Immunoassay for tetrachloroisophthalonitrile (chlorothalonil), its derivatives and breakdown products

• US 5,654,178 A
• Filed: 06/07/1995
• Issued: 08/05/1997
• Est. Priority Date: 03/29/1993
• Status: Expired due to Fees

• Current Assignee: GB Biosciences Corp. (China National Chemical Corporation)
• Original Assignee: ISK Biosciences Corp. (Astrazeneca PLC)
• Inventors: Petersen, Frank P., Fitzpatrick, Donna A., Kikani, Bharat B., Rittenburg, James H.
• Primary Examiner(s): Feisee, Lila
• Application Number:
  US08/484,687
• Time in Patent Office: 790 Days
• Field of Search: 435/240.27, 530/388.9, 530/300, 514/304
• US Class Current: 435/70.21
• CPC Class Codes: C07K 16/44 against material not provided for elsewhere , e.g. haptens, metals, DNA, RNA, amino acids
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Novel derivatives of chlorothalanil® (tetrachloroisophthalonitrile, TCPN), permit conjugation of the TCPN derivative to a carrier useful as an immunogen to induce antibodies specific for TCPN or a TCPN metabolite, or a screening antigen to detect such antibodies. Polyclonal and monoclonal antibodies specific for TCPN or a TCPN metabolite, as well as hybridoma cells producing such antibodies are disclosed. Also provided are immunoassay methods for detection and quantitation of pesticide chemicals, in particular, the fungicide TCPN or a TCPN metabolite, and kits containing a TCPN derivative screening antigen, an anti-TCPN or anti-TCPN metabolite antibody useful for performing the immunoassays.

Claims

1. An antibody useful in an immunoassay to detect or measure tetrachloroisophthalonitrile (TCPN) or a TCPN metabolite in a sample, wherein

i) said TCPN metabolite is 4-hydroxy-2,5,6-trichloroisophthalonitrile, or 3-carbamyl-2,4,5-trichlorobenzoic acid;

and

ii) said antibody specifically binds TCPN or said TCPN metabolite.

2. The antibody of claim 1, which specifically binds tetrachloroisophthalonitrile, 4-hydroxy-2,5,6-trichloroisophthalonitrile, and 3-carbamyl-2,4,5-trichlorobenzoic acid.

3. The antibody of claim 1, which is a polyclonal antibody, and which specifically binds tetrachloroisophthalonitrile and exhibits no greater than about 0.5% cross-reactivity with 4-hydroxy-2,5,6-trichloroisophthalonitrile in said immunoassay.

4. The antibody of claim 1, which is a polyclonal antibody, and which specifically binds tetrachloroisophthalonitrile, and exhibits less than about 0.01% cross-reactivity with 3-carbamyl-2,4,5-trichlorobenzoic acid in said immunoassay.

5. The antibody of claim 1, which specifically binds tetrachloroisophthalonitrile.

6. The antibody of claim 1, which is a polyclonal antibody and is capable of detecting tetrachloroisophthalonitrile at about 5.0 parts per billion of said sample in said immunoassay.

7. The antibody of claim 1, which specifically binds 4-hydroxy-2,5,6-trichloroisophthalonitrile.

8. The antibody of claim 1, which specifically binds 3-carbamyl-2,4,5-trichlorobenzoic acid.

13. An antibody according to any of claims 1-12 produced by immunizing an animal with a protein carrier conjugated with a compound selected from the group consisting of 5-hydroxy-2,4,6-trichloroisophthalonitrile, 5-amino-2,4,6-trichloroisophthalonitrile, 5-mercapto-2,4,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 4-hydroxy-2,5,6-trichloroisophthalonitrile, tetrachloroisophthalonitrile, 3-carbamyl-2,4,5-trichlorobenzoic acid.

14. The antibody of claim 13, wherein said carrier protein is bovine serum albumin or keyhole limpet hemocyanin.

15. A method for isolating from a complex mixture a compound capable of binding to the antibody of claim 1 or 2, wherein said compound is (i) 4-hydroxy-2,5,6-trichloroisophthalonitrile, or 3-carbamyl-2,4,5-trichlorobenzoic acid, or (ii) a protein conjugated with a chemical selected from the group consisting of 5-hydroxy-2,4,6-trichloroisophthalonitrile, 5-amino-2,4,6-trichloroisophthalonitrile, 5-mercapto-2,4,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 4-hydroxy-2,5,6-trichloroisophthalonitrile, tetrachloroisophthalonitrile, and 3-carbamyl-2,4,5-trichlorobenzoic acid, and said method comprises:

(a) immobilizing said antibody on a solid phase support or carrier;

(b) contacting said complex mixture with said immobilized antibody allowing said compound to bind to said antibody, and washing away any unbound material; and

(c) eluting said bound compound,

thereby isolating said compound.

9. A monoclonal antibody produced by the 6F9 hybridoma cell line having ATCC accession number HB 11395.

10. Hybridoma cell line 6F9 having ATCC accession number HB 11395.

17. A method for producing a monoclonal antibody that specifically binds tetrachloroisophthalonitrile (TCPN) or a TCPN metabolite, wherein said TCPN metabolite is 4-hydroxy-2,5,6-trichloroisophthalonitrile, or 3-carbamyl-2,4,5-trichlorobenzoic acid, and which method comprises:

(a) culturing a hybridoma cell line according to claim 10 under conditions which permit antibody production and secretion by said cell line; and

(b) obtaining the culture medium containing said antibody.

11. An antibody useful in an immunoassay to detect or measure a TCPN derivative in a sample, wherein

i) said TCPN derivative is a compound selected from the group consisting of 4-amino-2,5,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 5-hydroxy-2,4,6-trichloroisophthalonitrile, and 5-amino-2,4,6-trichloroisophthalonitrile;

and

ii) said antibody specifically binds the TCPN derivative.

12. The antibody of claim 11, which specifically binds 4-amino-2,5,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 5-hydroxy-2,4,6-trichloroisophthalonitrile, and 5-amino-2,4,6-trichloroisophthalonitrile.

16. A method for producing a hybridoma cell line that produces a monoclonal antibody that specifically binds tetrachloroisophthalonitrile (TCPN) or a TCPN metabolite, wherein said TCPN metabolite is 4-hydroxy-2,5,6-trichloroisophthalonitrile, or 3-carbamyl-2,4,5-trichlorobenzoic acid, and said method comprises:

(a) immunizing a donor animal with a protein carrier conjugated with a compound selected from the group consisting of 5-hydroxy-2,4,6-trichloroisophthalonitrile, 5-amino-2,4,6-trichloroisophthalonitrile, 5-mercapto-2,4,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 4-hydroxy-2,5,6-trichloroisophthalonitrile, tetrachloroisophthalonitrile, and 3-carbamyl-2,4,5-trichlorobenzoic acid;

(b) obtaining B lymphocytes from said immunized donor animal;

(c) fusing said B lymphocytes with cells of a fusion partner cell line to obtain hybrid cells; and

(d) selecting hybrid cells which produce antibodies that specifically binds TCPN or a TCPN metabolite by;

(i) culturing said hybrid cells;

(ii) screening the medium of said cultures for the presence of an antibody which binds to TCPN or a TCPN metabolite;

thereby detecting antibody producing hybrid cells; and

(e) growing said selected hybrid cells, thereby producing said hybridoma cell line.

18. A method for producing an antibody that specifically binds tetrachloroisophthalonitrile (TCPN), a TCPN metabolite or a TCPN derivative, which comprises

i) immunizing an animal with a macromolecular carrier conjugated with a compound selected from the group consisting of 5-hydroxy-2,4,6-trichloroisophthalonitrile, 5-amino-2,4,6-trichloroisophthalonitrile, 5-mercapto-2,4,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 4-hydroxy-2,5,6-trichloroisophthalonitrile, tetrachloroisophthalonitrile, and 3-carbamyl-2,4,5-trichlorobenzoic acid;

and

ii) screening the serum of the animal for an antibody that specifically binds tetrachloroisophthalonitrile (TCPN), a TCPN metabolite or a TCPN derivative;

wherein said TCPN metabolite is 4-hydroxy-2,5,6-trichloroisophthalonitrile or 3-carbamyl-2,4,5-trichlorobenzoic acid, and said TCPN derivative is a compound selected from the group consisting of 4-amino-2,5,6-trichloroisophthalonitrile, 4-mercapto-2,5,6-trichloroisophthalonitrile, 5-hydroxy-2,4,6-trichloroisophthalonitrile, and 5-amino-2,4,6-trichloroisophthalonitrile.

19. The method according to claim 18, wherein the macromolecular carrier is a protein.