Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-07-13
2001-08-14
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007930, C435S007950, C435S070210, C435S452000, C435S326000, C435S345000, C435S810000, C435S975000, C436S518000, C436S528000, C436S548000, C436S021000, C436S815000, C436S824000, C436S825000, C436S901000, C436S512000, C530S388900, C530S389800, C530S391100, C530S413000, C530S807000
Reexamination Certificate
active
06274334
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to a hybridoma cell line and monoclonal antibody produced therefrom which may be used to detect the &bgr;-adrenergic agonist, ractopamine and its metabolites, particularly in animal tissues, excreta, feeds, and in human excretions.
2. Description of the Prior Art
Ractopamine is a phenethanolamine leanness-enhancing agent recently approved as a feed additive for swine by the United States Food and Drug Administration Center for Veterinary Medicine (Federal Register, 65, 4111-4112, 2000; Muirhead,
Feedstuffs,
72,1, 2000). Hogs administered dietary ractopamine exhibit increased growth rates, feed efficiencies, and greater yields of boneless, closely trimmed retail cuts (Anderson et al.,
Fat and cholesterol reduced foods: technologies and strategies,
43-73, 1990; Stites et al.,
J. Anim. Sci.,
69, 3094-3101, 1991; Watkins et al.,
J. Anim. Sci.,
68, 3588-3595, 1990) relative to untreated control animals. The positive influence of ractopamine hydrochloride on these economically important traits should make the product attractive to swine growers, and perhaps to producers of livestock species for which ractopamine is not approved.
Phenethanolamine &bgr;-agonists have a history of being used for off-label purposes by livestock producers (Kuiper et al.,
J. Anim. Sci.,
76, 195-207, 1998; Mitchell et al.,
J. Anim. Sci.,
76, 208-211, 1998) hoping to improve the economics of livestock production; additionally, they have been used extensively by body-builders hoping to modify their phenotypic characteristics (Prather et al., 27, 1118-1121, 1995; Hausmann et al.,
Int. J. Legal Med.,
111:261-264, 1998; Ayotte et al.,
J. Toxicol.-Toxin Rev.,
18, 113-123, 1999). The most commonly abused &bgr;-agonist is clenbuterol hydrochloride, a highly potent phenethanolamine (Smith,
J. Anim. Sci.,
76, 173-194, 1998), that has not been approved for leanness-enhancing effects in livestock or in humans by any regulatory body worldwide.
The presence of drug residues in animal tissues is a concern for food safety, especially when the compound has been used illegally or in a manner proscribed by regulatory officials (off-label use). In an effort to combat the illicit use of &bgr;-agonist compounds, regulatory organizations worldwide test animal tissues or excreta for the presence of illicit drugs (Elliott et al.,
Vet. Quart.,
18, 41-44, 1996; Kuiper et al.,
J. Anim. Sci.,
76, 195-207, 1998; Mitchell et al.,
J. Anim. Sci.,
76, 208-211, 1998). For regulatory purposes, both screening and confirmatory assays are used to detect illegal residues. Immunoassays are convenient screening tools used to detect the presence of an analyte in various matrices, and have wide application for determination of the presence of environmental toxins (Sanborn et al.,
J. Agric. Food. Chem.,
46, 2407-2416, 1998), herbicides (Clegg et al.,
J. Agric. Food. Chem.,
47, 5031-5037, 1999), insecticides/pesticides (Wang et al.,
J. Agric. Food Chem.,
47, 3416-3424, 1999; Abad et al.,
J. Agric. Food. Chem.,
47, 2475-2485, 1999), and pharmaceuticals (Stanker et al.,
Food Agric. Immuno.,
10, 121-131, 1998; Brandon et al.,
J. Agric. Food Chem.,
46, 3653-3656, 1998). A successful screening assay should be quick, reliable, and relatively inexpensive. Positive samples from screening assays may then be assayed by more costly and complex instrumental methods such as GC-MS or LC-MS that unequivocally identify the analyte in the sample (eliminating false-positives). However, for screening, immunoassays provide the advantages of high throughput, portability, and sensitivity (detection limits in the ppb range). High sensitivities of immunoassays are particularly desirable for off-label drug monitoring because it may be desirable to detect the analyte even after extended withdrawal periods. Beta-adrenergic agonists immunoassays have been developed for clenbuterol (Yamamoto et al.,
J. Immunoassay,
3: 155-171, 1982), albuterol (salbutamol; Adam et al.,
J. Immunoassay,
11, 329-345, 1990), fenoterol (Haasnoot et al.,
Analyst,
119, 2675-2680, 1994), as well as ractopamine (Haasnoot et al.,
Analyst,
119, 2675-2680, 1994; Elliott et al.,
Analyst,
123, 1103-1107, 1998; Shelver et al.,
J. Immunoassay,
21, 1-23, 2000). Currently available immunoassay kits cross-react poorly with ractopamine (Wicker et al.,
Analyst,
120, 2879-2881, 1995), and the immunoassay previously reported for ractopamine was generated from polyclonal antibodies. Development of an immunoassay based on a monoclonal antibody is advantageous, relative to a polyclonal based immunoassay because a continuous supply of a homogeneous antibody can be assured, eliminating the batch-to-batch differences commonly encountered with polyclonal antibodies.
SUMMARY OF THE INVENTION
We have now discovered a hybridoma cell line designated 5G10 that produces and secretes a monoclonal antibody toward the &bgr;-adrenergic agonist ractopamine hydrochloride {(1R*, 3R*), (1R*, 3S*)-4-hydroxy-&bgr;-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methyl]-benzenemethanol hydrochloride}. This cell line secretes antibody with isotype IgGl
K
shown to be useful for the development of an immunoassay. The antibody is specific for racemic ractopamine, and also binds stereoselectively with the (1R,3R) isomer which is responsible for conferring biological activity to the molecule. Phenethanolamine &bgr;-agonists show low cross-reactivity.
The monoclonal antibody of this invention may be incorporated into kits for the detection and quantitative determination of low levels of ractopamine in samples, especially in human and animal tissue, body fluids and excreta, and also in feed material. Detection of ractopamine in sample materials is accomplished using immunosorbent assay procedures conventional in the art.
It is an object of this invention to provide a hybridoma cell line that produces and secretes a high affinity monoclonal antibody which is specific for ractopamine, stereoselective for the (1R,3R) isomer, and effective for detecting and quantifying ractopamine in sample materials at very low levels.
Another object of this invention is to provide immunoassay methods for determining off-label and illegal use of ractopamine and for detecting levels of ractopamine exceeding tolerance limits in samples.
A further object is to provide kits useful for the assay of ractopamine based on the monoclonal antibody described herein.
Yet another object is to provide a method for recovering or removing ractopamine from any material such as by affinity purification.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
DEPOSIT OF BIOLOGICAL MATERIAL
The cloned hybridoma cell line 5G10 was deposited on Jun. 20, 2000, under the conditions of the Budapest Treaty with the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA, and has been assigned number ATCC PTA-2103.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention we have created a hybridoma cell line that produces a monoclonal antibody that binds ractopamine, has an unexpectedly high stereoselectivity for the (1R,3R) isomer and is effective for detecting and quantifying very low levels of the &bgr;-agonist. The monoclonal antibody of the invention is capable of detecting ractopamine levels of 0.5 ng/mL [at 80% B/B
0
(IC
20
)] in competitive inhibition ELISA.
Preparation of the hybridomas may be accomplished using conventional techniques such as described by Kohler and Milstein (Nature, 256:495-497, 1975), Koprowski et al. (U.S. Pat. No. 4,196,265) or Wands (U.S. Pat. No. 4,271,145), the contents of each of which are incorporated by reference herein. Generally, the process of preparation comprises the steps of immunizing an animal with the antigen of interest, recovering splenocytes or lymphocytes from the animal, fusing the splenocytes or lymphocytes with continuously replicating myeloma cells to produce hybrid cells, an
Shelver Weilin L.
Smith David J.
Chin Christopher L.
Fado John D.
Grun James L.
Ribando Curtis P.
Silverstein M. Howard
LandOfFree
Monoclonal antibody, cell line and immunoassay for ractopamine does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Monoclonal antibody, cell line and immunoassay for ractopamine, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Monoclonal antibody, cell line and immunoassay for ractopamine will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2512032