U.S. patent application number 11/928632 was filed with the patent office on 2008-08-07 for cytoplasmic antigens for detection of candida.
This patent application is currently assigned to Rockeby Biomed (Australia) Pty. Ltd.. Invention is credited to Denis Ballantyne, John Warmington.
Application Number | 20080187988 11/928632 |
Document ID | / |
Family ID | 25284255 |
Filed Date | 2008-08-07 |
United States Patent
Application |
20080187988 |
Kind Code |
A1 |
Warmington; John ; et
al. |
August 7, 2008 |
CYTOPLASMIC ANTIGENS FOR DETECTION OF CANDIDA
Abstract
The present invention relates to a method and a means of
diagnosing Candida infection. In particular the present invention
relates to a method of diagnosing Candida infection by measuring
the levels of antibody to Candida cytoplasmic antigen present in a
biological sample taken from a subject at risk of, or suspected to
be suffering from a Candida infection.
Inventors: |
Warmington; John; (Bentley
WA, AU) ; Ballantyne; Denis; (Bentley WA,
AU) |
Correspondence
Address: |
FOLEY AND LARDNER LLP;SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Rockeby Biomed (Australia) Pty.
Ltd.
|
Family ID: |
25284255 |
Appl. No.: |
11/928632 |
Filed: |
October 30, 2007 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11316739 |
Dec 27, 2005 |
|
|
|
11928632 |
|
|
|
|
10682807 |
Oct 10, 2003 |
|
|
|
11316739 |
|
|
|
|
09841188 |
Apr 25, 2001 |
6916626 |
|
|
10682807 |
|
|
|
|
Current U.S.
Class: |
435/259 |
Current CPC
Class: |
A61P 31/10 20180101;
G01N 33/571 20130101; G01N 2333/40 20130101; G01N 33/56961
20130101; C07K 14/40 20130101 |
Class at
Publication: |
435/259 |
International
Class: |
C12N 1/06 20060101
C12N001/06 |
Claims
1. A method of preparing a Candida antigen composition comprising
the steps of: (a) providing Candida albicans yeast cells; (b)
disrupting said yeast cells to produce a cell lysate; (c)
clarifying said cell lysate to produce a soluble cytoplasmic
antigen fraction; (d) filtering and organically extracting said
soluble cytoplasmic antigen fraction; and (e) depleting soluble
cell wall mannoprotein from said soluble cytoplasmic antigen
fraction, to produce a soluble cytoplasmic antigen fraction
comprising antigens of molecular weights 55 kDa, 30 kDa and 20
kDa.
2. The method of claim 1, wherein the yeast cells are disrupted by
mechanical disruption.
3. The method of claim 1, wherein step (c) is performed by
centrifugation.
4. The method of claim 1, wherein the filtration of step (d) is
performed by using a filter that has a 0.45 .mu.m filter
membrane.
5. The method of claim 1, wherein the organic extraction of step
(d) is a chloroform extraction.
6. The method of claim 1, wherein step (e) is performed by Con
A-Sepharose chromatography.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a method and a means of
diagnosing Candida infection. In particular the present invention
relates to a method of diagnosing Candida infection which is both
sensitive and rapid.
BACKGROUND OF THE INVENTION
[0002] Candida is the most commonly identified causative agent of
oral or vaginal thrush. However, over the last few decades Candida
has emerged as a significant cause of life-threatening infections
in hospital patients. Ironically the increasing incidence of these
"invasive" or "systemic" Candida infections has been advances in
modern medicine. Patients that are now surviving major injuries,
surgery, cancers and organ transplants are vulnerable to
life-threatening Candida infections. In the United States, Candida
is now the fourth most common cause of blood infections in
hospitals.
[0003] The major problem with systemic Candida infections is that
there are few definitive clinical signs or symptoms. Treatment is
largely based on suspicion rather than a definitive diagnosis. Even
with the availability of anti-fungal drugs such as fluconazole a
high mortality rate (30 to 70%) is associated with systemic Candida
infections. The high rate of mortality is largely due to the rapid
onset of infection and a rapidly fatal outcome. Without an accurate
diagnosis the infection often goes unnoticed until it is too late
to effectively treat. This has led to a comment by clinicians that
Candida infections are usually diagnosed at autopsy. Accordingly,
there is a need for a rapid diagnostic assay that is capable of
early diagnosis of Candida infection so that appropriate treatment
may be instituted thereby reducing the mortality rate.
[0004] The main difficulty in the diagnosis of Candida infections
is that being a commensal, mere isolation of Candida from body
surfaces, or orifices, is not diagnostic of an infection. Culture
of Candida from blood or deep tissue is still the main method of
diagnosis of systemic Candida infections. However, it can take
several days for a culture to become positive, by then it may be
too late to effectively treat the infection. Also, false positives
may occur due to contamination from superficial body sites. Of more
importance, is the observation that in up to fifty percent of
autopsy proven cases of systemic candidiasis, blood cultures were
negative and therefore of no diagnostic value.
[0005] Nuclear magnetic resonance (NMR) and radioisotope scanning
have been used to detect Candida infections in tissues and organs.
However, those methods are not useful for early diagnosis.
[0006] Recently analysis of the Candida metabolite arabinitol was
proposed as a diagnostic tool. However, as arabinitol is produced
by the human body, further clinical studies have cast doubt on its
value.
[0007] The polymerase chain reaction (PCR) has also been used in
the diagnosis of invasive Candida infections. However, PCR has not
established itself as a useful diagnostic method for Candida for
the same reasons as outlined above ie Candida is a ubiquitously
present microorganism and false positives, due to superficial
contamination, are prevalent.
[0008] Immunoassays are the established procedures for the
diagnosis of many types of infectious diseases. Immunoassays have
the advantage that they are rapid and have a standardised assay
format. Immunoassays can be designed to either detect Candida
antigens, or host antibodies reactive against Candida antigens.
Several immunoassays are commercially available for the detection
of Candida antigens in sera or other body fluids. However, these
assays lack either sensitivity or specificity or both.
[0009] Immunoassays have been developed based on the detection of
immunodominant Candida antigens. Candida mannan is a highly
immunogenic cell wall antigen. However, as Candida is a commensal,
most individuals have antibody to Candida mannan, so its usefulness
in the diagnosis of systemic infection is limited. The applicant
has now surprisingly found that a more discriminatory assay for
Candida than previously used is the detection of cytoplasmic
antigen. The advantage of this diagnostic assay is that antibody to
this cytoplasmic antigen is only produced in response to an actual
infection. The applicant has further demonstrated that the use of a
combination of cytoplasmic antigen with other antigens is very
predicative of Candida infection.
[0010] Accordingly, the present invention overcomes or at least
alleviates the problems normally associated with diagnosing Candida
infection.
SUMMARY OF THE INVENTION
[0011] In its most general aspect, the invention disclosed herein
provides a simple and rapid method for diagnosis of Candida
infection. The method of diagnosis of Candida infection may be used
to screen large numbers of samples for possible infection.
[0012] Accordingly, in one aspect, the invention provides a method
of diagnosing Candida infection, comprising the steps of:
[0013] a). obtaining a biological sample from a subject at risk of,
or suspected to be suffering from, Candida infection, and
[0014] b). measuring the levels of antibody to Candida cytoplasmic
antigen present in the biological sample.
[0015] Antibody levels may be measured using known techniques of
immunology including enzyme-linked immunoassay (ELISA or EIA),
biligand binding (sandwich technique), fluorometric assay,
chemiluminescent assay, immunochromatography, radialimmunodiffusion
or radioimmunoassay (RIA). ELISA, immunochromatography or
chemiluminescent assay methods are particularly preferred, since
these are quick, sensitive, and specific, and are readily automated
for large-scale use. These methods also provide quantitative
determinations.
[0016] The diagnostic method utilises antigens expressed by
Candida, especially cytoplasmic antigen. The antigens isolated from
Candida as disclosed herein may, in certain embodiments of the
diagnostic method of the present invention, be immobilised on an
inert surface, embedded in a gel, or may be conjugated to a
molecule which imparts colour, fluorescence or radioactivity to the
antigen.
[0017] In a second aspect, the invention provides a method for
assessing the prognosis of Candida infection, comprising the steps
of measuring the levels of antibody to Candida cytoplasmic antigen
in a biological sample.
[0018] Persons skilled in the art will appreciate that the
techniques disclosed herein may be used on any type of biological
sample. Preferable the biological sample is selected from the group
consisting of bone marrow, plasma, spinal fluid, lymph fluid, the
external sections of the skin from respiratory, intestinal, and
genitourinary tracts, tears, saliva, milk, blood; both whole blood
and sera, blood cells, tumours and organs. Most preferably the
biological sample is sera.
[0019] Biological samples that may be analysed by the method of the
present invention can also be obtained via swabs, shunts or the
like. The biological samples may be analysed directly, or may be
treated prior to testing by, for example, concentration or pH
adjustment.
[0020] In a third aspect, the present invention further provides a
method of detecting the presence or absence of a Candida antibody
comprising the steps of:
[0021] a). exposing a biological sample, which may include a
Candida antibody, to an isolated cytoplasmic Candida antigen;
and
[0022] b). detecting the reaction between antibody and antigen.
[0023] In an especially preferred embodiment of the present
invention the diagnostic assay further utilises other Candida
antigens in combination with the cytoplasmic antigen. In particular
the cell wall antigen (including mannose) and/or purified
immunodominant antigen (enolase) are utilised.
[0024] Accordingly, in a forth aspect of the present invention
there is provided a method of diagnosing Candida infection,
comprising the steps of:
[0025] a). obtaining a biological sample from a subject at risk of,
or suspected to be suffering from, Candida infection, and
[0026] b). measuring the levels of antibody present in the
biological sample to Candida cytoplasmic antigen in combination
with measuring the levels of antibody to either cell wall antigen
or immunodominant antigen (enolase) or both.
[0027] The reagents and means of diagnosis of the present invention
may also be embodied in a kit for use in a diagnostics laboratory
or may be adapted and automated for analysing large numbers of
samples at a central receiving centre.
[0028] Accordingly, in a fifth aspect the invention provides a kit
when used for detecting the presence or absence of a Candida
antibody in a biological sample, comprising:
[0029] a). a biological sample collection device;
[0030] b). a cytoplasmic Candida antigen; and
[0031] c). means for detecting reaction between the antibody and
antigen in the sample.
[0032] Suitable buffering agents and ionic salts may also be
included in the kit.
[0033] In a sixth aspect the invention provides a method of
preparing a cytoplasmic antigen comprising the step of removing
lipoproteins by chloroform extraction.
BRIEF DESCRIPTION OF THE FIGURES
[0034] FIG. 1 shows a coomassie blue stained SDS-PAGE with major
protein bands of the Candida cytoplasmic antigen fraction observed
at 55 kDa, 35 to 45 kDa region, 30 kDa and 20 kDa.
[0035] FIG. 2 shows a single coomassie blue band of 48 kDa
corresponding to the expected size of the enolase antigen.
[0036] FIG. 3 shows a coomassie blue stained gel of the clarified
cell wall antigen preparation. A broad smear of stain can be seen
ranging in size from 90 kDa to 200 kDa
[0037] FIG. 4 shows a number of sera screened against the Candida
cytoplasmic antigen preparation.
[0038] FIG. 5 shows antibody reactivity to the three Candida
antigens--cytoplasmic, cell wall and immunodominant antigens, using
negative-control sera.
[0039] FIG. 6 shows antibody reactivity to the three Candida
antigens--cytoplasmic, cell wall and immunodominant antigens, using
sera from patients with superficial candidiasis.
[0040] FIG. 7 shows antibody reactivity to the three Candida
antigens--cytoplasmic, cell wall and immunodominant antigens, using
sera from patients with systemic candidiasis.
[0041] FIG. 8 shows the error bar of the Applicant antigen test
values in the different blood culture patients (95% CI).
[0042] FIG. 9 shows an error plot of the mean Candida antibody
values measured by the Applicant antigen test in both the blood
culture positive and negative groups of patients (95% confidence
interval).
[0043] FIG. 10 shows an error bar graph of the Applicant antigen
test data for invasive candidiasis and healthy controls.
[0044] FIG. 11 shows an immunoblot in which panel A shows the C.
albicans cytoplasmic (enolase) antigen disclosed in Buckley et al.
compared with panel B, C. albicans antigen disclosed in the present
application.
ABBREVIATIONS USED
[0045] EDTA Ethylenediaminetetraacetic acid EIA Enzyme immunoassay
ELISA Enzyme-linked immunosorbent assay
RIA Radioimmunoassay
[0046] BSA Bovine serum albumin DMSO Dimethyl sulfoxide .beta.-Me
.beta.-mercaptoethanol TMB 3,3',5,5'-tetramethyl-benzidine
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] The practice of the present invention employs, unless
otherwise indicated, conventional molecular biology, cellular
biology, and immunoassay techniques within the skill of the art.
Such techniques are well known to the skilled worker, and are
explained fully in the literature. See, e.g., Harlow and Lane,
"Antibodies: A Laboratory Manual" (1988); Maniatis, Fritsch &
Sambrook, "Molecular Cloning: A Laboratory Manual" (1982); "Animal
Cell Culture" (R. I. Freshney, ed., 1986); "Immobilised Cells and
Enzymes" (IRL Press, 1986); B. Perbal, "A Practical Guide to
Molecular Cloning" (1984); Sambrook, et al., "Molecular Cloning: a
Laboratory Manual" (1989) and Ausubel, F. et al., 1989-1999,
"Current Protocols in Molecular Biology" (Green Publishing, New
York).
[0048] In describing the present invention, the following
terminology is used in accordance with the definitions set out
below.
[0049] As used herein, a "biological sample" refers to a sample of
tissue or fluid isolated from a individual, including but not
limited to bone marrow, plasma, serum, spinal fluid, lymph fluid,
the external sections of the skin, respiratory, intestinal, and
genitourinary tracts, tears, saliva, milk, blood; both whole blood
and anti-coagulated whole blood, blood cells, tumours, organs, and
also includes samples of in vivo cell culture constituents,
including but not limited to conditioned medium resulting from the
growth of cells in cell culture medium, putatively Candida infected
cells, recombinant cells, and cell components.
[0050] "Human tissue" is an aggregate of human cells which may
constitute a solid mass. This term also encompasses a suspension of
human cells, such as blood cells, or a human cell line.
[0051] For the purposes of this specification it will be clearly
understood that the word "comprising" means "including but not
limited to", and that the word "comprises" has a corresponding
meaning.
[0052] It will be clearly understood that, although a number of
prior art publications are referred to herein, this reference does
not constitute an admission that any of these documents forms part
of the common general knowledge in the art, in Australia or in any
other country.
[0053] Persons skilled in the art will appreciate that any number
of different immunoassays may be used in the present invention. For
example, the Candida antigens disclosed herein may be used in
antibody capture assays, antigen capture assays, wherein the
antigen/antibody complex forms a "special" class of antigen or
two-antibody sandwich assays.
Techniques Used for Antigen Preparation
[0054] The term "Candida antigen" as used here means any one of the
three separate types of Candida antigen utilised in the present
invention, namely, cell wall antigen (including mannose), total
cytoplasmic antigen (mannose depleted) or purified immunodominant
antigen (enolase). Use of the term "Candida antigens" means that
all three antigens were involved or could be utilised. A number of
techniques may be used to prepare the Candida antigens including
biochemical extraction, column chromatography, Gel fractionation,
gene cloning, differential precipitation, filtration, dialysis or
centrifugation; however, the preferred techniques are those
disclosed herein. Briefly, these techniques involve either
mechanical, chemical or enzymatic lysis of Candida cells, followed
by separation of insoluble cell walls from soluble cytoplasmic
fraction by centrifugation, filtration and dialysis. Chemical
treatment of cell wall fraction to release cell wall antigens
followed by centrifugation and dialysis. Filtration and organic
extraction of soluble cytoplasmic cell extract. Separation of
mannoproteins by ConA affinity chromatography. Purification of the
immunodominant enolase antigen from the soluble cytoplasmic extract
by anion and cation affinity chromatography. It will be appreciated
by those skilled in the art that other techniques, or modifications
or variations of the above techniques, may be adopted without
adversely affecting the spirit of the present invention.
Techniques Used for Antibody Preparation and Labelling
[0055] Antiserum to the Candida antigens disclosed herein may be
produced in a host animal such as rabbit or sheep. The serum
fraction containing the antibody may be isolated by standard
techniques. This antiserum may be employed in several of the
embodiments of the invention hereinafter set forth, or a more
sensitive and specific antibody might be obtained by further
purification of the serum by electrophoresis, high-speed
centrifugation or the like. Ultimately, large quantities of highly
specific monoclonal antibody may be produced by means of the
hybrid-myeloma techniques by methods known to those skilled in the
art.
[0056] Certain embodiments of the present invention employ antibody
to the Candida antigens immobilised on cellulose, agarose, sephadex
or glass beads or other similar inert surfaces such as metal,
plastic or ceramic which do not interfere with subsequent reaction.
Adsorption, Br--CN activation or other techniques known in the art
may be employed to immobilise the antibody.
[0057] Other embodiments of the present invention employ the
antibody to the Candida antigens conjugated to a chromophoric
(highly coloured) molecule, an enzochromic (an enzyme which
produces colour upon addition of reagents) molecule, fluorochromic
(fluorescent) molecule or a luminogenic (luminescent) molecule.
[0058] The conjugate of antibody with enzyme is made using
techniques known in the prior art. (For references, see Avrameas,
S, and Uriel, J., in Comptes Rendus Hebdomadaires des Seances de
l'Academie des Sciences, vol. 262, p. 2543, (1966); Nakane, P. K.
and Pierce, G. B., in Journal of Histochemistry and Cytochemistry,
vol. 14, p. 929, (1966); Nakane, P. K., in Methods in Enzymology,
vol. 37, p. 133, (1975)).
[0059] Chromophoric molecules that may be used are
2,3-dinitrobenzene (DNB) salts, dinitrophenol (DNP) and methyl and
butyl orange. Other suitable chromophoric agents are well known in
the art. Enzochromic molecules that may be conjugated with the
antibody are enzymes that give colour with appropriate reagents.
Examples are alkaline phosphatase (ALP) which develops colour with
nitrophenyl phosphate (NPP), glucose oxidase with glucose, and
D-galactopyranoside. These and other examples are well known in the
art. Examples of fluorogenic agents are 2,4-dinitrofluorobenzene
and "pipsyl" derivatives. Luminogenic molecules may be conjugated
to antibodies by the method of Branchini, et al. (Biochem. Biophys.
Res. Commun. 97, 334 [1980]). The term "chromophoric" hereinafter
is intended to include "enzochromic" "fluorochromic" and
"luminogenic" molecules as well.
[0060] Certain embodiments of the invention also utilise Candida
antibody tagged with a radioactive element. I.sup.125 conjugated by
means of the chloramine-T procedure is a common example, but other
methods known in the art may also be employed.
Techniques Used for Antigen Immobilisation and Labelling
[0061] Antigen molecules may be immobilised on a solid carrier by a
variety of methods known in the art, including covalent coupling,
direct adsorption, physical entrapment and attachment to a
protein-coated surface. For references describing the methodology,
see Silman, I. H. and Katchalski, E. in Annual Review of
Biochemistry, Vol. 35, p. 873 (1966); Melrose, G. J. H., in Review
of Pure and Applied Chemistry, Vol. 21, p. 83, (1971); and
Cuatrecasas, P. and Anfinsen, C. B., in Methods in Enzymology, Vol.
22, (1971).
[0062] Lai et al. (German OS No. 2,539,657, U.S. Pat. No.
4,066,512) discloses a method of attachment to a protein-coated
surface. In this method, the internal and external surfaces of a
microporous membrane are first coated with a water-insoluble
protein such as zein, collagen, fibrinogen, keratin, glutelin,
polyisoleucine, polytryptophan, polyphenylalanine, polytyrosine, or
copolymers of leucine with p-amino phenylalanine. Such a coating
renders the membrane capable of immobilising a wide variety of
biologically active proteins including enzymes, antigens, and
antibodies. A microporous structure is defined as one having more
than 50% of its total volume in the form of pores ranging in size
from 25 nanometres to 25 micrometers, preferably from 25 nanometres
to 14 micrometers. A pore size range from 25 nanometres to 5
micrometers is employed in most applications herein. Uncoated
microporous membranes have as much as 70 to 75% of their volume as
pore space. The pores permit liquid flow through the membrane.
After being coated by zein, for example, the pore space is reduced
5 to 10% with the result that the structure retains its essential
properties of having a high proportion of its volume as pore space
and permitting liquid flow through the pores. The structure has a
large surface area in contact with any solution contained within
the pores.
[0063] Such a coated membrane, having immobilised antigen or
antibody, provides a compact, easy to manipulate carrier for the
immobilised antigen or antibody. Its integral structure permits
removal of bound from unbound components by simple mechanical
means.
[0064] Non-specific binding may be minimised by interposing a
second stage immobilisation step, in which an immunochemically
neutral protein is immobilised to the filter. Immobilisation
therefore occurs in two stages according to a preferred embodiment
of the invention: a first stage in which the desired immunochemical
component is immobilised, and a second stage, following the
completion of the first, in which an immunochemically neutral
protein such as fetal calf serum or bovine gamma globulin is next
immobilised. The term immunochemically neutral is defined in terms
of the specific components of the assay. Any protein, which does
not combine immunochemically with a component of the assay or with
one of the reagents, is considered immunochemically neutral, even
though such protein might be immunochemically reactive in another
system.
[0065] Where the substance to be detected is an antibody, the
immunochemically reactive moiety of the conjugate must be an
antibody capable of binding immunochemically with the antibody to
be tested. Such antibodies may be obtained by immunising an animal
with the antibody or immunoglobulin fraction of serum from the
animal in which the antibody to be tested originated. For example,
where the antibody to be tested is a human antibody, a goat
antibody against human antibody is obtained from the serum of a
goat immunised against human immunoglobulin (antibody). The enzyme
moiety may be any enzyme capable of catalysing a reaction which can
be detected by any method known to those skilled in the art, and
which retains its activity after conjugation with antibody.
Horseradish peroxidase is preferred because of its convenience and
suitability to a wide range of applications. It is well known that
the enzyme catalyses the oxidations of a variety of organic
compounds in the presence of hydrogen peroxide. Many such organic
substrates are chromogenic, ie. undergo a colour change upon
oxidation.
[0066] It has been found in the present invention that the purity
of the enzyme preparation used in the formation of conjugate has an
effect on the degree of non-specific binding. The greater the
purity of the enzyme preparation, the less the non-specific
binding. In part, the reduction is made possible because, the total
amount of conjugate protein required is reduced as the specific
activity of the enzyme is increased. The opportunity for
non-specific binding is therefore reduced as well. In the preferred
embodiment, the use of a highly purified peroxidase preparation has
been found to significantly reduce the amount of colour reaction
observed in control samples as compared with known positives.
Techniques Used for Candida Antibody Detection
Antibody Capture Technique
[0067] A Candida antigen prepared by the techniques disclosed
herein is immobilised, preferably on an inert surface such as PVC,
paper or a similar bibulous mat. The immobilised Candida antigen is
then put into contact with a sample suspected of containing Candida
antibody. In the case of aqueous samples such as blood or urine,
the solution is buffered and ionic salts may be present at optimum
concentration for Candida antibody-Candida antigen interaction.
TRIS or borate buffered phosphate at pH 7.5 to 9.0 and ionic
strength about 0.010 to 0.5, for example, are suitable buffering
agents and ionic salts. The inert surface with Candida antigen or
Candida antigen-Candida antibody complex thereon is next put into
contact with antibody to Candida antigen conjugated to a
chromophormic molecule. Preferably the Candida antigen is in
solution, buffered at pH from about 7.5 to 9.0 and ionic
concentration equivalent to about 0.01M to about 0.1M NaCl. After
careful rinsing under water or with suitable surfactants such as
Tween 20 to remove excess coloured antibody, the inert surface is
inspected for colour, fluorescence or luminescence directly or
after addition of colour-developing agents. Colour on the inert
surface indicates interaction between immobilised Candida
antigen-Candida antibody complex in solution. A control may be run
for colour comparison.
[0068] This technique may be adapted to clinical use by employing
Candida antigens tagged with radioactive elements and observing
either depletion of activity in solution or uptake on solid support
of radioactivity. This embodiment is highly sensitive and rapid and
suitable for large numbers of samples.
Enzyme-Linked Immunoassay-ELISA
[0069] A solution comprising Candida antibody conjugated to enzyme
which forms colour with developing reagents and buffer and ionic
salts suitable for reaction between Candida antigen and the Candida
antibody is put into contact and allowed to react with Candida
antigen immobilised, preferably, on an inert surface such as PVC,
paper strip or glass bead. The amount of enzochromic conjugated
Candida antibody is sufficient to saturate about 50% of the
reactive sites on the immobilized antibody. The inert surface with
antibody-Candida antigen enzyme complex is put into contact with
buffered sample suspected of containing Candida, said sample having
an unknown amount of Candida antibody. The colour of the resultant
immobilised antibody-Candida antigen-enzyme complex on the strip
after colour developing reagents are added is observed in
comparison to a control strip which has not been treated with
sample containing Candida antibody. Dilution in colour on inert
surface treated with sample means presence of Candida antibody in
the unknown sample.
[0070] This method may be adapted for clinical use by contacting
samples and immobilised enzyme, preferably in tubes which may be
centrifuged and watching developing colour spectrophotometrically.
This embodiment is very sensitive and rapid.
Radialimmunodiffusion-Precipitin Reaction
[0071] One of the Candida antigens is suspended in a softened
gelatinous medium such as agar or agarose along with buffers and
salts to maintain pH between about 6.0 to 9.0 and ionic strength
between about 0.01M to 0.5M for optimal antigen-antibody
interaction. The suspending medium of U.S. Pat. No. 4,259,207 is a
suitable example. The mixture is spread out to harden on a test
plate or, preferably, poured into a disc-shaped container such as
an Octolony plate. A small amount of sample is placed on the
solidified gel, preferably in a centre well and the plate or disc
is allowed to stand preferably covered for a period of hours.
Diffusion of sample into the surrounding area occurs during this
period. If the Candida antibody is present, it reacts with the
embedded Candida antigen and causes an opaque area in a radial
pattern about the point of application of sample. A control can be
run for comparison. Calibration of an amount of Candida antibody in
the sample, if desired, can be obtained by controlling temperature,
time and size of sample and comparing the resultant size of radial
area with one of known concentration.
Radioimmunoassay
[0072] A Candida antigen of the present invention is immobilised on
an inert surface such as glass beads in a separation column. A
portion of Candida antigen is conjugated to a radioactive element,
preferably 1125 and allowed to react with the immobilised Candida
antigen in an amount sufficient to, saturate 50% of the binding
sites. The immobilised Candida antigen-enzyme complex is put into
contact with a sample suspected of containing Candida antibody, the
sample being buffered between pH 6-9 and containing total ionic
salts about 0.05 to 0.5M for optimal reaction conditions for
formation of Candida antigen-antibody complex. The Candida antibody
is eluted from the antigen and the eluant is measured for
radioactivity. Loss of activity compared to a control indicates
Candida antibody in the sample.
Haemagglutination
[0073] Candida antibody may be assayed through standard
haemagglutination techniques with Candida antigen to antibody used
as sensitising agent.
[0074] It is to be understood that methods described hereinabove
for assay of Candida antibody employing coloured reagents have been
presented most specifically for application where neither trained
personnel nor sophisticated instruments are available. These
methods, however, may be adapted for use in a clinical setting
where large numbers of samples are to be assayed by substituting
radioactive elements for chromogenic conjugated molecules.
[0075] It is also to be understood that the term "colour" is not to
be interpreted as being limited to the narrow visible range of the
electromagnetic spectrum, but is meant to include wavelengths which
may be measured by standard spectrophotographic instruments such as
spectrophotometers and absorption and emission colourimeters in
both the uv and the ir range.
[0076] Although it is contemplated that the methods of the present
invention are to be applied to biological fluids themselves, the
sensitivity and specificity of the method can be improved by
culture of the fluids preferably on medium selective for Candida
prior to testing.
[0077] Sensitivity may also be improved by preliminary treatment of
biological samples with lysing agents such as isotonic solution,
sound, or lysozyme to release Candida antibody into the
extracellular environment. U.S. Pat. No. 4,166,765, for example,
discloses suitable lysing procedures for biological samples
containing bacteria. Any lysing agent may be employed which does
not interfere with subsequent enzyme activity.
Assays Embodied in Kit Form
[0078] The diagnostic method and means of the present invention may
be embodied in the form of a kit for use by individuals for
self-diagnosis of Candida in the privacy of their homes.
[0079] The kit comprises a means for sample collection, the Candida
antigen to Candida antibody and a means for detecting reaction
between sample and Candida antigen.
[0080] In embodiments adapted for clinical use, electrophoretic
separation techniques such as isoelectric focusing or zone
electrophoresis which are based on differences of both size and
charge distribution between products and reactants may likewise be
used to separate products from reactants. Products separated
electrophoretically may be detected by characteristic locations
compared to standards or may be identified by colour or
immunochemically. Resinous beads of charged surfaces may also be
used to separate products and reactants.
[0081] The means for detecting reaction in the case of immunoassay
in a preferred embodiment of the invention is a gelatinous medium
in which the Candida antigen to antibody is suspended. The
gelatinous medium is in a transparent glass or plastic container
and comprises buffer and ionic salts for optimal conditions for
formation of the Candida antigen-antibody complex. Reaction is
noted as a transparent area radiating from the central point at
which the sample is applied.
[0082] The means for detecting reaction in another preferred
embodiment comprising immunoassay is the Candida antigen to Candida
antibody conjugated to a chromophore in a sealed, sterile packet
along with buffer and ionic salts. For assay, the contents of the
packet are diluted with water in a marked tube supplied in the kit.
Included also in this embodiment is the antigen to Candida antibody
immobilised on an inert surface. For assay, the inert surface with
immobilised Candida antigen is put into contact with sample and
then with the solution of chromophore-conjugated Candida anti-IgA
antibody, protein A or protein G. The inert surface is inspected
for colour, which indicates Candida.
[0083] In a particularly preferred embodiment, the kit of the
present invention is provided in the form of an
immunochromatographic test strip device. There are many patents
that cover a number of technologies, formats, reagents and
materials that may be of great value in the development and
production of immunochromatographic test strip devices. For
example, U.S. Pat. No. 5,075,078, International Patent Application
No. WO95/16207, U.S. Pat. No. 5,654,162 and European Patent No.
0810436A1. The assay methods used with the devices disclosed in
these patents are essentially the same. A ligand specific for the
analyte (normally, but not necessarily an antibody [Ab]) is
immobilised to a membrane such as nitrocellulose. The detector
reagent, typically an antibody coupled to latex or colloidal metal,
is deposited (but remains unbound) into the conjugate pad. When
sample (urine, plasma, whole blood, etc.) is added to the sample
pad, it rapidly wets through to the conjugate pad and the detector
reagent is solubilised. The detector reagent begins to move with
the sample flow front up the membrane strip. Analyte that is
present in the sample will be bound by the antibody that is coupled
to the detector reagent. As the sample passes over the zone to
which the capture reagent has been immobilised, the analyte
detector reagent complex is trapped. Colour develops in proportion
to the amount of analyte present in the sample.
[0084] In the present case, while the above principles are the
same, rather than detecting analyte per se, the
immunochromatographic test strip device would detect antibody. In
such situations, it would be the antigen(s) disclosed herein which
would immobilised onto membranes, sample pads, reagent pads and
other porous media rather than antibody. There are a wealth of
information regarding the development of such devices including
methods of binding antigen/antibodies to nitrocellulose and the
like and detecting such bound material. See for example, Towbin et
al. 1979, Proc. Natl. Acad. Sci. USA 76:4350, the entirety of which
is included herein by reference.
[0085] Although the invention has been described with reference to
presently preferred embodiments, it should be understood that
various modifications can be made without departing from the spirit
of the invention. Moreover, the following examples are offered by
way of illustration only and are not intended to limit the
invention in any manner. All patent and literature references cited
herein are expressly incorporated.
Example 1
Preparation of Candida Antigen
[0086] The following three types of Candida antigen were
prepared:
[0087] 1). Cell wall antigen (including mannose);
[0088] 2). Total cytoplasmic antigen (mannose depleted); and
[0089] 3). Purified immunodominant antigen (enolase).
[0090] A clinical isolate of the Candida albicans, was obtained
from a patient with vaginal thrush. The identity of the Candida
species was confirmed with the use of an API 20C Auxonagram strip
(API System S.A., France). The C. albicans isolate was designated
KEMH5.
[0091] 200 ml YEPD culture medium (1% yeast extract, 2% peptone, 2%
D-glucose) was inoculated with the isolate as a starter culture and
incubated for 24 h at 30.degree. C. with aeration. The starter
culture was then used to inoculate a 10 L YEPD culture incubated
under similar conditions in a 23 L Bio-Flo Fermenter IV System (New
Brunswick Scientific, Edison, N.J.).
[0092] The Candida culture was harvested from the Bio-Flo fermenter
system and separated from culture medium with the use of a Pellicon
filtration cassette (Millipore, USA). Concentrated cells were
separated from residual medium by centrifugation in 500 ml
centrifuge flasks for 15 min at 1,660.times.g and 4.degree. C. The
supernatant was discarded and the pelleted cells were resuspended
in protein extraction buffer (20 mM bis-Tris, pH 6.5). The yeast
cells were then centrifuged as described previously, resuspended
and pooled for further processing.
[0093] Candida cells were ruptured mechanically with the use of a
Dynomill.RTM. (WAB, Switzerland). Milling was continued until 99%
cell disruption was obtained. The soluble Candida cell extracts
were collected and dispensed into 50 ml centrifuge tubes. The
extracts were centrifuged for 12 h at 8,517.times.g and 4.degree.
C. to precipitate insoluble cell walls. The supernatants containing
the soluble cytoplasmic antigen fraction were recovered and passed
through a 0.45 .mu.m filter membrane.
[0094] The filtrates were then extracted with an equal volume of
chilled chloroform. Following centrifugation at 4.degree. C. for 15
min at 1,036.times.g the upper aqueous phase was aspirated and
transferred to a dialysis tube. The soluble cytoplasmic protein
fractions were dialysed in column binding buffer (20 mM Tris/HCl,
pH 7.4, 0.5M NaCl, 1 mM MnCl.sub.2.4H.sub.2O, 1 mM CaCl.sub.2) for
12 h in preparation for chromatography.
[0095] The soluble cytoplasmic antigen fraction was depleted of
contaminating soluble cell wall mannoprotein by Con A-Sepharose
chromatography. The dialysed cytoplasmic antigen fraction was
filtered through a 0.45 .mu.m filter. 50 ml of the dialysed extract
was applied onto a Con A-Sepharose column (2.6.times.12.5 cm)
equilibrated in binding buffer at a flow rate of 4 ml/min. The
unbound flow-through fraction (non-glycosylated proteins) was
collected. Bound mannoproteins were eluted with 0.5M .alpha.-methyl
mannoside in binding buffer. This step was performed before the
next run and to clean the column before storage.
[0096] The soluble cytoplasmic antigen fraction was dialysed
overnight against 20 mM Tris.Cl, pH7.4. An estimate of the quantity
of protein in solution was performed using the Bio-Rad.RTM.
(Bradford) microassay procedure in accordance with the
manufacturers instructions. A portion of the cytoplasmic antigen
extract was analysed by SDS-PAGE.
[0097] As shown in FIG. 1 there was a number of major protein bands
observed which varyed in size from approx 20 kDa up to approx 60
kDa in size. The major staining bands being at 55 kDa, four bands
in the 35 to 45 kDa region, 30 kDa and 20 kDa. This was in stark
contrast to the large number of Coomassie blue staining bands in
the original crude lysate prior to organic extraction and Con
A-Sepharose chromatography.
[0098] Purification of the enolase antigen was conducted in the
same fashion as the soluble Candida cytoplasmic antigen except that
it was not subjected to Con A-Sepharose chromatography. Instead,
following dialysis and filtering through a 0.20 .mu.m syringe
filter (cellulose acetate), the filtered extracts were applied to a
Pharmacia Biotech XK 50/20 chromatography column packed with
Pharmacia Biotech Source 15Q quaternary ammonium anion exchanger
(Pharmacia LKB, Uppsala, Sweden). The column was equilibrated prior
to chromatography with column binding buffer `A` (20 mM bis-Tris,
pH 6.5). Anion exchange chromatography of the crude extracts was
controlled and recorded using the Bio-Rad.RTM. Econo.RTM. system
(Bio-Rad Laboratories, USA). Bound protein was eluted from the
column with a salt gradient of buffer `B` (1M NaCl in buffer `A`,
pH 6.5). The recovered fractionated proteins were analysed by an
enzyme activity assay.
[0099] The active enzyme enolase hydrolyses D(+)-2-phosphoglyceric
acid (PGA) to phosphoenolpyruvate (PEP). The production of PEP can
be monitored by spectrophotometry at 240 nm. 20 .mu.l of protein
solution was combined with 1 ml of enolase substrate solution (50
mM Tris-HCl pH 7.4, 2.7 mM magnesium acetate, 11.0 mM EDTA, 1.2 mM
D(+)-2-phosphoglyceric acid) in a quartz cuvette and the change of
absorbance recorded at 1 min intervals. The specific activity was
defined as the conversion of 1 .mu.mol of PGA to PEP per min per mg
protein. An estimate of the quantity of protein in solution was
performed using the Bio-Rad.RTM. (Bradford) microassay
procedure.
[0100] Eluate fractions containing enolase activity were selected
and dialysed for 12 h at 25.degree. C. in hpH.sub.2O. The dialysed
fractions were recovered and filtered through a 0.20 .mu.m syringe
filter. The filtrate was concentrated ten-fold by evaporation under
vacuum for 5 h. The concentrated samples were dialysed with binding
buffer `A` (10 mM sodium acetate, pH 4.7) immediately prior to
application to a Pharmacia Biotech Mono S HR10/10 chromatography
column packed with methyl sulphonate cation exchanger (Pharmacia
LKB, Uppsala, Sweden). Cation exchange chromatography was performed
using the Bio-Rad.RTM. Biologic system. Bound protein fractions
were eluted from the column with a salt gradient of buffer `B` (1M
NaCl in buffer `A`, pH 4.7). Fractions containing enolase activity
were identified by the enzyme activity assay described above.
[0101] FIG. 2 shows a single Coomassie blue band of 48 kDa
corresponding to the expected size of the enolase antigen. The
identification of the 48 kDa antigen as the glycolytic enzyme
enolase was confirmed by an enolase activity assay.
[0102] Purification of the cell wall antigen was conducted as
follows: the precipitated insoluble cell walls were collected
following centrifugation as described above. The cell walls were
washed with hpH.sub.2O then collected by centrifugation at 6,000
rpm. This step was repeated three times or until the supernatant
was no longer cloudy. This ensured any residual soluble cytoplasmic
antigen was removed from the cell wall preparation. The washed cell
wall pellet was then resuspend in 10 mM Phosphate buffer pH7.4
containing 1% v/v .beta.-Me and incubated for 30 min at 37.degree.
C. in a shaker to solubilise the cell wall antigens. The sample was
then centrifuged for 5 min at 8,000 rpm and the pellet was then
discarded. The supernatant was transferred into a fresh tube and
recentrifuged (5 min at 8,000 rpm). The supernatant containing the
solubilised cell wall antigen was then dialysed in hpH.sub.2O for
48 h at 4.degree. C. (four changes of water), or until no odour was
detected. Following dialysis the sample was centrifuged three times
5 min at 8,000 rpm to remove any residual particular matter.
[0103] Following clarification the cell wall antigen preparation
was analysed by SDS-PAGE. The resulting Coomassie blue stained gel
is presented in FIG. 3. A broad smear of stain is seen ranging in
size from 90 kDa to 200 kDa. The lack of discrete protein bands is
typical of mannoproteins, where differences in the number of
mannose groups added to the protein base results in a variety of
molecular weights.
Example 2
Enzyme Linked Immunosorbent Assays (Elisas)
[0104] A serum panel was collected from 1998 to 2000 from various
patients with Candida infections. Negative control (Control) sera
(n=20) were obtained from the Red Cross Blood Bank, Perth,
Australia and was obtained from healthy males in the 19 to 25 year
age group. Sera (n=13) from patients with recurrent vulvo vaginal
candidiasis (VVC) were obtained from King Edward Memorial Hospital,
Perth, Australia. Sera (n=108) from patients with oral candidiasis
were obtained from Clinipath Ltd and the UWA Dental School, Perth,
Australia. Sera (n=39) from patients (n=28) with systemic
candidiasis were obtained from Princess Margaret Hospital, Perth,
Australia and Prince of Wales Hospital, Sydney, Australia.
[0105] In the case of patients with oral and vaginal Candida
infection, confirmation of infection was made by physical
examination and by culture of Candida organisms from the relevant
body site. In the case of patients with systemic infection,
confirmation of infection was through positive blood culture or
biopsy. In all cases the immune status of the patient was
unknown.
[0106] Sera from patients with either superficial or systemic
candidiasis were screened by ELISA using trays coated with the
Candida cytoplasmic antigen. The protein content of each antigen
preparation was determined using a commercial assay (BioRad) with
BSA as a standard. A series of ELISAs were performed to determine
the optimal coating concentration for each antigen (data not
shown). The optimal coating concentration being that which gave the
greatest discrimination between a positive and a negative control
serum. For each antigen the optimum coating concentration was
determined to be 2 .mu.g/ml.
[0107] A 96 well C8 strip microtitre plate (Greiner GmbH, Germany),
was coated with either Candida cell wall antigen, cytoplasmic
antigen, or purified enolase antigen as prepared in Example 1. 50
.mu.l of a 2.0 .mu.g/ml solution of the antigen was diluted in
coating buffer (0.1M NaHCO.sub.3, pH 9.3) and added to individual
wells. The plates were incubated for 12 h at 4.degree. C. then
equilibrated to ambient temperature. After equilibrating the plates
to ambient temperature, coating solution was decanted and the plate
tapped dried. Plates were inverted on paper towel to drain.
Alternatively excess coating solution was aspirated by the
automated plate washer (Dynatech Laboratories, Chantilly Va., USA).
It was important not to wash the plate at this stage.
[0108] A volume of 300 .mu.l of blocking solution (PBS pH 7.3, 2%
(w/v) BSA (ICN, Australia), 0.01% (w/v) Tween 20), was applied to
each well and incubated at 25.degree. C. for 90 min. Blocking
solution was decanted and the plate tapped dried. Plates were
inverted on paper towel to drain and tapped dried for a second
time. At this stage plates were either used immediately, or dried
for storage. Plates to be dried were placed inverted in a sealable
container such as a plastic food container with a number of silica
gel desiccant sachets for 48 h. The inclusion of approximately 20
small desiccant sachets was adequate for the drying of 6 coated
ELISA micro-well trays. Dried plates were sealed into heat-sealed
packets with a single desiccant sachet and labelled. Plates were
stored at 4.degree. C. until required. Packets containing plates
were equilibrated to ambient temperature before opening.
[0109] Human test sera diluted 1/100 in blocking solution was
dispensed into wells in 50 .mu.l aliquot's and incubated at
37.degree. C. for 30 min. The primary antibody solution was
aspirated and wells were washed six times in PBS-Tween 20. The
plates were inverted on paper towels and allowed to drain for 10
min. The plates were then tapped dried.
[0110] A volume of 100 .mu.l of a horseradish peroxidase anti-human
IgG conjugate diluted 1/10,000 in blocking solution was dispensed
to each well. Secondary antibody solution was incubated at
37.degree. C. for 30 min. The secondary antibody solution was
aspirated and wells were washed six times in PBS-Tween 20. Plates
were inverted on paper towel to drain for 10 min and then tapped
dried. Plates were inverted on paper towel for a second time and
allowed to drain for 5 min. Plates were then tapped dried.
Particular care was employed to ensure that all traces of secondary
conjugate solution was removed as residual conjugate was
established as the major factor responsible for disparity of
results (Dynatech Laboratories Inc, USA).
[0111] A volume of 100 .mu.l of TMB liquid substrate solution was
dispensed into each well and developed at 25.degree. C. for 10 min.
The reaction was terminated with the addition of 100 .mu.l of 1M
phosphoric acid or 1M H.sub.2SO.sub.4. The absorbance values for
each well were measured at 450 nm, reference 620 nm with a MRX
automated plate reader.
[0112] Each immunoassay was performed in triplicate and the mean
value of absorbance was used. The absorbances are shown as a
Scatter diagram in FIG. 4. Three groups of patients with Candida
infections were analysed. The first group were patients with
systemic candidiasis (Systemics), the second group had oral
candidiasis (Oral) and the third group had vulvovaginal candidiasis
(VVC). Blood bank sera (Control) from males in the 19 to 25 year
age group, who were at low risk of having an undetected or
subclinical Candida infection were used as a control. The cut-off
absorbance (OD.sub.450=0.22) was the mean value of the negative
control sera. From these data the cytoplasmic antigen ELISA had a
sensitivity of 89% and a specificity of 95%. This is higher than
that reported for other Candida serological tests (Zoller et al.,
1991. J. Clin. Micro. 29:1860-1867).
[0113] To further increase the sensitivity of the Candida ELISA
multiple antigens were used. These were the cell wall, cytoplasmic
and native enolase (described above).
[0114] The use of multiple antigens increased the sensitivity of
the Candida ELISA. It also provided greater discrimination between
superficial and systemic infection. Six negative control sera
(serum obtained from healthy males in the 19 to 25 year age group)
were used in ELISAs with microtitre tray wells individually coated
with the three Candida antigens. For each serum the antibody titre
to each of the three antigens was below that of the cut-off line
(FIG. 5). This line is the cut off value assigned based on a
comparison of the average antibody titres of sera from control
patients versus those of candidiasis patients. The value plotted on
the y-axis of the graph is the ratio of the cut-off absorbance
divided into the absorbance of the test serum.
[0115] Serum obtained from 6 patients with superficial candidiasis
was then reacted in the ELISA. Again the absorbance value of each
serum was divided by the absorbance of the cut-off (FIG. 6). The
characteristic antibody response of the sera from patients with
superficial candidiasis was a high titre against the cell wall
antigen preparation (1.5 to 2 times the cut-off value). The
antibody reactivity to the complete cytoplasmic antigen preparation
was positive in most cases (1 to 1.5 times the cut-off). In
contrast the antibody titre to the enolase antigen was below or
equal to that of the cut-off. There is a correlation between the
antibody titre to the internal Candida antigens (cytoplasmic and
enolase) and the severity of the superficial infection (data not
shown). However, the severity of the infection in the six patients
analysed was not known.
[0116] Six sera taken from patients with systemic candidiasis
(confirmed by positive blood culture) were analysed by ELISA. The
results are presented in FIG. 7. In the case of the patients with
systemic candidiasis the antibody response to the cell wall antigen
preparation was positive (1.5 to 2 times the cut-off value). Also,
the antibody titres to the internal Candida antigens (cytoplasmic
and enolase) were also positive (1.5 to 2.5 times cut-off
value).
Conclusions
[0117] The Candida mannan depleted cytoplasmic antigen preparation
disclosed herein can be used to identify patients with Candida
infections. The sensitivity and specificity using an ELISA with
microtitre trays coated with this antigen is greater than that
obtained by other Candida diagnostic tests. Further, the ELISA
assay format disclosed herein is easier to perform, more robust and
more rapid than formats used in other available Candida diagnostic
assays. The ELISA format also has the advantage that it is
quantifiable. This enables the patient to be monitored over a
period of time and changes in the titre of the antibody response to
the Candida antigens recorded. The ability of the test to monitor
overtime the antibody titre to Candida antigens has a prognostic
value in terms of measuring the patient's response to antifungal
drugs and in the overall survival prospects of the patient. Another
advantage of the cytoplasmic antigen preparation is that the method
developed to produce the antigen is simpler and more rapid than
other available procedures (eg. compare with that of Zoller et al.,
1991, supra).
Example 3
Clinical Evaluation in France
[0118] Clinical evaluation of the triple antigen test kit as
described in Examples 1 and 2 was undertaken in the Department of
Parasitology and Medical Mycology at the University of Grenoble
Faculty of Medicine, Grenoble, France using stored sera.
[0119] Sera from two groups of patients were analysed: those that
were blood culture positive and those that were blood culture
negative. When possible, sera were taken before, at the time of and
after the first day of positive blood culture to be tested. The
blood culture negative group was divided into 3 subgroups: Patients
that were colonised with Candida and were serology positive,
patients that were colonised with Candida and were serology
negative, and patients that were not colonised with Candida and
serology negative. The sera were obtained from patients
hospitalised between 1998 and 2000.
[0120] The triple antigen ELIZA test ("the Applicant antigen test")
was performed according to Example 2. The cut-off calibrator sera
was obtained by pooling sera taken from males in 19 to 25 age group
who had no history of Candida infections.
[0121] Table 1 shows that the Applicant antigen test was positive
in 15 out of 19 patients who had a positive blood culture.
TABLE-US-00001 TABLE 1 APPLICANT TRIPLE ANTIGEN TEST AS USED IN THE
FRENCH STUDY Applicant Date of Triple Ag Applicant serum Abs/Cut
Triple Ag Serology- relative to Applicant off Score immunofluo-
Patient Patient Serum Candida first +ve Triple Ag (0.46) (0.46
rescence IEP IEP Ag Group ID ID species culture Abs ratio cut-off)
IFI Pasteur FSK emie Candidemia AMI C1 C.g -4 1.597 3.5 +++ +++ AMI
C2 +3 1.519 3.3 +++ ++ +++ +++ BRIG C3 C.a -2 0.385 0.8 - - BRIG C4
+10 0.325 0.7 - - COE C5 C.t -13 0.406 0.9 - - COE C6 +1 0.734 1.6
+ +++ COE C7 +29 0.632 1.4 + - COH C8 C.g -14 0.597 1.3 + - COH C9
+9 0.661 1.4 + - COH C10 +65 0.391 0.9 - - COM C11 C.g +1 1.806 3.9
+++ + +++ ++ COM C12 +19 1.862 4.0 +++ ++ ++ +++ CON C13 C.g -27
0.732 1.6 + - + CON C14 +1 0.5 1.1 (+) - ++ CON C15 +8 0.367 0.8 -
- ++ DA SI C17 C.a +2 1.805 3.9 +++ + ++++ ++ DA SI C18 +70 1.277
2.8 ++ ++ ++ (+) FER C19 C.a -35 0.693 1.5 + - FER C20 +2 0.368 0.8
- - - FER C21 +16 0.229 0.5 - - FON C22 C.a -46 0.51 1.1 (+) - FON
C23 +3 1.899 4.1 +++ +++ FON C24 +27 1.854 4.0 +++ +++ HAM C25 C.a
+1 1.083 2.4 ++ + + + HAM C26 +31 1.168 2.5 ++ + ++ (+) HEN C27 C.t
+2 0.324 0.7 - - HEN C28 +7 0.646 1.4 + - HEN C29 +40 0.432 0.9 - -
KHA C30 C.a -13 0.332 0.7 - - KHA C31 +2 1.553 3.4 +++ + KHA C32
+27 1.393 3.0 +++ + LON C33 C.a -2 0.341 0.7 - - LON C34 +6 0.447
1.0 (+) - LON C35 +61 0.35 0.8 - - MAN C36 C.a -28 0.505 1.1 (+) -
MAN C37 +5 0.288 0.6 - - MAN C38 +72 0.199 0.4 - - NI C39 C.t &
+3 0.223 0.5 - - NI C40 C.k +9 0.368 0.8 - ++ + + PAS C41 C.a +5
0.865 1.9 + + ++ + PASe C42 +32 1.279 2.8 ++ + ++ ++ + PIL C43 C.p
-2 0.495 1.1 (+) - PIL C44 +51 0.831 1.8 + + RAM C45 C.t +5 1.414
3.1 +++ ++ RAM C46 +23 1.114 2.4 ++ + NOI C47 ? 0 0.611 1.3 + +++
Hospital ABE D21 No No info. 0.748 1.6 + + patients info. that are
FRE D22 No No info. 0.454 1.0 - - colonised info. but have BEN D23
C.a urine 0.331 0.7 - - Negative BER D24 C.t mouth/ 0.463 1.0 - -
Candida fae serology BOM D25 C.a broncal/ 1.046 2.3 ++ ++ fae CAP
D26 No No info. 0.658 1.4 + - info. CAR D27 C.a trachea 0.933 2.0
++ + CHE D28 C.t urine 1.376 3.0 +++ - FER D29 C.a urine/ 0.363 0.8
- - faeces GIN D30 C.g urine/ 0.663 1.4 + - faeces PER D31 C.a
thorax 0.378 0.8 - - drain Hospital BEN D32 C. spp urine 0.469 1.0
- - patients BON D33 -- 0.44 1.0 - - that are CIA D34 C.a urine
0.92 2.0 ++ ++ non- DAVID D35 -- 0.651 1.4 + - colonised CAR and
have PEL D36 -- 0.752 1.6 + - negative DI M D37 -- 0.489 1.1 (+) -
Candida FEU D38 -- 0.633 1.4 + - serology FOG D39 C.a thorax 1.095
2.4 ++ + drain MOR D40 0.38 0.8 - - GO D41 0.677 1.5 + - Hospital
ALL D42 C. spp urine 1.177 2.6 ++ + patients BAR D43 C.g, septic
1.375 3.0 +++ +++ that are C.a, shock colonised C.t and have BOE
D44 C.g urine/ 1.096 2.4 ++ ++ positive mouth Candida BUI D45 No No
info. 1.125 2.4 ++ ++ serology info. COL D46 No No info. 1.062 2.3
++ +++ info. DAG D47 No No info. 0.705 1.5 + - info. BE D48 C.a
& mouth/ 1.123 2.4 ++ +++ C.t trachea GEN D49 No No info. 1.426
3.1 +++ ++++ info. GEN D50 No No info. 1.489 3.2 +++ ++++ info. LEC
D51 No No info. 1.668 3.6 +++ +++++ info. LECr D52 No No info. 1.62
3.5 +++ +++++ info. Legend: Candida Culture Sp. Applicant Kb IFI
IEP Pasteur IEP FSK Ag-emie C.a = C. albicans <10 = - <20 = -
1 arc = + 1 arc = + 1/2 dil = + C.g = C. glabrata 10-20 = + 20 = +
2 arc = ++ 2 arc = ++ 1/4 dil = ++ C.k = C. kefir 20-30 = ++ 40 =
++ 3 arc = +++ 3 arc = +++ C.p = C. parapsilosis 30-40 = +++ 80 =
+++ 4 arc = ++++ 4 arc = ++++ C.t = C. tropicalis 160 = ++++ 320 =
+++++
Of the 12 patients who had sera taken before or on the day of the
first positive blood culture, 8 gave a positive (or low positive)
result. When compared with other serology tests used by the French
group 12 out of 19 patients were positive by the immunofluorescence
(IFI) serology test. All but one of these was positive using the
Applicant antigen test. One patient was also positive by the
Applicant antigen test, but negative by IFI. All of the 5 patients
that tested positive by IEP Pasteur, IEP FSK or Ag-emie serology
tests were also positive by the Applicant antigen test.
[0122] It is possible that some of the patients that were negative
by both the Applicant and the IFI test may have had a transient
candidemia due to central line contaminations.
[0123] Six of 11 patients that were know to be colonised, but had
negative serology were positive by the Applicant antigen test. Two
of the positive patients were also positive by IFI. Of the 10
non-colonised hospital patients with negative serology six were
positive by the Applicant antigen test, two of these positive
patients were also positive by IFI. All nine patients that were
colonised patients with positive serology were positive by the
Applicant antigen test. These data compared to 8 out of 9 patients
that were positive by IFI. The only IFI negative sample was a low
positive by the Applicant antigen test.
[0124] The statistical analysis of these data is presented in FIG.
8 and Table 2.
TABLE-US-00002 TABLE 2 Mean 95% Confidence Category (Units)
Interval Candidemia Patients 21.79.sup.a,b 16.25-27.33 Colonised +
negative 14.55.sup.a,c 9.67-19.42 Serology Non-colonised +
14.2.sup.b,d 10.68-17.72 negative serology Colonised + positive
27.27.sup.c,d 23.12-31.43 serology .sup.ap = 0.71 .sup.bp = 0.58
.sup.c&.sup.dp < 0.01
In the candidemia patients with positive blood culture to Candida,
the mean of their Candida antibody levels detected by Applicant
antigen test was 21.79 (16.25-27.33 95% CI). Using the Independent
Samples T-test, the p value was 0.71 between the candidemia group
and the colonised group with negative serology to Candida. The p
value was 0.58 between the means of the candidemia group and the
non-colonised group that was negative for Candida serology.
[0125] For the negative blood culture patients, the patients in the
groups that were negative for Candida serology had generally lower
Candida antibody levels detected by the Applicant antigen test. The
mean antibody levels were 14.55 units in the colonised group
(9.67-19.42, 95% CI) and 14.2 units in the non-colonised group
(10.68-17.72, 95% CI). These levels were significantly lowered
(p<0.01) from the mean antibody levels in the group of patients
that had positive Candida colonisation culture results and positive
Candida serology, mean 27.27 (23.12-31.43, 95% CI). These are
clearly seen in the error bars in FIG. 8.
[0126] Overall, there was a good correlation with the Applicant
antigen test and other tests used. There was also a good
correlation with the titre of antibody detected by the Applicant
antigen test and the level of positiveness of the other tests ie.,
a patient that had a high positive result with the Applicant
antigen test also had a similar result with the other tests used
(eg, patients AMI, COM, DA SI, FON, PAS and RAM). Similarly
patients that were negative or low positive with the Applicant
antigen test were also negative or weak positive by the other tests
(eg, patients BRIG, FER, HEN, LON and MAN). It was noted that some
of the blood culture negative patients were positive by the
Applicant antigen test, which demonstrated the great sensitivity of
the Applicant antigen test.
Example 4
Clinical Evaluation in Spain
[0127] A similar clinical evaluation to that undertaken in Example
3 was conducted by Professors Guillermo Quindos, MD, PhD, Maria
Dolores Moragues, PhD, and Jose Ponton, PhD, Department of
Immunology, Microbiology and Parasitology at the Faculty of
Medicine, University of Pais Vasco, Bilbao, Spain.
[0128] The retrospective study sera were obtained from 11 patients
(Table 3--Patients 1.1 to 1.32) with invasive candidiasis as
defined by positive blood culture or by histology and positive
tissue biopsy. The "blood culture negative" group consisted of sera
from 12 patients (Table 4--Patients 2.2 to 2.53) selected on the
basis of the patients have a risk of invasive candidiasis but
having negative blood cultures. Between 3 and 5 sera were tested
per patient. For patients with microbiologically proven candidiasis
the sera were taken before, at the time of and after the positive
blood culture. For the blood culture negative group the sera were
taken at various times during hospitalisation. As well as sera from
hospital patients, sera from three healthy blood donors were also
tested (Table 5). Also a group of fresh sera were prospectively
tested from 5 patients, two with positive Candida blood cultures
and 3 without (Table 5).
TABLE-US-00003 TABLE 3 Patients With Positive Blood Culture Day
Triple Platelia Platelia Spanish Spanish Candida species Patient
extraction Ag Ag Ab Anti-B Anti-GT & Outcome 1.01 -21 + + +
++++ + C. albicans 1.01 -11 + + + ++++ ++ 1.01 0 + + + ++++ ++ 1.01
7 + ++ ++ +++ +++ 1.01 13 +++ + ++ +++ +++ 1.11 -1 +++ ++ +++ ++++
++ C. glabrata 1.11 8 +++ ++ +++ ++++ +++ 1.11 21 +++ + + +++ +++
1.11 29 +++ + +++ ++++ ++++ Exitus d 45 1.17 -11 +++ ++ +++ ++++ -
C. albicans 1.17 -4 +++ + +++ +++ + 1.17 0 +++ + ++ +++ + 1.17 3 +
+ + +++ + 1.17 5 + + + +++ + Exitus d 15 1.18 -6 ++ (+) + +++ - C.
parapsilosis 1.18 2 ++ + + ++ - 1.18 6 + +++ +++ +++ - 1.18 13 +
+++ - + + Exitus d 13 1.19 -3 - - - + - C. albicans 1.19 0 - + - +
- 1.19 4 - + - + - 1.19 7 + + - ++ - Exitus d 7 1.22 -11 + + + + +
C. albicans 1.22 -7 + + - + + 1.22 0 + + + ++ + 1.22 2 + + + + +
1.22 7 + + + + + Discharge d 7 1.25 -2 - + - ++ - C. albicans 1.25
1 + - + +++ - 1.25 4 + - - ++ - 1.25 14 + (+) - ++ - 1.25 26 + + -
++ - 1.26 -9 - + (+) ++ - C. parapsilosis 1.26 -5 + + + +++ - 1.26
0 + + + +++ - 1.26 9 + ++ + +++ - 1.26 16 + + + ++ - 1.30 -25 + (+)
? +? C. alibicans 1.30 -4 + + +++ ++++ 1.30 0 + - +++ ++++ 1.30 3 +
- ? +? 1.30 56 + + ? -? Exitus d 70 1.31 -4 ++ - ++ +? C. albicans
1.31 -1 + + ++ ++++ 1.31 6 ++ (+) ++ ++++ 1.31 13 ++ - ++ ++++
Exitus d 32 1.32 -19 - - + - C. parapsilosis 1.32 -17 - + + - 1.32
0 - - + - 1.32 7 - + + - 1.32 15 - + + - Discharge d 33 Scoring:
Applicant Ab Platelia Ag Platelia Ab Spanish B-Ab Spanish GT Ab
0-10 = - <0.5 = - <1 = - <20 = - <20 = - 10-20 = +
0.5-5 = + 1-10 = + 20-80 = + 20-200 = + 20-30 = ++ 5-10 = ++ 10-20
= ++ 80-600 = ++ 200-600 = ++ >30 = +++ >10 = +++ >20 =
+++ 600-5000 = +++ 600-1200 = +++ >5000 = ++++ >1200 =
++++
TABLE-US-00004 TABLE 4 Patients With Negative Blood Culture Day
Applicant Platelia Platelia Spanish Spanish Patient extraction
Triple Ag Ag Ab Anti-B Anti-GT 2.2 1 ++ + - 2.2 5 ++ + - 2.2 6 ++ +
(+) 2.4 1 + (+) ND 2.4 15 ++ + ND 2.4 17 + + ND 2.7 1 + - - + - 2.7
4 ++ + + +++ - 2.7 8 + + + +++ - 2.7 11 + + + +++ - 2.7 15 + + +
+++ - 2.10 1 + - +++ - 2.10 3 ++ - +++ - 2.10 7 ++ - +++ - 2.10 11
++ - ++++ - 2.10 15 ++ - +++ - 2.14 1 ++ (+) + +++ - 2.14 3 ++ - +
+++ - 2.14 6 ++ - + +++ - 2.14 9 ++ (+) + +++ - 2.14 12 ++ + + +++
- 2.18 1 - - - 2.18 4 + + - 2.18 8 + - - 2.18 12 + + - 2.18 22 + +
+ 2.26 1 + - - 2.26 9 + + + 2.26 16 + - + 2.26 23 + + + 2.26 30 + +
+ 2.49 1 - - - 2.49 11 + - - 2.49 15 - - - 2.49 18 - - (+) 2.49 27
+ - + 2.50 1 + (+) + 2.50 9 + + (+) 2.50 15 + - + 2.50 22 + - +
2.50 26 + - + 2.51 1 - - - 2.51 11 - - (+) 2.51 18 - + - 2.51 22 -
+ - 2.51 29 + - - 2.52 1 + - (+) 2.52 8 + - - 2.52 11 + - (+) 2.52
15 + - (+) 2.52 18 + - (+) 2.53 1 + - - 2.53 8 + - - 2.53 11 + +
(+) 2.53 18 + - - 2.53 22 + - - Scoring: Rockeby Ab Ag Platelia Ab
Platelia Spanish B-Ab Spanish GT Ab 0-10 = - <0.5 = - <1 = -
<20 = - <20 = - 10-20 = + 0.5-5 = + 1-10 = + 20-80 = + 20-200
= + 20-30 = ++ 5-10 = ++ 10-20 = ++ 80-600 = ++ 200-600 = ++ >30
= +++ >10 = +++ >20 = +++ 600-5000 = +++ 600-1200 = +++
>5000 = ++++ >1200 = ++++
TABLE-US-00005 TABLE 5 New Patients and Blood Donor Controls Day
Blood Applicant Platelia Platelia Spanish Spanish Patient
extraction Culture RESULT Ag Ab Anti-B Anti-GT B. Donor 1 N/A - -
ND ND ND ND B. Donor 2 N/A - - ND ND ND ND B. Donor 3 N/A - - ND ND
ND ND New Patients 1 N/A - - ND ND ND ND 2 N/A - (+) ND ND ND ND 3
N/A Asperg + ND ND ND ND 4 ? C.g + ND ND ND ND 4 ? C.g ND ND ND ND
5 ? C.a ++ ND ND ND ND 5 ? C.a ND ND ND ND Legend: Culture Species
Applicant Ab C.a = C. albicans 0-10 = - C.g = C. glabrata 10-20 = +
20-30 = ++ >30 = +++
[0129] Table 6 summarises the original Spanish data split into the
two groups of patients, one blood culture positive and the other
blood culture negative. Of the blood culture positive group, the
Applicant antigen test identified 8/11 patients as positive before
they became blood culture positive. Ultimately 10/11 patients were
positive with the Applicant antigen test. Only one patient (1.32)
remained negative. This patient was also negative by the Spanish
germ tube antibody test and was only transiently positive by the
Platelia (BioRad) mannan antigen test. It may be possible that this
patient had a transient candidemia.
TABLE-US-00006 TABLE 6 Summary of Spanish Data Applicant Platelia
Platelia Spanish Spanish Triple Ag Ag Ab Anti-B Anti-GT Culture
Positive Patients Negative result 1 0 1 0 4 Positive before culture
8 11 9 8 6 Positive after culture 2 1 0 1 Total Patients tested 11
11 11 8 11 Culture Negative Patients Negative result 0 3 0 0 6
Positive result 12 9 2 3 5 Total Patients tested 12 12 2 3 11
Patient ID 1.01 pos pos pos pos pos 1.11 pos pos pos pos pos 1.17
pos pos pos pos pos 1.18 pos pos pos pos (pos) 1.19 (pos) pos neg
pos neg 1.22 pos pos pos pos pos 1.25 pos pos (pos) pos neg 1.26
pos pos pos pos neg 1.30 pos pos pos ND pos 1.31 pos pos pos ND pos
1.32 neg pos pos ND neg Patient ID 2.02 pos pos neg 2.04 pos pos
2.07 pos pos pos pos neg 2.10 pos neg pos neg 2.14 pos (pos) pos
pos neg 2.18 pos pos (pos) 2.26 pos pos pos 2.49 pos neg (pos) 2.50
pos (pos) pos 2.51 (pos) pos neg 2.52 pos neg neg 2.53 pos (pos)
neg
[0130] The Applicant antigen test identified all 12 of the blood
culture negative patient group as being positive for Candida
antibody. In comparison, the Platelia Mannan antigen test
identified 9/12 patients as being positive compared to 5/11 by the
Spanish Germ tube antibody test. The main problem with the blood
culture negative patient group was that there was no other
confirmation of diagnosis.
[0131] Overall, there was good correlation with the results of the
Applicant antigen test and that of the other serology tests used by
the Spanish group. Where a patient was strongly positive by the
Applicant antigen test (ie.: patient's 1.11, 1.17, 1.18 and 1.31),
they were also strongly positive by the other tests. Also, where
sera were negative or low positive by the Applicant antigen test,
they are also usually negative or weakly positive by the other
tests. For example, patients 1.19, 1.22, 1.25, 1.26, 1.32, 2.18,
2.26, 2.49, 2.50, 2.51, 2.52 and 2.53.
[0132] Where fresh sera was analysed (Table 5), there was a perfect
correlation with the Applicant antigen test and whether the sera
was blood culture positive or negative.
[0133] The statistical analysis of these data is presented in FIG.
9 and Table 7. From the error plot diagram in FIG. 9, it is evident
that the group of patients with positive blood culture have a
higher Candida antibody levels detected by Syscan3 (mean 25.86, 95%
CI: 16.28-35.44) as compared to the patients with negative blood
culture as a group (mean 17.30, 95% CI: 13.42-21.19). Comparing the
means using the Independent Samples T-Test, the difference between
the two groups is statistically significant at p=0.087.
TABLE-US-00007 TABLE 7 Mean and 95% Confidence Interval of Mean of
Applicant Antigen Test Scores 95% Confidence Category Mean (Units)
Interval Positive Blood 25.86* 16.28-35.44 Culture Negative Blood
17.30* 13.42-21.19 Culture *p = 0.087
Example 5
Clinical Evaluation in Australia
[0134] Sera collected from patients with invasive candidiasis was
obtained from an Australian hospital (1997 to 1998), the patients
had haematological malignancies (n=24). Control sera were collected
from males 18 to 25 years of age (n=20) with no history of Candida
infection. The patient sera were tested with the Applicant antigen
test as described in Example 2. Each sera was tested in triplicate
and the average reading used. The average absorbance reading for
each serum was divided by that of the "cut-off" calibrator serum
supplied with the Applicant antigen test. This value was then
multiplied by 10 to give a value in arbitrary units.
[0135] The results of the Applicant antigen test using a value of
20 units (two times the cut-off calibrator serum value) or above as
defining a positive sample is presented in Table 8.
TABLE-US-00008 TABLE 8 Results of the Applicant antigen test using
20 units as a cut-off Invasive Healthy Candidiasis Controls Total
Test Positive 20 0 20 Test Negative 4 20 24 Total 24 20 44
[0136] With the Applicant antigen test using 20 units as cut-off,
the specificity of the test was 100% and sensitivity was
83.3%.--Positive predictive value was 83.3% and negative predictive
value was 100%. When the value of the test considered positive was
set at 10 units or 1 times the value of the cut-off sera
absorbance, the specificity of the test decreased, but the
sensitivity increased (Table 9). The specificity was 90%,
sensitivity 87.5%. Positive predictive factor increased to 91.3%,
while negative predictive factor decreased to 85.7%.
TABLE-US-00009 TABLE 9 Results of the Applicant antigen test using
10 units as a cut-off Invasive Healthy Candidiasis Controls Total
Test Positive 21 2 23 Test Negative 3 18 21 Total 24 20 44
[0137] The results of the Applicant antigen test using sera from
patients with invasive candidiasis are presented in Table 10. Only
one of the negative results came from a patient who was blood
culture positive. Two of the four negative samples were from
patients with central line contaminations. Three of the four
negative test results came from patients with Candida parapsilosis
infections, the other being C. albicans.
TABLE-US-00010 TABLE 10 Sera from Patients with Invasive
Candidiasis Test with Applicant Antigen Test Pa- Abs tient (420 nm)
Units Result Site of isolation Candida spp. A 1.69 61 Positive
blood culture parapsilosis B 0.15 5 Negative blood culture
parapsilosis C 1.16 42 Positive peritoneal cavity guillermondii D
1.85 67 Positive blood culture albicans E 1.16 42 Positive blood
culture albicans F 1.49 54 Positive peritoneal cavity glabrata G
0.56 20 Positive peritoneal cavity parapsilosis I 1.24 45 Positive
cathater albicans K 0.98 35 Positive oesophagus albicans L 3.46 124
Positive peritoneal cavity albicans M 0.19 7 Negative central line
albicans N 1.44 52 Positive wound albicans P 0.55 20 Positive
sputum glabrata Q 1.12 40 Positive sputum tropicalis R 1.02 37
Positive central line albicans S 0.88 32 Positive blood culture
albicans T 1.6 58 Positive blood culture glabrata U 0.22 8 Negative
central line parapsilosis V 0.55 20 Positive urine parapsilosis W
0.31 11 Negative peritoneal cavity parapsilosis X 0.59 21 Positive
central line albicans Y 0.85 31 Positive blood culture albicans Z
1.06 38 Positive bronch albicans ZA 1.05 38 Positive urine
tropicalis
[0138] The Applicant antigen test data for the invasive candidiasis
group and healthy controls are presented in the error bar diagram
as seen in FIG. 10 and Table 11. In FIG. 10, the group with
invasive candidiasis has a higher mean (31.45 units) as compared to
the healthy blood donor group (7.52 units). This difference was
statistically significant (p<0.01). The 95% confidence interval
range of the means was higher as well in the invasive candidiasis
group (23.57-39.33 units), as compared to the healthy donor group
(6.92-8.12 units).
TABLE-US-00011 TABLE 11 Mean and 95% Confidence Interval of the
Mean of Patients with Invasive Candidiasis and healthy blood donors
95% CI of Group Mean (Units) mean (Units) Invasive 31.45
23.57-39.33 Candidiasis Healthy blood 7.52 6.92-8.12 donors
[0139] In this study the Applicant antigen test was used to test
sera from patients with invasive candidiasis, superficial
candidiasis (oral or vaginal thrush) and healthy male controls. As
a commensal organism, healthy individuals can have a measurable
antibody titre to Candida antigens. In order to differentiate
between normal and infection associated antibody levels a cut-off
calibrator serum was supplied. The absorbance of the serum being
tested was divided by the cut-off calibrator serum absorbance and
multiplied by 10 to give an arbitrary unit value. Using a value of
20 units or above as an indicator of a positive test gave the
greatest discrimination between the patient group with invasive
candidiasis and the healthy controls (positive predictive value of
83%, negative predictive value 100%). If the value at which a
sample was considered positive was lowered to 10 units (ie. the
cut-off calibrator value), the positive predictive value increased
slightly to 87.5% but the negative predictive value decreased to
90%.
[0140] Only one patient with a positive blood culture returned a
negative test result with the Applicant antigen test. Two out of
the four negative sera were from patients with a central line
contamination. This could therefore reflect a transient infection
in these patients, which may not provoke an antibody response. It
is of interest that 3 of the 4 negative tests were due to C.
parapsilosis infections. This organism is frequently associated
with biofilms, which may shield it from the host immune
response.
[0141] In conclusion, the Applicant antigen test is a rapid,
reliable and easy test to perform. It showed good sensitivity and
specificity in the diagnosis of invasive and severe superficial
Candida infections.
Example 6
Comparative Test of Cytoplasmic Antigen of Present Application
Against Antigens Disclosed in U.S. Pat. No. 4,806,465
[0142] U.S. Pat. No. 4,806,465 in the name of Buckley et al.
discloses mannan-depleted cytoplasmic extract of mycelium,
fractionated by ion exchange chromatography. In contrast, as
described in the Examples above, the cytoplasmic antigens of the
present invention are isolated from blastospore. Moreover, the
antigens isolated in the present invention had molecular weights of
55 kDa, 30 kDa and 20 kDa. In contrast, the molecular weights of
the antigens isolated by Buckley et al. are 120 to 135 Kd, 48 to 52
Kd and 35 to 38 Kd.
[0143] Notwithstanding, we decided to test the antigens prepared in
the present invention with the monoclonal antibodies described in
Buckley et al. that specifically bound the antigens prepared by
Buckley et al.
[0144] Samples of the hybridoma cell lines ATCC #HB-8397 and ATCC
#HB-8398, were obtained. These hybridomas produce monoclonal
antibodies that are monospecific to the cytoplasmic antigens
disclosed in U.S. Pat. No. 4,806,465 (see abstract of U.S. Pat. No.
4,806,465). These monoclonal antibodies were tested against samples
of the antigens disclosed in the Examples above and briefly, none
of these reacted with any of the antigens disclosed. FIG. 11, shows
the results of the experiments. Panel A shows the immunoblot of C.
albicans cytoplasmic (enolase) antigen disclosed in Buckley et al.,
while panel B shows the C. albicans antigen as disclosed in the
present application.
* * * * *