U.S. patent application number 11/982410 was filed with the patent office on 2008-10-02 for process and materials for the rapid detection of streptococcus pneumoniae employing purified antigen-specific antibodies.
This patent application is currently assigned to Binax, Inc.. Invention is credited to Mary Kathleen Fent, Vladimir Andrei Koulchin, Elena Valentin Molokova, Norman James Moore.
Application Number | 20080241191 11/982410 |
Document ID | / |
Family ID | 33449075 |
Filed Date | 2008-10-02 |
United States Patent
Application |
20080241191 |
Kind Code |
A1 |
Moore; Norman James ; et
al. |
October 2, 2008 |
Process and materials for the rapid detection of streptococcus
pneumoniae employing purified antigen-specific antibodies
Abstract
Disclosed is a cell wall C-polysaccharide antigen of
Streptococcus pneumoniae which contains not more than 10% by weight
of protein, and preferably less which has been purified with 0.1N
NaOH prior to deproteinizing. Also disclosed are polyvalent
antibodies raised against Streptococcus pneumoniae which have been
affinity purified by passing them over a chromatographic affinity
matrix to which is coupled the purified and at least partially
deproteinized antigen to render them antigen-specific.
Inventors: |
Moore; Norman James; (North
Berwick, ME) ; Fent; Mary Kathleen; (Cumberland
Center, ME) ; Koulchin; Vladimir Andrei; (Portland,
ME) ; Molokova; Elena Valentin; (Portland,
ME) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP (w/ISA)
155 SEAPORT BLVD.
BOSTON
MA
02210-2600
US
|
Assignee: |
Binax, Inc.
Portland
ME
|
Family ID: |
33449075 |
Appl. No.: |
11/982410 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11740738 |
Apr 26, 2007 |
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11982410 |
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10989050 |
Nov 16, 2004 |
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11740738 |
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09397110 |
Sep 16, 1999 |
6824997 |
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10989050 |
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09156486 |
Sep 18, 1998 |
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09397110 |
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Current U.S.
Class: |
424/244.1 ;
530/389.5 |
Current CPC
Class: |
G01N 2021/757 20130101;
G01N 2021/752 20130101; G01N 2021/7759 20130101; G01N 2400/10
20130101; C07K 16/1275 20130101; G01N 21/78 20130101; G01N
2333/3156 20130101; G01N 33/56944 20130101; G01N 2469/10
20130101 |
Class at
Publication: |
424/244.1 ;
530/389.5 |
International
Class: |
A61K 39/09 20060101
A61K039/09; C07K 16/12 20060101 C07K016/12 |
Claims
1. A cell wall C-polysaccharide antigen of Streptococcus pneumoniae
which contains not more than 10% by weight of protein which has
been obtained by a) subjecting a culture of S. pneumoniae to a
treatment which kills the bacterial cells, b) separating the killed
bacteria cells as a cell pellet of weakly alkaline pH in the order
of from about 7.0 to 7.4, c) subjecting the pellet of step (b) to
incubation with a sufficient quantity of 0.1 N NaOH to elevate its
pH to a value in excess of 12.0 for a period of at least 45
minutes, d) deproteinizing the mixture, and e) separating out a
purified cell wall C-polysaccharide antigen containing not more
than about 10% by weight of protein.
2. A cell wall C-polysaccharide antigen of Streptococcus pneumoniae
according to claim 1 containing from 5 to 8% by weight of protein
wherein the bacterial cells in step (a) were killed by a sodium
azide treatment.
3. Polyvalent antibodies raised in an animal against Streptococcus
pneumoniae bacteria or their cell wall C-polysaccharide antigen
which have been affinity purified and thereby rendered antigen
specific by passing them over a chromatographic affinity column on
which the affinity gel is wholly comprised of a non-ion exchange
material to which has been coupled the cell wall C-polysaccharide
antigen of claim 1.
4. Polyvalent antibodies raised in an animal against Streptococcus
pneumoniae bacteria or their cell wall C-polysaccharide antigen
which have been affinity purified and thereby rendered antigen
specific by passing them over a chromatographic affinity column on
which the affinity gel is wholly comprised of a non-ion exchange
material to which has been coupled the cell C-polysaccharide
antigen of claim 2.
Description
PARENT APPLICATION
[0001] This application is a division of U.S. application Ser. No.
09/397,110 filed Sep. 16, 1999, which is in turn a continuation in
part of U.S. application Ser. No. 09/156,486 filed Sep. 18, 1998 in
the names of the same inventors.
BRIEF DESCRIPTION OF THE INVENTION
[0002] The present invention relates to a purified carbohydrate
antigen of S. pneumoniae and its use in affinity purifying
antibodies raised in an animal against S. pneumoniae bacteria or
against the carbohydrate antigen. The affinity purified antibodies
are especially useful in a specific and sensitive
immunochromatographic ("ICT") assay, performable within about 15
minutes, for the detection of Streptococcus pneumoniae in a bodily
fluid, such as urine or cerebrospinal fluid, of a patient showing
clinical signs of an infection caused by S. pneumoniae.
BACKGROUND OF THE INVENTION
[0003] Streptococcus pneumoniae ("S. pneumoniae") is a leading
causative organism of pneumonia-type illnesses and other lower
respiratory tract infections such as bronchitis, as well as of
upper respiratory tract infections, including infectious otitis
media and sinusitis and of disseminated invasive infections,
including bacteremia and meningitis. When not properly diagnosed
and treated, S. pneumoniae pneumonic infection may lead to any of
pericarditis, empyema, purpura fulminans, endocarditis or at least
one type of arthritis, where S. pneumoniae is the causative
organism in each instance. Such pneumonic infection is also often a
precursor of bacteremia or meningitis. To now, it nevertheless is
common for pneumonia arising from S. pneumoniae to be diagnosed and
treated somewhat empirically.
[0004] To a significant extent, this is because the tests presently
available for the detection of S. pneumoniae are either (1) time
consuming, labor intensive and in need of instrumental assistance
for reading results, or (2) lacking in sensitivity and/or
specificity. Because of problems associated with lack of
sensitivity and/or specificity, e.g., physicians tend toward
conservatively prescribing expensive, broad spectrum antibiotics
for patients with pneumonia-type respiratory infections in lieu of
prescribing a less expensive antibiotic specific to S. pneumoniae
where it would adequately cure the infection. This and other
liberal prescribing of broad spectrum antibiotics is, of course, a
major cause of today's well-publicized medical crisis consequent
from the increasing resistance of many types of infectious bacteria
to previously highly efficacious antibiotics. This crisis and the
potential untoward consequences for at least some patients of
empirical diagnosis and treatment are among many reasons why a
reliable and rapid assay for detecting S. pneumoniae in human body
fluids is needed.
[0005] Pneumonia caused by S. pneumoniae is a serious disease,
estimated to occur at the rate of one to five cases per 1,000
persons per year in the United States alone. Depending upon the age
and state of health (based on unrelated factors) of patients
infected with S. pneumoniae-caused pneumonia, the disease has a
mortality rate of between 4 percent and 30 percent of infected
patients.
[0006] The most time-honored methods of attempting to diagnose S.
pneumoniae-caused diseases, and especially pneumonia, involve the
Gram stain and culture of expectorated sputum of patients suspected
of harboring the disease, followed by biochemical identification
methods. This procedure requires in the order of one to four days
from start to finish. It has proved to be an unsatisfactory
diagnostic tool because (1) other bacteria present in the patient's
saliva often overgrow the sputum culture, and (2) S. pneumoniae
frequently is present in the human upper respiratory tract even
when no sign of disease attributable to this bacterium is present
in the individual. For example, it is estimated that some 30
percent of U.S. children are habitual carriers of S. pneumoniae.
Adults, too may become colonized by S. pneumoniae without
themselves entering a disease state. The carriage rates of the
organism by both children and adults increase with crowding
conditions and during winter months.
[0007] Co-agglutination, latex particle agglutination and
counter-immunoelectrophoresis methods for detecting the
polysaccharide capsular antigens of S. pneumoniae in sputum
specimens have been developed and are rapid, but they have not been
shown to exhibit reliable sensitivity or specificity, probably
because there are some 83 serotypes of S. pneumoniae, each of which
may vary in immunogenicity and in other respects. The commercial
polyvalent anti-serum developed and used for these tests contains
antibodies to all 83 of the S. pneumoniae serotype antigens, but it
nevertheless may fail to detect the less immunogenic antigen
serotypes. This polyvalent antiserum also has shown
cross-reactivity with other streptococci and some other infectious
bacteria, e.g., Haemophilus influenzae. Hence both false-negative
and false-positive reactions may occur randomly when these tests
are used on sputum samples.
[0008] Several enzyme-immunoassays ("EIA") have been developed
which are based on detection of the pneumococcal C-polysaccharide
antigen that has been found to be present in the pneumococcal cell
wall of all of the S. pneumoniae serotypes. See, e.g., Parkinson,
A. J., Rabiego, M. E., Sepulveda, C., Davidson, M. and Johnson, C.,
30 J. Clin. Microbiol. 318-322 (1992). This C-polysaccharide
antigen is a phosphocholine-containing polysaccharide derived from
teichoic acid. These EIA assays are of acceptable specificity and
sensitivity even though most often performed on sputum samples.
Each such assay, however, requires two to three hours performance
time after sample collection as well as the use of instrumentation
normally available primarily in clinical laboratories. In addition,
these assays need to be run by, or under close supervision of,
trained personnel.
[0009] Reliance upon sputum samples to diagnose S. pneumoniae
infections is frequently less than satisfactory in achieving a
diagnosis of S. pneumoniae-caused pneumonia, and not just because
of the potential for contamination of the sample by other bacteria
in the mouth and/or by indigenous upper respiratory tract S.
pneumoniae. Sputum is often difficult to collect; more-over, once
medication of the patient is commenced, the number of viable S.
pneumoniae in sputum rapidly decreases. In particular, the presence
of the C-polysaccharide antigen in sputum may rapidly become
difficult to detect if an antibiotic therapy is used that attacks
the cell wall of the S. pneumoniae microorganism. When S.
pneumoniae causes infectious otitis media, meningitis and various
other aforementioned infectious disease states, sputum samples are
of no aid in diagnosis.
[0010] Collection of blood cultures from patients suspected of S.
pneumoniae infection eliminates the contamination problems that
attend sputum samples. Where blood serum samples are found to
contain S. pneumoniae, diagnosis of various diseases of which it is
causative may readily be made. The drawback here is that only about
20 percent of all pneumonia patients infected by S. pneumoniae
become bacteremic; therefore, relying solely on blood samples to
diagnose S. pneumoniae-caused pneumonia may yield false-negative
results.
[0011] Urine samples have been found to be the most reliable and
convenient ones to use in detecting S. pneumoniae-caused pneumonia
because they can be non-invasively obtained; they will not be
contaminated with oral microflora; and the presence of the
bacterium in urine persists, albeit at a constantly decreasing
level of concentration, even after patient therapy has been
initiated, so that daily monitoring of patient urine samples to
assess the efficacy of a prescribed therapy may yield useful
information. It should be noted that human carriers of S.
pneumoniae who show no disease symptoms often do not have
sufficient pathogen present to have S. pneumoniae antigens present
in their urine.
[0012] A very recent article describes the successful diagnosis of
meningitis caused by S. pneumoniae using an EIA method to test
samples of cerebrospinal fluid. In the EIA, a monoclonal
immunoglobulin A antiphosphoryl-choline antibody was employed to
detect the C-polysaccharide antigen. See Stuertz, K, Merx, I,
Eiffert, H., Schmutzhard, E., Mader, M. and Nau, R., 36 J. Clin.
Microbiol. 2346-2348. The results obtained compared favorably with
those reported by Yolken, R. H., Davis, D., Winkelstein, J.,
Russell, H. and Sippel, J. E., 20 J. Clin. Microbiol. 802-805
(1984) obtained in an EIA in which two antibodies for S. pneumoniae
in cerebrospinal fluid were used--a horse antibody to the
pneumococcal C-polysaccharide antigen, bound to microtiter plates,
and a pooled rabbit antiserum to the polysaccharide capsular
antigen in the liquid phase.
BRIEF DESCRIPTION OF THE INVENTION
[0013] According to the present invention, antibodies to the
C-polysaccharide antigen of S. pneumoniae raised in rabbits are
affinity purified with isolated and purified C-polysaccharide
antigen having less than about 10% protein content.
[0014] These affinity purified antibodies are conjugated to an
agent which produces a color reaction upon the formation of a
sandwich with S. pneumoniae C-polysaccharide antigen from a test
sample and additional affinity purified C-polysaccharide antibody
immobilized upon a nitrocellulose matrix.
[0015] The test is conducted in a disposable immunochromatographic
test device and requires no instrumentation to interpret the
result. It can easily and successfully be performed by persons who
have no training in laboratory techniques.
[0016] The preferred test sample for diagnosis of S.
pneumoniae-caused pneumonia is patient urine, but the test also
works with other bodily fluid samples that contain S. pneumoniae,
including serum and sputum. Diagnosis of S. pneumoniae-caused
meningitis may be readily made using patient cerebrospinal fluid as
the test sample.
[0017] This invention for the first time offers the benefit of a
test for S. pneumoniae that is performable within a 15-minute time
span and is of at least equal specificity and sensitivity to EIA
tests requiring eight to twelve times as long and much more work,
to obtain a result. The test is easy to perform, requires no
special training, equipment, or instrumentation and it enables a
rapid diagnosis of pneumonia caused by S. pneumoniae. It can be
readily performed in a doctor's office, thus permitting the patient
to be immediately placed on a S. pneumoniae-specific therapeutic
regimen. It can, of course, be performed in a clinical laboratory,
but it can also easily be performed in a geriatric center, in a
patient's home or in any environment where S. pneumoniae-caused
pneumonia or other pathogenic condition is suspected to be
epidemic.
[0018] The test of this invention is important to administer when
disease states such as otitis media, bronchitis or sinusitis appear
because once it can be established that any of these is due to S.
pneumoniae rather than another infectious agent, appropriate
therapy can promptly be initiated. Small children are especially
prone to otitis media because of the shorter length and smaller
diameter of their Eustachian tubes, so that early detection of S.
pneumoniae if present may well forestall the onset of a more
serious, or even life-threatening, disease state. Papers by Norris
et al, J. Pediatrics, 821-827 (1966) and Hongeng et al, 130 J.
Pediatrics, No. 5 (May 1997) indicate that children with sickle
cell disease are highly susceptible to S. pneumoniae infection,
with S. pneumoniae sepsis being the most common invasive infection
among this populace and those once so infected having a much
heightened risk of recurrence and subsequent death. Clearly,
employing the ICT test of this invention to test the urine of these
patients on a regular basis may be helpful in diminishing the need
for the unremitting penicillin prophylaxis that the second of these
papers recommends.
[0019] The ease of performance of the test and its ability to
detect the C-polysaccharide antigen of S. pneumoniae in urine
suggests that this test should prudently be performed on patients
without overt clinical signs of related infection who report
feeling substantially under par. Any such patient in whom it is
established that S. pneumoniae is present in significant enough
quantities to give a positive urine ICT test is a predictable
candidate for developing a more severe infection--and the ability
to forestall the disease development before it becomes severe by
administering appropriate therapy is newly presented by this
invention.
[0020] As a part of developing the rapid test of this invention,
applicants have also developed a novel purified form of the
C-polysaccharide cell wall antigen that is indigenous to all
serotypes of S. pneumoniae. This novel purified antigen is of
special use in affinity purifying polyvalent antibodies raised in
an animal against S. pneumoniae bacteria or against the crude
C-polysaccheride antigen.
DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1 and related FIGS. 1A, 1B and 1C hereof show the
structure of a typical ICT device which has been adapted to perform
the S. pneumoniae assay as hereinafter described in detail.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Broadly speaking, the ICT assay for S. pneumoniae as herein
described may be designed and configured to be run on any known
disposable ICT device disclosed in the art. Preferably it is
designed to be conducted, and is conducted, using an ICT device of
the type disclosed in copending U.S. patent Application Serial No.
now U.S. Pat. No. 6,168,956 Ser. No. 07/706,639, of Howard
Chandler, or one of its continuation-in-part applications, all of
which are assigned to Smith-Kline Diagnostics, Inc. but are
exclusively licensed to Binax, Inc. (which is entitled to
assignment of this application), in a wide area of use fields that
includes diagnoses of human respiratory system diseases.
[0023] The preferred device is suitably impregnated in one region
thereof with antigen-specific polyvalent antibodies to the
C-polysaccharide antigen of S. pneumoniae. Labeled antigen-specific
antibodies are applied to another area of the device. The test
sample suspected of containing S. pneumoniae is contacted first
with the labeled antigen-specific antibodies, which then flow with
the sample to the device area containing unlabeled bound
antigen-specific antibodies, whereupon if S. pneumoniae is present
in the sample, the labeled antibody:C-polysaccharide antigen
conjugate already formed by contact binds to the immobilized
unlabeled affinity purified antibodies, whereupon a visible color
reaction is produced. The label may be any substance known in the
art to produce visible color upon the reaction of a labeled
antibody:antigen complex with bound unlabeled antibodies. Such
labels include various finely divided metallics, various organic
molecules, and various molecular combinations such as enzyme
combinations with another color-producing molecule. In this
invention, colloidal gold particles constitute the preferred
label.
[0024] It is of major importance in designing the test device, that
the concentration of antibody present at each of the two sites of
the test device where reaction occurs be sufficient to insure that
antigen present in the test sample will be captured by the labelled
antibodies as the test sample contacts them and that labelled
antibody: antigen conjugate will be readily captured and held by
the bound antibodies at the sample capture line. Experimental work
undertaken in connection with this invention has shown that active
antibody to the C-polysaccharide antigen of S. pneumoniae must be
present at each site of a test device at which antigen: antibody
reaction is to occur, in a concentration of between 7.7
nanograms/sq. mm. of surface area and 385 nanograms/sq. mm. of
surface area. If antibody concentrations lower than 7.7
nanograms/sq. mm. are present at a site where reaction is intended
to occur, false negative results are likely.
[0025] Various methods of affinity purification of antibodies to
the C-polysaccharide antigen of S. pneumoniae, are known. The one
hereinafter described is preferred in the present invention, but
others may be substituted. It is noted, however, that the
affinity-purified antibodies of this invention are to be sharply
distinguished from the "affinity-purified antibody preparation"
which is described by Sjogren and Holme, 102 J. Immunol. Methods
93-100 (1987). These authors describe obtaining a hot
phenol-purified C-polysaccharide antigen of S. pneumoniae
containing 17% protein and absorbing it on an ion exchange gel, DE
AE--Sepharose CL6B. After 48 hours incubation this preparation was
packed into columns at approximately neutral pH of 7.2. The binding
efficiency of the antigen to the gel is said to be about 60%.
Antibodies were passed over these columns and incubated for 30
minutes, followed by elution of the columns with 0.5 M Na Cl in
PBS. It is known that leakage of antigen from ion-exchange columns
is a frequent occurrence. In this system, it is reasonable to
hypothesize that the product eluted from the gel was an in
situ-formed immune complex of antibodies and antigen rather than a
preparation of the purified antigen of this invention. It should
particularly be noted that, in this invention, the purified antigen
containing less than 10% protein is covalently coupled to a spacer
molecule such as BSA--hydrazine conjugate, and the resulting labile
antigen-conjugate ligand is then covalently coupled to a
chromatographic gel--e.g., the Formyl Spherilose of Example 4,
which is then applied to a column. The antibodies are added and
eluted, with strongly acidic buffer, from the immobilized antigen
on the column.
[0026] The antibody herein preferred is raised by conventionally
injecting a rabbit with S. pneumoniae strain R6, a non-encapsulated
S. pneumoniae strain available from the American Type Culture
Collection under ATCC No. 39938 which is subjected to heat-killing
of the cells before injection into the animal. After an appropriate
time period, the animal is bled to obtain serum containing the
desired antibodies, followed by purification thereof. Other
antibodies to the S. pneumoniae C-polysaccharide antigen may be
substituted for those specifically described herein without
departing from this invention.
[0027] The antibody should initially be tested for cross reactivity
to other common infectious bacteria. The preferred antibody
referred to herein was tested, using the ELISA method, for
cross-reactivity with each of the following: Citrobacter freundii,
Staphylococcus aureus, Enterobacter cloacae, Enterobacter faecalis,
Streptococcus Group B Type III, E. coli, Neisseria meningitidis,
Salmonella cubana, Salmonella paratyphi A, Klebsiella pneumoniae,
Streptococcus Group B type II, Staphylococcus epidermidis,
Salmonella enteritidis, Streptococcus Group A, Serratia marcescens,
Candida albicans, Haemophilus influenzae, Moraxella catarrhalis,
Corynebacterium kutscheri, Pseudomonas putida, Proteus vulgaris,
Enterococcus avium, Acinetobacter baumannii, Klebsiella oxytoca,
Acinetobacter lwoffli, Pseudomonas aeroginosa, Staphylococcus
saphrophyticus, Enterococcus durans, Corynebacterium bovis, Proteus
mirabilis, Pseudomonas stutzeri, Pseudomonas cepacia, Salmonella
typhi, Streptococcus Group F, Streptococcus Group B type 1a,
Candida stellatoides, Streptococcus parasanguis, Streptococcus
Group G, Streptococcus Group C, Streptococcus mutans, Morganella
morganii, Staphylococcus haemolyticus, Haemophilus influenzae type
B, Stenotrophomonas maltophilia, Haemophilus influenzae type D,
Gardnerella vaginalis, Streptococcus mitis, Haemophilus
parainfluenzae, Streptococcus sanguis, and H. influenzae
nontypeable.
[0028] The only significant cross reactivity found was with
Streptococcus mitis and Staphylococcus aureus. The first, S. mitis,
is a causative agent for endocarditis, the overt patient symptoms
of which physicians can readily distinguish clinically from those
of an S. pneumoniae lung infection. S. mitis contains the same
C-polysaccharide antigen as S. pneumoniae and the two share the
ability to cause endocarditis, albeit S. pneumoniae normally does
so in patients whose primary pneumonia has not been appropriately
treated and who may then develop bacteremia and/or endocarditis or
another pathogenic secondary infection. S. mitis, by contrast, is
not a causative agent for pneumonia; endocarditis attributable to
S. mitis normally develops independently of any other infection. It
is accordingly believed that suspected cases of primary
endocarditis caused by S. mitis can be confirmed, when needed,
using the assay of this invention. It should be noted, however,
that S. mitis is less likely to be present in urine than S.
pneumoniae and hence, an assay of blood serum may be more likely to
yield confirmatory information in that instance.
[0029] Some strains of S. aureus are known to secrete Protein A, a
non-specific protein which indiscriminately binds IgG, and hence,
all antibodies. The suspected presence of these S. aureus entities
may be readily confirmed or ruled out by running other simple tests
well known in the art. (As shown in Example 9, S. aureus strains in
which protein A is not present show no cross reactivity to the
antibody of this invention.) A minor cross-reaction with
Haemophilus influenzae was observed, but is not believed to be
significant enough to cause a problem in the detection of S.
pneumoniae in urine samples.
[0030] The following examples illustrate the preferred mode of
affinity purification of the antibody, including the preliminary
separation and purification of the antigen used to effect antibody
purification, thus yielding an antigen-specific polyvalent antibody
preparation.
EXAMPLE #1
Bacterial Growth Conditions
[0031] S. pneumoniae strain R6 (ATCC No. 39938) was grown in S.
pneumoniae broth supplemented with 20 mM of Hepes buffer. The broth
had the following composition per liter:
TABLE-US-00001 Pancreatic digest of casein 17.0 g. Glucose 10.0 g.
NaCl 5.0 g. Papain digest of soybean meal 3.0 g. Yeast extract 3.0
g. K.sub.2 HPO.sub.4 2.5 g. HEPES 20 mM
This broth had an initial pH of 7.2.+-.0.2 at 26.degree. C. It was
autoclaved for 15 minutes at 15 psi and 121.degree. C. and set
aside to cool.
[0032] Frozen aliquots of S. pneumoniae strain R6 (ATCC No. 39938)
were inoculated onto 5% sheep blood agar plates and allowed to
grow. Growth from the plates was harvested in smaller aliquots of
the seed broth and this seed broth was inoculated into three
flasks, each containing 1,700 ml of supplemented S. pneumoniae
broth of the composition shown above and further grown at
37.degree. C. in an atmosphere of 5 percent CO.sub.2, with
agitation but not aeration. When the pH of the broth fell below 5.5
(its late log phase) the flasks were removed from the incubator,
the cells were killed with 0.1 percent sodium azide and the pH was
adjusted to above 7.0 to prevent autolysis. The flasks were then
stored at 4.degree. C. overnight. The following day, the suspension
from each flask was centrifuged at 8,000 rpm for 60 minutes. The
pellets were then combined and recentrifuged at 13,000 rpm for 30
minutes. The wet weight of the pellet was recorded and it was
stored at -20.degree. C.
EXAMPLE #2
Isolation of S. pneumoniae
C-Polysaccharide Antigen Containing Less than 10% Protein
[0033] Cells grown, treated and stored as in Example 1 were thawed
at room temperature and suspended in phosphate-buffered saline
solution ("PBS") of pH 7.2 with 0.2 percent of sodium azide in a
ratio of 1.2 ml. of buffer to 1 gram of wet cells and left at room
temperature for two days.
[0034] Eleven ml per gram of the wet cells of 0.1 N NaOH was then
added to the S. pneumoniae suspension (in phosphate buffered
saline), resulting in a pH of 12.34 (as measured by pH meter) and
incubated for 45 minutes at about 30.degree. C. The pH of the
suspension was then adjusted to 2.75 (measured by pH meter) with 2
N HCl, followed by centrifuging the suspension at 3,500 rpm for 25
minutes. The supernatant was then separated and its pH was adjusted
to 7.0-7.1 with 1 N NaOH. This essentially neutralized supernatant
was dialyzed at 4.degree. C. against water for two days in dialysis
tubing (obtained from Spectra/Por) having a molecular weight
cut-off of 12,000 to 14,000. The dialyzed supernatant was
concentrated 25 to 40 times on a vacuum rotary evaporator.
[0035] Proteinase K (from Boehringer Mannheim) in the amount of
0.20 mg. per gram of wet cells, was added and the mixture was
allowed to stand at 37.degree. C. for three and one-half to four
hours and then at room temperature overnight and the next day.
[0036] Following digestion with Proteinase K, the resulting
supernatant was dialyzed at 4.degree. C. against water in the
dialysis tubing from Spectra/Por having a molecular weight cut-off
of 12,000-14,000. The dialyzed supernatant was thereupon divided
into 12 aliquots, each of which was placed in a 30 ml glass tube
and mixed with an equal volume of 90 percent phenol. The tubes were
closed and incubated for 23 minutes at 68-72.degree. C. in a
thermal water bath wherein the water level was slightly above that
of the mixture level in the tubes. The suspension in each of the
tubes was occasionally stirred with a glass Pasteur pipette to make
the suspension more nearly homogeneous to the naked eye. After this
incubation, the suspension was allowed to stand at room temperature
for 30 minutes and then was centrifuged at 5,000 rpm for 40 minutes
at a temperature of 15.degree. C.
[0037] The upper water phase in each tube was then carefully
withdrawn with a glass syringe; its volume was carefully measured
for each individual tube and it was replaced with an equal volume
of fresh water. The steps of incubation of the suspension at
68-72.degree. C. followed by centrifugation at 5,000 rpm for 40
minutes at 15.degree. C., was performed again and repeated then
repeated once.
[0038] The lower phenol phase in each of the tubes was then
carefully withdrawn with a glass syringe, leaving the intermediate
(mixed water-phenol) and upper (water) phases in the tubes.
[0039] Meanwhile a flask containing cold ethanol, in a volume ratio
of about 10:1 relative to the combined extracted phenol phase from
the tubes, was placed in an ice bath. To this flask the phenol
phase was slowly added, drop by drop, with intensive stirring.
After all of the phenol phase was added, stirring was continued for
10 to 15 minutes, whereupon the mixture was placed in a
refrigerator at 4.degree. C. and left overnight to foster pelleting
of the C-polysaccharide antigen. The following day the mixture was
subjected to centrifugation at 12,000 rpm for 20 minutes at
4.degree. C. The resulting pellet of C-polysaccharide antigen was
suspended in about 0.4 ml per gram of wet cells of water and
dialyzed against distilled water at 4.degree. C. overnight, using
the Spectra/Por tubing with molecular weight cut-off of
12,000-14,000 referred to above. The resulting aqueous solution of
C-polysaccharide antigen was lyophilized and weighed. Its protein
concentration was evaluated by the Lowry Method; its composition
was checked on SDS-PAGE (12 percent gel) by Western immunoblot
assay and its C-polysaccharide antigen activity was checked by
ELISA.
[0040] This operation was repeated a number of times. It was found
that the overall yield of S. pneumoniae C-polysaccharide antigen
was from 1.2 to 1.4 percent per gram of wet cells of S. pneumoniae
strain R6, while its protein content was between about 5 and about
8 percent.
[0041] It should be noted that, in general, C-polysaccharide
antigen preparations with a protein content exceeding 10% are less
likely to perform satisfactorily in this invention than
preparations of less than 10% protein content.
EXAMPLE #3
Preparation of BSA Conjugate of the Antigen
[0042] For coupling of the purified S. pneumoniae strain R6
C-polysaccharide antigen to a chromatographic column to permit
affinity purification of rabbit anti-S. pneumoniae strain R6
antibodies, a BSA-hydrazine conjugate was selected. Other known
materials having similar functions may be selected and conjugated
to accomplish this coupling function.
[0043] The BSA-hydrazine conjugate was prepared as follows:
[0044] Hydrazine dihydrochloride obtained from Aldrich Chemical Co.
was dissolved in water to produce an 0.5 M solution. The pH was
adjusted to 5.2 with dry NaOH and dry bovine serum albumin ("BSA")
from Sigma Chemical Co. was added to produce a final concentration
of BSA of 25 mg per ml of solution. After complete dissolution of
BSA, N-(dimethylamino-propyl)-N.sup.1-ethylcarbodiimide
hydrochloride (from Fluka Chemical Co.) was added in a quantity to
produce a final concentration of 2.5 mg per ml of solution. This
reaction mixture was incubated at room temperature, with continuous
stirring, overnight. The next day, it was intensively dialyzed
against distilled water at 4.degree. C. Concentration of conjugate
was measured (as BSA concentration) at 280 nm on a Beckman DU 640
spectrophotometer.
[0045] To couple this conjugate to S. pneumoniae strain R6
C-polysaccharide antigen, the procedure was as follows:
[0046] The dry preparation of the antigen was dissolved, in the
amount of 1.1 mg per ml, in distilled water. Using diluted HCl, the
solution pH was adjusted to 5.0-6.0. BSA:hydrazine conjugate in
aqueous solution in a concentration of 23 mg per ml was treated
with dilute HCl to bring its pH to between 4.0 and 5.0, and this
solution was then slowly added to the antigen solution in a volume
ratio of about 1:6.65 (about 3:1 by weight). After three minutes of
stirring, N-(dimethylaminopropyl)-N'-ethylcarbodiimide
hydrochloride (from Fluka Chemical Co.) in about 100 to 200 mcl of
distilled water was added to the reaction mixture in a
N-ethylcarbodiimide hydrochloride to C-polysaccharide antigen
weight ratio of about 1 to 1.92.
[0047] After stirring for two hours at room temperature, the pH of
the resulting mixture was adjusted to about 9.0 with dilute NaOH.
The incubation was thereupon continued at room temperature for one
hour and then at 4.degree. C. overnight.
EXAMPLE #4
Affinity Column Preparation and Antibody Purification
[0048] To the ligand solution from Example 3, dilute HCl was added
to bring its pH to 7.0. Formyl Spherilose from Isco, Inc. was
selected as the matrix for the immunoadsorbent gel. The ligand of
C-polysaccharide antigen was coupled to this matrix using known
procedures, e.g., as described in Spherilose applications, ISCO
Applications Bulletin 78 at pages 28-35. Other known matrices and
coupling procedures may be substituted.
[0049] The gel was packed into the column and washed alternatively
with distilled water, 0.2 M glycine-HCL solution of pH 2.5, triple
strength phosphate-buffered saline of pH 7.2 and regular strength
PBS of pH 7.2, using 5 to 10 volumes per volume of the gel of each
solution.
[0050] The resulting activated column was then used for affinity
purification of antibodies, thus producing antigen-specific
antibodies, as follows:
[0051] Rabbit antiserum to S. pneumoniae Strain R6 (ATCC No. 39938)
directed to whole heat-killed cells, was mixed with dry NaCl to a
final concentration of 0.5 M. This mixture was centrifuged at 8,500
rpm for 20 minutes and the supernatant was filtered through cotton
wool. The filtrate was applied on the affinity column. The unbound
components were washed from the column with triple strength
phosphate buffered saline of pH 7.2 and with regular strength
phosphate buffered saline of pH 7.2. The antibodies were eluted
from the column with 0.2 M glycine-HCl buffer of pH 2.5. The eluent
was monitored at 280 nm on the Beckman spectrophotometer and the
fractions containing antibodies were pooled in a flask which was
placed in an icewater bath. The pooled fractions were neutralized
with aqueous 0.5 M NaH.sub.2PO.sub.4 of pH 9.0.
[0052] The concentration of antibodies was evaluated from the
absorbance value at 280 nm on the spectrophotometer.
[0053] The antibody solution was dialyzed against PBS of pH 7.2 and
concentrated on a PM-10 filter obtained from Amicon until a
concentration of 0.8-1.5 mg/ml of antibody was achieved.
[0054] It was found that 18-20 mg of affinity purified antibodies
were recovered from each 25 ml of rabbit antiserum to S. pneumoniae
strain R6 so treated.
[0055] These affinity purified antibodies were utilized in an ICT
test specific for S. pneumoniae C-polysaccharide antigen as
described in the ensuing example.
EXAMPLE #5
ICT Device and its Preparation
[0056] A. Preparation of Test Device:
[0057] A test device comprising a hinged cardboard housing equipped
with a window to allow the viewing of both the test results and
control results was prepared as shown in FIG. 1. The device has a
recess into which is placed a preformed plastic swab well for
receiving the sample-wetted swab on the right-hand (labeled 1 in
the drawing). An overlabel shown in FIG. 1A is then placed over the
entire right-hand side of the device. The overlabel has been
equipped with two holes--a lower one (marked B on FIG. 1A) into
which the saturated swab is to be inserted and an upper one (marked
B on FIG. 1A) toward which the swab will be pushed after insertion
thereof into the hole B. The position of the overlabel with its
holes A and B, and the swab well cooperate to hold the swab in a
proper position during the assay and to promote the expulsion of
sorbed liquid from the swab.
[0058] A preassembled test strip (marked C on FIG. 1) described
below, is inserted into the recess (labeled 2 on FIG. 1) and held
in place by an adhesive applied to the bottom thereof. An overlabel
shown in FIG. 1B is placed atop the left-hand side. It has been
equipped with a single hole (marked D in FIG. 1B) which mates to
the right-hand side hole A when the device is closed for
performance of the assay.
[0059] The assembled device is stored in a sealed pouch with
desiccant until it is used. Prior to sealing the pouch and storing,
a lightly adhesive tape is placed on the outer edge of the
right-hand half of the device.
[0060] B. Construction and Preparation of the Test Strip:
[0061] FIG. 1C shows the construction of the preassembled strip. It
is comprised of a conjugate pad of sorbent material in which a
conjugate of gold particles and the antigen-specific rabbit anti-S.
pneumoniae C-polysaccharide antigen antibodies described above have
been impregnated. A bridge pad of Ahistrom 1281 (not shown)
connects the conjugate pad to a nitrocellulose pad onto which a
capture line for the sample which reacts with the conjugate has
been established by embedding a stripe of antigen-specific rabbit
anti-S. pneumoniae C-polysaccharide antigen antibodies, prepared as
described above. The nitrocellulose pad also has a downstream
control line established by striping the pad with goat anti-rabbit
immunoglobulin (IgG). Following the nitrocellulose pad, the strip
is ended by an absorbent pad which serves as a reservoir for
liquid. All of these pads are backed by an adhesive strip and
placed into a device.
[0062] The conjugate pad is normally made from non-woven polyester
or extruded cellulose acetate. To prepare this pad for use in the
assay, gold particles of 50 nm. diameter are conjugated to
affinity-purified rabbit anti-S. pneumoniae C-polysaccharide
antibodies prepared as described above. The conjugation is effected
using a known method such as that described by DeMay in Polak, J.
M. and Van Norden, S. (Eds.), Immunochemistry: Modern Methods and
Application, (Wright, Bristol, England, 1986). The gold conjugate
particles are mixed with a drying agent consisting of aqueous 5 mM
sodium tetraborate of pH 8.0 containing 1.0 percent BSA, 0.1
percent Triton X-100, 2.0 percent Tween 0.20, 6.0 percent sucrose
and 0.02 percent sodium azide. The pad is heated sufficiently to
remove all of the liquid present and stored in a low-humidity
environment pending assembly of the test strip. These pads and
their treatment are especially chosen so that the pads will hold
the dry conjugate and will release it only when later wetted by
sample.
[0063] The nitrocellulose pad is first treated by embedding a
stripe of affinity purified rabbit anti-S. pneumoniae
C-polysaccharide antibodies in a first portion thereof, using a
carrier solution of phosphate buffered saline. These antibodies act
as the capture line. In a second portion of the pad downstream of
the first one in the assembled test device, the control line is
established by striping goat anti-rabbit IgG in the same carrier
solution on the surface of the pad. The nitrocellulose pad is then
subjected to desiccation at 18-25.degree. C. to promote permanent
absorption of the protein stripes thereto.
[0064] The absorbent pad used is of a commercially available
cellulosic material sold under the name Ahlstrom 939. This pad
requires no special treatment.
[0065] C. Kit Preparation:
[0066] As sold in commerce, the test device containing the finished
test strip is assembled. In practice, a number of devices are
packaged with a commensurate number of swabs fashioned from fibrous
Dacron and a bottle of "Reagent A" equipped with a top adapted to
deliver Reagent A dropwise. "Reagent A" is a solution of 2.0
percent Tween 20, 0.05 percent sodium azide and 0.5 percent sodium
dodecyl sulfate in a 0.05 M sodium citrate-sodium phosphate buffer
of pH 6.5. Positive and negative controls are also included in each
kit.
EXAMPLE #6
Conducting the ICT Assay
[0067] In practice, the swab furnished with each device is dipped
into the liquid sample, completely immersing the swab head. The use
of the swab to act as a filter for undissolved solids, semisolids
and colloids present in liquid biological samples such as urine,
blood, lymph, etc., and also in liquid environmental samples is the
subject of copending, commonly assigned, application Ser. No.
09/044,677, now U.S. Pat. No. 6,548,309, of Norman Moore and
Vincent Sy filed Mar. 19, 1998, which is assigned to Binax, Inc.
The swab is inserted into the hole at the bottom of the device
(hole B of FIG. 1A) and gently pushed upward so that the swab tip
is visible in the top hole (hole A of FIG. 1A). The Reagent A vial
is held vertically above hole B and three drops of Reagent A are
slowly added. The adhesive liner is then immediately peeled from
the right edge of the device and the device is closed and securely
sealed, thus pressing the swab in the swab well against the gold
conjugate pad. After 15 minutes, the result can be read in the
window of the device. A negative sample--i.e., one not containing
identifiable S. pneumoniae C-polysaccharide antigen--will exhibit
only the control line in the top half of the window. A positive
sample containing the target antigen will show two lines, the lower
one of which is the patient (or sample) line; even a faint sample
line indicates the presence of the target antigen in the sample. If
no line appears in the window after 15 minutes, or only a sample
line appears in the lower part of the window, the test is invalid
and must be repeated.
[0068] Using the procedure described above, the devices prepared as
described in Example 5 were tested using the ICT procedure just
described against 146 patient urine samples obtained from Centers
for Disease Control. Since the patient diagnoses relative to the
presence of S. pneumoniae infection were based on a variety of
different indications including blood culture, Gram stain, sputum
culture, Autolysin PCR and Pneumolysin PCR, but urine assay results
had not been made, each of these samples was tested in our
laboratory with ICT as herein described and also with ELISA, for
the presence of S. pneumoniae C-polysaccharide antigen.
[0069] Personnel performing the ICT and ELISA assays were not
apprised of the Centers for Disease Control classifications of the
urine samples as having been collected from patients diagnosed as
positive or negative for S. pneumoniae infection. It was found that
the ICT and ELISA results were, at the very least, comparable in
terms of both sensitivity and specificity in some 134 instances. It
is noted, however, that in some instances neither the ELISA nor the
ICT tests could be fully correlated to the patient diagnoses
supplied by Centers for Disease Control. It is believed that the
art-recognized inadequacies of culture evaluation as a basis for
diagnosis of S. pneumoniae infection and the fact that no
information was available concerning either the therapy
administered to any of the patients, or the time therapy was
commenced relative to the time the urine sample was collected are
factors that preclude any completely meaningful comparison of all
the results.
[0070] The substantial comparability of ICT and ELISA results in
134 instances confirms the considerable advantages that the
15-minute ICT test of this invention offers in terms of rapid
diagnosis of S. pneumoniae-caused infection and consequent early
institution of the most effective patient therapy.
EXAMPLE #7
Clinical Trials
[0071] Using the devices prepared as described in Example 5 and the
ICT procedure as described in Example 6, first paragraph, clinical
studies were conducted at three sites, using a bank of
characterized specimens. These included 273 urine specimens
collected from hospitalized patients and outpatients. Among the 273
patients, 35 gave positive blood culture results and 238 gave
negative blood culture results. (It should be noted that culture
methods often vary substantially from place to place. The urine
samples of the blood culture positive patients were presumed to
have been collected within 24 hours of both blood collection and
initial administration of antibiotics. Of the 238 urine samples
from patients with blood that tested negative in blood culture
tests, 28 were collected from bacteremic patients, 4 from patients
with empyema, 53 from patients with pnemonia and 153 from patients
with urinary tract infections).
[0072] In addition, 100 urine samples, which were collected from
individuals with no known infection, were assayed in the test of
this invention, involving the devices prepared as in Example 5 and
the ICT procedure described in Example 6. Blood samples from these
individuals gave negative results in culture tests.
[0073] Of the 35 urines from patients testing positive for S.
pneumoniae in blood culture tests, 30 gave positive results in the
test of this invention and 5 gave negative results. Of the 338
urine samples from patients, all of whom tested negative in blood
culture tests and 100 of whom were presumed negative, 21 tested
positive in the ICT test of this invention and 317 tested negative.
The sensitivity of the ICT test was calculated as 86%, the
specificity as 94% and the accuracy as 93%.
[0074] It should be noted that of the patients whose urine tested
positive for S. pneumoniae by ICT and whose blood cultures gave
negative results for S. pneumoniae, it was established by other
tests that of those with urinary tract infections 5 had E. coli
infections, 2 had Enterobacter cloacae infections, 3 had
lactobacillus infections, 1 was infected with Providencia Stuartii,
1 with Staphylococcus aureus, 1 with Streptococcus (non A, nonB)
and 1 with a Streptococcus (nonD) infection. Two of those who had
pneumonia also were infected with Mycobacterium tuberculosis and
one with Mycobacterium kansasii. One bacteremic patient was
infected also with Proteus mirabilis. Four patients with no known
infection had urine samples that tested positive with the ICT test
of this invention.
EXAMPLE #8
Clinical Trials
[0075] Tests were conducted at seven hospitals, six in the United
States and one in Spain to evaluate 215 urine specimens from both
hospitalized and outpatients with at least one of lower respiratory
symptoms and sepsis symptoms or who were otherwise suspected of
harboring pneumococcal pneumonia. In these tests, the device
prepared in accordance with Example 5 was utilized in the procedure
of Example 6 and the results were compared with blood culture
results conducted on blood specimens from the same patients. No
effort was made to assure uniformity of culture methods among the
participating institutions.
[0076] The blood culture results yielded 31 positive assessments
for S. pneumoniae and 184 negative assessments. Of the 31 patients
whose blood culture results were positive, the ICT test of this
invention conducted on urine samples showed 28 positives and 3
negatives. Of the 184 patients whose blood culture results were as
assessed negative, 45 provided urine samples that tested positive
in the ICT test of this invention while 139 urine samples from
these patients tested negative. Sensitivity in this trial for the
test of the invention was calculated as 90%, specificity as 76% and
accuracy as 78%.
[0077] The results obtained with the ICT tests of this invention in
Examples 7 and 8 must be considered in the light of the well-known
problems associated with culture tests and the known likelihood
that about 80% of patients infected with pneumococcal pneumonia
will not produce blood specimens that contain S. pneumoniae. It is
believed that further experience with the assay of this invention
will demonstrate convincingly that its specificity, sensitivity and
accuracy are understated in Examples 7 and 8 due to the use of
blood culture tests for comparison purposes.
EXAMPLE #9
Further Cross-Reactivity Testing
[0078] Using the device prepared as in Example 5 and the procedure
of Example 6, some 144 organisms at concentrations of 106 to
10.sup.9 CFU/mL were tested. Each of the organisms tested was grown
on appropriate agar and incubated at 37.degree. C. in 5% CO.sub.2
overnight, whereupon the plates were checked for purity and well
isolated colonies of each organism were selected for testing.
[0079] Of the 144 organisms, only one--S. mitis--A.T.C.C. #49456,
gave a positive test and hence was cross-reactive. This was
expected, as noted above, because S. mitis is known to contain the
C-polysaccharide cell wall antigen which the test of this invention
is designed to detect.
[0080] Negative results in the assay of the invention were obtained
with each of the following: Acinetobacter anitratus (ATCC #49139)
Acinetobacter baumanii (ATCC # 1906-T), Acinetobacter calcoaceticus
(ATCC No. 49466), Acinetobacter haemolyticus (A.T.C.C. # 19002),
Adenovirus 2 and 3 (pooled pure culture sample obtained from Center
for Disease Control), Alcaligenes faecalis (A.T.C.C. # 6633),
Bordetella pertussis (A.T.C.C. # 3467), Branhamella catarrhalis
(A.T.C.C. # 25238-T), Blastonyces dernatitidis (pure culture
obtained from Center for Disease Control, strain number unknown),
Candida albicans (A.T.C.C. #'s e10231, 14053 and 60193, each tested
separately), Candida stellatoides (A.T.C.C. #11006), Citrobacter
freundii (A.T.C.C. #375GT), Coccidiodes immitis (pure culture from
Center for Disease Control, strain number unknown), Corynebacterium
kutscheri (A.T.C.C. # 15677-T), Corynebacterium matruchotii
(A.T.C.C. # 14266-T), Corynebacterium pseudodipheriticum (A.T.C.C.
10700-T), Enterobacter cloacae (A.T.C.C. #'s 13047-T, 23355, 35030
and 49141, each tested separately), Enterococcus avium (A.T.C.C.
No. 49462), Enterococcus durans (A.T.C.C. # 49135), Enterococcus
faecalis (A.T.C.C. #'s 19433-T, 29212, 49477, 49478, 49149 and
51299, each tested separately), Escherichia coli (A.T.C.C. #'s
23513, 8739, 23514, 25922, 35218, 1173GT, 35421 and 15669 and one
unnumbered sample, each tested separately), Escherichia hermannii
(A.T.C.C. #'s 33650-T and 4648GT, each tested separately),
Flavobacterium indologenes (A.T.C.C. # 49471), Flavobacterium
meningosepticum (A.T.C.C. # 49470), Gardnerella vaginalis (A.T.C.C.
# 14018-T), Haemophilus influenzae, a (A.T.C.C. # 9006),
Haemophilus influenzae, b (A.T.C.C. #'s 9795 and 33533, each tested
separately), Haemophilus influenzae, c (A.T.C.C. # 9007),
Haemophilus influenzae, d (A.T.C.C. # 9008), Haemophilus
influenzae, e (A.T.C.C. # 8142), Haemophilus influenzae, f
(A.T.C.C.# 9833, Haemophilus influenzae, NT (A.T.C.C. #'s 49144,
49247 and 49766, each tested separately), Haemophilus
parainfluenzae (A.T.C.C. # 3339Z-T, obtained as a pure culture from
Center for Disease Control), Histoplasma capulatum (Two separate
pure cultures from Center for Disease Control, strains unknown,
each tested separately), Klebsiella oxytoca (A.T.C.C. #'s 43086 and
49131, each tested separately), Klebsiella pneumoniae (A.T.C.C. #'s
13882, 13883-T and 49472, each tested separately), Lactobacillus
acidophilus (A.T.C.C. # 4356), Lactobacillus casei (A.T.C.C. #
393), Lactobacillus gasseri (A.T.C.C. # 33323), Lactobacillus
jensenii (A.T.C.C. # 25258), Legionella pneumophila (A.T.C.C. #
33152), Listeria monocytogenes (A.T.C.C. # 7644), Micrococcus
luteus (A.T.C.C. #'s 9341 and 49732, each tested separately),
Moraxella ostoensis (A.T.C.C. # 15276), Morganella morganii
(A.T.C.C. # 25830-T), Mycoplasma genitalium (A.T.C.C. # 33530,
obtained as a pure culture from Center for Disease Control),
Mycoplasma hominis (A.T.C.C. # 27545, obtained as a pure culture
from Center for Disease Control), Mycoplasma pneumoniae (FH Type 2,
obtained as a pure culture from Center for Disease Control),
Neisseria cinerea (A.T.C.C. # 14685), Neisseria gonorrheae
(A.T.C.C. #'s 8660, 19424-T and 27631, each tested separately),
Neisseria lactamica (A.T.C.C. # 23970-T), Neisseria meningitidis
(A.T.C.C. # 13077-T), Neisseria subflava (A.T.C.C. # 49275),
Nocardia farcinia (obtained as a pure culture from Center for
Disease Control), Paracoccidiodes brasiliensis (strain # unknown,
obtained as a pure culture from Center for Disease Control),
Parainfluenzae Type 1 (strain C 39, obtained as a pure culture from
Center for Disease Control), Parainfluenzae Type 2 (strain H A
47885, obtained from Center for Disease Control as a pure culture),
Proteus mirabilis (A.T.C.C. #'s 7002 and 12453, each tested
separately), Proteus vulgaris (A.T.C.C. #'s 13315-T and 49132, each
tested separately), Providencia stuartii (A.T.C.C. # 49809),
Pseudomonas aeruginosa (A.T.C.C. #'s 15442 and 27853, each tested
separately), Pseudomonas cepacia (A.T.C.C. # 25416-T), Pseudomonas
pickettii (A.T.C.C. # 49129), Pseudomonas putida (A.T.C.C. #
49128), Pseudomonas putrefaciens (A.T.C.C. # 49138), Pseudomonas
stutzeri (A.T.C.C. # 17588-T), Respiratory Syncitial Virus, pooled
(Pooled sample of Strain A2 and A.T.C.C. # 18573, each obtained
from Center for Disease Control as a pure culture), Rhinovirus
(A.T.C.C. #'s 088 and 077, each obtained as a pure culture from
Center for Disease Control and each tested separately), Salmonella
cubana (A.T.C.C. # 12007), Salmonella enteritidis (A.T.C.C. #
13076-T), Salmonella paratyphi A (A.T.C.C. # 9150), Salmonella
typhi (A.T.C.C. # 6539), Serratia marcescens (A.T.C.C. # 13880-T),
Sphingobacterium multivorum (A.T.C.C. # 35656), Staphylococcus
aureus (A.T.C.C. #'s 12598, 6538P, 25923, 29213, 43300 and 49476,
each tested separately), Staphylococcus epidermidis (A.T.C.C. #'s
12228, 14990-T, 49134, and 49461, each tested separately),
Staphylococcus haemolyticus (A.T.C.C. # 29970-T), Staphylococcus
saprophyticus (A.T.C.C. #'s 15305-T and 49907, each tested
separately), Staphylococcus xylosis (A.T.C.C. # 49148),
Stenotrophomonas maltophilia (A.T.C.C. # 13637-T), Streptococcus
anginosus (A.T.C.C. # 9895), Streptococcus bovis (A.T.C.C. #
49133), Streptococcus Group A (A.T.C.C. #'s 1357, and 19615, each
tested separately), Streptococcus Group B (A.T.C.C. #'s 13813-T,
12386, 12400, 12401, 27591, 12973, 12403, and 31475, each tested
separately), Streptococcus Group C (A.T.C.C. # 12388),
Streptococcus Group F (A.T.C.C. # 12392), Streptococcus Group G
(A.T.C.C. 12394), Streptococcus mutans (Shockman strain),
Streptococcus parasanguis (A.T.C.C. # 15909), Streptococcus sanguis
(A.T.C.C. # 10556-T), Trichomonas vaginalis (A.T.C.C. #'s 085 and
520, each obtained as a pure culture from Center for Disease
Control and tested separately).
EXAMPLE #10
Clinical Trial with Healthy and Sick Children
[0081] An as yet unfinished clinical trial with healthy and sick
children as participants is in progress. Preliminary spot results
show that, using devices prepared as described in Example 5 and
following the procedure described in Example 6, S. pneumoniae was
detected in urine of 2 of 3 children diagnosed with sinusitis. It
is believed that the sinusitis case wherein the child's urine
tested negative may involve a different causative agent.
[0082] In the same trials, S. pneumoniae was detected with the
device and method of this invention in the cerebrospinal fluid of
the only child who exhibited overt signs of meningitis, enabling
prompt and effective therapeutic treatment of this individual.
EXAMPLE #11
Detection of S. Pneumoniae Antigen in Urine of Meningitis
Patients
[0083] Two patients exhibiting overt clinical symptoms of
meningitis were hospitalized. One had received antimicrobial
therapy prior to admission; the other had not. From each,
cerebrospinal fluid was obtained and subjected to a culture test.
The test results were negative, and so were blood culture
results.
[0084] As a last diagnostic resort, devices prepared according to
Example 6 were utilized in the procedure described in Example 7 on
urine samples obtained from each patient. In each case, the urine
samples tested positive for the S. pneumoniae C-polysaccharide cell
wall antigen.
[0085] These preliminary results strongly suggest that urine
samples may be routinely utilized in lieu of cerebrospinal fluid to
test for S. pneumoniae-caused meningitis. The ability to substitute
urine for cerebrospinal fluid as a test medium, if confirmed by
further clinical experience, will be of great benefit to patients
and medical practitioners alike. Spinal taps, by which
cerebrospinal fluid must be obtained, are painful for patients and
somewhat hazardous as well. For medical practitioners, spinal taps
are time consuming and require concentrated attention to
detail.
[0086] Those skilled in the art of immunochemistry generally, and
especially those skilled in immunoassays, will recognize that other
materials and ingredients and at times, other procedural steps, can
readily be substituted for those specifically recommended herein. A
vast array of literature, both patent and non-patent, discusses the
design and use of reliable, one-time-use, disposable immunoassay
test devices that could be substituted for the preferred ICT device
described and recommended herein. It is not intended that the
present invention should be limited with respect to substitutable
assay devices, materials, ingredients or process steps except
insofar as the following claims may so limit it.
* * * * *