Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells

• US 20050042685A1
• Filed: 05/17/2002
• Published: 02/24/2005
• Est. Priority Date: 09/06/2001
• Status: Active Grant

• Current Assignee: AdnaGen AG
• Original Assignee: AdnaGen AG
• Inventors: Albert, Winfried, Steffens, Pia, Waschuetza, Stefanie, Krehan, Alf-Andreas
• Granted Patent: US 7,507,528 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/57492 involving compounds localized on the membrane of tumor or cancer cells

Abstract

This invention relates to a method for selecting and/or for qualitative and/or quantitative detection of predetermined biological cells from or in a sample containing biological cells, the sample being mixed with a predetermined combination of at least two antibodies and/or antibody derivatives, which bind preferentially with their binding sites to different epitopes of the cells to be selected or detected, and/or with at least one biospecific antibody and/or antibody derivative, which binds preferentially with its two binding sites to different epitopes of the cells to be selected or detected, which are separated from the sample with cells marked with at least one of the antibodies and/or antibody derivatives, and the separated cells being tested with a predetermined combination of at least two molecular-biological detection reagents, the at least two detection reagents reacting preferentially with at least one component of the cells to be selected or detected.

Claims

1. A method for selecting and/or for qualitative and/or quantitative detection of predetermined biological cells from or in a sample containing biological cells, which method comprises:

mixing the sample with (a) a predetermined combination of at least two antibodies and/or antibody derivatives, that bind with their binding sites to different epitopes of the cells to be selected or detected, and/or with (b) at least one bispecific antibody and/or antibody derivative, that binds with its two binding sites to different epitopes of the cells to be selected or detected,

separating the cells marked with at least one of the antibodies and/or antibody derivatives from the sample,

and testing the separated cells with a predetermined combination of at least two molecular-biological detection reagents, for the expression of a predetermined combination of at least two mRNA portions the expression of which is effected at least in the cells to be selected or detected.

2. The method according to claim 1, wherein at least two detection reagents are used which react with different components of the cells to be selected or detected.

4. The method according to claim 1, wherein antibodies or antibody derivatives coupled to solid phases are used in order to separate the target cells from the sample.

5. The method according to claim 1, wherein the antibodies or antibody derivatives are marked with fluorophores, and the separation of the marked cells from the sample is effected by means of flow cytometry.

6. The method according to claim 1, wherein the antibodies or antibody derivatives are coupled to magnetic or pseudo-magnetic particles, and

in order to separate the marked cells from the sample, the magnetic or pseudo-magnetic antibody-coupled particles after mixing with the sample are separated magnetically from the sample.

7. The method according to claim 1, wherein the antibodies or antibody derivatives have binding sites which bind to tumor cells.

8. The method according to claim 1, wherein the antibodies or antibody derivatives have binding sites which bind to cells of one or more specific tumor types or sub-types.

9. The method according to claim 1, wherein, in order to separate tumor cells or cells of a specific tumor type or sub-type, the antibodies or antibody derivatives have binding sites which bind to epitopes of an epithelial antigen, of an epithelial membrane antigen, of the antigen MUC1 and/or of the antigen PLAP.

10. The method according to claim 1, wherein at least one of the antibodies is selected from the group of antibodies consisting of GP1.4, MOC-31, Ber-EP 4, HMPV.2, 8B6, E29 and 131-11741.

11. The method according to claim 1, wherein the, a combination of antibodies comprises Ber-EP 4 and MOC31 or comprises at least two antibodies selected from the group of antibodies consisting of HMPV.2, GP1.4 and Ber-EP 4.

12. The method according to claim 1, wherein the, combination of antibodies comprises at least two antibodies selected from the group of antibodies consisting of 131-11741, GP1.4, E29 and HMPV.2, or comprises at least two antibodies selected from the group of antibodies consisting of HMEIV.2, GP1.4 and Ber-EP 4, whereby breast tumor cells are separated from the sample.

13. The method according to claim 1, wherein the, combination of antibodies comprises the antibodies Ber-EP 4 and MOC-31, whereby colon tumor cells are separated from the sample.

14. The method according to claim 1, wherein the, combination of antibodies comprises at least two antibodies selected from the group of antibodies consisting of MOC-31, Ber-EP 4 and 8B6, whereby testicular tumor cells are separated from the sample.

17. The method according to claim 1, wherein, in order to detect tumor cells or cells of a specific tumor type or sub-type, a combination of mRNA portions is tested, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of GA733.2, EGFR, CEA, HER2/neu, claudin-7 (CLDN7), GCAP (ALPPL2)/ALPP, GRPR, HMGIC, CK20, MAGE3, MUC1 and stanniocalcin (STC1).

18. The method according to claim 17, wherein, in order to detect tumor cells or cells of a specific tumor type or sub-type, a combination of mRNA portions is tested, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of EGFR, GA733.2 and HER-2/NEU.

19. The method according to claim 17, wherein, in order to detect breast tumor cells, a combination of mRNA portions is used, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of GA733.2, MUC1, Her-2/neu, claudin 7, CK20, PIAGE-3, stanniocalcin, EGFR and CEA.

20. The method according to claim 17, wherein, in order to detect breast tumor cells, a combination of mRNA portions is used, which combination comprises mRNA portions corresponding to sequence portions of (a) the genes GA733.2 and MUC1, (b) the genes Her-2/neu and claudin7, (c) at least two genes selected from the group consisting of CK20, MAGE-3 and MUC 1. (d) and/or at least two of the genes selected from the group consisting of stanniocalcin, EGFR and CEA.

21. The method according to claim 17, wherein, in order to detect colon tumor cells, a combination of mRNA portions is used, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of CK20, EGFR, GA733.2, CEA and stanniocalcin.

22. The method according to claim 21, wherein, in order to detect colon tumor cells, a combination of mRNA portions is used, which combination comprises mRNA portions corresponding to sequence portions of (a) at least two genes selected from the group consisting of CK20, EGFR, CEA and stanniocalcin and/or (b) at least two genes selected from the group consisting of EGFR, CEA and GA733.2.

23. The method according to claim 17, wherein, in order to detect testicular tumor cells, a combination of mRNA portions is used, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of ALPP/ALPPL2 (GCAP), GA733.2 (=EGP-40), HMGI-C, and GRPR.

24. The method according to claim 1, wherein the mRNA portions are multiplied and/or detected using polymerase chain reaction (PCR), LCR, NASBA RT-PCR and/or hybridization methods.

25. The method according to claim 1, wherein the mRNA of the separated cells is transcribed reversely into cDNA, the cDNA is multiplied and subsequently the presence or absence of the mRNA portion to be detected is detected.

26. The method according to claim 25, wherein the multiplied cDNA is digested by restriction enzymes and the presence or absence of the mRNA to be detected is detected by means of the produced cDNA fragments.

27. The method according to claim 25, wherein the cDNA corresponding to the mRNA to be detected is determined by means of fluorescence-based real time-PCR.

28. The method according to claim 1, wherein the mRNA that codes for the protein β

-actin is detected as an internal control.

3. The method according to wherein the separation of the marked cells is effected in liquid phase or solid phase.

15-16. -16. (Cancelled)

29. A diagnosis kit for selecting and/or for qualitative and/or quantitative detection of predetermined biological cells from or in a sample containing biological cells, which kit comprises:

a predetermined combination of at least two antibodies and/or antibody derivatives that, bind with their binding sites to different epitopes of the cells to be selected or detected,

and/or at least one bispecific antibody and/or antibody derivative that, binds with both its binding sites to different epitopes of the cells to be selected or detected,

and at least two molecular-biological detection reagents, for detecting the expression of a predetermined combination of at least two mRNA portions,

the expression of these at least two mRNA portions being effected at least in the cells to be selected or detected.

31. The diagnosis kit according to claim 29, wherein the antibodies or antibody derivatives are marked with fluorophores.

32. The diagnosis kit according to claim 29, wherein the antibodies or antibody derivatives are coupled to solid phases.

33. The diagnosis kit according to 32, wherein the antibodies or antibody derivatives are coupled to magnetic or pseudo-magnetic particles.

34. The diagosis kit according to claim 29, wherein the antibodies or antibody derivatives have binding sites which bind to tumor cells.

35. The diagnosis kit according to claim 29, wherein the antibodies or antibody derivatives have binding sites which bind to cells of one or more specific tumor types or sub-types.

36. The diagosis kit according to claim 29, characterized in that, in order to separate tumor cells or a specific type or sub-type of tumor cells, the antibodies or antibody derivatives have binding sites which bind to epitopes of an epithelial antigen, of an epithelial membrane antigen, of the antigen MUC1 and/or of the antigen PLAP.

37. The diagosis kit according to claim 29, wherein the kit comprises at least one antibody selected from the group of antibodies consisting of GP1.4, MOC-31, Ber-EP4, HMPV.2, 8B6, E29 and 131-11741.

38. The diagnosis kit according to claim 29, wherein the kit comprises, the antibodies Ber-EP4 and MOC31 or at least two antibodies selected from the group of antibodies consisting of HMPV.2, GP1.4 and Ber-EP4.

39. The diagnosis kit according to claim 29, wherein the kit comprises at least two antibodies selected from the group of antibodies consisting of 131-11741, GP1.4, E29 and HMPV.2, or at least two antibodies selected from the group of antibodies consisting of HMPV.2, GP1.4 and Ber-EP4.

40. The diagnosis kit according to claim 29, wherein the kit comprises the antibodies Ber-EP4 and MOC-31.

41. The diagnosis kit according to claim 29, wherein the kit comprises at least two antibodies selected from the group of antibodies consisting of MOC-31, Ber-EP4 and 8B6.

42. The diagnosis kit according to claim 29, wherein the detection reagents are suitable for detecting sequence portions of different nucleic acids, DNA, RNA, and/or of different sequence portions of the same nucleic acids, DNA, RNA, and/or different alleles of the same nucleic acid, DNA, RNA.

43. The diagnosis kit according to claim 29, characterized in that the detection reagents detect the expression of a predetermined combination of at least two mRNA portions, the expression of the at least two mRNA portions being effected at least in the cells to be selected or detected.

44. The diagnosis kit according to claim 43, wherein the detection reagents detect the expression of a combination of mRNA portions, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of GA733.2, EGFR, CEA, HER-2/neu, claudin-7 (CLDN7), ALPPL2 (GCAP)/ALPP (PLAP), GRPR, HMGIC, CK20, MAGE-3, MUC1 and stanniocalcin (STC1).

45. The diagnosis kit according to claim 44, wherein the detection reagents detect the expression of a combination of mRNA portions, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of EGFR, GA733.2 and HER-2/NEU.

46. The diagnosis kit according to claim 44, wherein, in order to detect breast tumor cells, the detection reagents detect the expression of a combination of mRNA portions, which combination comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of GA733.2, HER-2/neu, claudin7, CK20, MAGE-3, MUWC, stanniocalcin, EGFR and CEA.

47. The diagnosis kit according to claim 44, wherein, in order to detect breast tumor cells, the detection reagents detect the expression of a combination of mRNA portions, which combination comprises mRNA portions corresponding to sequence portions of (a) the genes GA733.2 and MUC1, (b) the genes Her-2/neu and claudin7, (c) at least two genes selected from the group consisting of CK20, MAGE-3 and WC 1 (d) and/or at least two genes selected from the group consisting of stanniocalcin, EGFR and CEA.

48. The diagnosis kit according to claim 44, wherein, in order to detect colon tumor cells, the detection reagents detect the expression of a combination of mRNA portions, which combination comprises mRNA portions corresponding to sequence portions of at least two of the gene products selected from the group consisting of CK20, EGFR, GA733.2, CEA and stanniocalcin.

49. The diagnosis kit according to claim 44, wherein, in order to detect colon tumor cells, the detection reagents detect the expression of a combination of mRNA portions, which combination comprises mRNA portions corresponding to sequence portions of (a) at least two genes selected from the group consisting of CK20, EGFR, CEA and stanniocalcin, and/or (b) at least two genes selected from the group consisting of EGFR, CEA and GA733.2.

51. The diagnosis kit according to claim 29, wherein the detection reagents for detecting the expression of mRNA portions have at least two oligonucleotide pairs, wherein each oligonucleotide pair is a primer pair for multiplying the cDNA which is complementary respectively to one of the mRNA portions.

52. The diagnosis kit according to claim 51, wherein at least one oligonucleotide of at least one primer pair is marked with a fluorophore.

53. The diagnosis kit according to claim 51, wherein, in order to detect tumor cells or cells of a specific tumor type or sub-type, the primer pairs are suitable for multiplying cDNA portions corresponding to sequence portions of at least two genes selected from the group consisting of GA733.2, EGFR, CEA, HER-2/NEU, claudin-7, ALPPL2 (GCAP)/ALPP, GRPR, HMGI-C, CK20, MAGE3, MUC1 and stanniocalcin.

54. The diagnosis kit according to claim 29, wherein the kit comprises a reverse transcriptase, a polymerase, a taq-polymerase and/or restriction enzymes.

30. (Cancelled)

50. (Cancelled)

55-57. -57. (Cancelled)