Claims for Patent: 6,416,962

Summary for Patent: 6,416,962

Title:	Method and device for identifying a mycobacterium species responsible for a mycobacterial infection
Abstract:	The invention relates to a method for identifying a Mycobacterium species responsible for a mycobacterial infection in human or animal, comprising selecting a suitable mycobacterial species and strain; preparing at least one mycobacterial antigen, respectively antigen preparation; binding the antigen, respectively the antigen preparation to a suitable carrier; causing the binding antigen to react with antibodies from serum of an individual infected with a Mycobacterium species; making visible antigen-antibody reactions for a suitable antibody (sub-)class; and identifying the responsible Mycobacterium species on the basis of the reactions which are made visible. The invention further provides a diagnostic kit which takes the form of a dip-stick on which is arranged a carrier strip with mycobacterial antigens binding thereto, and means for visualizing antigen-antibody reactions occurring on the carrier after contact with the serum for testing. In another embodiment the diagnostic kit comprises a microtiter plate, in the wells of which a specified antibody is arranged, and means for making visible antigen-antibody reactions occurring in the wells after contact with the serum for testing. The third embodiment is an immunoblot with mycobacterial antigens separated by electrophoresis binding thereto, and means for visualizing antigen-antibody reactions occurring on the immunoblot after contact with the serum for testing.
Inventor(s):	Das; Pranab Khumar (Castricum, NL), Van Es; Remco Maria (Koog aan de Zaan, NL), Houthoff; Hendrik Jan (Amsterdam, NL)
Assignee:	Kreatech Biotechnology B.V. (Amsterdam, NL)
Application Number:	09/166,663
Patent Claims:	1. A method for identifying a Mycobacterium species responsible for a mycobacterial infection in a patient by testing a sample comprising antibodies from the patient, the method comprising the steps of: (a) providing an antigenic preparation of a Mycobacterium species, wherein the antigen preparation comprises two or more immuno-cross-reactive antigen components (ImCRACs); wherein the ImCRACs are bound to a solid carrier to form carrier-bound ImCRACs; (b) contacting the carrier-bound ImCRACs with the sample under conditions suitable for antibody-antigen binding, to provide a set of carrier-bound antibody-ImCRACs; and (c) detecting the set of carrier-bound antibody-ImCRACs; wherein the set of carrier-bound antibody-ImCRACs is characteristic of the Mycobacterium species. 2. The method according to claim 1, wherein the sample comprising antibodies from the patient is a serum sample. 3. The method according to claim 1, wherein the set of the carrier-bound antibody-ImCRACs identifies the Mycobacterium species according to one of the following: a set of antibody-ImCRACs consisting of ImCRACs 10 Kda, 14 Kda, 16 Kda, 10-16 Kda, 22 Kda, 22-28 Kda, 29/33 Kda, 31 Kda, 33 Kda, 33-38 Kda, 38-40 Kda, 58-60 Kda, 68 Kda and 64/65 Kda identifies M. tuberulosis; a set of antibody-ImCRACs consisting of the ImCRAC 10-6 Kda identifies M. bovis, a set of antibody-ImCRACs consisting of ImCRACs 10-16 Kda, 58-60 Kda and 68 Kda identifies M. avium, a set of antibody-ImCRACs consisting of ImCRACs 29/33 Kda and 64/65 Kda identifies M. leprae, a set of antibody-ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 27 Kda, 29/33 Kda, 31 Kda, 45/48 Kda, 664/65 Kda and 66 Kda identifies Bacillus Calmette-Guerin, and a set of antibody-ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 45/48 Kda and 66 Kda identifies RIVM 7114. 4. The method according to claim 1, wherein the infection by the Mycobacterium species is associated with one or more of the following diseases: tuberculosis, Johne's disease, Crohn's disease, rheumatoid arthritis, tuberculous leprosy or lepromatous leprosy. 5. The method according to claim 1, wherein the antigen preparation of step (a) is separated by electrophoresis and the carrier is a membrane to which the ImCRACS are transferred by electroblotting. 6. The method according to claim 5, wherein the membrane is a nitrocellulose membrane. 7. The method according to claim 5, wherein the set of the carrier-bound antibody-ImCRACs comprise a band, a doublet, an extra band or a smear. 8. The method according to claim 1, wherein the antigen preparation of the Mycobacterium species is a total protein preparation. 9. The method according to claim 8, wherein the antigen preparation is a KP-100 fraction or an SP-100 fraction of the total protein preparation. 10. The method according to claim 1, wherein the solid carrier is a surface of a microtiter plate well. 11. The method according to claim 10, wherein each of the two or more ImCRACs of the antigen preparation are separated and each is bound to a surface of a separate well of the microtiter plate. 12. The method according to claim 11, wherein a step of: removing the antibodies that do not bind, is incorporated between steps (b) and (c). 13. The method according to claim 1, wherein the ImCRAC is characterized by molecular weight. 14. The method according to claim 13, wherein the set of the carrier-bound antibody-ImCRACs is a set comprising antibody-ImCRACs including an antigen having a molecular weight selected from the group consisting of 10-16 Kda, 14 Kda, 20-28 Kda, 27 Kda, 29/33 Kda (doublet), 31 Kda, 31-40 Kda, 38-40 Kda, 45/48 Kda (doublet), 58 60 Kda, 64/65 Kda (doublet), 66 Kda and 68 Kda. 15. The method according to claim 13, wherein the two or more sets of carrier-bound antibody-ImCRACs characteristic of particular Mycobacterium species comprise a Mycobacterium species selected from the group consisting of M. tuberculosis, M. bovis, M. avium, Bacillus Calmette-Guerin and RIVM 7114. 16. The method according to claim 1, wherein the carrier-bound antibody-ImCRACs are detected by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM and IgA. 17. The method according to claim 16, wherein the antibody-enzyme conjugate acts on a fluorogenic compound to produce a detectable fluorescent compound. 18. The method according to claim 16, wherein the carrier-bound antibody-ImCRACs are detected by an immunometric assay. 19. The method according to claim 16, wherein the antibody-enzyme conjugate acts on a chromogenic compound to produce a detectable colored compound. 20. The method according to claim 19, wherein the enzyme of the antibody-enzyme conjugate is selected from the group consisting of peroxidase, beta-galactosidase and alkaline phosphatase. 21. The method according to claim 16, wherein the solid carrier is a dip-stick. 22. The method according to claim 21, wherein the set of the carrier-bound antibody-ImCRACs detected on the dipstick is a set of ImCRACs comprising a band or doublet selected from the group consisting of the 10-16 Kda band, the 14 Kda band, the 20-28 Kda band, the 27 Kda band, the 29/33 Kda doublet, the 31 Kda band, the 31-40 Kda band, the 38-40 Kda band, the 45/48 Kda doublet, the 58-60 Kda band, the 64/65 Kda doublet, the 66 Kda band and the 68 Kda band. 23. The method according to claim 21, wherein the set of ImCRACs identifies the Mycobacterium species according to one of the following: a set of ImCRACs consisting of ImCRACs 10 Kda, 14 Kda, 16 Kda, 10-16 Kda, 22 Kda, 22-28 Kda, 29/33 Kda, 31 Kda, 33 Kda, 33-38 Kda, 38-40 Kda, 58-60 Kda, 68 Kda and 64/65 Kda identifies M. tuberculosis; a set of ImCRACs consisting of the ImCRAC 10-16 Kda identifies M. bovis, a set of ImCRACs consisting of ImCRACs 10-16 Kda, 58-60 Kda and 68 Kda identifies M. avium, a set of ImCRACs consisting of ImCRACs 29/33 Kda and 64/65 Kda identifies M. leprae, a set of ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 27 Kda, 29/33 Kda, 31 Kda, 45/48 Kda, 664/65 Kda and 66 Kda identifies Bacillus Calmette-Guerin, and a set of ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 45/48 Kda and 66 Kda identifies RIVM 7114. 24. A method for detecting or identifying an antibody reactive with a Mycobacterium species in a sample, comprising: (a) providing a solid phase carrier having bound thereto an antigen preparation from a culture of the Mycobacterium species, wherein the antigen preparation comprises two or more immuno-cross-reactive antigen components (ImCRACs), forming carrier-bound ImCRACs; (b) contacting the carrier-bound ImCRACs with the sample under conditions suitable for binding of antibodies in the sample to the carrier-bound ImCRACs to provide a set of carrier-bound antibody-ImCRACs; and (c) detecting the set of carrier-bound antibody-ImCRACs; wherein the binding of the carrier-bound ImCRACs to antibodies of the sample exhibits a set of antibody-ImCRACs characteristic of the Mycobacterium species. 25. The method according to claim 24, wherein the antigen preparation is separated by electrophoresis and the carrier is a membrane to which the ImCRACS are bound by electroblotting. 26. The method according to claim 24, wherein the membrane is a nitrocellulose membrane. 27. The method according to claim 24, wherein the antigen preparation of the Mycobacterium species is a total protein preparation. 28. The method according to claim 24, wherein the antigen preparation is a KP-100 or SP-100 fraction of the total protein preparation. 29. The method according to claim 24, wherein the set of the carrier-bound antibody-ImCRAC comprises an ImCRAC selected from the group consisting of: 10-16 Kda, 14 Kda, 20-28 Kda, 27 Kda, 29/33 Kda, 31 Kda, 31-40 Kda, 38-40 Kda, 45/48 Kda, 58-60 Kda, 64/65 Kda, 66 Kda and 68 Kda. 30. The method according to claim 24, wherein the set of ImCRACs identifies the Mycobacterium species according to one of the following: a set of ImCRACs consisting of ImCRACs 10 Kda, 14 Kda, 16 Kda, 10-16 Kda, 22 Kda, 22-28 Kda, 29/33 Kda, 31 Kda, 33 Kda, 33-38 Kda, 38-40 Kda, 58-60 Kda, 68 Kda and 64/65 Kda identifies M. tuberulosis; a set of ImCRACs consisting of ImCRAC 10-16 Kda identifies M. bovis, a set of ImCRACs consisting of ImCRACs 10-16 Kda, 58-60 Kda and 68 Kda identifies M. avium, a set of ImCRACs consisting of ImCRACs 29/33 Kda and 64/65 Kda identifies M. leprae, a set of ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 27 Kda, 29/33 Kda, 31 Kda, 45/48 Kda, 664/65 Kda and 66 Kda identifies Bacillus Calmette-Guerin, and a set of ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 45/48 Kda and 66 Kda identifies RIVM 7114. 31. The method according to claim 24, wherein the library of two or more sets of carrier-bound Ab-ImCRAC complexes characteristic of particular Mycobacterium species comprises a Mycobacterium species selected from the group consisting of M. tuberculosis, M. bovis, M. avium, Bacillus Calmette-guerin and RIVM 7114. 32. The method according to claim 24, wherein the solid phase carrier is a surface of a well of a microtiter plate. 33. The method according to claim 32, wherein each of the two or more ImCRACs of the antigen preparation are separated and each is bound to a surface of a separate well of the microtiter plate. 34. The method according to claim 33, wherein a step of: removing the antibodies that do not bind, is incorporated between steps (b) and (c). 35. The method according to claim 24, wherein the substrate-bound antibody-ImCRAC complexes are made visualizable by using at least one antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM and IgA. 36. The method according to claim 35, wherein the antibody-enzyme conjugate acts on a chromogenic compound to produce a detectable colored compound. 37. The method according to claim 36, wherein the enzyme of the antibody-enzyme conjugate is selected from the group consisting of peroxidase, beta-galactosidase and alkaline phosphatase. 38. The method according to claim 35, wherein the antibody-enzyme conjugate acts on a fluorogenic compound to produce a detectable fluorescent compound. 39. The method according to claim 24, wherein the solid carrier is a dip-stick. 40. The method according to claim 39, wherein the set of the carrier-bound antibody-ImCRACs detected on the dipstick is an antibody-ImCRAC set comprising a band or doublet selected from the group consisting of the 10-16 Kda band, the 14 Kda band, the 20-28 Kda band, the 27 Kda band, the 29/33 Kda doublet, the 31 Kda band, the 31-40 Kda band, the 38-40 Kda band, the 45/48 Kda doublet, the 58-60 Kda band, the 64/65 Kda doublet, the 66 Kda band and the 68 Kda band. 41. The method according to claim 39, wherein the antibody-ImCRAC set identifies the Mycobacterium species according to one of the following: a set of ImCRACs consisting of ImCRACs 10 Kda, 14 Kda, 16 Kda, 10-16 Kda, 22 Kda, 22-28 Kda, 29/33 Kda, 31 Kda, 33 Kda, 33-38 Kda, 38-40 Kda, 58-60 Kda, 68 Kda and 64/65 Kda identifies M.tuberulosis; a set of ImCRACs consisting of ImCRAC 10-16 Kda identifies M. bovis, a set of ImCRACs consisting of ImCRACs 10-16 Kda, 58-60 Kda and 68 Kda identifies M. avium, a set of ImCRACs consisting of ImCRACs 29/33 Kda and 64/65 Kda identifies M. leprae, a set of ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 27 Kda, 29/33 Kda, 31 Kda, 45/48 Kda, 664/65 Kda and 66 Kda identifies Bacillus Calmette-Guerin, and a set of ImCRACs consisting of ImCRACs 22 Kda, 25 Kda, 45/48 Kda and 66 Kda identifies RIVM 7114. 42. A diagnostic kit for the identification of antibodies elicited by a Mycobacterium species, comprising: (i) an antigenic preparation of a Mycobacterium species, wherein the antigen preparation comprises two or more immuno-cross-reactive antigen components (ImCRACs); wherein the ImCRACs are bound to a solid carrier; and wherein binding of the carrier-bound ImCRACs to antibodies reactive with the ImCRACs exhibits a set of antibody-ImCRACs characteristic of particular Mycobacterium species; and (ii) an antibody-enzyme conjugate directed against at least one antibody of an isotype selected from the group consisting of IgG, IgM and IgA. 43. The diagnostic kit according to claim 42, wherein the solid carrier is a dip-stick, a microtiter well, a nitrocellulose membrane or a nylon membrane. 44. The diagnostic kit according to claim 42, further comprising a chromogenic substrate or a fluorogenic substrate suitable for detection of the antibody-enzyme conjugate. 45. The diagnostic kit according to claim 42, to identify M. bovis reactivity, wherein the ImCRACs comprise an ImCRAC with a molecular weight of 10-16 Kda. 46. The diagnostic kit according to claim 42, to identify M. avium, wherein the ImCRACs comprise ImCRACs with molecular weights 10-16 Kda, 58-60 Kda and 68 Kda. 47. The diagnostic kit according to claim 42, to identify M. leprae. wherein the ImCRACs comprise ImCRACs with molecular weights 29/33 Kda and 64/65 Kda. 48. The diagnostic kit according to claim 42, to identify Bacillus Calmette-Guerin, wherein the ImCRACs comprise ImCRACs with molecular weights 22 Kda, 25 Kda, 27 Kda, 29/33 Kda, 31 Kda, 45/48 Kda, 664/65 Kda and 66 Kda. 49. The diagnostic kit according to claim 42, to identify RIVM 7114, wherein the ImCRACs comprise ImCRACs with molecular weights 22 Kda, 25 Kda, 45/48 Kda and 66 Kda. 50. The diagnostic kit according to claim 42, wherein the ImCRACs comprise ImCRACs with molecular weights 10-16 Kda, 14 Kda, 20-28 Kda, 27 Kda, 29/33 Kda, 31 Kda, 31-40 Kda, 38-40 Kda, 45/48 Kda, 58-60 Kda, 64/65 Kda, 66 Kda and 68 Kda. 51. The diagnostic kit according to claim 42, wherein the enzyme of the antibody-enzyme conjugate is selected from the group consisting of peroxidase, beta-galactosidase and alkaline phosphatase. 52. The diagnostic kit according to claim 51, wherein the ImCRACs are characterized by molecular weight. 53. The diagnostic kit according to claim 52, wherein the ImCRACs comprise two or more antigens selected from the group consisting of ImCRACs of molecular weight 10 Kda, 14 Kda, 16 Kda, 10-16 Kda, 22 Kda, 22-28 Kda, 29/33 Kda, 31 Kda, 33 Kda, 33-38 Kda, 38-40 Kda, 58-60 Kda, 68 Kda and 64/65 Kda.

Details for Patent 6,416,962

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Teknika Llc	TICE BCG	bcg live	For Injection	102821	06/21/1989		2012-12-17