Method of protein extraction from cells
Methods for producing a protein extract from cells, such as cells containing viral proteins, are provided. In general terms, the methods involve: increasing the pH of the cells to a pH of at least about pH 10.0 to produce an intermediate composition, and then, in the presence of a non-ionic detergent, neutralizing the pH of the intermediate composition to produce the protein extract. Kits and compositions for practicing the subject methods are also provided.
1. A method for producing a protein extract and detecting a protein in said protein extract from fixed human epithelial cells, wherein said protein is a viral protein, comprising:
a) contacting said fixed human epithelial cells with an extraction reagent to produce an intermediate composition having a pH of greater than pH 10.0, wherein said extraction reagent comprises a non-ionic detergent, NaOH, and a buffer;
b) contacting said intermediate composition with a neutralizing reagent to neutralize said pH of said intermediate composition to a pH from pH 7 to pH 8, wherein said neutralizing agent comprises a source of hydrogen ions, and to produce said protein extract; and
c) contacting said protein extract with an antibody and thereby detecting said viral protein in said protein extract.
2. The method of claim 1 , further comprising:
receiving a cellular sample comprising said fixed human epithelial cells prior to step a).
3. The method of claim 1 , wherein said fixed human epithelial cells are fixed cervical cells.
4. The method of claim 1 , wherein said fixed human epithelial cells are present in an ethanol-based or methanol-based transport medium.
5. The method of claim 2 , wherein said cellular sample is received from a remote location.
6. The method of claim 1 , wherein said pH in step (a) is in the range of pH 11.0 to pH 13.
7. The method of claim 1 , wherein said non-ionic detergent is octylphenolpoly(ethyleneglycolether) x or a polysorbate detergent.
8. The method of claim 1 , wherein said detecting employs a capture agent for said protein.
9. The method of claim 1 , wherein said detecting uses an enzyme linked immunosorbent assay (ELISA).
10. The method of claim 1 , wherein said protein is a human papillomavirus (HPV) protein.
11. The method of claim 1 , wherein said protein is an HPV E6 protein.
12. The method of claim 1 , wherein said fixed human epithelial cells are exfoliated cervical cells.
13. The method of claim 1 , wherein said viral protein is encoded by a pathogenic virus.
14. The method of claim 13 , wherein said pathogenic virus is selected from the group consisting of HIV, Ebola virus, Marburg virus, hepatitis virus, respiratory syncytial virus (RSV), herpes simplex virus (HSV), human papilloma virus (HPV).
15. The method of claim 1 , wherein said viral protein is E6 or E7 protein of HPV.
16. The method of claim 15 , wherein said HPV is HPV strain 4, 11, 20, 24, 28, 36, 48, 50, 16, 18, 31, 35, 30, 39, 45, 51, 52, 56, 59, 58, 33, 66, 68, 69, 26, 53, 73, or 82.
17. The method of claim 15 , wherein said HPV is an oncogenic HPV strain selected from the group consisting of HPV 26, HPV 53, HPV 66, HPV 73, HPV 82, HPV 16, HPV 18, HPV 31, HPV 35, HPV 30, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 59, HPV 58, HPV 33, HPV 66, HPV 68, HPV 69, and HPV 82.
18. The method of claim 8 , wherein said viral protein is E6 protein of HPV; and the capture agent is an antibody against the E6 protein.
19. The method of claim 8 , wherein said viral protein is E 6 protein of HPV; and said the capture agent comprises a polypeptide containing a PDZ domain.
20. The method of claim 19 , wherein said the PDZ domain is the second domain of MAGI-1, the PDZ domain of DLG or TIP 1.
21. The method of claim 1 , wherein said fixed human epithelial cells are from a cell sample comprising further contains mucous or blood.
22. The method of claim 1 , wherein said fixed human epithelial cells were fixed with a chemical fixative.
23. The method of claim 22 , wherein said chemical fixative is selected from the group consisting of alcohols, aldehydes, ketones, osmium tetroxide, acetic acid, picric acid, heavy metal ion salts, and propylene glycol.
24. The method of claim 23 , wherein said alcohol is methanol or ethanol; said the aldehyde is gluteraldehyde or formaldehyde; and said the ketone is acetone.
25. The method of claim 1 , wherein said neutralizing reagent comprises hydrochloric acid or acetic acid.