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Immunochemical Methods and Biosensors for pollutants determination (General principles and application)

Immunochemical Methods and Biosensors for pollutants determination (General principles and application). Danila Moscone Department of Chemical Science and Technology University "Tor Vergata" Rome, Italy danila.moscone@uniroma2.it.

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Immunochemical Methods and Biosensors for pollutants determination (General principles and application)

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  1. Immunochemical Methods and Biosensors for pollutants determination(General principles and application) Danila Moscone Department of Chemical Science and Technology University "Tor Vergata"Rome, Italy danila.moscone@uniroma2.it

  2. Immunoassays (IAs) are techniques based on the formation of a thermodynamically stable antigen – antibody complex. These methods already play an important role, especially in clinical chemistry, being used for the fast and safe detection of proteins, hormones, and pharmaceutical agents.

  3. Immunoassays become important when: • Fast measurement and evaluation are required  • Highest possible detection strength is required  • Large numbers of samples are to be expected  • Only complex and expensive analytical methods are otherwise available. • The greatest potential for the use of immunoassays in environmental analytical chemistry is in SCREENING i.e., for the selection of contaminated and uncontaminated samples for further validation analysis.

  4. Terminology Antigen: Original - Substance able to generate antibody. More general - Substance that can be recognized by antibody or T cells Immunogen: Substance able to generate an immune response Hapten: Non-immunogenic substance. Usually low molecular weight. Induces antibody formation when coupled to a larger “carrier” molecule. Can bind antibody

  5. Hapten - DNP Immunize with Antibodies formed DNP None BSA Anti-BSA DNP-BSA Anti-DNP Anti-BSA Anti-DNP-BSA Protein Carrier - Bovine Serum Albumin

  6. Antibody structure Antigen binding sites Light Chain Heavy Chain ANTIBODY (immunoglobulin) Abiological molecule (protein) that specifically recognizes a foreign substance (antigen) as a means of natural defence .

  7. Antibodies: production and labelling • PRODUCTION • Animals have a large number of antibody producing cells, all producing a different antibody. When an animal (rabbit) is injected with antigen, proliferation of the corresponding antibody producing cell is promoted. Blood from the rabbit contains antibodies, originating from different cells with slight variations. LABELLING Radio-isotopes, Enzymes, Fluorescent, probes (Quantum dots), Chemi-luminescent probes, Metal tags

  8. Antibodies Polyclonal Monoclonal Individual B lymphocyte hybridoma is cloned and cultured. Secreted antibodies are collected from culture media Antibodies that are collected from sera of exposed animal recognize multiple antigenic sites of injected biochemical. recognize ONE antigenic site of injected biochemical

  9. Antigen-antibody Interactions

  10. Features of the Antigen-Antibody Interaction • Reversibility • Non-covalent Interactions • Affinity • Measure of the strength of the binding • Ease of association or dissociation • Avidity • Increase in affinity due to multivalent binding • The summation of multiple affinities

  11. Non-covalent binding

  12. Affinity and Avidity

  13. Antibody-based assays

  14. Enzyme-Linked Immunosorbent Assay ELISA

  15. immobilisation surface Specific Ab antigen- enzyme conjugate E Ag E E Direct competitive immunoassay (I) Coating Incubation E E E S Enzym. reaction Product measurement P Affinity reaction

  16. E E E E E E E E Direct competitive immunoassay (II) I.No analyte - high detection signal II. Analyte present - detection signal reduced

  17. ENZYMATIC REACTION SECONDARY LABELLED Ab FREE Ag and SPECIFIC Ab ADDITION ANTIGEN COATING BLOCKING S P Indirect competitive ELISA format The enzymatic product concentration is inversely proportional to the analyte (standard or sample) amount

  18. Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y enzyme produces colour ELISA SANDWICH FORMAT Y Y Y Antibody 2nd antibody with enzyme Y Y Y Antibody/Antigen

  19. Functional concentration range Signal (enzyme activity) Antigen concentration signal/concentration curve

  20. ELISA PLATES ELISA PLATE WASHER SPECTROPHOTOMETER ADAPTED FOR ELISA PLATES

  21. Lateral Flow Strips (Dipsticks) Apply sample solution, upon application of sample biochemicals dissolve Positive: no antigen Immobilised Antibody area Control area Negative: antigen present • Immunochromatography (Lateral Flow) • Biochemical components are separated across an absorbent membrane into discrete distinct regions.

  22. Predator support Sample pad QUALITATIVE TEST Test line analyte Ab-colloidal gold

  23. Sample pad Ab-colloidal gold Analyte QUALITATIVE TEST:Analyte absent in the sample Test line

  24. Ab-colloidal gold Analyte If the analyte is ABSENT in thesample the line will be colored Test line Sample pad

  25. Ab-colloidal gold Analyte Analyte PRESENT in the sample Test line Sample pad

  26. Sample pad Ab-colloidal gold Analyte Test line

  27. Ab-colloidal gold Analyte Test line Sample pad

  28. If the analyte is present in the samplethe line will be not colored Test line Ab-colloidal gold Analyte Sample pad

  29. We can use these immunochemical elements to assemble a special kind of biosensors called Immunosensors

  30. Signal transducer Recorder Biological component biosensor Analyte

  31. What do they have in common? Nose Eye Small molecules / olfactory membrane / nerve cells / brain Visible light / rods and cones / nerve cells / brain Biosensor Analyte / bioreceptor / transducer / processor

  32. Staphylococcus aureus gram-positive, non spore-forming bacterium able to synthetise: Enterotoxins: A, B, C, D, E (thermostable); • Coagulase; • Thermonuclease. 100-200 ng of enterotoxins are sufficient to cause toxinfection in immuno-compromised subjects.

  33. DEVELOPED TEST: Conventional ELISAProteina A Conventional ELISAS.aureus ELISA/AMPLI S.aureus ELIMCS.aureus ELIMES.aureus

  34. p-NITROPHENOL p-NPP AP Secondary antibody-AP Specific antibody (MAb o PAb) ProteinA/S.aureus Human IgG Spectrophotometric ELISA

  35. ELISAProtein A (a – d) + d y = b x 1 + c MAb LOD LOD 0.6 ng/mL 0.07 ng/mL IgG 10 mg/mL Sensitivity Sensitivity MAb 1:10000 7.6 ng/mL 0.6 ng/mL Ab2-AP 1:1000 PAb y = <x0> + 3s IgG 10 mg/mL PAb 1:10000 Ab2-AP 1:1000 Sensitivity was calculated as tha amount of protein A needed to produce a 25% increase in the signal

  36. LOD LOD 2 106 cell/mL 2 104 cell/mL Sensitivity Sensitivity 9 106 cell/mL 2 105 cell/mL ELISAS.aureus MAb PAb IgG 10 mg/mL IgG 10 mg/mL MAb 1:10000 PAb 1:10000 Ab2-AP 1:1000 Ab2-AP 1:1000 No cross-reactivity

  37. AMPLI Q NADPH Alkaline phosphatase Pi INT Acetaldehyde NADH Alcohol deydrogenase Diaphorase FORMAZAN NAD+ Ethanol DAKO, Handbook for AmpliQ, 1997

  38. ELISAS.aureus AMPLIQ MAb PAb LOD LOD 6 104 cell/mL 7 102 cell/mL IgG 10 mg/mL IgG 10 mg/mL Sensitivity Sensitivity MAb 1:10000 PAb 1:10000 2 105 cell/mL 6 103 cell/mL Ab2-AP 1:1000 Ab2-AP 1:1000

  39. Magnetic Beads Magnetic particles are particles constituted from a dispersion of magnetic material (Fe2O3 and Fe3O4) and then covered with a thin shell of polymer which contains the magnetic material and also serves to define a surface area for the absorption or coupling of a large variety of other molecules. Good results in immunological field Ø1-5 µm Measurements on real samples

  40. ELIMC (Enzyme Linked ImmunoMagnetic Colorimetry) AP All reactions were carried out in eppendorf tubes No intermediate washings p-NITROPHENOL Microtitre ELISA p-NPP

  41. AP ELIME (Enzyme Linked ImmunoMagnetic Electrochemistry) a-naphthol + NaH PO 2 3 a-naphthyl phosphate DPV Potential range 0-600 mV Scan speed 100 mV/s Pulse width 50 ms Modulation time 60 ms Interval time 0.16 s • Selectivity Ag-Ab; • Sensibility of electrochemical detection; • Possibility of concentrating magnetic particles on the electrode surface. +

  42. Addition of Enzymatic substrate for Electrochemical measurement Magnetic tube

  43. MAb LOD 1 104 cells/mL IgG0.5 mg/mL Sensitivity Mab 1:50000 MAb LOD 2 105 cells/mL Ab2-AP 1:300 1 103 cells/mL IgG 1.2 mg/mL Sensitivity MAb 1:1000 2 104 cells/mL Ab2-AP 1:100 ELIMCS.aureus ELIMES.aureus

  44. Analysis Time LOD Sensitivity Mab ELISA prot. A 7.6 ng/mL 0.6 ng/mL 22 h Pab ELISA prot A 0.07 ng/mL 0.6 ng/mL 22 h Mab ELISA S.a 6 6 9 10 cell/mL 2 10 cell/mL 22 h Pab ELISA S.a 5 2 10 cell/mL 4 22 h 2 10 cell/mL Mab ELISA AmpliQ 5 4 2 10 cell/mL 22 h 6 10 cell/mL Pab ELISA AmpliQ 3 2 6 10 cell/mL 22 h 7 10 cell/mL Mab ELIMC 5 4 2 10 cell/mL 4 h 1 10 cell/mL 3 4 Mab ELIME 1 10 2 10 cell/mL 4 h cell/mL

  45. Air samples Two air samples from hospital rooms Sampling carried out by a SAS air-sampler. Flow rate 35 litri/min, for 30 minuts, collin 30 ml of buffer

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