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GENE EXPRESSION STUDY ON PROTEIN LEVEL

This study explores the use of protein expression methods, such as ELISA and Western blot, for the detection of HIV infection. The clinical application of these methods is demonstrated through a fictive case scenario. Other molecular genetic techniques, such as RT-PCR, are also discussed.

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GENE EXPRESSION STUDY ON PROTEIN LEVEL

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  1. GENE EXPRESSION STUDY ON PROTEIN LEVEL EPh 2017

  2. studies of protein expression are based on specific antigen-antibody interactions • the majority of currently used laboratory diagnostic methods are still based on the detection of proteins

  3. Methods 1. Immunohistochemistry 2. ELISA 3. Western blot

  4. antigen Polyclonal antibodies recognize different epitopes of an antigen (e.g. a protein) Monoclonal antibodies are specific for a single epitope Hybridoma cells

  5. Immunohistochemistry direct indirect

  6. ELISA (Enzyme linked immunosorbent assay) 1st antibody Coating washes blocking washes 2nd antibody Reading washes

  7. ELISA types

  8. Western blot 1. Proteins are separated by SDS polyacrylamide gel electrophoresis (SDS PAGE) 2. Proteins (bands) are transfered to a membrane (blot) 3. Immunodetection

  9. SDS polyacrylamide gel electrophoresis (SDS PAGE)

  10. Coomassie staining

  11. In the next few slides we show you an example for the clinical application of the protein expression methods

  12.    Fictive case: Patient (35) with two transient partners. Is she HIV positive? If yes, did she also infect her partners? B C A

  13. ELISA • for the detection of HIV-specific IgG antibodies • very sensitive, but not 100% specific • reproducible, usually detects both HIV-1 and HIV-2 • can be applied for the detection of circulating antibodies as early as within 3 months after HIV infection

  14. 1. Coating

  15. 2. Blocking of free binding surfaces of the well with an indifferent protein

  16. 1st antibody: we incubate the plate with the serum sample

  17. Unbound antibodies are removed by washes

  18. 2nd antibody: the plate is incubated with anti-human antibody conjugated with an enzyme

  19. Unbound 2nd antibody is removed by washes

  20. Addition of substrate

  21. Addition of substrate

  22. Enzyme conjugated to the 2nd antibody cleaves the substrate and a colored reaction product is formed

  23. The result of the color reaction is read at a given wavelength

  24. Positive ELISA Negative ELISA

  25. - + C B A A B C Based on the results of the ELISA, who is infected by? (see optical densities measured at 405 nm) Negative controlPositive control A B C 0.153 1.689 0.055 0.412 1.999

  26. In order to reconfirm the results of the ELISA test, Western blot is used in HIV diagnostics

  27. Are individuals A,B and C HIV-positive? No band: Negative p31 or p24 and gp160 or gp120 bands simultaneously: Positive there are bands, however criteria of positivity are not fulfilled uncertain 1, HIV+ serum (pozitive control) 2, HIV- serum (negative control) A B C

  28. B - + C B A A B C + -         A C What kind of other molecular genetic technique can be applied to prove the HIV infection?

  29. WB+ ELISA + Potential exposure p24 protein Anti-HIV Ab Day 0 Day 9 Day 24 Day 28 Viral RNA RT –PCR +

  30. Facultative

  31. César Milstein, a molecular biologist and winner of the 1984 Nobel Prize for Physiology or Medicine. This photograph was taken in the Medical Research Council Laboratory of Molecular Biology, Cambridge, where Milstein and his colleagues developed monoclonal antibodies, the achievement for which his Nobel Prize was awarded

  32. Why should we use serial dilutions of a serum sample? sera 1/10 1/100 1/1000 1/10000

  33. Few questions related to ELISA studies of HIV infected(?) patients

  34. What has been measured with this ELISA method? A. HIV-specific antibodies B. HIV-specific antigens C. Free, circulating virus in the patient D. HLA-specific antibodies A

  35. What will happen if we forget to wash out the anti-human Ig-conjugate prior to addition of the substrate? A. There will be no color reaction upon addition of the substrate B. The same results of the ELISA will be obtained C. All wells will show equally strong color reaction D. A and B. C

  36. What would happen if we forgot to add the serum sample to the plate, while all other steps were carried out properly? A. Since the anti-human Ig-conjugate would not bind, we remove it by washing the plate. B. The anti-human Ig-conjugate would bind non-specifically to the plate. C. Optical density values would be close to positive control in all wells D. A and C. A

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