Determination of the number of fluorescent molecules on calibration beads for flow cytometry

• US 5,620,842 A
• Filed: 03/29/1995
• Issued: 04/15/1997
• Est. Priority Date: 03/29/1995
• Status: Expired due to Term

• Current Assignee: Becton, Dickinson & Co
• Original Assignee: Becton, Dickinson & Co
• Inventors: Davis, Kenneth A., Bishop, James E.
• Primary Examiner(s): Knode, Marian C.
• Assistant Examiner(s): FREED, RACHEL
• Application Number:
  US08/413,045
• Time in Patent Office: 748 Days
• Field of Search: 435/967, 435/4, 435/7.5, 435/180, 436/528, 436/518, 436/524, 436/531, 436/8, 436/10, 436/800
• US Class Current: 435/4
• CPC Class Codes: G01N 15/1012 Calibrating particle analysers; References therefor
• CPC Class Codes: G01N 15/14 Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 33/585 with a particulate label, e.g. coloured latex
• CPC Class Codes: G01N 33/96 involving blood or serum control standard
• CPC Class Codes: Y10S 435/967 Standards, controls, materials, e.g. validation studies, buffer systems
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/101666 Particle count or volume standard or control [e.g., platelet count standards, etc.]

Abstract

A rapid, simple method for preparing beads for calibrating flow cytometers which contain a known number of fluorophores per bead is presented. Briefly, the invention utilizes beads coated with a stable complex of a fluorophore and an enzyme. the enzymatic activity of a known number of beads gives an accurate measure of fluorophore density on those beads.

Claims

1. A method for preparing calibration beads for use in calibrating a flow cytometer and for determining the concentration of fluorophore on the calibration beads comprising:

(i) preparing a stable complex consisting of fluorophore and enzyme such that the enzymatic activity is correlated to fluorophore concentration;

(ii) coating a bead with said fluorophore-enzyme complex;

(iii) experimentally measuring the activity of the enzyme in the bead-fluorophore-enzyme complex; and

(iv) determining the concentration of fluorophore per bead using the activity of the enzyme determined in (iii).

2. The method of claim 1 wherein the fluorophore is selected from the group consisting of phycoerythrin, APC, Per-CP, and fluoresceinisothiocyanate.

3. The method of claim 1 wherein the enzyme is alkaline phosphatase.

The method of claim 1 wherein the bead is made of polymethylemethacrylate which possesses amino groups on the surface.

4. The method of claim 3 wherein a ratio of the fluorophore to the alkaline phosphatase is 1:

1 (mole/mole).

5. The method of claim 1 wherein the bead is made of polymethylemethacrylate which possesses amino groups on the surface.

6. The method of claim 5, wherein the bead is first complexed with biotin and then coated with streptavidin.

7. The method of claim 6, wherein the fluorophore-enzyme complex is biotinylated and then bound to streptavidin coated biotinylated beads.

8. A method for preparing calibration beads for use in calibrating a flow cytometer and for determining the concentration of fluorophore on the calibration beads comprising:

(i) preparing a conjugate of enzyme-fluorophore-biotin such that the enzymatic activity is correlated to fluorophore;

(ii) admixing said conjugate with a streptavidin coated bead to form a bead-streptavidin-biotin-fluorophore-enzyme complex;

(iii) experimentally measuring the activity of the enzyme in the complex; and

(iv) determining the concentration of fluorophore in the complex using the activity of the enzyme determined in (iii).

9. The method of claim 8 wherein the fluorophore is selected from the group consisting of phycoerythrin, APC, Per-CP, and fluoresceinisothiocyanate.

10. The method of claim 8 wherein the enzyme is alkaline phosphatase.

11. The method of claim 8 wherein a ratio of fluorophore to alkaline phosphatase is 1:

1 (mole/mole).

12. The method of claim 8 wherein the bead is made of polymethylemethacrylate which possesses amino groups on the surface.

13. The method of claim 12, wherein the bead is first complexed with biotin and then coated with streptavidin.

14. The method of claim 13 wherein the fluorophore-enzyme complex is biotinylated and then bound to streptavidin coated biotinylated beads.