Monoclonal antibodies to hygromycin B and the method of making the same

• US 5,620,890 A
• Filed: 03/14/1995
• Issued: 04/15/1997
• Est. Priority Date: 03/14/1995
• Status: Expired due to Fees

• Current Assignee: United States Department Of Agriculture
• Original Assignee: United States Department Of Agriculture
• Inventors: Kamps-Holtzapple, Carol, Stanker, Larry H., DeLoach, John R.
• Primary Examiner(s): Eisenschenk, Frank C.
• Application Number:
  US08/404,007
• Time in Patent Office: 763 Days
• Field of Search: 530/388.9, 530/389.1, 530/389.8, 435/240.27, 935/104, 935/103
• US Class Current: 435/345
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: Y10S 435/975 Kit

Abstract

Hybridoma cell lines have been produced which secrete monoclonal antibodies that bind hygromycin B and are effective to detect hygromycin B levels as low as about 0.7 ppm. These monoclonal antibodies may be used for the detection and quantitative determination of trace amounts of hygromycin B in samples, especially in animal tissue and feed material.

Claims

1. A hybridoma cell line which produces and secretes monoclonal antibodies which bind hygromycin B and are effective to detect hygromycin B levels at about 1.4 ppm or lower.

5. The hybridoma cell line of claim 1, wherein said cell line is HB-13.

6. A monoclonal antibody produced by the hybridoma cell line of claim 1.

8. A monoclonal antibody produced by the hybridoma cell line of claim 5.

9. A kit for detecting hygromycin B in animal tissue or feed material comrising the monoclonal antibody of claim 6 and a reagent for detecting said antibody.

10. A kit for detecting hygromycin B in animal tissue or feed material comprising the monoclonal antibody of claim 8 and a reagent for detecting said antibody.

2. A hybridoma cell line which produces and secretes monoclonal antibodies which bind hygromycin B and are effective to detect hygromycin B levels at about 1.4 ppm or lower, wherein the hybridoma cell line is produced using an immunization preparation comprising hygromycin B conjugated to an immunogenic carrier through a spacer of more than 5 atoms, wherein th spacer prevents the masking of hygromycin B by the carrier protein, and and immunization protocol comprising at least three injection over a period of two months.

3. The hybridoma cell line of claim 2 wherein said spacer is 16 atoms.

4. The hybridoma cell line of claim 3 wherein said preparation is formed by the process comprising reacting hygromycin B with sulfo-succinimidyl-4-(p-maleidophenyl)-butyrate (sulfo-SMPB) in the presence of a protein carrier derivatized with 2-iminothiolane•

HCl under conditions effective to form hygromycin B-SMPB-S-carrier protein conjugate.

7. A monoclonal antibody produced by the hybridoma cell line of claim 4.

11. A kit for detecting hygromycin B in animal tissue or feed material comprising the monoclonal antibody of claim 7 and a reagent for detecting said antibody.