Claims for Patent: 7,148,029

Summary for Patent: 7,148,029

Title:	Immunoassay for neonicotinyl insecticides
Abstract:	The present invention provides immunogens for generating anti-neonicotinoid antibodies as well as antibodies, methods, reagents and kits for determining the presence of one or more neonicotinoid insecticides in a sample by immunoassay. The present invention has particular application to determination of the concentration of neonicotinoid insecticides applied to a plant propagation materials such as seed.
Inventor(s):	Brady; James Francis (Summerfield, NC), Simmons; Dana Philip (Summerfield, NC), Wilson; Timothy Edward (Summerfield, NC)
Assignee:	Syngenta Crop Protection, Inc. (Greensboro, NC)
Application Number:	10/149,512
Patent Claims:	1. A protein conjugate of the formula ##STR00053## wherein A is 2-chloropyrid-5-yl or 2-chlorothiazol-5-yl; R.sup.3 is hydrogen or C.sub.1 C.sub.4alkyl; R.sup.1 and R.sup.2 independently are hydrogen or C.sub.1 C.sub.4alkyl or R.sup.1 and R.sup.2 are taken together with the nitrogen atoms to which they are attached to form an imidazoline or an oxadiazine ring; n is an integer of from 1 to 4; X is N--NO.sub.2 or N--CN; and PR an immunogenic is a protein residue. 2. An antibody useful in an immunoassay of a sample to determine the amount of a neonicotinoid insecticide, wherein said antibody (I) is a polyclonal antibody produced by immunizing an animal with a neonicotinoid hapten conjugated to an immunogenic carrier protein and (II) specifically binds to the neonicotinoid insecticide and wherein the antibody is selective for a compound of the formula ##STR00054## wherein A is 2-chloroDyrid-5-yl or 2-chlorothiazol-5-yl; R and R.sup.3 independently are hydrogen or C.sub.1 C.sub.4alkyl; R.sup.1 are R.sup.2 independently are hydrogen or C.sub.1 C.sub.4alkyl or R.sup.1 and R.sup.2 are taken together with the nitrogen atoms to which they are attached to form an imidazoline or an oxadiazine ring; and X is N--NO.sub.2 or N--CN; wherein the antibody is prepared using the immunogen of claim 1. 3. The antibody according to claim 2, wherein said antibody is selective for a compound selected from the group consisting of imidacloprid, nitenpyram, acetamiprid, thiamethoxam, thiacloprid and clothiadin. 4. A compound of the formula ##STR00055## wherein A is 2-chloropyrid-5-yl or 2-ch lorothiazol-5-yl; R.sup.3 is hydrogen or C.sub.1 C.sub.4alkyl; R.sup.1 and R.sup.2 independently are hydrogen or C.sub.1 C.sub.4alkyl or R.sup.1 and R.sup.2 are taken together with the nitrogen atoms to which they are attached to form an imidazoline or an oxadiazine ring; n is an integer from 1 to 4; and X is N--NO.sub.2 or N--CN. 5. A compound according to claim 4 of the formula ##STR00056## 6. A compound according to claim 5 of the formula ##STR00057## 7. A compound according to claim 4 of the formula ##STR00058## 8. A compound according to claim 7 of the formula ##STR00059## 9. A protein conjugate according to claim 1 of the formula ##STR00060## 10. A protein conjugate according to claim 1 of the formula ##STR00061## 11. A method of determining the concentration of a neonicotinoid insecticide in a sample, comprising the steps of: (a) providing a solid phase with an immobilized antibody selective for said neonicotinoid insecticide wherein the said neonicotinoid insecticide is a compound of the formula ##STR00062## wherein A is 2-chloropyrid-5-yl or 2-chlorothiazol-5-yl; R and R.sup.3 independently are hydrogen or C.sub.1 C.sub.4alkyl; R.sup.1 are R.sup.2 independently are hydrogen or C.sub.1 C.sub.4alkyl or R.sup.1 and R.sup.2 are taken together with the nitrogen atoms to which they are attached to form an imidazoline or an oxadiazine ring; and X is N--NO.sub.2 or N--CN, wherein the antibody is prepared using the immunogen of claim 1; (b) contacting said sample with the immobilized antibody in the presence of a known amount of a neonicotinoid insecticide hapten-enzyme conjugate; (c) washing the solid phase of step (b) to remove any unbound hapten-enzyme conjugate or sample; (d) reacting a chromogenic substrate specific for said hapten-enzyme conjugate with the washed solid phase of step (c) in order to generate a chromogen; and (e) measuring the amount of the chromogen produced by step (d) in order to determine the amount of antibody-bound hapten-enzyme conjugate and hence the amount of neonicotinoid insecticide in said sample. 12. A method according to claim 11 wherein said sample is obtained by extracting a plant propagation material in a suitable solvent. 13. A method according to claim 12 wherein said plant propigation material is a seed. 14. A method according to claim 13 wherein said seed is selected from the group consisting of canola, sorghum, wheat, cotton, field corn or sweet corn. 15. A method according to claim 11 wherein said antibody is a polyclonal antibody produced by immunizing an animal with a neonicotinoid insecticide conjugated to an immunogenic carrier protein. 16. A method according to claim 15 wherein said immunogenic carrier protein is a carrier molecule selected from the group consisting of a purified protein derivative (PPD, Tuberculin) from Diptheria virus, bovine serum albumin, cationized bovine serum albumin, human serum albumin, ovalbumin and keyhole limpet hemocyanin. 17. A kit useful for an immunoassay of a sample to determine the amount of a neonicotinoid insecticide, wherein the kit comprises in combination in one or more containers: (a) a solid phase with an immobilized antibody selective for said neonicotinoid insecticide wherein the said neonicotinoid insecticide is a compound of the formula ##STR00063## wherein A is 2-chloropyrid-5-yl or 2-chlorothiazol-5-yl; R and R.sup.2 independently are hydrogen or C.sub.1 C.sub.4alkyl; R.sup.1 are R.sup.2 independently are hydrogen or C.sub.1 C.sub.4alkyl or R.sup.1 and R.sup.2 are taken together with the nitrogen atoms to which they are attached to form an imidazoline or an oxadiazine ring; and X is N--NO.sub.2 or N--CN; wherein the antibody is prepared using the immunogen of claim 1; (b) an enzyme conjugate comprising an enzyme conjugated to a neonicotinoid hapten. 18. The kit of claim 17 wherein the antibody is a polyclonal antibody produced by immunizing an animal with a neonicotinoid conjugated to an immunogenic carrier protein. 19. The kit of claim 18 wherein the enzyme is horseradish peroxidase. 20. The kit of claim 17 wherein the antibody specifically binds to a neonicotinoid insecticide selected from the group consisting of imidacloprid, nitenpyram, acetamiprid, thiamethoxam, thiacloprid and clothiadin. 21. The protein conjugate according to claim 1, wherein the immunogenic protein residue is a carrier molecule selected from the group consisting of a purified protein derivative (PPD, Tuberculin) from Diptheria virus, bovine serum albumin, cationized bovine serum albumin, human serum albumin, ovalbumin and keyhole limpet hemocyanin. 22. The protein conjugate according to claim 1, wherein the immunogenic protein residue is an enzyme residue selected from the group consisting of alkaline phosphatase, horseradish peroxidase and beta-galactosidase.

Details for Patent 7,148,029

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Grifols Therapeutics Llc	ALBUKED, PLASBUMIN-20, PLASBUMIN-25, PLASBUMIN-5	albumin (human)	For Injection	101138	10/21/1942		2020-12-06
Baxalta Us Inc.	BUMINATE, FLEXBUMIN	albumin (human)	Injection	101452	03/03/1954		2020-12-06
Csl Behring Ag	ALBURX	albumin (human)	Injection	102366	07/23/1976		2020-12-06
Grifols Biologicals Llc	ALBUTEIN	albumin (human)	Injection	102478	08/15/1978		2020-12-06
Instituto Grifols, S.a.	HUMAN ALBUMIN GRIFOLS	albumin (human)	Injection	103352	02/17/1995		2020-12-06
Instituto Grifols, S.a.	HUMAN ALBUMIN GRIFOLS	albumin (human)	Injection	103352	06/11/2003		2020-12-06
Csl Behring Llc	ALBUMINAR, ALBUMINAR-20, ALBUMINAR-25, ALBUMINAR-5	albumin (human)	Injection	103955	03/17/2000		2020-12-06