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Determination of Melamine by ELISA Technique

15 March 2007. 2. . Outline. Principle of ELISAELISA kitApplication and qualitycontrol for melamine determination. 15 March 2007. 3. . Principle of ELISA . What is ELISA ?How does ELISA work ?. 15 March 2007. 4. . What is ELISA ? . EIA:Enzyme Immuno AssayELISA :Enzyme Linked Immuno Sorben

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Determination of Melamine by ELISA Technique

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    1. 15 March 2007 1 Determination of Melamine by ELISA Technique ????? ??????????????????

    2. 15 March 2007 2 Outline Principle of ELISA ELISA kit Application and qualitycontrol for melamine determination

    3. 15 March 2007 3 Principle of ELISA What is ELISA ? How does ELISA work ?

    4. 15 March 2007 4 What is ELISA ? EIA: Enzyme Immuno Assay ELISA : Enzyme Linked Immuno Sorbent Assay

    5. 15 March 2007 5 Principle of ELISA Specificity of antigen- antibody (Ag-Ab) interaction The Ab /Ag are immobilized on solid support The Ag / Ab are interacted with their binding sites Enzyme-conjugate = Tracer Analyte conc. correspond signal intensity

    6. 15 March 2007 6 How does ELISA work ? ELISA Techniques: Non-Competitive ELISA Direct Competitive ELISA Indirect Competitive ELISA

    7. 15 March 2007 7 ELISA Techniques

    8. 15 March 2007 8 ELISA Techniques Direct Competitive ELISA

    9. 15 March 2007 9 Application of ELISA for Melamine determination ELISA kits ELISA assay Quality control Handing and storage

    10. 15 March 2007 10 ELISA Kits Microtitre plate (12 x 8 strips) Standards Melamine-HRP conjugate Wash solution concentrate Substrate solution (TMB) Stop solution

    11. 15 March 2007 11 ELISA ASSAY Sample preparation ELISA assay procedure Interpretation of results

    12. 15 March 2007 12 Sample preparation Whole milk Sample 1 ml centrifuge at 10,000-13,000 x g, 5 min Dilute a portion of the middle layer 1:3 in 10% MeOH/20mM PBS

    13. 15 March 2007 13 Sample preparation Milk powder Sample 8.6 g 60 ml 10% MeOH/20mM PBS Centrifuge at 10,000-13,000 x g, 5 min Dilute a portion of the middle layer 1:5 in 10% MeOH/20mM PBS

    14. 15 March 2007 14 Sample preparation Yogurt & Ice scream Sample 5 g 5 ml 10% MeOH/20mM PBS mix by vortex 1 min centrifuge at 3000 rpm, 10 min Dilute supernatant 1:4 in 10% MeOH/20mM PBS

    15. 15 March 2007 15 Sample preparation Milk chocolate Sample 5 g 10 ml distilled water Warm at 46-60oC in water bath Mix by vortex 1 min centrifuge at 3,000 rpm, 10 min Dilute a portion of the middle layer 1:62.5 in 10% MeOH/20mM PBS

    16. 15 March 2007 16 Sample preparation White chocolate Sample 5 g 10 ml distilled water Warm at 46-60oC in water bath Mix by vortex 1 min centrifuge at 3,000 rpm, 10 min Dilute a portion of the middle layer 1:40 in 10% MeOH/20mM PBS

    17. 15 March 2007 17 ELISA ASSAY Antigen-Antibody interaction Sequential adding Incubation Rinsing steps Manual & Automate rinsing Form colour Substrate Stop reaction & Read absorbance

    18. 15 March 2007 18 ELISA Assay Calibration curve construction

    19. 15 March 2007 19 ELISA Assay Quantitative determination

    20. 15 March 2007 20 Sensitivity Dose response curve

    21. 15 March 2007 21 ELISA Assay

    22. 15 March 2007 22 ELISA Assay

    23. 15 March 2007 23 Quality control of ELISA ELISA Kit Performance of kit Interference & Contamination Handing and storage Instruments Personal skills

    24. 15 March 2007 24 ELISA Kit Sensitivity Range of standard curve Accuracy Spiked sample, recovery Precision Duplication sample, wells Contamination Method Blank, reagent

    25. 15 March 2007 25 ??????????????????????? Melamine ??? ELISA ???????????????????? Melamime ????????????????????????????? ??. Matrix ??????????????????? ????????????????????????????????????????????????? ???? Chocolate ???????????????????????? ???????????????????????????????????????? ??????????????????????????????????????????????????????????????

    26. 15 March 2007 26 ??????????????????????? Melamine ??? ELISA ?????????????????????? (??????? 311) ?.?. 2551 ?????? ????????????????????? ?????? ??????????? “ ??????????????????????????? ????????????????????( ????????????? ????????? ??????????) ????????????????????????????????????????????????????????????????? ???????????????????? ?????? ???????????”

    27. 15 March 2007 27 ??????????????????????? Melamine ??? ELISA ????????????????????????????? ????? ?????? ????????????????????????????????????? ???????????????????? (??????? 2) “ -??????? 1.0 ???????????????????? ????????????????????????? ??????????????????????????????????????????? ? - ??????? 2.5 ???????????????????? ???????????????????????????????????????????????? ”

    28. 15 March 2007 28 References Hennion MC. and Barcelo D. (1998). Analytica Chimica Acta. 362 p. 3-34 Oubina A., Gascon J. and Barcelo D. (1997). Analytica Chimica Acta. 347 p. 121-130 Abraxis Melamine Plate Kit Cat.#50005B Instruction Booklet. Abraxis LLC U.S.A

    29. 15 March 2007 29 Q & A

    30. 15 March 2007 30

    31. 15 March 2007 31 ??????? ???????? ?????????????????? ?????????????????????? ?????????????????? ?????????????????????????????? ?????????????????????? ??????????????? ??????????????? ???????????????????????? ?????????? ???????????????????????????????????????? ?????????????????????????????? ??????????????????????

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