precipitin) has incubated with the sample and reacted with its respective antigen to form an insoluble aggregate • Particle immunoassays This allows rapid and sensitive detection of antibodies that are markers of such diseases, as infectious mononucleosis and rheumatoid arthritis. • Immunonephelometry.. The antigen concentration can be determined within minutes of the reaction.

5. Purpose • The purpose of an immunoassay is to measure (or, in a qualitative assay, to detect) an analyte. Immunoassay is the method of choice for measuring analytes normally present at very low concentrations that cannot be determined accurately by other less expensive tests • Common uses include measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury. • They are also used to detect antigens associated with organisms that are difficult to culture, such as hepatitis B virus and Chlamydia trichomatis. 

6. How They Are Collected • Blood samples are collected by vein puncture with a needle. • Urine samples are acceptable for drug assays

7. Homogenous & Heterogeneous Methods • Homogeneous immunoassays do not require separation of unbound complexes from the bound complexes, and thus are faster and easier to perform then heterogeneous immunoassays. • Heterogeneous immunoassays require the separation of unbound complexes, often utilizing a solid phase reagent such as a magnetic particle or plastic bead. 

8. Normal Results • Immunoassays which are qualitative are reported as positive or negative. Quantitative immunoassays are reported in mass units • Although immunoassays are both highly sensitive and specific, false positive and negative results may occur. • False-negative results may be caused by improper sample storage or treatment, reagent deterioration, or improper washing technique. • False-positive results are sometimes seen in persons who have certain antibodies, especially to mouse immunoglobulins (immune cells) that may be used in the test. • Immunoassay is anin vitroprocedure, and is therefore not associated with complications

9. Advantages of Immunoassays • High Sensitivity – Low detection limit • High Specificity - Detect specific compound • Safe and simple • Fast Tests (between 5 minutes and 1 hour) • Cost effective • Field-portable tests • Tests can yield quantitative or qualitative data

10. Disadvantages of Immunoassays: • They have limitations • Negative results don’t always rule out the presence of a drug • May not be sensitive to be certain compounds • Some chemists are reluctant to use immunoassays due to its biological basis and their unfamiliarity with it

11. Common Immunoassay Types: • Enzyme-linked immunosorbent assay (ELISA) • Western blot • Immunofluorescent antibody assay (IFA) • Electrochemoluminescence • Magnetic immunoassays

12. Advantage of Each Type • ELISA- Widely-used, high-throughput, inexpensive • Western Blot- Highly sensitive and specific • IFA- simple to perform • Electrochemoluminescence- very high sensitivity • Magnetic immunoassays- produce quantitative results

13. Disadvantage of Each Type: • ELISA- Requires temperature control, more qualitative results, time-and-labor intensive • Western Blot- Has a complicated lab analysis • IFA- less sensitive than ELISA, also requires use of a microscope • Electrochemoluminescence- More applicable to commercial labs • Magnetic immunoassays- requires a magnetic reader that limits its field application

14. Case #1 • Immunoassay is used in examining hair for drug investigations. - Hair analysis was performed in order to determine and establish inter and intraday variabilities.

15. Case #1 • Being examined: - 2 suicide cases • 1 allege date rape • Benzodiazepines user • Results: All hair specimens screened positive for benzodiazepines using micro-plate enzyme immunoassay. • More Results: 2 cases (including date rape) were negative for FN and 7-AFN (aminoflunitrazepam). 2 post mortem samples were positive for FN and its metabolite.

16. Case #2 • Post mortem false positive reactivity • Tool Utilized: Enzyme-multiplied urine phencyclidine (PCP) immunoassay • Why? A single-agent fatal tramadol overdose (Tramadol level of 14.0 mg/L while therapeutic range is 0.1 to 0.3 mg/L) • Results: The urine yielded positive for PCP at 88 mAU/min.

17. Case #2 • BUT… • The family was like, “Nawwww”. • False positive • Conclusion: Immunoassay of tramadol and its metabolites in aggregate is responsible for the PCP immunoassay interference and false-positive result.

18. Works Cited • Wild, David E. Immunoassay Handbook, third edition. Oxford: Elsevier Ltd, 2005. online • "What Is the History of Immunoassays?" Apoptosis, Cell Death, Caspases, Necrosis, Assay Development, Blocking Buffer, ELISA Buffer. N.p., n.d. Web. 02 Apr. 2013. • "Surge" Immunoassay Tests. N.p., n.d. Web. 02 Apr. 2013. • http://www.seedtechnology.net/genetic_wrk/2006%20IAWG%20-%20Basics%20of%20Immunoassay%20Presentation.pdf • http://www.bvgh.org/Biopharmaceutical-Solutions/Global-Health-Primer/Targets/cid/ViewDetails/ItemID/13.aspx • http://www.princeton.edu/~ota/disk2/1988/8830/883006.PDF • https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=2&ved=0CDkQFjAB&url=http%3A%2F%2Fwww.cap.org%2Fapps%2Fdocs%2Fnewspath%2F1112%2Fbenzodiazepines.doc&ei=-5FbUd79DqSCyAHGqYHoAg&usg=AFQjCNHlIsalpYnszYIY2mudvn2k7dP-FQ&sig2=wWCzEOSPPk_bKJypEmanqg&bvm=bv.44697112,d.aWM&cad=rja

19. Review Questions • What do immunoassay tests detect? • What are some advantages/disadvantages of immunoassays? • What is the difference between homogenous and heterogeneous immunoassays? • What are normal results of immunoassays? • What are the common immunoassay types?