Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Chemical modification or the reaction product thereof – e.g.,...
Reexamination Certificate
2001-09-27
2003-01-14
Ceperley, Mary E. (Department: 1641)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Chemical modification or the reaction product thereof, e.g.,...
C530S388900, C530S403000, C530S404000, C530S405000, C435S007920, C435S007930, C435S345000, C435S810000, C436S528000, C436S548000
Reexamination Certificate
active
06506885
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to hybridoma cell lines and monoclonal antibodies produced therefrom which may be used to detect tilmicosin.
2. Description of the Prior Art
Tilmicosin is a semi-synthetic macrolide antibiotic that is approved for veterinary use in cattle and swine to combat respiratory disease in the US, and in Canada for the treatment of pneumonia in lambs. Bovine respiratory disease associated with
Mannheimia (Pasteurella) haemolytica
can be treated and controlled by a single subcutaneous injection of tilmicosin. Tilmicosin also has in vitro activity against several gram-negative bacteria associated with respiratory disease, including
Actinobacillus pleuropneumoniae, Pasteurella multocida, P. haemolytica,
and Mycoplasma spp. The administration of tilmicosin may also be a convenient way to reduce mastitis infection.
Tilmicosin is derived from the macrolide antibiotic tylosin, produced by
Streptomyces fradiae.
Synthesis of tilmicosin was described by Debono et al. (U.S. Pat. No. 4,820,695). Briefly, in a two step process, tylosin is first hydrolyzed to yield desmycosin, which is then treated with dimethyl piperidine to produce tilmicosin.
It is known that most antimicrobial agents have limited ability for cellular penetration. However, tilmicosin has been shown to concentrate in bovine lung tissue. Even after serum levels have dropped below therapeutic concentrations, lung concentrations have been shown to exceed the
P. haemolytica
MIC for at least 72 hours after dosage.
Attempts have been made to produce a quick and economical competitive enzyme-linked immunosorbent assay (cELISA) method for determining tilmicosin residues in feeds and tissues. Jackman et al. (1997, J. Vet. Pharmacol. Therap., 20 (suppl. 1):131-132) reported the preparation of polyclonal antibodies to tilmicosin using desmycosin and lactenocin conjugated to carrier proteins. Silverlight et al. (1999, Food and Agricultural Immunology, 11:321-328) later reported that the antibodies raised against desmycosin and lactenocin conjugates bound both tilmicosin and tylosin. However, tilmicosin could not be used in an immunogenic preparation.
Despite these advances, the need persists for a monoclonal antibody to tilmicosin having improved sensitivity and specificity.
SUMMARY OF THE INVENTION
We have now discovered hybridoma cell lines which produce and secrete monoclonal antibodies which selectively bind to tilmicosin. We have unexpectedly found that these hybridomas may be obtained by using as an immunization agent or immunogen, 23-deoxo-23-demycinosyl tilmicosin which has been conjugated to an immunogenic carrier. Tilmicosin in biological samples may be detected and quantified by contacting the sample with the antibodies to form a tilmicosin/antibody immunocomplex when tilmicosin is present, which immunocomplex may then be detected. The monoclonal antibodies also may be incorporated into kits for the detection and quantification of tilmicosin.
It is an object of this invention to provide hybridoma cell lines that produce and secrete high affinity monoclonal antibodies which selectively bind to tilmicosin.
A further object of this invention is to provide monoclonal antibodies which selectively bind to tilmicosin but not tylosin.
Another object of this invention is to provide immunoassay methods for the measurement of tilmicosin in biological samples.
A further object is to provide kits useful for the assay of tilmicosin which include the monoclonal antibodies described herein.
Yet another object is to provide an immunization agent which may be used to produce hybridoma cell lines that produce and secrete high affinity monoclonal antibodies which selectively bind to tilmicosin.
Other objects and advantages of this invention will become readily apparent from the ensuing description.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with this invention we have created hybridoma cell lines that produce monoclonal antibodies that bind tilmicosin and are effective for detecting and quantifying levels of this antibiotic. We have unexpectedly discovered that by use of a novel immunogen, monoclonal antibodies may be produced which possess improved specificity and increased affinity for tilmicosin. The antibodies of this invention may be used to rapidly and accurately detect and quantify tilmicosin, providing an indicator of the level of this antibiotic in biological samples.
Traditionally, preparation of 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], Wands [U.S. Pat. No. 4,271,145], or Stanker et al. [U.S. Pat. No. 5,466,784], the contents of each of which are incorporated by reference herein. Briefly, 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, and screening the resultant hybrid cells for the production of antibodies to the antigen.
Often, the compound of interest is a relatively small molecule, and hence is itself incapable or only poorly capable of stimulating the immune system to produce antibodies. To render such compounds immunogenic, they are generally conjugated to an immunogenic carrier in such a manner that the resultant immunogen is capable of stimulating the immune system of an animal to produce specific antibodies that are capable of binding the unconjugated compound. Application of this traditional protocol for the generation of monoclonal antibodies to a small compound such as tilmicosin, would logically dictate an immunogen prepared by conjugation of tilmicosin to a carrier protein. However, in a departure from established practice, we describe here the preparation of monoclonal antibodies using significantly different, novel immunogens.
The method of preparing the hybridomas comprises the following steps:
Immunogen. The immunization agent of this invention is not constructed from tilmicosin, but is derived from 23-demycinosyl tilmicosin (which may also be referred to as 20-deoxo-20-[3,5-dimethyl-piperidin-1-yl]desmycosin). The structures of tilmicosin and 23-demycinosyl tilmicosin are shown in formulas I and II, respectively:
The immunization agent is prepared by covalently conjugating an immunogenic carrier to 23-deoxo-23-demycinosyl tilmicosin at carbon atom number 23 thereof. Immunogenic carriers are defined herein as any compound to which the haptens may be attached to render them immunogenic. Suitable carriers are well known and may be readily determined by the practitioner skilled in the art. Without being limited thereto, preferred carriers include proteins such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), ovalbumin, glucose oxidase, and human thyroglobulin.
The immunogenic carrier may be conjugated to the 23-deoxo-23-demycinosyl tilmicosin molecule directly or through an optional crosslinking agent or spacer. In accordance with the preferred embodiment, the immunogen is created by conjugating the carrier to 23-deoxo-23-demycinosyl tilmicosin which has been modified by reaction at the C
23
thereof with a crosslinking agent. A variety of diamine crosslinking agents are suitable for use herein, with diamino hydrocarbons and/or thiols being preferred. Suitable diamino hydrocarbons include, but are not limited to straight or branched chain, cyclic, saturated or unsaturated, diamino hydrocarbons, such as 1,3-diaminopropane, 1,4-diaminobutane, or hexamethylenediamine.
In a particularly preferred embodiment which is described in greater detail in Example 1, to selectively bind the crosslinking agent to C
23
of the hapten, the C
23
of 23-demycinosyl tilmicosin (II) is first halogenated, for example, by treatment with Cl
2
, F
2
, or Br
2
, and preferably I
2
. In this initial reaction, only the C
23
is halogenated to a signifi
Ceperley Mary E.
Deck Randall E.
Fado John D.
Silverstein M. Howard
The United States of America as represented by the Secretary of
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