Granted Patent B2

US 12,455,287 · App. 17/441,177 · Granted Oct 28, 2025

Inventors: Katherine D. Shives (Aurora, CO); Antje Schickert (Arvada, CO); Rebecca K. Montange (Louisville, CO); Jeffrey W. Steaffens (Broomfield, CO)

G01N33/56983G01N1/30G01N15/1459G01N2015/0038

Abstract

Claims (29)

1. A method for flow cytometry evaluation of a biological material for unassociated non-enveloped viral particles having a non-enveloped viral capsid, the method comprising:

preparing a fluorescently-stained fluid sample for flow cytometry evaluation, the fluorescently-stained fluid sample comprising:

an aqueous liquid medium;

a sample of biological material to be evaluated by flow cytometry for the unassociated non-enveloped viral particles having the non-enveloped viral capsid;

at least one fluorescent stain to fluorescently stain the unassociated non-enveloped viral particles to prepare unassociated labeled particles of virus size dispersed in the aqueous liquid medium, wherein each said unassociated labeled particle comprise a said unassociated non-enveloped viral particle stained with the at least one fluorescent stain, and wherein each said fluorescent stain has a fluorescent emission response when the unassociated labeled particle is subjected to a stimulation radiation;

after the preparing, subjecting the fluorescently-stained fluid sample to flow cytometry evaluation in a flow cytometer, the flow cytometry evaluation comprising flowing the fluorescently-stained fluid sample through an investigation zone of the flow cytometer and in the investigation zone subjecting the fluorescently-stained fluid sample to the stimulation radiation and detecting for the fluorescent emission response from the investigation zone and counting identified occurrences of the unassociated labeled particles; and

the preparing the fluorescently-stained fluid sample comprising fluorescent staining the biological material in a fluid sample composition with a said fluorescent stain to prepare a stained fluid sample composition at an acidic pH in an acidic pH range of from pH 3.0 to pH 6.5.

2. The method of claim 1 , wherein the acidic pH range is from pH 4.0 to pH 6.0.

3. The method of claim 1 , wherein the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles.

4. The method of any one of claim 3 , wherein the fluorogenic dye is a first fluorogenic dye and the at least one fluorescent stain comprises a second fluorogenic dye for staining of protein content of the viral capsid.

5. The method of claim 3 , wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a pH in the acidic pH range.

6. The method of claim 1 , wherein the preparing the fluorescently-stained fluid sample comprises diluting a preliminary fluid sample with aqueous sample dilution liquid to prepare a diluted preliminary fluid sample, wherein the aqueous sample dilution liquid has a pH in the acidic pH range.

7. The method of claim 6 , wherein the diluted preliminary fluid sample has a pH in the acidic pH range and the fluorescent staining comprises adding the said fluorescent stain to the diluted preliminary fluid sample as the fluid sample composition.

8. The method claim 1 , wherein the at least one fluorescent stain comprises a fluorescent antibody stain for binding with an epitope of the viral capsid of the unassociated non-enveloped viral particle.

9. The method of claim 1 , wherein as fed to the flow cytometer the fluorescently-stained fluid sample has a pH in the acidic pH range.

10. The method of claim 1 , wherein:

the at least one fluorescent stain comprises a fluorogenic dye for staining of nucleic acid content inside the viral capsid of the non-enveloped viral particles;

the fluorescent staining is a first fluorescent staining the biological material and the stained fluid sample composition is a first stained fluid sample composition, and the preparing the fluorescently-stained fluid sample comprises second fluorescent staining the biological material after the first fluorescent staining; and

the second fluorescent staining comprises staining the biological material with a fluorescent antibody stain to prepare a second stained fluid sample composition including both the fluorescent antibody stain and the fluorogenic dye for staining of nucleic acid content, wherein the second stained fluid sample composition is at a second pH that is larger than the acidic pH by at least 0.5 pH unit, and the second pH is pH 5.5 or larger.

11. The method of claim 1 , wherein the stained fluid sample comprises dissolved disaccharide.

12. The method of claim 3 , wherein as fed to the flow cytometer the fluorescently-stained fluid sample is the stained fluid sample composition from the fluorescent staining.

13. The method of claim 10 , wherein the second pH is pH 6.0 or larger.

14. The method of claim 1 , wherein the unassociated non-enveloped viral particles are virions.

15. The method claim 1 , wherein the unassociated non-enveloped viral particles are virus-like particles.

16. The method of claim 1 , wherein the unassociated non-enveloped viral particles are genetically modified.

17. The method of claim 1 , wherein the viral capsid is of a bacteriophage.

18. The method of claim 1 , wherein the viral capsid is of virus family Adenoviridae or of virus family Parvoviridae.

19. The method of claim 1 , wherein the unassociated non-enveloped viral particles have a particle size of from 30 nanometers to 1 micron.

20. The method of claim 11 , wherein the disaccharide comprises trehalose.

Assignments (7)

Continuity (2)

References Cited (176)