Assay for JC virus antibodies
The disclosure relates to methods and reagents for analyzing samples for the presence of JC virus antibodies. Disclosed is a method that includes obtaining a biological sample from a subject (e.g., plasma, serum, blood, urine, or cerebrospinal fluid), contacting the sample with highly purified viral-like particles (HPVLPs) under conditions suitable for binding of a JCV antibody in the sample to an HPVLP, and detecting the level of JCV antibody binding in the sample to HPVLP. In one embodiment, determining the level of anti-JCV antibodies in the subject sample provides a method of identifying PML risk in a subject.
1. A set of reaction mixtures comprising a first reaction mixture in contact with a first substrate and a second reaction mixture in contact with a second substrate, wherein
the first reaction mixture comprises:
i) a first aliquot of a sample from a subject;
ii) soluble, non-immobilized, Highly Purified Viral-Like Particles (HPVLPs) comprising VP1 polypeptides of the JC Virus (JCV); and
iii) HPVLPs immobilized to the first substrate, and
the second reaction mixture comprises:
i) a second aliquot of the sample from the subject;
ii) substantially no soluble, non-immobilized, HPVLPs; and
iii) HPVLPs immobilized to the second substrate, wherein
the sample was tested in an ELISA to detect the presence or absence of anti-JCV antibodies and was classified as indeterminate for the presence or absence of anti-JCV antibodies.
2. The set of reaction mixtures of claim 1 , further comprising a third reaction mixture, wherein the third reaction mixture comprises an aliquot of a sample isolated from an individual known to be infected with JC virus and no soluble HPVLPs.
3. The set of reaction mixtures of claim 1 , further comprising a fourth reaction mixture, wherein the fourth reaction mixture does not comprise the sample or HPVLPs.
4. The set of reaction mixtures of claim 1 , wherein one or more of the reaction mixtures further comprise a diluent.
5. The set of reaction mixtures of claim 4 , wherein the diluent is selected from a solution that includes BSA, phosphate buffered saline (PBS), or PBS containing polyoxy ethylene (20)-sorbitane mono-laurate.
6. The set of reaction mixtures of claim 1 , wherein the sample aliquots are at a 1:200 dilution in the reaction mixtures.
7. The set of reaction mixtures of claim 1 , wherein the sample is plasma, serum, blood, urine, or cerebrospinal fluid.
8. The set of reaction mixtures of claim 1 , wherein the soluble HPVLPs are at a concentration of 1 μg/ml.
9. The set of reaction mixtures of claim 1 , wherein at least 20% of the HPVLPs comprise more than five VP1 polypeptides in an HPVLP.
10. The set of reaction mixtures of claim 1 , wherein at least 70% of the HPVLPs comprise more than five VP1 polypeptides in an HPVLP.
11. The set of reaction mixtures of claim 1 , wherein an HPVLP consists essentially of VP1 polypeptides.
12. The set of reaction mixtures of claim 1 , wherein an HPVLP further comprises at least one of a VP2 or a VP3 of the JCV.
13. The set of reaction mixtures of claim 1 , wherein the VP1 in the HPVLPs is a recombinant VP1.
14. The set of reaction mixtures of claim 1 , wherein at least one VP1 in the HPVLPs is a mutant VP1.
15. The set of reaction mixtures of claim 1 , wherein each HPVLP is composed of about 5, 10, 20, 30, 40, 50, 60, 70 or 72 VP1 pentamers.
16. The set of reaction mixtures of claim 1 , wherein each HPVLP is composed of about 50, 150 or 360 VP1 polypeptides.
17. The set of reaction mixtures of claim 1 , wherein the sample is from a subject from whom the level of anti-JCV antibodies have previously been determined at an earlier date.
18. The set of reaction mixtures of claim 17 , wherein the subject was assigned a high, moderate, or low risk of PML based on the level of anti-JCV antibodies in the subject on the first date.
19. The set of reaction mixtures of claim 1 , wherein the sample is from a subject who tested positive for JCV DNA in urine.
20. The set of reaction mixtures of claim 1 , wherein the sample is from a subject who previously tested positive for anti-JCV antibodies.
21. The set of reaction mixtures of claim 1 , wherein the sample is from a subject who previously tested negative for anti-JCV antibodies.
22. A substrate having a first and second chamber, wherein:
i) the first chamber comprises:
a first aliquot of a sample from a subject,
soluble, non-immobilized, Highly Purified Viral-Like Particles (HPVLPs) comprising VP1 polypeptides of the JC Virus (JCV), and
ii) the second chamber comprises:
a second aliquot of the sample from the subject;
is substantially free of soluble, non-immobilized, HPVLPs, and
wherein the sample was tested in an ELISA to detect a presence or absence of anti-JCV antibodies and was classified as indeterminate for the presence or absence of anti-JCV antibodies.
23. The substrate of claim 22 , further comprising a third chamber, wherein the third chamber comprises an aliquot of a sample isolated from an individual known to be infected with JC virus and is substantially free of soluble HPVLPs.
24. The substrate of claim 22 , further comprising a fourth chamber, wherein the fourth chamber comprises no sample and is substantially free of soluble HPVLPs.
25. The substrate of claim 22 , wherein the substrate is a microtiter plate, a slide, a bead, or a column.
26. The substrate of claim 22 , wherein the chambers further comprise a nonspecific protein that is antigenically neutral selected from bovine serum albumin (BSA), casein, or a solution of milk powder.
27. The substrate of claim 22 , wherein one or more of the chambers further comprises adiluent.
28. The substrate of claim 22 , wherein the diluent is selected from a solution that includes BSA, phosphate buffered saline (PBS), or PBS containing polyoxyethylene (20) sorbitane mono-laurate.
29. The substrate of claim 22 , wherein the sample aliquots are at a 1:200 dilution in the respective chambers.
30. The substrate of claim 22 , wherein the sample is plasma, serum, blood, urine, orcerebrospinal fluid.
31. The substrate of claim 22 , wherein the HPVLP is at a concentration of 1 μg/ml.
32. The substrate of claim 22 , wherein at least 20% of the HPVLPs comprise more than five VP1 polypeptides in an HPVLP.
33. The substrate of claim 22 , wherein at least 70% of the HPVLPs comprise more than five VP1 polypeptides in an HPVLP.
34. The substrate of claim 22 , wherein an HPVLP consists essentially of VP1 polypeptides.
35. The substrate of claim 22 , wherein an HPVLP further comprises at least one of a VP2 or a VP3 of the JCV.
36. The substrate of claim 22 , wherein the VP1 in the HPVLPs is a recombinant VP1.
37. The substrate of claim 22 , wherein at least one VP1 in the HPVLPs is a mutant VP1.
38. The substrate of claim 22 , wherein each HPVLP is composed of about 5, 10, 20, 30, 40, 50, 60, 70 or 72 VP1 pentamers.
39. The substrate of claim 22 , wherein each HPVLP is composed of about 50, 150 or 360 VP1 polypeptides.
40. The substrate of claim 22 , wherein the sample is from a subject from whom the level of anti-JCV antibodies have previously been determined at an earlier date.
41. The substrate of claim 40 , wherein the subject was assigned a high, moderate, or low risk of PML based on the level of anti-JCV antibodies in the subject on the first date.
42. The substrate of claim 22 , wherein the sample is from a subject who tested positive for JCV DNA in urine.
43. The substrate of claim 22 , wherein the sample is from a subject who previously tested positive for anti-JCV antibodies.
44. The substrate of claim 22 , wherein the sample is from a subject who previously tested negative for anti-JCV antibodies.
45. A kit comprising the set of reaction mixtures of claim 1 .
46. A kit comprising the substrate of claim 22 .
47. The set of reaction mixtures of claim 1 , wherein the sample is from a subject taking an immunomodulator, a subject considering taking an immunomodulator, or a subject suspected of having Progressive Multifocal Leukoencephalopathy (PML).
48. The substrate of claim 22 , wherein the sample is from a subject taking an immunomodulator, a subject considering taking an immunomodulator, or a subject suspected of having Progressive Multifocal Leukoencephalopathy (PML).
49. The substrate of claim 22 , wherein the substrate further comprises HPVLPs immobilized on the substrate.
50. The set of reaction mixtures of claim 1 , wherein the first and second reaction mixtures are configured to distinguish cross-reactive antibodies in the sample from JCV-specific antibodies in the sample.
51. The set of reaction mixtures of claim 1 , wherein the first and second reaction mixtures identify a presence or absence of anti-JCV antibodies in the sample.
52. The substrate of claim 49 , wherein the substrate is configured to distinguish cross-reactive antibodies in the sample from JCV-specific antibodies in the sample classified as indeterminate by ELISA.
53. The substrate of claim 49 , wherein the substrate is configured identify a presence or absence of anti-JCV antibodies in the sample classified as indeterminate by ELISA.