BIOSENSORS BLOCKS TO PROGRESS: 1. TECHNICAL PROBLEMS Low concentrations of analytes. 2. BUSINESS CONSTRAINTS Multi-analyte panels

Transcription

1 BIOSENSORS BLOCKS TO PROGRESS: 1. TECHNICAL PROBLEMS Low concentrations of analytes 2. BUSINESS CONSTRAINTS Multi-analyte panels 3. REGULATORY ISSUES Must incorporate internal controls

2 Analyte conc n moles / litre Glucose Digoxin Theophylline Oxytocin Aldesterone Cortisol Insulin hcg IgM Rh Influenza (elderly) Influenza (Paediatric) Hepatitis a - foetoprotein Metabolites Therapeutic drugs Small hormones Protein hormones Antibodies Cellular antigens Viral antigens Tumour antigens Analyte molecules per cm 3 Water = 3.3x10 22

3 ANTIBODY BASED SENSORS AND IMMUNOASSAY LEADING ON TO OPTICAL WAVEGUIDE SENSORS

4 ANTIBODIES Infectious Agent ANTIGEN ANTIBODIES ANTIBODIES

5 THE ANTIBODY MOLECULE (IgG)

6 Antibody (IgG) Antigen Binding site specific for only one species of antigen -s-s- -s-s- 5 nm

7 Disulphide link Cysteine S S

8 Antibody (IgG) Antigen Binding site specific for only one species of antigen -s-s- -s-s-

9 ANTIBODY - SELECTIVITY Corresponding antigen, e.g. on the surface of a measles virus _ + A different antigen, e.g. on the surface of a rubella virus _ + Another different antigen, e.g. on the surface of an influenza virus + _ + _ + _ + _ Antibody s binding site, e.g. that recognises a measles antigen Key and lock analogy

10 Small analyte Antibody-Analyte Binding + + Large analyte + Steric hinderance

11 + Ab + An AbAn Dissociation Constant, K d K Ab An Fractional K p d d T T [Ab][An] [AbAn] [Ab] [An] K Ab d saturation, T - [AbAn] [AbAn] [An] [An] [AbAn] [AbAn] p [An] [AbAn] Ab T

12 ANTIBODY-ANALYTE BINDING p [Free Analyte] K [Free Analyte] d K d Dissociation Constant Fractional saturation Can be as low as M K d [FREE ANALYTE]

13 ENZYME: ANALYTE CONC n v. REACTION RATE K m ~ M ANTIBODY: ANALYTE BINDING CURVE K d M

14 Analyte conc n moles / litre Glucose Digoxin Theophylline Oxytocin Aldesterone Cortisol Insulin hcg IgM Rh Influenza (elderly) Influenza (Paediatric) Hepatitis a - foetoprotein Metabolites Therapeutic drugs Small hormones Protein hormones Antibodies Cellular antigens Viral antigens Tumour antigens Analyte molecules per cm 3 Water = 3.3x10 22

15 GenSci 2000L Fermentor GeneScience Pharmaceuticals Co, Shanghai

16 ANTIBODY PRODUCTION

17 DNA and PROTEINS DNA Code for Pro Code for Asp Code for Try Code for Gly Code for Arg Code for Phe Code for Leu Code for Glu Code for Val Code for Asp Code for Ala Cellular Protein synthesis Pro Asp Try Protein Gly Arg Phe Leu Glu Val Asp Ala

18 8 10 variants GENE DNA

19 ANTIBODIES Infectious Agent ANTIGEN ANTIBODIES ANTIBODIES Several of the 10 8 variants will fortuitously have a key and lock fit for one or more of the infectious agent outer proteins

20 MONOCLONAL ANTIBODIES We can now produce antibodies commercially that can recognise and combine with our molecules of interest:

21 GenSci 2000L Fermentor GeneScience Pharmaceuticals Co, Shanghai

22 Dear Michael Flanagan, Sigma Life Science is pleased to announce that it now has over 25,000 monoclonal and polyclonal antibodies available! JUST RELEASED: Over 1100 New Goat Polyclonal Antibodies 100 New Stem Cell Specific Monoclonal Antibodies Over 350 New IHC Validated Mouse Polyclonal Antibodies 3000 New Rabbit Polyclonal Antibodies

23 Key immunological terms for immunoassay Antigen A substance that elicits an antibody response. Generally, only large molecules can elicit an immune response in the body. This prevents the body reacting against essential small molecules - e.g. metabolites formed on ingesting and digesting food Epitope An alternative term for an antigenic determinant. These are particular chemical groups on a molecule that are antigenic, i.e. that elicit a specific immune response.

24 Key immunological terms for immunoassay Hapten A compound, usually of low molecular weight, that is not itself immunogenic but, when bound to a carrier protein or cell, becomes immunogenic and induces antibodies, which can bind the hapten either alone or in the presence of a carrier. An important aspect of immunology, for assay and sensor developers, as many potential analytes are too small to be antigenic in their own right.

25 IMMUNOASSAY FORMATS SANDWICH COMPETITIVE

26 Microtitre Plate

27

28 Abbott ImmunoAnalyser

29

30 FLUORESCENT SANDWICH IMMUNOASSAY FLUORESCEN T SAN DWICH IMMUN OASSAY CAPTURE AN TIBOD Y 10 nm SUBSTRATE (e.g. glass or plastic)

31 FLUORESCENT SANDWICH IMMUNOASSAY Sample, FLUORESCEN e.g. blood, T SAN DWICH thought IMMUN to contain OASSAY the analyte, added AN ALYTE CAPTURE AN TIBOD Y SUBSTRATE (e.g. glass or plastic)

32 FLUORESCENT SANDWICH IMMUNOASSAY Analyte binds to the capture antibody FLUORESCEN T SAN DWICH IMMUN OASSAY AN ALYTE CAPTURE AN TIBOD Y SUBSTRATE (e.g. glass or plastic)

33 FLUORESCENT SANDWICH IMMUNOASSAY Fluorescently labelled antibody added FLUORESCEN T SAN DWICH IMMUN OASSAY LABELLED AN TIBOD Y FLUOROPHORE AN ALYTE CAPTURE AN TIBOD Y SUBSTRATE (e.g. glass or plastic)

34 FLUORESCENT SANDWICH IMMUNOASSAY Fluorescently labelled antibody binds to analyte FLUORESCENT SANDWICH IMMUNOASSAY UNBOUND LABELLED ANTIBODY FLUOROPHORE BOUND LABELLED ANTIBODY ANALYTE CAPTURE ANTIBODY SUBSTRATE (e.g. glass or plastic)

35 FLUORESCENT SANDWICH IMMUNOASSAY Excess FLUORESCEN fluorescently T SAN DWICH labelled IMMUN antibody OASSAY washed off BOUN D LABELLED AN TIBODY AN ALYTE CAPTURE AN TIBOD Y SUBSTRATE (e.g. glass or plastic)

36 FLUORESCENT SANDWICH IMMUNOASSAY Fluorescent FLUORESCEN label T SAN excited DWICH IMMUN - florescent OASSAY emission measured proportional to analyte present Exciting light BOUN D LABELLED AN TIBODY AN ALYTE CAPTURE AN TIBOD Y SUBSTRATE (e.g. glass or plastic) Fluorescence

37 Fluorescence response FLUORESCENT SANDWICH IMMUNOASSAY Unknown sample response read off a standard curve Response of a sample of unknown analyte concentration Responses measured at known analyte concentrations Concentration of analyte in sample Analyte concentration

38 IMMUNOASSAY FORMATS SANDWICH COMPETITIVE

39 COMPETITIVE IMMUNOASSAY Capture Antibody

40 Analyte Labelled analyte COMPETITIVE IMMUNOASSAY Fluorophore Capture Antibody

41 Analyte COMPETITIVE IMMUNOASSAY Labelled analyte Capture Antibody

42 Capture Antibody COMPETITIVE IMMUNOASSAY

43 COMPETITIVE IMMUNOASSAY Exciting light Capture Antibody Fluorescence

44 Fluorescence response FLUORESCENT COMPETITIVE IMMUNOASSAY Unknown sample response read off a standard curve Response of a sample of unknown analyte concentration Concentration of analyte in sample Responses measured at known analyte concentrations Analyte concentration