Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1995-08-11
2002-03-12
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S005000, C435S007100, C435S007200, C435S188000, C436S512000, C436S527000, C436S538000
Reexamination Certificate
active
06355445
ABSTRACT:
More specifically, the invention relates to the detection of the pathogen
Brucella abortus
using the virus
Brucella bacteriophage.
Brucellosis is a disease caused by the bacterial genus, Brucella, named after Dr. David Bruce who discovered the organism in 1887. The disease is zoonotic, although different species are usually found in specific domestic animals, such as cattle (
B. abortus
), swine (
B. suis
), sheep (
B. ovis
), goats (
B. melitensis
) and dogs (
B. canis
). The manifestations of these bacteria in animals are usually reproductive complications (aborted fetuses, inflamed uterus or orchitis). While vaccinations in animals have proven partially effective in offering protection, the vaccines are pathogenic for other animals and humans. Infection is passed to humans through the ingestion of milk, milk products, the handling of contaminated carcases or aborted fetuses, and by the contact of infected tissues or body fluids. The disease is rarely passed from human to human, and then usually by exposure to contaminated blood specimens. Brucella is the number one cause of laboratory acquired infection. The great majority of patients with the disease survive, but only a small percentage ever recover completely. Usually the people infected are subject to relapses of recurrent, or undulant, fever, incapacitation, nausea and arthritis.
Brucella is a highly infective organism which causes debilitating symptoms, and which can persist in the environment for months under the right conditions. There are no effective vaccines and only limited therapeutic recourses to the bacteria. In other words, Brucella is potentially a bacterial warfare agent. Accordingly, there is a need for an effective detection assay.
Methods are available for the detection of pathogenic bacteria, but these have limitations. Culturing bacteria from clinical specimens is sensitive but often requires selective media, several days of incubation and the right nutrients or conditions (Brucella needs 5-10% carbon dioxide). Common serological techniques are usually insensitive. The enzyme-linked immunosorbent assay is usually rapid, sensitive and specific but gives false-positive for
Staphylococcus aureus
protein A, requires a source of antibodies which is difficult to raise, and may not detect different strains of the same species.
The object of the present invention is to meet the above defined need for an effective detection assay for Brucella (specifically
Brucella abortus
) in the form of an assay for the detection of pathogenic bacteria by using bacteriophages, a type of virus that is specific for host bacteria.
Accordingly, the present invention relates to a method of detecting the presence of a pathogenic bacteria in a liquid sample using a bacteriophage specific to the bacteria comprising the steps of producing a bacteriophage stock; conjugating the bacteriophage stock to an enzyme; mixing the conjugated bacteriophage with a sample suspected of containing the bacteria; and detecting any changes resulting from a reaction of the conjugated bacteriophage with the bacteria.
More specifically, the invention relates to a method of detecting the presence of the bacteria
Brucella abortus
in a sample using virus Brucella comprising the steps of producing a stock of Brucella bacteriophage, conjugating the Brucella bacteriophage to the enzyme urease; mixing the conjugated Brucella bacteriophage with a sample suspected of containing the bacteria
Brucella abortus
; and detecting any changes resulting from a reaction of the conjugated Brucella bacteriophage wit h the
Brucella abortus.
MATERIALS AND METHODS
(1) Bacteria and Bacteriophages:
Brucella abortus
30
, B. abortus
2308
, B. melitensis
16M,
B. suis
144 and bacteriophages WB1 (Webridge) and BK (Berkeley) were acquired from Agriculture Canada, Animal Diseases Research institute (ADRI-Nepean), Nepean, Ontario,
Francisella tularensis
LVS was acquired from Dr. F. Jackson, Dept. Medical Bacteriol., University of Alberta, Edmonton, Alberta, who in turn acquired it from the American Type Culture Collection.
Escherichia coli
1511 was acquired from the Dept. Microbiology & Infectious Diseases, University of Calgary at Calgary, Alberta.
(2) Antibodies: To compare methods of conjugating enzymes to other proteins, antibodies were used as the Other protein. Mouse anti-
Brucella abortus
antisera were raised by immunizing mice (100 ug smooth-lipopolysaccharide/0.2 ml/mouse, given on weeks 0, 1, 5 at two sites intramuscular (i.m.) in the thigh and two sites subcutaneous (s.c.) under the skin on the back, blood taken by heart puncture on week 5, sera removed and pooled). Mouse monoclonal antibody Ys-T9-2 (3 mg antibody/ml ascites fluid) was acquired from D. R. Bundle of the National Research Council of Canada. Mouse anti-bacteriophage WB1 antisera were raised by immunization with 0.2 ug bacteriophage/0.2 ml/mouse [in a partially purified preparation that has 1.2×10
9
plaque forming units, 1 ug bacteriophage protein, and 160 ug total protein (growth medium proteins,
Brucella abortus
lysate debris also present) per ml] given on weeks 0, 1 and 2 both i.m. and s.c. as before, blood was taken on week 3 and the sera removed and pooled. Urease conjugated anti-mouse IgG goat antiserum was from the Sigma Chemical Co. (St. Louis, Mo.).
(3) Antigens:
Brucella abortus
2308 and
B. melitensis
16M were grown in Brucella broth (under an atmosphere with a 5% CO
2
),
Escherichia coli
1511 was grown in nutrient broth, and
E. tularensis
LVS was grown in Chamberlain's synthetic broth. The cells were killed with 2.0% phenol, removed by centrifugation, tested for sterility, washed in saline, then dispensed into vials so that after lyophilization there was 10 mg/vial.
(4) Chemicals: Urease (type VII), urea substrate tablets and bromcresol purple indicator tablets were obtained from the Sigma Chem. Co., Cesium chloride was obtained from Boehringer Mannheim GmbH, West Germany, and m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) was obtained from Pierce Chemical Co., Rockford, Ill.
Brucella abortus
Bacteriophage Preparations
Bacteriophages WB1 (Weybridge) and BK (Berkeley) were initially diluted 10
4
and 10
3
RTD (routine test dilution, highest dilution producing lysis on the propagating strain). Of the Brucella species and strains tested with both bacteriophages,
B. abortus
30 was the most sensitive (i.e. the best propagating host) to the bacteriophages, and WB1 appeared more lytic than BK. Bacterial cells grown on agar plates for a day did not appear to be lyzed by a bacteriophage inoculum. Plates that were freshly inoculated with
B. abortus
30 (a suspension that gave an O.D.
620
of 0.1 and 10
9
bacteria, a 1:100 dilution of this was made and 0.1 ml of the latter was plated onto Brucella agar plate with crystal violet), then with 10
3
plaque forming units (PFU), and incubated at 37° C., 5% CO
2
, showed extensive lysis. Small colonies of resistant bacteria (likely lysogenic) had to be removed. The plaques were cut and removed aseptically with an inoculating needle, placed in 50 ml sterile saline in a 250 ml flask, agitate (150 rpm., 1 h, 37° C.), and the liquid was filtered through a 0.22 um filter.
(a) In the first two attempts to produce a bacteriophage stock, the above described bacteriophage filtrate was simply added to early cultures of
B. abortus
30 (10
8
bacteria in 2 liters of Brucella broth in a 6 liter flask, 16 h, 37° C., 5% CO
2
, 150 rpm). The culture was shaken for 24 hours. The bacteria were removed by centrifugation and the supernatant was filtered through a 0.45 um filter (changing after every 250 ml volume). The yield was 3.2×10
7
PFU of bacteriophage/ml in 1200 ml.
240 ml (one-fifth of supernatant) of 34% w/v) Carbowax—PEG 6000, 1% dextran sulfate (w/v) 7.5% NaCl (w/v) was added to 1200 ml of culture supernatant to concentrate the phages. The solution was gently mixed and stored overnight at 4° C. Most of the supernatant was poured off, but the bottom 300 ml was centrifuged at 12,000×g, 4° C. for 15 min. The
Cherwonogrodzky John W.
Lotfali Kamil
Her Majesty the Queen in right of Canada as represented by the
Nixon & Vanderhye P.C.
Swartz Rodney P
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