Claims
- 1. A particle light scatter-based immunoassay for measuring an analyte in a fluid sample, comprising the steps of:
a) combining with said fluid sample, first binding molecule-coated monodisperse microspheres having a resolvable light scatter signal and second binding molecule-coated colloidal particles, or a prior-prepared immunocomplex comprising said monodisperse microspheres, said analyte and said colloidal particles, to form a mixture and to allow a reaction to occur, said reaction being said formation or said decomplexation of said immunocomplex, respectively, so that said thus-reacted mixture includes relative amounts of non-colloidal particles selected from said group consisting of said immunocomplex and uncomplexed microspheres, the relative amounts of said immunocomplex and said uncomplexed microspheres being dependent upon said presence or amount of analyte in said fluid sample; b) illuminating said non-colloidal particles after said reaction with an incident light source to produce individual light scatter signals for each of said non-colloidal particles; and c) determining a statistical distribution of said thus-produced light scatter signals so that said distribution can be correlated with said presence or amount of said analyte in said fluid sample.
- 2. A method according to claim 1, further comprising said step of correlating said light scatter signal measurement with said presence or amount of said analyte in said fluid sample.
- 3. A method according to claim 2, further comprising said step of determining a statistical distribution of said light scatter signal produced by a control.
- 4. A method according to claim 3, wherein said control comprises said first binding molecule-coated monodisperse microspheres.
- 5. A method according to claim 3, wherein said control comprises said prior-prepared immunocomplex.
- 6. A method according to claim 3, wherein said correlating step comprises normalizing the statistical distribution of said light scatter signal with respect to said control light scatter signal determination.
- 7. A method according to claim 1, wherein said illuminating step produces substantially low angle forward light scatter signals.
- 8. A method according to claim 1, wherein said illuminating step comprises generating forward light scatter signals of from about 10° to about 80°.
- 9. A method according to claim 1, wherein said illuminating step produces substantially right angle light scatter signals.
- 10. A method according to claim 1, wherein said illuminating step comprises generating light scatter signals which are backscatter signals of from about 100° to about 170°.
- 11. A method according to claim 1, wherein said determining step includes the step of determining a distribution of the pulse amplitudes of the thus-produced signals.
- 12. A method according to claim 11, wherein said determining step includes the step of generating a histogram of the pulse amplitudes.
- 13. A method according to claim 1, wherein said determining step includes the step of determining a distribution of the integrated pulse areas of the thus-produced signals.
- 14. A method according to claim 13, wherein said determining step includes the step of generating a histogram of the integrated pulse areas.
- 15. A method according to claim 1, wherein said illuminating step includes the step of directing said incident light source onto each non-colloidal particle and monitoring light scattered therefrom to produce a signal for each particle representing a ratio between the amplitude of light scattered at a relatively low angle by the particle and the amplitude of light scattered at a relatively high angle by such particle.
- 16. A method according to claim 1, wherein said determining step comprises the step of determining a distribution of pulse width times the maximum amplitude of the thus-produced signals.
- 17. A method according to claim 16, wherein said determining step comprises generating a histogram of the pulse width times the maximum amplitude of the thus-produced signals.
- 18. A method according to claim 1, wherein said illuminating step comprises directing a stream of the thus-reacted mixture through an optical sheath flow particle analyzer comprising a sheath flow cell having a predetermined central bore width or diameter.
- 19. A method according to claim 18, wherein said predetermined central bore width or diameter is from about 100 μm to about 500 μm.
- 20. A method according to claim 19, wherein said predetermined central bore width or diameter is about 250μ.
- 21. A method according to claim 18, wherein said stream has a substantially constant diameter.
- 22. A method according to claim 21, wherein said stream diameter is from about 3μ to about 10μ.
- 23. A method according to claim 1, wherein said incident light source is a laser light source.
- 24. A method according to claim 1, wherein said analyte is an antigen, antibody, hapten, nucleic acid or ligand.
- 25. A method according to claim 1, wherein said first binding molecule and said second binding molecule are complementary to said analyte.
- 26. A method according to claim 25, wherein said first binding molecule is an antibody specific to said analyte.
- 27. A method according to claim 25, wherein said second binding molecule is an antibody specific to said analyte.
- 28. A method according to claim 25, wherein said first binding molecule is a monoclonal antibody specific to said analyte.
- 29. A method according to claim 25, wherein said second binding molecule is a monoclonal antibody specific to said analyte.
- 30. A method according to claim 25, wherein said first binding molecule is a monoclonal antibody specific to a first epitope on said analyte, and said second binding molecule is a monoclonal antibody specific to a second epitope on said analyte.
- 31. A method according to claim 1, wherein said first binding molecule is said analyte.
- 32. A method according to claim 1, wherein said second binding molecule is complementary to said analyte.
- 33. A method according to claim 32, wherein said second binding molecule is an antibody specific to said analyte.
- 34. A method according to claim 33, wherein said antibody is a monoclonal antibody.
- 35. A method according to claim 1, wherein said monodisperse microspheres comprise a synthetic polymeric material.
- 36. A method according to claim 35, wherein said polymeric material comprises polybutadiene, polystyrene or a derivative thereof.
- 37. A method according to claim 1, wherein said monodisperse microspheres are glass microspheres.
- 38. A method according to claim 1, wherein said monodisperse microspheres comprise microscopic oxide powders.
- 39. A method according to claim 1, wherein said monodisperse microspheres have an average diameter of from about 0.25 μm to about 10 μm.
- 40. A method according to claim 39, wherein said average diameter is from about 0.25 μm to about 10 μm.
- 41. A method according to claim 39, wherein said average diameter is from about 0.5 μm to about 5.0 μm.
- 42. A method according to claim 1, wherein said colloidal particles are polydisperse.
- 43. A method according to claim 1, wherein said colloidal particles comprises a metal or a metal compound.
- 44. A method according to claim 43, wherein said metal compound is an oxide, hydroxide or a salt.
- 45. A method according to claim 43, wherein said metal is gold, platinum, silver or copper.
- 46. A method according to claim 45, wherein said metal is gold.
- 47. A method according to claim 1, wherein said colloidal particles have diameters ranging from about 20 nm to about 120 nm.
- 48. A method according to claim 47, wherein said diameters range from about 50 nm to about 80 nm.
- 49. A method according to claim 1, the ratio of the average diameter of said monodisperse microspheres to the average diameter of said colloidal particles is from about 15:1 to about 30:1.
- 50. A method according to claim 1, wherein said colloidal particles and said monodisperse microspheres are present in said solution in a ratio effective to reduce interference by non-specific binding substances.
- 51. A method according to claim 50, wherein the ratio of said colloidal particles to said monodisperse microspheres is from about 2:1 to about 100,000:1.
- 52. A method according to claim 51 wherein said ratio is from about 1,000:1 to about 10,000:1.
- 53. A method according to claim 1, further comprising the step of separating the immunocomplex and the uncomplexed microspheres from the thus-reacted mixture prior to said step of illuminating.
- 54. A particle light scatter-based immunoassay for simultaneously measuring two or more analytes in a single fluid sample, comprising the steps of:
(a) combining with said fluid sample for each of said first and second analytes, first binding molecule-coated monodisperse microspheres having a resolvable light scatter signal and second binding molecule-coated colloidal particles, or a prior-prepared immunocomplex comprising said monodisperse microspheres, said analyte and said colloidal particles to form a mixture and to allow a reaction to occur, said reaction being the formation or the decomplexation of the first and second immunocomplexes, respectively, so that the thus-reacted mixture includes relative amounts of non-colloidal particles selected from the group consisting of first and second immunocomplexes and first and second uncomplexed microspheres, the relative amounts of said first immunocomplex and said first uncompleted microspheres, and the relative amounts of the second immunocomplex and the second uncomplexed microspheres being dependent upon the presence or amounts of said first and second analytes, respectively, in said fluid sample; (b) illuminating the non-colloidal particles with an incident light source to produce light scatter signals for each of the non-colloidal particles; and (c) determining a statistical distribution of the thus-produced light scatter signals so that the distributions can be correlated with the presence or amount of said first and second analytes, respectively, in said fluid sample.
- 55. Apparatus for measuring the light scattering characteristics of particles, said apparatus comprising:
(a) a flow cell defining a bore; (b) means for passing a stream of liquid bearing said particles along said bore, wherein said means includes sheath flow means for passing a stream of a sheath liquid along said bore and sample stream means for passing a stream of a sample liquid bearing said particles along said bore within said stream of sheath liquid; (c) means for directing light through said flow cell along a beam path transverse to said bore so that said light impinges on particles in said stream; (d) detector means for detecting light scattered by said particles and providing signals representing strength of the detected light; and (e) cell position feedback control means for automatically adjusting the relative position of said flow cell and said beam path in response to said signals from said detector means so as to maximize the strength of said detected light as represented in said signals.
- 56. Apparatus as claimed in claim 55, wherein said cell position feedback control means includes means for moving said beam path in an adjustment direction transverse to said bore.
- 57. Apparatus as claimed in claim 56, wherein said cell position feedback control means includes means for moving said flow cell.
- 58. Apparatus as claimed in claim 55, wherein said detector means includes a photodetector and means for providing said signals so that said signals represent amplitudes of responses provided by said photodetector.
- 59. Apparatus for measuring the light scattering characteristics of particles, said apparatus comprising:
(a) a flow cell defining a bore; (b) sheath flow means for passing a stream of a sheath liquid bearing said particles along said bore; (c) sample stream means for passing a stream of a sample liquid bearing said particles along said bore within said stream of sheath liquid; (d) means for directing light through said cell along a beam path transverse to said bore so that said light impinges on particles in said stream of sample liquid; (e) detector means for detecting light scattered by said particles and providing signals representing at least one characteristic of the detected light; (f) dispersion sensing means for providing a dispersion signal representing dispersion in said characteristic as represented in said signals from said detector means; and (g) sample stream feedback control means for automatically adjusting said sample stream means to alter the rate of flow of said sample liquid in response to said dispersion signal so as to reduce said rate of flow when said dispersion signal is above a preselected maximum.
- 60. Apparatus for measuring the light scattering characteristics of particles, said apparatus comprising:
(a) a flow cell defining a bore; (b) sheath flow means for passing a stream of a sheath liquid bearing said particles along said bore; (c) sample stream means for passing a stream of a sample liquid bearing said particles along said bore within said stream of sheath liquid; (d) means for directing light through said cell along a beam path transverse to said bore so that said light impinges on particles in said stream of sample liquid; (e) detector means for detecting light scattered by said particles and providing signals representing at least one characteristic of the detected light; (f) dispersion sensing means for providing a dispersion signal representing dispersion in said characteristic as represented in said signals from said detector means; and (g) sheath liquid flow feedback control means for automatically adjusting said sheath flow to alter the rate of flow of said sheath liquid in response to said dispersion signal so as to reduce said rate of flow when said dispersion signal is above a preselected maximum.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. patent application Ser. No. 08/286,778, filed Aug. 5, 1994, which is a continuation of U.S. patent application Ser. No. 07/994,903, filed Dec. 22, 1992, abandoned, the contents of which are hereby incorporated by reference in their entireties.
Divisions (1)
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Number |
Date |
Country |
Parent |
08473187 |
Jun 1995 |
US |
Child |
09750375 |
Dec 2000 |
US |
Continuations (1)
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Number |
Date |
Country |
Parent |
07994903 |
Dec 1992 |
US |
Child |
08286778 |
Aug 1994 |
US |
Continuation in Parts (1)
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Number |
Date |
Country |
Parent |
08286778 |
Aug 1994 |
US |
Child |
08473187 |
Jun 1995 |
US |