BIOMAN 2010

1. 1 BIOMAN 2010 Immunoassays Barbara Bielska Northampton Community College Tannersville, PA

2. 2 Antibody-mediated immunity Antibody-mediated immunity is part of acquired or adaptive immunity Antigen Antibody-mediated immunity involves B cells, and T cells (lymphocytes).

3. 3 What is antigen Any substance that can elicit an immune response is said to be immunogenic and is called an immunogen. There is a clear operational distinction between an immunogen and an antigen. An antigen is defined as any substance that can bind to a specific antibody. All antigens therefore have the potential to elicit specific antibodies, but some need to be attached to an immunogen in order to do so. This means that although all immunogens are antigens, not all antigens are immunogenic.

4. 4 Immunogen: adjuvant

5. 5 What Is Antibody

6. 6 What Is Antibody

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8. 8 Immunoglobulins Immunoglobulins – Antibodies IgD surface of many cells (mostly B) IgM surface of B cells IgG blood; the most abundant Ab IgA digestive and respiratory systems IgE skin, digestive and respiratory tract; responsible for allergic reactions

9. 9 IgG: immunoglobulin G

10. 10 Antibodies

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12. 12 Vaccine is made from an antigen isolated or produced from the disease-causing microorganism. The vaccine is injected into the blood stream. The B cells in the blood stream respond to the antigen by producing antibodies. The antibodies bind to the antigen to "neutralize“ or inactivate it. In addition, memory cells are produced and remain ready to mount a quick protective immune response against subsequent infection with the same disease causing agent.

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14. 14 Antibodies: Applications Diagnostic tests Research tool Drug delivery system

15. 15 Immunoassays Diagnostic and research tools Allow to detect very minute amounts of the analytes Highly sensitive and specific Multibillion dollar business Examples: HIV test, hepatitis test, tests for numerous infectious diseases and other disorders (thyroid dysfunction), test for H. pylori, numerous QC tests.

16. 16 Polyclonal and Monoclonal Antibodies Polyclonal Ab recognize the same antigen but different epitopes (antigenic determinants) heterogeneous made by the B cells Monoclonal Ab recognize the same antigen and the same epitope, homogenous clones made by mice hybridomas (tissue culture)

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18. 18 Monoclonal Ab 

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24. 24 Types of immunoassays ELISA: enzyme linked immuno-sorbent assay Radio-immunoassays Immunoassays with color or fluorescent tags (colloidal gold, polymers, crystals) Basic formats Direct assay Indirect assay Sandwich assay Competitive assay Multiplex assay Rapid assay: lateral flow chromatography

25. 25 Microtiter plate

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31. 31 ELISA: Enzyme Linked Immunoassay

32. 32 ELISA steps

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34. 34 Competitive immunoassay

35. 35 Competitive immunoassay

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37. 37 Multiplex ELISA protein array

38. 38 Rapid test

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40. 40 Test device component categories Porous materialsSample pads Conjugate pads Membranes Absorbent pads ReagentsCapture antibodies and/or antigens (test line and control line) Conjugate ligand (antibody or antigen) Detector particle (e.g., colloidal gold) Blocking agents, detergents, surfactants, stabilizers, buffers, etc. Housing and lamination materials Back laminate (for holding porous components together) Top laminate (optional, to act as a "splash guard" or prevent evaporation and back-migration of detector reagent) Device housing (optional—if used, the housing typically comprises two, snap-fit plastic pieces that envelop the reagent-loaded porous media assembly and facilitate sample addition) 

41. 41 Rapid immunoassay

42. 42 Rapid immunoassay

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44. 44 Rapid lateral flow test

45. 45 Pregnancy test

46. 46 Specifications for a pregnancy test Following are the development guidelines given to the research and development manager responsible for designing, developing, and scaling up to manufacturing a hypothetical lateral-flow diagnostic test device for detecting human chorionic gonadotropin (hCG) in urine. The hCG hormone is measured in milli-international units per milliliter (mIU/ml). More than 99% of specimens containing 25 mlU/ml will produce a visible signal at the test line. More than 99% of specimens containing 5 mlU/mL will not produce a visible signal at the test line. The test will have a control line that will always produce a visible signal if the test is performed correctly and if all of the test components are functional. If the sample is positive (contains 25 mIU/ml hCG), the test must produce a visible signal at the test line within 3 minutes of sample addition. The test result must be stable (not change) for at least 30 minutes after the sample is added. The test device will be contained in a plastic housing and designed to be compatible with "in-stream" sampling protocols. The packaged (unused) product must be stable for at least 18 months at ambient conditions (15°–30°C). Total incremental manufacturing cost per test (excluding licensing and royalties) can not exceed $0.25 US.

47. 47 Rapid test for drugs of abuse

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