METHODS FOR DETECTING AND MEASURING AGGREGATION

• US 20140045170A1
• Filed: 07/17/2013
• Published: 02/13/2014
• Est. Priority Date: 07/18/2012
• Status: Active Grant

• Current Assignee: Labrador Diagnostics LLC (SoftBank Group Corp.)
• Original Assignee: Theranos Incorporated
• Inventors: Anekal, Samartha, Holmes, Elizabeth A., Patel, Paul, Gibbons, Ian
• Granted Patent: US 9,389,229 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: B01L 3/5021 Test tubes specially adapted for centrifugation purposes centrifuges B04B5/04
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/556 Fixed or stabilised red blood cell
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/86 involving blood coagulating time or factors, or their receptors

Abstract

Methods, compositions, systems, and devices are provided for performing and analyzing agglutination assays. In one aspect, methods for image analysis of agglutination assays are provided. In another aspects, methods for performing agglutination assays are provided. In one aspect, the methods may be used for the detection of various molecules, including viruses or antibodies against a virus. In another aspect, the methods can be used to determine effective immunization of a subject.

Claims

1. A method for determining the presence of an antibody in a biological sample, wherein said antibody binds selectively to a viral particle, the method comprising:

(a) incubating a mixture of erythrocytes, the viral particle, and the biological sample suspected of containing said antibody, under conditions permitting agglutination of the erythrocytes via interaction with said viral particle; and

(b) detecting whether said agglutination occurs in said mixture, wherein the absence of said agglutination indicates the presence of said antibody,

wherein said steps (a)-(b) take place in less than one hour.

2. The method of claim 1, wherein said erythrocytes are pre-fixed.

6. The method of claim 1, wherein said biological sample comprises plasma or serum.

8. The method of claim 1, wherein said biological sample is pre-treated with neuraminidase.

9. The method of claim 8, wherein said pre-treatment is carried out by incubating said biological sample with neuraminidase, and wherein the final concentration of the neuraminidase in the mixture is between 0.1 and 1.5 U/L.

10. The method of claim 8, wherein said pre-treatment is carried out by incubating said sample with active neuraminidase for less than 30 minutes.

11. The method of claim 1, wherein said biological sample is serially diluted prior to step (a).

13. The method of claim 1, wherein the presence of said agglutination is evidenced by formation of erythrocyte-viral particle clusters, and wherein said clusters are captured in an imaging region of an optical device, and wherein said detecting step comprises:

(i) capturing a plurality of images of said clusters at different locations of said imaging region with the optical device; and

(ii) detecting the occurrence of said agglutination based on analysis of said images.

14. The method of claim 13, wherein said optical device comprises a camera.

15. The method of claim 13, wherein said optical device comprises a microscope.

17. The method of claim 13, wherein said analysis comprises calculating the size of said clusters based on the center-to-center distance of individual erythrocytes captured in each of said images.

18. The method of claim 1, wherein said step (a) comprises incubating a mixture of said viral particle and said biological sample before adding said erythrocytes.

3-5. -5. (canceled)

7. (canceled)

12. (canceled)

16. (canceled)

19. A method for determining the presence of an antibody in a biological sample, where said antibody binds selectively to a viral particle, the method comprising:

(a) incubating a mixture of erythrocytes, the viral particle, and the biological sample suspected of containing said antibody, under conditions permitting agglutination of the erythrocytes via interaction with said viral particle; and

(b) capturing with the aid of an optical device an image of said mixture, wherein the presence of an erythrocyte-viral particle cluster in said image indicates the occurrence of said agglutination and lack of detectable amount of said antibody, and wherein the absence of said cluster indicates the lack of said agglutination and the presence of detectable amount of said antibody.

20. The method of claim 19, wherein the formation of said erythrocyte-viral particle cluster is determined by assessing center-to-center distance between individual erythrocytes captured in said image.

21. (canceled)

22. A method for determining the presence of a viral particle in a biological sample, comprising:

(a) incubating a mixture of erythrocytes and a biological sample suspected of containing said viral particle, under conditions permitting agglutination of the erythrocytes via interaction with said viral particle; and

(b) detecting whether said agglutination occurs in said mixture, wherein the presence of said agglutination indicates the presence of said viral particle,

and wherein steps (a)-(b) take place in less than one hour.

28. The method of claim 22 wherein said erythrocytes are pre-fixed.

50. The method of claim 22 wherein the presence of said agglutination is evidenced by formation of erythrocyte-viral particle clusters, and wherein said clusters are captured in an imaging region of an optical device, and wherein said detecting step comprises:

(i) capturing a plurality of images of said clusters at different locations of said imaging region with the optical device; and

(ii) detecting the occurrence of said agglutination based on analysis of said images.

51. The method of claim 50, wherein said optical device comprises a camera.

52. The method of claim 50, wherein said optical device comprises a microscope.

54. The method of claim 50, wherein said analysis comprises calculating the size of said clusters based on the center-to-center distance of individual erythrocytes captured in each of said images.

23-27. -27. (canceled)

29-49. -49. (canceled)

53. (canceled)

55-80. -80. (canceled)