Immunoassays for differential detection of clostridium difficile

Patent metadata

Field	Value
Publication number	US-11434264-B2
Application number	US-201615550625-A
Country	US
Kind code	B2
Filing date	Feb 12, 2016
Priority date	Feb 13, 2015
Publication date	Sep 6, 2022
Grant date	Sep 6, 2022

Abstract

Ultrasensitive and quantitative assays for detecting toxins of Clostridium difficile, which may involve digital ELISA, are provided. Also provided herein are differential detection assays that allow for distinguishing toxin B of highly virulent Clostridium difficile strains from toxin B of less virulent strains.

First claim

What is claimed is:

1. A method for measuring a level of multiple toxins of Clostridium difficile ( C. difficile ) in a stool sample suspected of containing a C. difficile strain and/or a toxin thereof, comprising:
(i) providing the stool sample suspected of containing a C. difficile strain and/or a toxin thereof,
(ii) measuring a level of toxin B of the C. difficile in the stool sample using an optical interrogation-based ELISA having a limit of detection of no greater than 20 pg/mL for toxin B in stool, and
(iii) measuring a level of toxin A of the C. difficile in the stool sample using an optical interrogation-based ELISA having a limit of detection of no greater than 10 pg/mL for toxin A in stool.

2. A method for treating Clostridium difficile ( C. difficile ) infection in a subject, comprising: selecting a subject having a diagnosis of C. difficile infection, the diagnosis a made by a method comprising:
(i) determining that the subject manifests a symptom associated with C. difficile infection; and (ii) determining a level of toxin A, toxin B, or both in a stool sample obtained from the subject, using an optical interrogation-based assay having a limit of detection of about: (a) no greater than 10 pg/mL for toxin A in stool, and (b) no greater than 20 pg/mL for toxin B in stool; and administering a suitable anti- C. difficile drug to the subject.

3. The method of claim 2 , wherein step (ii) is performed at a level of detection of about:
(a) 0.3 to 10 pg/mL for toxin A in stool, and
(b) 1 to 20 pg/mL for toxin B in stool.

4. The method of claim 3 , wherein the level of toxin B determined being higher than 20 pg/ml indicates that the subject has C. difficile infection.

5. The method of claim 3 , wherein the symptom is diarrhea.

6. The method of claim 3 , wherein step (ii) is performed by a differential detection method to determine whether the sample contains a highly virulent strain or a less virulent strain.

7. The method of claim 1 , wherein the stool sample is processed via dilution and filtration prior to step (ii) and step (iii).

8. The method of claim 3 , wherein the method on which the diagnosis is based further comprises detecting the presence of a nucleic acid that encodes toxin A, a nucleic acid that encodes toxin B, or both, in a sample obtained from the subject.

9. The method of claim 3 , wherein the stool sample is processed via dilution and filtration prior to step (ii).

10. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 10 pg/mL for toxin A in stool.

11. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 5 pg/mL for toxin A in stool.

12. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 1 pg/mL for toxin A in stool.

13. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 0.45 pg/mL for toxin A in stool.

14. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 0.3 pg/mL for toxin A in stool.

15. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 20 pg/mL for toxin B in stool.

16. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 15 pg/mL for toxin B in stool.

17. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 5 pg/mL for toxin B in stool.

18. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 1.5 pg/mL for toxin B in stool.

19. The method of claim 3 , wherein step (ii) is performed at a level of detection of about 1 pg/mL for toxin B in stool.

20. The method of claim 3 , wherein step (ii) is not performed using a cytotoxicity assay.

21. The method of claim 1 , wherein:
(a) step (ii) is performed at a limit of detection of about 1 to 20 pg/mL for toxin B in stool, and
(b) step (iii) is performed at a limit of detection of about 0.3 to 10 pg/mL for toxin A in stool.

22. The method of claim 21 , wherein step (ii) and step (iii) each comprises detecting a fluorescence signal.

23. The method of claim 3 , wherein step (ii) comprises detecting a fluorescence signal.

24. The method of claim 21 , wherein the measuring a level of toxin B of the C. difficile in the stool sample using an optical interrogation-based ELISA comprises:
(a) exposing a plurality of capture objects that each include a binding surface having affinity for toxin B molecules to the stool sample;
(b) associating at least some of the toxin B molecules from the stool sample with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one toxin B molecule and a statistically significant fraction of the capture objects do not associate with any toxin B molecules;
(c) determining, via optical interrogation, a measure indicative of a number of the plurality of capture objects that associated with a toxin B molecule in step (b); and
(d) determining a level of toxin B in the stool sample based at least in part on the measure indicative of a number determined in step (c).

25. The method of claim 21 , wherein the determining a level of toxin A of the C. difficile in the stool sample using an optical interrogation-based ELISA comprises:
(a) exposing a plurality of capture objects that each include a binding surface having affinity for toxin A molecules to the stool sample;
(b) associating at least some of the toxin A molecules from the stool sample with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one toxin A molecule and a statistically significant fraction of the capture objects do not associate with any toxin A molecules;
(c) determining, via optical interrogation, a measure indicative of a number of the plurality of capture objects that associated with a toxin A molecule in step (b); and
(d) determining a level of toxin A in the stool sample based at least in part on the measure indicative of a number determined in step (c).

26. The method of claim 3 , wherein step (ii) comprises
(a) exposing a plurality of capture objects that each include a binding surface having affinity for toxin A molecules and/or toxin B molecules to the stool sample;
(b) associating at least some of the toxin A molecules and/or toxin B molecules from the stool sample with respect to the plurality of capture objects such that at least some of the capture objects associate with at least one toxin A molecule and/or toxin B molecule and a statistically significant fraction of the capture objects do not associate with any toxin A molecules or toxin B molecules;
(c) determining, via optical interrogation, a measure indicative of a number of the plurality of capture objects that associated with a toxin A molecule and/or a toxin B molecule in step (b); and
(d) determining a level of toxin A and/or a level of toxin B in the stool sample based at least in part on the measure indicative of a number determined in step (c).

27. The method of claim 3 , wherein the anti- C. difficile drug is an antibiotic drug.

28. The method of claim 27 , wherein the antibiotic drug comprises metronidazole, fidaxomicin, and/or vancomycin.

29. A method for treating Clostridium difficile ( C. difficile ) infection in

Assignees

• Quanterix Corp
• Beth Israel Deaconess Medical Ct Inc

Inventors

• Pollock Nira
• Kelly Ciaran
• Duffy David C
• Song Linan
• Zhao Mingwei

Classifications

• C07K14/33 Primary

  from Clostridium (G) · CPC title

• C07K2317/33

  Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity · CPC title

• C07K16/1282

  Clostridium (G) · CPC title

• G01N33/56911

  Bacteria · CPC title

• G01N2333/33

  from Clostridium (G) · CPC title