Multiplex flow assays preferably with magnetic particles as solid phase

• US 20050260686A1
• Filed: 07/11/2005
• Published: 11/24/2005
• Est. Priority Date: 11/18/1997
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Edwards, Richard B., Watkins, Michael I.
• Granted Patent: US 7,205,160 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: G01N 33/537 with separation of immune complex from unbound antigen or antibody
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54333 Modification of conditions of immunological binding reaction, e.g. use of more than one type of particle, use of chemical agents to improve binding, choice of incubation time or application of magnetic field during binding reaction
• CPC Class Codes: G01N 33/545 Synthetic resin
• CPC Class Codes: G01N 33/56994 Herpetoviridae, e.g. cytomegalovirus, Epstein-Barr virus
• CPC Class Codes: G01N 33/582 with fluorescent label
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: Y10S 435/973 Simultaneous determination of more than one analyte
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 428/2984 Microcapsule with fluid core [includes liposome]
• CPC Class Codes: Y10T 428/2987 Addition polymer from unsaturated monomers only
• CPC Class Codes: Y10T 428/2989 Microcapsule with solid core [includes liposome]
• CPC Class Codes: Y10T 428/2991 Coated

Abstract

Heterogeneous assays for different analytes in a single biological sample are performed simultaneously in a multiplexed assay that combines flow cytometry with the use of magnetic particles as the solid phase and yields an individual result for each analyte. The particles are distinguishable from each other by characteristics that permit them to be differentiated into groups, each group carrying an assay reagent bonded to the particle surface that is distinct from the assay reagents of particles in other groups. The magnetic particles facilitate separation of the solid and liquid phases, permitting the assays to be performed by automated equipment. Assays are also disclosed for the simultaneous detection of antibodies of different classes and a common antigen specificity or of a common class and different antigen specificities. Each type is accomplished by immunological binding at the surfaces of two distinct solid phases in a sequential manner with dissociation of the binding and washing of the solid phase in between the binding steps.

Claims

1-29. -29. (canceled)

30. A method for the analysis of a sample to simultaneously yet individually detect antibodies of different classes that have a single common antigen specificity, said method comprising:

contacting said sample with a first solid phase coated with said antigen to cause antibodies in said sample to bind to said antigen;

washing said first solid phase to isolate said first group from unbound species;

suspending said first solid phase thus washed in a liquid suspending medium and releasing said antibodies from said antigen into said medium to form a supernatant containing unbound multiple-class antibodies specific to said antigen;

isolating said supernatant from said first solid phase and contacting said supernatant with a second solid phase, said second solid phase comprising one or more portions each coated with an immunological binding member with specific binding affinity for a single antibody class, and each portion being capable of differentiation from other such portions; and

individually detecting the occurrence of immunological binding between said antibody classes and said immunological binding members while differentiating between said portions.

31. A method in accordance with claim 30 in which said first solid phase is a plurality of microparticles.

32. A method in accordance with claim 30 in which said second solid phase is a plurality of microparticles.

33. A method in accordance with claim 30 in which said first and second solid phases are each a plurality of microparticles.

34. A method in accordance with claim 30 in which said occurrence of immunological binding is detected using labeled class-specific antibodies.

35. A method in accordance with claim 30 in which said occurrence of immunological binding is detected using phycoerythrin-labeled class-specific antibodies and individual detection is achieved by fluorescence detection.

36. A method in accordance with claim 30 in which said occurrence of immunological binding is detected using labeled analogs of said antigen.

37. A method in accordance with claim 30 in which said occurrence of immunological binding is detected using phycoerythrin-labeled analogs of said antigen and individual detection is achieved by fluorescence detection.

38. A method in accordance with claim 31 in which said microparticles are of magnetically responsive material, and said washing is facilitated by magnetically separating said first group of microparticles from said sample.

39. A method in accordance with claim 31 in which said microparticles are of magnetically responsive material, and both said washing of said first group of microparticles and said isolating of said supernatant from said first solid phase are facilitated by magnetically separating said microparticles from said sample and said supernatant.

40. A method in accordance with claim 32 in which said portions are subgroups of microparticles differing from each other by particle size, and differentiation between said subgroups is achieved by flow cytometry.

41-49. -49. (canceled)