Method and diagnosis kit for selecting and or qualitative and/or quantitative detection of cells
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.
1. A method for detection of a predetermined biological cell in a sample containing biological cells comprising:
(i) mixing the sample with (a) a predetermined combination of at least two antibodies and/or antibody derivatives that bind to two different epitopes of the cell to be detected, or with (b) at least one bispecific antibody and/or antibody derivative that binds to two different epitopes of the cell to be detected, wherein said predetermined combination of at least two antibodies or bispecific antibody provides an increased sensitivity in terms of the number of cells that can be detected in a given sample volume as compared to the number of cells that can be detected in said sample volume using a single monospecific antibody that binds to one of said epitopes,
(ii) binding the cell with at least one of the antibodies and/or antibody derivatives thereby marking the cell,
(iii) separating the marked cell from the sample, and
(iv) testing the separated cell for the expression of a predetermined combination of at least two mRNAs, the expression of which is effected in the cell to be detected, wherein the expression of at least one of the mRNAs indicates the predetermined cell is detected,
wherein said increased sensitivity is achieved without a decrease in specificity.
2. The method according to claim 1 , wherein the separation of the marked cell is effected in liquid phase.
3. The method according to claim 1 , wherein antibodies or antibody derivatives coupled to solid phases are used in order to separate the marked cell from the sample.
4. The method according to claim 1 , wherein the antibodies or antibody derivatives are marked with fluorophores, and the separation of the marked cell from the sample is effected by means of fluorescence-activated cell sorting.
5. The method according to claim 1 , further comprising coupling the antibodies or antibody derivatives to magnetic particles to produce magnetic antibody-coupled particles, and magnetically separating the magnetic antibody-coupled particles from the sample after mixing with the sample.
6. The method according to claim 1 , wherein the antibodies or antibody derivatives have binding sites which bind to tumor cells.
7. 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.
8. The method according to claim 1 , wherein the antibodies or antibody derivatives comprise binding sites which bind to epitopes of an epithelial antigen, an epithelial membrane antigen, the antigen MUC1 and/or the antigen PLAP.
9. 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.
10. The method according to claim 1 , wherein the 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.
11. 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 HMPV.2, GP1.4 and Ber-EP 4, whereby breast tumor cells are detected in the sample.
12. 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 detected in the sample.
13. 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 detected in the sample.
14. The method according claim 1 , further comprising testing 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, EGER, CEA, HER2/neu, claudin-7 (CLDN7), GCAP (ALPPL2)/ALPP, GRPR, HMGIC, CK20, MAGE3, MUC1 and stanniocalcin (STCI), whereby cells of a specific tumor type or subtype are detected.
15. The method according to claim 14 , wherein the combination of mRNA portions comprises mRNA portions corresponding to sequence portions of at least two genes selected from the group consisting of EGFR, GA733.2 and HER2/NEU, where cells of a specific tumor type or subtype are detected.
16. The method according to claim 14 , wherein the combination of mRNA portions 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, MAGE-3, stanniocalcin, EGFR and CEA, whereby breast tumor cells are detected.
17. The method according to claim 14 , wherein the combination of mRNA portions comprises mRNA portions corresponding to sequence portions of (a) the genes GA733.2 and MUC1, (b) the genes Her-2/neu and claudin 7, (c) at least two genes selected from the group consisting of CK20, MAGE-3 and MUC1, and/or (d) at least two genes selected from the group consisting of stanniocalcin, EGFR and CEA, whereby breast tumor cells are detected.
18. The method according to claim 14 , wherein the combination of mRNA portions 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, whereby colon tumor cells are detected.
19. The method according to claim 18 , wherein the combination of mRNA portions 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 lest two genes selected from the group consisting of EGFR, CEA and GA733.2, whereby colon tumor cells are detected.
20. The method according to claim 14 , wherein the combination of mRNA portions 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, whereby testicular tumor cells are detected.
21. 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.
22. The method according to claim 1 , further comprising reverse transcribing the mRNA of the separated cells into cDNA, multiplying the cDNA and subsequently detecting the presence or absence of the mRNA portion to be detected.
23. The method according to claim 22 , further comprising digesting the multiplied cDNA by restriction enzymes to produce cDNA fragments, and detecting the presence or absence of the mRNA to be detected by means of the produced cDNA fragments.
24. The method according to claim 22 , wherein the cDNA corresponding to the mRNA to be detected is determined by means of fluorescence-based real time-PCR.
25. The method according to claim 1 , wherein the mRNA that codes for the protein β-actin is detected as an internal control.
26. The method according to claim 1 , wherein the separation of the marked cell is effected with a solid phase.