Assay procedures and apparatus

• US 7,326,573 B2
• Filed: 01/10/2003
• Issued: 02/05/2008
• Est. Priority Date: 01/10/2003
• Status: Active Grant

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Inventors: Bell, Michael L.
• Primary Examiner(s): Warden; Jill
• Assistant Examiner(s): Ramillano; Lore
• Application Number:
  US10/340,278
• Publication Number:
  US 20040137635A1
• Time in Patent Office: 1,852 Days
• Field of Search: 436/63, 436/172, 435/7.92
• US Class Current: 436/63
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 2021/6421 Measuring at two or more wavelengths
• CPC Class Codes: G01N 2021/6441 with two or more labels
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53

Abstract

In cytometry and like procedures, particles containing signal dyes are exposed to a laser, and the resulting fluorescence is assessed in two different wavelength bands. When the particle is a single-assay particle, or when the particle carries only a single dye, the fluorescences in the different wavelength bands are combined in order to assess the concentration of the signal dye.

Claims

1. A method of examining a composition which comprises a plurality of particles, each of the particles

(i) having a coding characteristic,

(ii) containing analyte-interaction sites and/or corresponding analyte-bearing sites which are the same as said analyte-interaction sites except that they have interacted with one or more analytes and have associated therewith one or more fluorochromic signal dyes, and

(iii) belonging to one only of a plurality of defined categories, each of the particles in each defined category

(a) having the same coding characteristic, and

(b) containing the same analyte-interaction sites and/or corresponding analyte-bearing sites;

the combination of the coding characteristic and the analyte-interaction sites and/or corresponding analyte-bearing sites on the particles in each category being different from the combination of the coding characteristic and the analyte-interaction sites and/or corresponding analyte-bearing sites on the particles in other categories;

the method comprising examining, one at a time, each particle of a representative sample of the particles, the examination comprising;

(A) determining the coding characteristic of the particle, including whether the particle is a single-assay particle or a dual-assay particle;

(B) subjecting the particle to radiation from a laser which causes fluorescence of any fluorochromic signal dye associated with analyte-bearing sites on the particle;

(C) assessing the fluorescence caused by the laser and falling within a first wavelength band;

(D) assessing the fluorescence caused by the laser and falling within a second wavelength band; and

(E) when step (A) determines that the particle is a single-assay particle, combining the fluorescence in the first wavelength band and the fluorescence in the second wavelength band, and

when step (A) determines that the particle is a dual-assay particle, recording separately the fluorescence in the first wavelength band and the fluorescence in the second wavelength band.

2. A method according to claim 1 wherein

(A) the plurality of particles includes

(i) dual-assay particles containing analyte-bearing sites which

(a) have interacted with a first analyte and have associated therewith a first fluorochromic signal dye, and/or

(b) have interacted with a second analyte and have associated therewith a second fluorochromic signal dye;

and

(ii) single-assay particles containing analyte-bearing sites which have interacted with an analyte and have associated therewith a fluorochromic signal dye which is the same as the first or the second signal dye or is different therefrom;

(B) the laser (i) causes the first fluorochromic signal dye, if present, to fluoresce in a first spectrum, (ii) causes the second fluorochromic signal dye, if present, to fluoresce in a second spectrum which is different from but overlaps the first spectrum, and (iii) causes the fluorochromic signal dye or dyes associated with the single-assay particles, if present, to fluoresce in a spectrum which includes at least one of the first wavelength band and the second wavelength band.

3. A method according to claim 2 wherein the fluorescence from the first signal dye has a peak at a first peak frequency;

the fluorescence from the second signal dye has a peak at a second peak frequency;

the first wavelength band includes the first peak frequency; and

the second wavelength band includes the second peak frequency.

4. A method according to claim 2 wherein the first and second wavelength bands are separated by an intermediate wavelength band whose width is 0 to 1 times the sum of the widths of the first and second wavelength bands.

5. A method according to claim 4 wherein the first wavelength band has an upper limit and the second wavelength band has a lower limit which is the same as the upper limit of the first wavelength band.

6. A method according to claim 2 which includes assessing the fluorescence in an intermediate wavelength band between the first and second wavelength bands, and, when step (A) determines that the particle is a single-assay particle, combining the fluorescences in the first, second and intermediate wavelength bands.

7. A method according to claim 2 wherein the single assay particles in one category interact with thyroid stimulating hormone.

8. A method of examining a composition which comprises a plurality of particles, each of the particles

(i) having a coding characteristic,

(ii) containing analyte-interaction sites and/or corresponding analyte-bearing sites which are the same as said analyte-interaction sites except that they have interacted with one or more analytes and have associated therewith one or more fluorochromic signal dyes, and

(iii) belonging to one only of a plurality of defined categories, each of the particles in each defined category

(a) having the same coding characteristic, and

(b) containing the same analyte-interaction sites and/or corresponding analyte-bearing sites;

the combination of the coding characteristic and the analyte-interaction sites and/or corresponding analyte-bearing sites on the particles in each category being different from the combination of the coding characteristic and the analyte-interaction sites and/or corresponding analyte-bearing sites on the particles in other categories;

the method comprising examining, one at a time, each particle of a representative sample of the particles, the examination comprising;

(A) determining the coding characteristic of the particle, including whether the particle is a single-assay particle or a dual-assay particle;

(B) subjecting the particle to radiation from a laser which causes fluorescence of any fluorochromic signal dye associated with analyte-bearing sites on the particle;

(C) assessing the fluorescence caused by the laser and falling within a first wavelength band;

(D) assessing the fluorescence caused by the laser and falling within a second wavelength band; and

(E) when the fluorescence assessed in steps (C) and (D) results from only a single signal dye, combining the fluorescence in the first wavelength band and the fluorescence in the second wavelength band, and

when the fluorescence assessed in steps (C) and (D) results from two or more signal dyes, recording separately the fluorescence in the first wavelength band and the fluorescence in the second wavelength band.

9. A method according to claim 8 wherein

(A) the plurality of particles includes

(i) dual-assay particles containing analyte-bearing sites which

(a) have interacted with a first analyte and have associated therewith a first fluorochromic signal dye, or

(b) have interacted with a second analyte and have associated therewith a second fluorochromic signal dye;

and

(ii) single-assay particles containing analyte-bearing sites which have interacted with an analyte and have associated therewith a fluorochromic signal dye which is the same as the first or the second signal dye or is different therefrom;

(B) the laser (i) causes the first fluorochromic signal dye, if present, to fluoresce in a first spectrum, (ii) causes the second fluorochromic signal dye, if present, to fluoresce in a second spectrum which is different from but overlaps the first spectrum, and (iii) causes the fluorochromic signal dye or dyes associated with the single-assay particles, if present, to fluoresce in a spectrum which includes at least one of the first wavelength band and the second wavelength band.

10. A method according to claim 9 which includes assessing the fluorescence in an intermediate wavelength band between the first and second wavelength bands, and, when the fluorescence assessed in steps (C) and (D) results from only a single signal dye, combining the fluorescences in the first, second and third wavelength bands.