Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Bacterium or component thereof or substance produced by said...
Reexamination Certificate
2001-03-16
2003-04-22
Smith, Lynette R. F. (Department: 1645)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Bacterium or component thereof or substance produced by said...
C424S130100, C424S141100, C424S150100, C424S164100, C530S387100, C530S388100, C530S388200, C530S388400, C530S387500, C435S070200, C435S070210
Reexamination Certificate
active
06551599
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to a method of preparing an immunogen comprising outer membranes from
Campylobacter jejuni
(hereafter designated Cj) and
Campylobacter coli
(hereafter designated Cc), inoculating animals with the immunogen, and detecting the desired hybridoma-producing antibodies; and to a composition comprising the immunogen. The invention is drawn further to hybridoma cell lines developed by this method to produce (1) monoclonal antibodies specific to Cj only and (2) monoclonal antibodies that recognize both Cj and Cc exclusively, and uses thereof.
BACKGROUND OF THE INVENTION
Campylobacters cause more human gastroenteritis than any other food-borne pathogen. Campylobacter enteritis is caused by two closely related species, Cj and Cc, but Cj is responsible for greater than 98% of human disease and produces more severe symptoms. Cj, has been further subdivided into Cj subspecies
jejuni
(Cjj) and Cj subspecies
doylei
(Cjd). (In the remainder of this application, the term Cjj will be used to designate
C. jejuni
subspecies
jejuni
and Cjd to designate
C. jejuni
subspecies
doylei
. The term Cj (
Campylobacter jejuni
) will be used to describe both
C. jejuni
subspecies
jejuni
and
C. jejuni
subspecies
doylei
collectively.) Cjj and Cjd are closely related; however, Cjj is the predominant subspecies causing human illness and isolated from food sources. Cj and Cc cause acute diarrhea in humans with associated enteritis, particularly in developing countries. These infections are prevalent in infants under 1 year in age and rank as the third most common cause of acute diarrhea after Rotavirus and enterotoxigenic
Escherichia coli
. The various clinical patterns of disease suggest that Campylobacter spp. and, more specifically, Cj strains are diverse and may possess more than one type of virulence factor. In addition, Cj, unlike Cc, are more often associated with symptomatic infections and with bloody diarrhea.
Since Campylobacter enteritis is a considerable world health problem contributing to morbidity in developed countries and to high mortality rates in children in developing countries, it is of clinical importance to develop a specific and rapid diagnostic assay to identify pathogenic Campylobacter in the stool of enteritis patients. The available culture methods for detecting the organism generally are extremely time consuming and costly. Several studies have reported production of anti-Campylobacter MAbs and their use in immunoassays to detect Campylobacter infection (Chaiyaroj et al., 1995).
Campylobacter was not recognized as a human pathogen until the mid 1970's. Previously, the principal food-borne pathogen of concern had been Salmonella spp. Since the 1970s, the food industry and the regulatory agencies have become aware of the importance of other food-borne pathogens, such as
Yersinia enterocolitica, Escherichia coli
O157:H7, and
Listeria monocytogenes
. Very recently, Cj has become recognized as the most frequent cause of gastroenteritis in the U.S. and has been observed as the most common pathogen associated with Guillain-Barre syndrome in humans. It is estimated that about 1 in 1,000 Campylobacter infections results in this serious illness (Nachamkin et al., 1992).
Campylobacter is very prevalent in poultry (Madden et al., 1998) and contaminates milk (Docherty et al., 1996). Hazard analysis critical control point (HACCP) systems for poultry are being implemented internationally (Notermans et al., 1994), and beginning in 1997 in the U.S., large poultry processors have been required to meet performance standards for the frequency and amount of Salmonella in their product (Anonymous, 1996). It is anticipated that similar performance standards will be established for Campylobacter spp. as better culture and quantitative detection methods are developed. The development of rapid, reliable, and cost-effective methods for the detection of food-borne pathogens is crucial to accurately assess the safety of a food product, the effectiveness of new control measures to minimize pathogens in a production or processing environment, and the basic biology and ecology of pathogens in the food environment.
Campylobacters are known to be widespread in the environment and contaminate soil and water (Stanley et al., 1998). Environmental monitoring requires detection methods that are field-based, portable, rapid, and capable of analyzing multiple samples. Biosensors that contain specific immobilized antibodies to capture a pathogen for subsequent detection by a laser light or other sensitive detection system are being developed (Zhou et al., 1998). The specificity, sensitivity, and affinity of the antibody in a biosensor are key factors for success. The following U.S. patents are incorporated by reference.
U.S. Pat. No. 4,404,194 discloses a 90 kDa protein from
C. jejuni
that has immuno-suppressive activity.
U.S. Pat. No. 4,785,086 discloses a DNA probe for detecting
C. jejuni.
U.S. Pat. No. 4,882,271, discloses a 300-700 kDa antigen from
Campylobacter pylori
and its use in various assays.
U.S. Pat. No. 4,942,126 discloses a method of identifying the presence of Campylobacter spp. in fecal specimens.
U.S. Pat. No. 5,200,344 discloses a method of identifying the presence of antibodies to
C. jejuni
or
C. coli
in test samples.
U.S. Pat. No. 5,374,531 discloses a method of analyzing particulate analytes, such as whole cells, in immunoassays.
U.S. Pat. No. 5,470,958 discloses an antigenic composition and antisera raised against a purified PEB 1 antigen from
C. jejuni.
U.S. Pat. Nos. 5,491,068 and 5,695,946 disclose a method of capturing specific bacterial cells with specific antibodies immobilized on magnetic beads.
U.S. Pat. No. 5,665,582 discloses a method for reversibly anchoring a biological material, such as a plastid, chromosome, nucleic acid, or protein to a solid support for use in an immunoassay.
U.S. Pat. No. 5,821,066 discloses a method of detecting, identifying, and quantifying respiring microorganisms in an immunoassay.
BRIEF SUMMARY OF THE INVENTION
The present invention provides for an immunogen comprising outer membrane complexes from multiple strains of
C. jejuni
and
C. coli
, the immunogen inducing an immune response in animals, such as mice, enhancing the production of hybridomas specific for
C. jejuni
and
C. coli
epitopes, wherein the immunogen induces a broad range of antibodies against many outer membrane molecules of
C. jejuni
and
C. coli
, but not against other gram-negative enteric bacteria or non-
jejuni/coli
Campylobacter. Monoclonal antibodies of the present invention made by using such an immunogen are specific for epitopes expressed on
C. jejuni
and
C. coli
, wherein the monoclonal antibody binds the
C. jejuni
and
C. coli
porin protein or carbohydrate bound to porin protein, the porin comprising the 45 kD and 35 kD monomeric forms and the trimeric form of the porin. The invention also provides for monoclonal antibodies which bind specifically to a 43 kD protein expressed only on
C. jejuni
strains, or to carbohydrate bound to a 43 kD protein expressed only on
C. jejuni
strains.
The invention provides for a method of testing a sample for the presence of
C. jejuni
and/or
C. coli
comprising the steps of:
(a) exposing a sample suspected of containing
C. jejuni
and/or
C. coli
to a MAb which specifically binds
C. jejuni
and
C. coli;
(b) detecting MAb-antigen binding, said binding being indicative of the presence of
C. jejuni
and/or
C. coli
in said sample; and
(c) capturing
C. jejuni
or
C. coli
with MAb-conjugated-magnetic beads or MAb-conjugated-polystyrene beads or MAb conjugated or bound to a solid matrix, and detection of bound
C. jejuni
or
C. coli
with a method selected from: a second specific MAb, a PCR-specific assay, mass spectrometry (MALDI-TOF), reporter phage specific for
C. jejuni
or
C. coli
, optical sensing, or electronic sensing.
The invention also provides for a method of immunizing animals by:
(a) preparing an immunogenic comp
Bates Anna H.
Brandon David L.
Mandrell Robert E.
Connor Margaret A.
Fado John
Portner Ginny Allen
Smith Lynette R. F.
The United States of America as represented by the Secretary of
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