Claims
- 1. A method for isolating a target bioentity from a biological sample by means of colloidal magnetic particles with reduced aggregation of said magnetic particles, comprising:
a) obtaining a biological specimen suspected of containing said target bioentity together with non-target bioentities and endogenous aggregating factors; b) contacting said biological specimen with a reagent effective to inactivate any endogenous aggregating factors present in said specimen; c) preparing an immunomagnetic suspension comprising a mixture of said specimen and colloidal, magnetic particles coupled to a biospecific ligand having binding affinity for at least one characteristic determinant present on said target bioentity; and d) subjecting said immunomagnetic suspension to a magnetic field to obtain a target bioentity- enriched fraction.
- 2. A method as claimed in claim 1, further comprising the steps of
e) purifying said target bioentity from said enriched fraction; and f) analyzing said purified target bioentity.
- 3. A method as claimed in claim 1, further comprising the steps of:
e) adding to said target bioentity enriched fraction at least one biospecific reagent which has binding affinity for at least one additional characteristic determinant on said target bioentity; f) separating said target bioentity in a magnetic field to remove unbound biospecific reagent from said enriched fraction.
- 4. A method as claimed in claim 3, further comprising the steps of:
g) purifying said separated target bioentity; and h) analyzing said purified target bioentity.
- 5. A method as claimed in claim 3, further comprising the steps of:
g) adding a non-cell exclusion agent to said separated target bioentities to allow exclusion of non-nucleated entities present in the sample; h) purifying said target bioentity; and i) analyzing said purified target bioentities.
- 6. A method as claimed in claim 1, wherein said target bioentity is selected from the group consisting of tumor cells, virally infected cells, fetal cells in maternal circulation, virus particles, bacterial cells, white blood cells, myocardial cells, epithelial cells, endothelial cells, proteins, hormones, DNA, and RNA.
- 7. A method as claimed in claim 2, wherein said target bioentities are analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, manual cell analysis and automated cell analysis.
- 8. A method as claimed in claim 1, said aggregation inhibiting agent being at least one selected from those consisting of a reducing agent, an animal serum protein, an immune-complex, a carbohydrate, a chelating agent, an unconjugated ferrofluid, and diamino butane.
- 9. A method as claimed in claim 5, wherein said aggregation inhibiting agent is a reducing agent selected from the group consisting of mercapto ethane sulfonic acid [MES], mercapto propane sulfonic acid [MPS] and dithiothreitol [DTT].
- 10. A method as claimed in claim 1, wherein said biospecific ligand is a monoclonal antibody.
- 11. A method as claimed in claim 9, wherein said biospecific ligand is a monoclonal antibody having affinity for epithelial cell adhesion molecule.
- 12. A method for isolating target bioentities from a biological sample by means of colloidal magnetic particles with controlled aggregation of said magnetic particles, comprising:
a) obtaining a biological specimen suspected of containing said target bioentities together with non-target bioentities and endogenous aggregating factors; b) contacting said biological specimen with a reagent effective to inactivate any endogenous aggregating factors present in said specimen; c) preparing an immunomagnetic suspension comprising a mixture of colloidal, magnetic particles coupled to a biospecific ligand having affinity for at least one characteristic determinant present on said target bioentity, said magnetic particles being further coupled to a first exogenous aggregation enhancing factor which comprises one member of a specific binding pair; d) adding a second exogenous aggregation enhancing factor to said immunomagnetic suspension to increase aggregation of said particles, said second aggregating enhancing factor comprising the other member of said specific binding pair; and e) subjecting said sample to a magnetic field to obtain a target bioentity-enriched fraction.
- 13. A method as claimed in claim 12, further comprising the steps of:
f) adding to said immunomagnetic suspension at least one biospecific reagent having binding affinity for at least one additional characteristic determinant on said target bioentity; g) separating said target bioentities in a magnetic field to remove unbound biospecific reagent; and h) adding a non-cell exclusion agent to said separated bioentities to allow exclusion of non-nucleated entities present in the sample; i) purifying said target bioentities; and j) analyzing said separated and purified target bioentities.
- 14. A method as claimed in claim 12, further comprising examining said purified target bioentity-enriched fraction to determine the degree of aggregation mediated by said first and second members of said specific binding pair.
- 15. A method as claimed in claim 12, wherein one or the other member of said specific binding pair is added to said purified bioentity fraction to reverse aggregation of said sample, thereby facilitating analysis of said target bioentities.
- 16. A method as claimed in claim 12, wherein said specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-streptavidin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.
- 17. A method as claimed in claim 12, wherein said biospecific ligand is a monoclonal antibody.
- 18. A method as claimed in claim 17, wherein said biospecific ligand is an antibody having affinity for epithelial cell adhesion molecule.
- 19. A method as claimed in claim 12 wherein said at least one biospecific reagent is selected from the group of consisting of monoclonal antibodies, polyclonal antibodies, detectably labeled antibodies, antibody fragments, and single chain antibodies.
- 20. A method as claimed in claim 12 wherein said target bioentities are analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, manual cell analysis and automated cell analysis.
- 21. A method as claimed in claim 12, wherein said immunomagnetic suspension is incubated for less than 30 minutes.
- 22. A method as claimed in claim 12, wherein said purification of said sample is performed in a magnetic field gradient of less than 6.3 kGauss/cm.
- 23. A method as claimed in claim 12, wherein said colloidal magnetic particle concentration in said immunomagnetic suspension is less than 10 μg per milliliter.
- 24. A method for isolating low antigen density tumor cells from a biological sample by means of colloidal magnetic particles with controlled aggregation of said magnetic particles, comprising:
a) obtaining a biological specimen suspected of containing said tumor cells together with non-tumor cells and endogenous aggregating factors; b) preparing an immunomagnetic suspension comprising a mixture of said specimen and colloidal, magnetic particles coupled to a biospecific ligand having binding affinity for at least one characteristic determinant present on said tumor cell, said magnetic particles being further coupled to a first exogenous aggregation enhancing factor, said factor comprising one member of a specific binding pair; c) adding a second exogenous aggregating enhancing factor to said immunomagnetic suspension to increase aggregation of said particles, said second aggregating enhancing factor comprising the other member of said specific binding pair; and d) purifying said sample in a magnetic field to obtain a tumor cell-enriched fraction.
- 25. A method as claimed in claim 24, further comprising the steps of:
e) adding to said fraction, at least one biospecific reagent having binding affinity for at least one additional characteristic determinant on said tumor cell; f) separating said tumor cells in a magnetic field to remove unbound biospecific reagent; g) adding a non-cell exclusion agent to said separated cells to allow exclusion of non-nucleated entities present in the sample; and h) analyzing said separated tumor cells to assess at least one of tumor cell number and type.
- 26. A method as claimed in claim 25, wherein a member of said specific binding pair is added to the separated cells to reverse aggregation of said sample, thereby facilitating analysis of said cells.
- 27. A method as claimed in claim 24, wherein said specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.
- 28. A method as claimed in claim 24, wherein said biospecific ligand is a monoclonal antibody.
- 29. A method as claimed in claim 28, wherein said biospecific ligand is an antibody having binding affinity for epithelial cell adhesion molecule.
- 30. A method as claimed in claim 25, wherein said at least one biospecific reagent is selected from the group of consisting of monoclonal antibodies, polyclonal antibodies, detectably labeled antibodies, antibody fragments and single chain antibodies.
- 31. A method as claimed in claim 25 wherein said tumor cells are analyzed by a process selected from the group consisting of multiparameter flow cytometry, immunofluorescent microscopy, laser scanning cytometry, bright field base image analysis, capillary volumetry, manual cell analysis and automated cell analysis.
- 32. A method as claimed in claim 24 wherein said biological sample is pretreated with an aggregation inhibiting agent to inactivate endogenous aggregation factors present in the sample prior to the preparation of said immunomagnetic suspension.
- 33. A method as claimed in claim 25, wherein said purified tumor cell-enriched fraction is examined to determine the degree of aggregation mediated by said first and second members of said specific binding pair.
- 34. A kit for inhibiting endogenous aggregation of colloidal magnetic particles in processing of biological material for isolation of target bioentities from such materials, comprising:
a) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said target bioentity, said antibody being coupled, directly or indirectly, to said base coating material; b) at least one antibody having binding specificity for a second characteristic determinant of said target bioentity; c) an endogenous aggregation inhibiting factor; and d) a non-cell exclusion agent for excluding non-nucleated cells from analysis.
- 35. A kit for isolating low antigen density tumor cells from a biological sample with controlled aggregation of colloidal magnetic particles, comprising:
a) a reagent effective to inactivate endogenous aggregating factors; b) coated magnetic nanoparticles comprising a magnetic core material, a protein base coating material, and an antibody that binds specifically to a first characteristic determinant of said tumor cell, said antibody being coupled, directly or indirectly, to said base coating material; said magnetic particles being further coupled to a first exogenous aggregation enhancing factor, said factor comprising one member of a specific binding pair; c) at least one antibody having binding specificity for a second characteristic determinant of said tumor cell; d) a second exogenous aggregation enhancing factor, said second aggregation factor comprising the other member of said specific binding pair; and e) a non-cell excluding agent for excluding non-nucleated cells from analysis.
- 36. A kit as claimed in claim 35, wherein said specific binding pair is selected from the group consisting of biotin-streptavidin, antigen-antibody, receptor-hormone, receptor-ligand, agonist-antagonist, lectin-carbohydrate, Protein A-antibody Fc, and avidin-biotin, biotin analog-avidin, desthiobiotin-streptavidin, desthiobiotin-avidin, iminobiotin-streptavidin, and iminobiotin-avidin.
- 37. A kit as claimed in claim 35, said kit further comprising a reagent for reversing the effect of said exogenous aggregation factor.
- 38. A kit as claimed in claim 35, said reagent effective to inhibit endogenous aggregation factor being at least one selected from the group consisting of a reducing agent, an animal serum protein, an immune-complexes, a carbohydrate, a chelating agent, an unconjugated ferrofluid, and diamino butane.
- 39. A method as claimed in claim 38, wherein said said endogenous aggregation factor inhibiting reagent is a reducing agent selected from the group of agents consisting of mercapto ethane sulfonic acid [MES], mercapto Propane Sulfonic acid [MPS] and dithiothreitol [DTT].
PRIORITY INFORMATION
[0001] The present application is a continuation of U.S. application Ser. No. 09/351,515 filed on Jul. 12, 1999.
Continuations (1)
|
Number |
Date |
Country |
Parent |
09351515 |
Jul 1999 |
US |
Child |
10449355 |
May 2003 |
US |