Monoclonal antibodies and detection methods for enzymes that confer resistance to 2,4-dichlorophenoxyacetic acid
Described herein are monoclonal antibodies and methods useful for determining and quantitating the presence of AAD-1 (aryloxyalkanoate dioxygenase) enzyme. These monoclonal antibodies are surprisingly well suited for detecting AAD-1 transgenic event gene products in a variety of plants and plant tissues. The invention further provides quantitative and qualitative immunoassays using the immunoglobulins of the invention.
1. An isolated monoclonal antibody that specifically binds to an aryloxyalkanoate-1 dioxygenase enzyme (AAD-1), wherein the monoclonal antibody is produced by a hybridoma on deposit with the American Type Culture Collection (ATCC) under an Accession Number selected from the group consisting of PTA-11251 (473F185.2#18), PTA-11252 (473F225.2#7), PTA-11253 (73F274.21136) (473F274.2#36), PTA-11254 (473H46.2), and PTA-11255 (473H172.3).
2. The monoclonal antibody of claim 1 produced by the hybridoma having Accession Number PTA-11251 (473F185.2#18).
3. The monoclonal antibody of claim 1 produced by the hybridoma having Accession Number PTA-11252 (473F225.2#7).
4. The monoclonal antibody of claim 1 produced by the hybridoma having Accession Number PTA-11253 (473F274.2#36).
5. The monoclonal antibody of claim 1 produced by the hybridoma having Accession Number PTA-11254 (473H46.2).
6. The monoclonal antibody of claim 1 produced by the hybridoma having Accession Number PTA-11255 (47311172.3).
7. A hybridoma cell line that produces a monoclonal antibody of claim 1 and is on deposit with the American Type Culture Collection (ATCC) under an Accession Number selected from the group consisting of PTA-11251 (473F185.2#18), PTA-11252 (473F225.2#7), PTA-11253 (473F274.2#36), PTA-11254 (4731146.2), and PTA-11255 (473H172.3).
8. The hybridoma cell line of claim 7 deposited under ATCC Accession Number PTA-11251 (473F185.2#18).
9. The hybridoma cell line of claim 7 deposited under ATCC Accession Number PTA-11252 (473F225.2#7).
10. The hybridoma cell line of claim 7 deposited under ATCC Accession Number PTA-11253 (473F274.2#36).
11. The hybridoma cell line of claim 7 deposited under ATCC Accession Number PTA-11254 (473H46.2).
12. The hybridoma cell line of claim 7 deposited under ATCC Accession Number PTA-11255 (473H172.3).
13. A method for identifying the presence of AAD-1 enzyme, comprising:
a) immobilizing a first monoclonal antibody of claim 1 onto an assay surface, and then washing said assay surface;
b) contacting said assay surface with a liquid suspected of containing AAD-1 enzyme for a period of time sufficient to allow binding, and then washing said assay surface;
c) contacting said assay surface with a different second antibody of claim 1 conjugated to a reporting group for a period of time sufficient to allow binding of said second conjugated monoclonal antibody, and then washing said assay surface; and,
d) detecting the presence or absence of said reporting group, wherein the presence of said reporting group indicates the presence of AAD-1 enzyme in the liquid.
14. A method for the quantitative determination of AAD-1 enzyme, comprising:
a) immobilizing an AAD-1 antiserum onto an assay surface;
b) contacting said assay surface with a liquid suspected of containing AAD-1 enzyme for a period of time sufficient to allow binding, and then washing said assay surface;
c) contacting said assay surface with a monoclonal antibody of claim 1 conjugated to a reporting group for a period of time sufficient to allow binding of said conjugated monoclonal antibody, and then washing said assay surface; and
d) quantifying the presence of said reporting group by comparison to a calibration curve, wherein the presence of said reporting group indicates the presence of AAD-1 enzyme in the liquid.
15. The method of claim 14 wherein the conjugated monoclonal antibody is produced by the hybridoma cell line having ATCC Accession Number PTA-11251 (473F1852#18).
16. A monoclonal antibody that specifically binds to an aryloxyalkanoate-1 dioxygenase enzyme (AAD-1) labeled with a reporting group, wherein the monoclonal antibody is produced by a hybridoma on deposit with the American Type Culture Collection (ATCC) under an Accession Number selected from the group consisting of PTA-11251 (473F185.2418), PTA-11252 (473F225.2#7), PTA-11253 (473F274.2#36), PTA-11254 (473H46.2), and PTA-11255 (473H172.3).
17. The monoclonal antibody of claim 16 , wherein said reporting group is a radioisotope or an enzyme.
18. An isolated monoclonal antibody that specifically binds to an aryloxyalkanoate-1 dioxygenase enzyme (AAD-1) immobilized on a surface, wherein the monoclonal antibody is produced by a hybridoma on deposit with the American Type Culture Collection (ATCC) under an Accession Number selected from the group consisting of PTA-11251 (473F185.2#18), PTA-11252 (473F225.2#7), PTA-11253 (473F274.2#36), PTA-11254 (473H46.2), and PTA-11255 (47311172.3).
19. The isolated antibody of claim 18 , wherein said monoclonal antibody is covalently coupled to said surface.