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

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

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

15. 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

16. 1. Coating

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

18. 1st antibody: we incubate the plate with the serum sample

19. Unbound antibodies are removed by washes

20. 2nd antibody: the plate is incubated with anti-human antibody conjugated with an enzyme

21. Unbound 2nd antibody is removed by washes

22. Addition of substrate

23. Addition of substrate

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

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

26. Positive ELISA
Negative ELISA

27. Based on the results of the ELISA, who is infected by
Based on the results of the ELISA, who is infected by? (see optical densities measured at 405 nm)
Negative control Positive control A B C
C B A A B C

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

29. 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

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

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

32. Facultative

34. 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

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

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

39. A. HIV-specific antibodies B. HIV-specific antigens
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

40. 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

41. 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