Rapid stick test for the diagnosis of bovine leukemia virus infection from serum or milk

• US 5,077,194 A
• Filed: 06/27/1988
• Issued: 12/31/1991
• Est. Priority Date: 06/20/1988
• Status: Expired due to Fees

• Current Assignee: University of Guelph Guelph Ontario Canada N1G 2W1
• Original Assignee: University of Guelph
• Inventors: Heeny, Jonathan L., Valli, Victor E. O.
• Primary Examiner(s): Rosen, Sam
• Application Number:
  US07/211,769
• Time in Patent Office: 1,282 Days
• Field of Search: 436/810, 436/528, 436/530, 436/532, 435/5, 435/7.1, 435/810
• US Class Current: 435/5
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/81 Tube, bottle, or dipstick

Abstract

A method for the detection of the presence of anti-viral or anti-bacterial antibodies in a specimen and a kit for the same. The method comprises the use of a solid phase support dipstick having attached thereto suitable antigen-binding material to which viral antigens or bacterial antigens are bound. The antibodies specific for the bound antigens bind to the antigen and a second-antibody labelled with a means for visually detecting those antibodies which have formed immunocomplexes with the bound nitrogen when in the presence of suitable reactive agents. Also, a method of using such a dipstick in this method and in the test kit is disclosed. Specifically, boving leukemia virus is tested for; a dipstick attached to a removable closure is employed; and the dipstick has two antigen binding areas, one of which is a control.

Claims

1. A test kit for the detection of antibodies to bovine leukemia virus in cow'"'"'s milk which comprises as individual components:

(a) a solid phase support dipstick having attached thereon at least two-spaced apart suitable antigen-binding substances;

a first of said antigen-binding substances having bound thereon one or more bovine leukemia virus antigens capable of forming immuno-complexes with the antibodies which may be present in the specimen, and

a second of said antigen-binding substances having bound thereon one or more control antigens for the detection of non-specific background;

(b) a series of five sealed containers with removable closures containing in series;

(i) an empty sterile container,

(ii) a first wash solution,

(iii) a solution containing enzyme conjugated to a second-antibody, said second-antibody being capable of forming an immunocomplex with the specimen antibodies,

(iv) a second wash solution,

(v) a substrate which reacts chemically with said enzymes so as to form a colored reaction product thereby indicating the presence of anti-viral antibodies in said specimen;

said dipstick being attached to a removable closure of one of said containers.

2. A kit according to claim 1 wherein said solid phase support dipstick is made of plastic.

3. A kit according to claim 2 wherein said antigenbinding substances are selected from the group consisting of nitrocellulose, polyamides and polyvinylidenes.

4. A kit according to claim 2 wherein said antigenbinding substance is nitrocellulose.

5. A kit according to claim 4 wherein said dipstick is prepared by mounting at least two nitrocellulose matrix square onto a plastic strip.

6. A kit according to claim 4 wherein said wash solution is PBS.

7. A kit according to claim 4 wherein said wash solutions are mixtures of polyoxyethylenesorbitan monolaurate and PBS.

8. A kit according to claim 4 wherein said enzyme is alkaline phosphatase.

9. A kit according to claim 8 wherein said substrate is selected from the group consisting of 5-bromo-4-chloro-3-indolyl-phosphate in the presence of nitroblue tetrazolium, naphthal AS-MX and AS-B1 phosphate in the presence of Fast Red or Fast Blue.

10. A kit according to claim 4 wherein said enzyme is horeradish peroxidase.

11. A kit according to claim 4 wherein said enzyme is β

-galactosidase and said substrate is 5-bromo-4-chloro-3-indolyl-β

-D-galactopyranoside.

12. A kit according to claim 4 wherein said second-antibody is monoclonal.

13. A kit according to claim 4 wherein said second-antibody is polyclonal and affinity purified.

14. A kit according to claim 12 wherein said monoclonal second-antibody specifically recognizes the IgG immunoglobulin molecules of the animal specie from which the specimen was taken.

15. A kit according to claim 12 wherein said monoclonal second-antibody specifically recognizes the IgA immunoglobulin molecules of the animal specie from which the specimen was taken.

16. A kit according to claim 13 wherein said monoclonal second-antibody specifically recognizes the IgM immunoglobulin molecules of the animal specie from which the specimen was taken.

17. A kit according to claim 13 wherein said polyclonal second-antibody specifically recognizes the IgG immunoglobulin molecules of the animal specie from which the specimen was taken.

18. A kit according to claim 13 wherein said polyclonal second-antibody specifically recognizes the IgA immunoglobulin molecules of the animal specie from which the specimen was taken.

19. A kit according to claim 13 wherein said polyclonal second-antibody specifically recognizes the IgM immunoglobulin molecules of the animal specie from which the specimen was taken.

20. A kit according to claim 4 wherein the bovine leukemia virus antigens are bound to the nitrocellulose so as to form a spot of antigen concentration at the distal end of the dipstick.