U.S. patent application number 10/174494 was filed with the patent office on 2003-08-14 for method and device for identifying a mycobacterium species responsible for a mycobacterial infection.
Invention is credited to Das, Pranab K., Houthoff, Hendrik Jan, Van Es, Remco Maria.
Application Number | 20030153019 10/174494 |
Document ID | / |
Family ID | 23803394 |
Filed Date | 2003-08-14 |
United States Patent
Application |
20030153019 |
Kind Code |
A1 |
Das, Pranab K. ; et
al. |
August 14, 2003 |
Method and device for identifying a mycobacterium species
responsible for a mycobacterial infection
Abstract
The invention relates to a method for identifying a
Mycobacterium species responsible for a mycobacterial infection in
human or animal, comprising selecting a suitable mycobacterial
species and strain; preparing at least one mycobacterial antigen,
respectively antigen preparation; binding the antigen, respectively
the antigen preparation to a suitable carrier; causing the binding
antigen to react with antibodies from serum of an individual
infected with a Mycobacterium species; making visible
antigen-antibody reactions for a suitable antibody (sub-)class; and
identifying the responsible Mycobacterium species on the basis of
the reactions which are made visible. The invention further
provides a diagnostic kit which takes the form of a dip-stick on
which is arranged a carrier strip with mycobacterial antigens
binding thereto, and means for visualizing antigen-antibody
reactions occurring on the carrier after contact with the serum for
testing. In another embodiment the diagnostic kit comprises a
microtiter plate, in the wells of which a specified antibody is
arranged, and means for making visible antigen-antibody reactions
occurring in the wells after contact with the serum for testing.
The third embodiment is an immunoblot with mycobacterial antigens
separated by electrophoresis binding thereto, and means for
visualizing antigen-antibody reactions occurring on the immunoblot
after contact with the serum for testing.
Inventors: |
Das, Pranab K.; (Castricum,
NL) ; Van Es, Remco Maria; (Koog aan de Zaan, NL)
; Houthoff, Hendrik Jan; (Amsterdam, NL) |
Correspondence
Address: |
HOFFMANN & BARON, LLP
6900 JERICHO TURNPIKE
SYOSSET
NY
11791
US
|
Family ID: |
23803394 |
Appl. No.: |
10/174494 |
Filed: |
June 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10174494 |
Jun 18, 2002 |
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09166663 |
Oct 5, 1998 |
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6416962 |
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09166663 |
Oct 5, 1998 |
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08454122 |
Nov 20, 1995 |
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5817473 |
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Current U.S.
Class: |
435/7.32 |
Current CPC
Class: |
G01N 33/6854 20130101;
G01N 2333/35 20130101; G01N 33/5695 20130101; G01N 2469/20
20130101 |
Class at
Publication: |
435/7.32 |
International
Class: |
G01N 033/554; G01N
033/569 |
Claims
What is claimed is:
1. A method for detecting or identifying a Mycobacterium species in
a biological sample, comprising: (a) selecting a standard
Mycobacterium; (b) preparing an antigen preparation from a culture
of the standard Mycobacterium, wherein the antigen preparation
comprises at least two immuno-cross-reactive antigen components
(ImCRACs); (c) separating the ImCRACs of the antigen preparation
according to molecular weight; (d) binding the separated IMCRACS of
the antigen preparation to a solid carrier; (e) contacting the
bound ImCRACs with the biological sample under conditions that
permit antibodies in the biological sample to bind with the
carrier-bound ImCRACs to provide a pattern of carrier-bound
antibody-ImCRAC complexes; (f) detecting the pattern of
carrier-bound antibody-ImCRAC complexes; and (g) comparing the
pattern of carrier-bound antibody-ImCRAC complexes from the sample
to a library of standard patterns of carrier-bound antibody-ImCRAC
complexes characteristic of particular Mycobacterium species,
wherein matching of the pattern of carrier-bound antibody-ImCRAC
complexes from the sample to one of the standard patterns of
carrier-bound antibody-ImCRAC complexes from the library permits
detection or identification of the Mycobacterium species in the
sample.
2. The method of claim 1, wherein the antigen preparation is
separated by electrophoresis prior to step (d) and the carrier is a
membrane to which the ImCRACS are bound by means of
electroblotting.
3. The method of claim 2, wherein the membrane is a nitrocellulose
membrane.
4. The method of claim 1, wherein the antigen preparation of the
standard Mycobacterium is a total protein preparation.
5. The method of claim 4, wherein the antigen preparation is a
KP-100 or SP-100 fraction of the total protein preparation.
6. The method of claim 1, wherein the carrier is a microtiter
plate.
7. The method of claim 1, wherein the pattern of the carrier-bound
antibody-ImCRAC complexes is a banding pattern consisting of 29/33
KDa, 45/48 KDa, 64 KDa and combinations thereof.
8. The method of claim 1, wherein the carrier-bound antibody-ImCRAC
complexes are made visualizable by using at least one
antibody-enzyme conjugate directed against at least one antibody of
an isotype selected from the group consisting of IgG, IgM, IgA and
combinations thereof.
9. The method of claim 8, wherein the enzyme of the antibody-enzyme
conjugate is peroxidase.
10. The method of claim 1, wherein the carrier is a dip-stick.
11. A method for detecting or identifying a Mycobacterium species
in a biological sample, comprising: (a) providing a test substrate
having bound thereto an antigen preparation from a culture of the
standard Mycobacterium, wherein the antigen preparation comprises
at least two immuno-cross-reactive antigen components (ImCRACs)
such that the ImCRACs of the antigen preparation separate according
to molecular weight; (b) contacting the bound ImCRACs of the test
substrate with the biological sample under conditions that permit
antibodies in the biological sample to bind with the
substrate-bound ImCRACs to provide a pattern of substrate-bound
antibody-ImCRAC complexes; (c) detecting the pattern of
substrate-bound antibody-ImCRAC complexes; and (d) comparing the
pattern of substrate-bound antibody-ImCRAC complexes from the
sample to a library of standard patterns of substrate-bound
antibody-ImCRAC complexes characteristic of particular
Mycobacterium species, wherein matching of the pattern of
substrate-bound antibody-ImCRAC-complexes from the sample to one of
the standard patterns of substrate-bound antibody-ImCRAC complexes
from the library permits detection or identification of the
Mycobacterium species in the sample.
12. The method of claim 11, wherein the antigen preparation is
separated by electrophoresis and the substrate is a membrane to
which the IMCRACS are bound by means of electroblotting.
13. The method of claim 12, wherein the membrane is a
nitrocellulose membrane.
14. The method of claim 11, wherein the antigen preparation of the
standard Mycobacterium is a total protein preparation.
15. The method of claim 14, wherein the antigen preparation is a
KP-100 or SP-100 fraction of the total protein preparation.
16. The method of claim 11, wherein the substrate is a microtiter
plate.
17. The method of claim 11, wherein the pattern of the
substrate-bound antibody-ImCRAC complexes is a banding pattern
consisting of 29/33 KDa, 45/48 KDa, 64 KDa and combinations
thereof.
18. The method of claim 11, wherein the substrate-bound
antibody-ImCRAC complexes are made visualizable by using at least
one antibody-enzyme conjugate directed against at least one
antibody of an isotype selected from the group consisting of IgG,
IgM, IgA and combinations thereof.
19. The method of claim 18, wherein the enzyme of the
antibody-enzyme conjugate is peroxidase.
20. The method of claim 11, wherein the substrate is a
dip-stick.
21. Diagnostic kit comprising: (1) a test substrate having bound
thereto an antigen preparation from a culture of standard
Mycobacterium, wherein the antigen preparation comprises at least
two immuno-cross-reactive antigen components (ImCRACs) that are
separable according to molecular weight, and wherein when the
ImCRACs of the test substrate are contacted with a biological
sample under conditions that permit antibodies in the biological
sample to bind with the substrate-bound ImCRACs, a pattern of
substrate-bound antibody-ImCRAC complexes is provided; and (2) a
means for visualizing the pattern substrate-bound antibody-ImCRAC
complexes.
22. The diagnostic kit of claim 21, wherein the test substrate is
selected from the group consisting of a dip-stick, a microtiter
plate, and an immunoblot.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending U.S.
application Ser. No. 08/454,122 filed on Jun. 7, 1995, the
disclosure of which is incorpored herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a method for identifying a
Mycobacterium species responsible for a mycobacterial infection in
human or animal. The invention further relates to diagnostic kits
for use in the method.
[0004] 2. Description of the Related Art
[0005] The genus Mycobacterium, which contains about 50 species, is
responsible for a number of human and animal diseases which are
known collectively as the mycobacterioses. The best known of these
in humans are leprosy, caused by M. leprae, which affects more than
ten million people worldwide, and tuberculosis, usually caused by
M. tuberculosis, at least ten million new cases of which occur each
year. Most other mycobacteria normally occur only as environmental
saprophytes but can also cause opportunist diseases. This happens
usually, but not only, in the case of people who have problems with
their immune system, such as AIDS patients and people undergoing
immunosuppression. These opportunist types comprise the
slow-growing species M. avium, and the closely related M.
intracellulare and M. scrofulaceum (often referred to together as
MAIS complex), M. kansasi, M marinum and M. ulcerans, and the
fast-growing species M. chelonae and M. fortuitum. Although once
rare, the incidence of opportunist mycobacterial diseases and
tuberculosis shows a parallel increase in the western world with
the incidence of AIDS. In addition there is limited but increasing
evidence that mycobacteria or antigens thereof play a direct or
indirect part in the etiology of a plurality of other diseases such
as sarcoidosis and Crohn's disease and different auto-immune
diseases such as auto-immune dermatitis, rheumatoid arthritis and
diabetes. This could be attributed to a structural mimicry between
epitopes of mycobacteria and those of the host.
[0006] The cell walls of mycobacteria are very complex and contain
many lipids, some with structures unique to the genus. These
structures comprise mycolinic acids and esters, peptido-glycolipde,
arabino-galactane and lipo-arabino-manane. The lipid-rich
mycobacterial cell walls are responsible for the characterizing
coloring properties of the mycobacteria. They also enable
mycobacteria to counter an attack by the immune system of the host.
A number of species, once taken up into macrophages, are capable of
surrounding themselves with a thick layer of secreted lipids.
[0007] Many different components of the mycobacteria begin an
interaction with the immune system. These components comprise
protein and hydrocarbon antigens, which can either be actively
secreted by the mycobacteria or can form part of the cell wall or
cell membrane. In addition they may be present in the cytoplasm,
for instance in the cytoplasmic matrix, ribosomes and enzymes.
Mycobacteria also possess innuno-modulating components such as
inmmunosuppressing compounds and adjuvants. Consequently, a single
mycobacterial species can induce a large variety of immune
responses in different forms and with diverse specificities. It is
therefore difficult to distinguish immune responses against
species-specific components from cross reactions. For this reason
it has therefore been found difficult to derive protein antigens
suitable for the detection of species-specific humoral responses as
a basis for a very sensitive and specific sero-diagnostic test for
tuberculosis. Because the mycobacteria occur a great deal in the
environment, human serum nearly always contains anti-mycobacterial
antibodies.
[0008] In view of the problems with the specificity of protein
antigens, a number of researchers, including the present inventors,
have focused their attention on species-specific glycolipid
antigens for the detection of specific humoral immune responses.
This approach is for example illustrated in Vega-Lopez et al., J.
Clin. Microbiol. 26(12), 2474-2479 (1988) and Roche et al., Int. J.
Mycobact. Dis. 60(2), 201-207 (1992) who both stated that in
serodiagnosis species-specific antigens and antibodies are
required. Although the immune reactivity against mycobacteria is of
the cell-mediated type and the humoral immune responses probably
play a minor part in the total effector mechanism of mycobacterial
immunity and immunopathology, studies in the antibody response to
immuno-dominant mycobacterial cross-reactive antigen components
(referred to hereinafter as "Im-CRAC") could shed light on the
varying capability of the host to recognize different mycobacterial
antigens. They could therefore provide indirect information
relating to the nature of the immune recognition of, and response
to, a specific mycobacterial pathogen.
SUMMARY OF THE INVENTION
[0009] It has now been found that the clinical manifestation of
mycobacterial diseases appears to be related to the varying
capability of an individual host to produce a humoral response to
different mycobacterial immuno-cross-reactive antigen components
(Im-CRAC). Each mycobacterial infection generates its own specific
antibody response to a number of specified antigens. Analysis of
the antibody-response by means of immunoblotting has demonstrated
that the immuno-dominant Im-CRAC vary in accordance with the
immunopathological manifestation of the mycobacterial diseases. It
has been found that the sera of individuals which are infected with
different Mycobacterium species cause different and distinguishing
band patterns on immunoblots of mycobacterial antigens.
[0010] This discovery forms the basis of the present invention,
whereby a method is provided for identifying a Mycobacterium
species responsible for a mycobacterial infection in human or
animal, comprising the steps of:
[0011] (a) selecting a suitable mycobacterial species and
strain;
[0012] (b) preparing an antigen preparation comprising at least one
mycobacterial antigen;
[0013] (c) binding the antigen, respectively the antigen
preparation to a suitable carrier;
[0014] (d) causing the binding antigen to react with antibodies
from serum of an individual infected with a Mycobacterium
species;
[0015] (e) making visible antigen-antibody reactions for a suitable
antibody (sub-)class; and
[0016] (f) identifying the responsible Mycobacterium species on the
basis of the reactions which are made visible.
[0017] In preference, the antigen preparation is separated by
electrophoresis prior to step (c) and the carrier is a membrane to
which the antigen is bound by means of electroblotting. This
process is called Western blotting.
[0018] The present invention is also a method for detecting or
identifying a Mycobacterium species in a biological sample. The
method includes:
[0019] (a) selecting a standard Mycobacterium;
[0020] (b) preparing an antigen preparation from a culture of the
standard Mycobacterium, wherein the antigen preparation comprises
at least two immuno-cross-reactive antigen components
(ImCRACs);
[0021] (c) separating the ImCRACs of the antigen preparation
according to molecular weight;
[0022] (d) binding the separated InCRACS of the antigen preparation
to a solid carrier;
[0023] (e) contacting the bound ImCRACs with the biological sample
under conditions that permit antibodies in the biological sample to
bind with the carrier-bound InCRAds to provide a pattern of
carrier-bound antibody-InCRAC complexes;
[0024] (f) detecting the pattern of carrier-bound antibody-ImCRAC
complexes; and
[0025] (g) comparing the pattern of carrier-bound antibody-ImCRAC
complexes from the sample to a library of standard patterns of
carrier-bound antibody-ImCRAC complexes characteristic of
particular Mycobacterium species,
[0026] wherein matching of the pattern of carrier-bound
antibody-ImCRAC complexes from the sample to one of the standard
patterns of carrier-bound antibody-ImCRAC complexes from the
library permits detection or identification of the Mycobacterium
species in the sample.
[0027] The present invention is also a method for detecting or
identifying a Mycobacterium species in a biological sample. The
method includes:
[0028] (a) providing a test substrate having bound thereto an
antigen preparation from a culture of the standard Mycobacterium,
wherein the antigen preparation comprises at least two
immuno-cross-reactive antigen components (ImCRACs) such that the
ImCRACs of the antigen preparation separate according to molecular
weight;
[0029] (b) contacting the bound ImCRACs of the test substrate with
the biological sample under conditions that permit antibodies in
the biological sample to bind with the substrate-bound ImCRACs to
provide a pattern of substrate-bound antibody-ImCRAC complexes;
[0030] (c) detecting the pattern of substrate-bound antibody-ImCRAC
complexes; and
[0031] (d) comparing the pattern of substrate-bound antibody-ImCRAC
complexes from the sample to a library of standard patterns of
substrate-bound antibody-ImCRAC complexes characteristic of
particular Mycobacterium species,
[0032] wherein matching of the pattern of substrate-bound
antibody-ImCRAC complexes from the sample to one of the standard
patterns of substrate-bound antibody-ImCRAC complexes from the
library permits detection or identification of the Mycobacterium
species in the sample.
[0033] In one preferred embodiment of the present invention, the
antigen preparation is separated by electrophoresis prior to step
(d) and the carrier or substrate can be a membrane to which the
lmCRACS are bound by means of electroblotting. In addition, the
membrane is a nitrocellulose membrane.
[0034] In another preferred embodiment of the present invention,
the antigen preparation of the standard Mycobacterium can be a
total protein preparation. Additionally, the antigen preparation
can be, but not limited to, a KP-100 or SP-100 fraction of the
total protein preparation.
[0035] In another preferred embodiment of the present invention,
the carrier and substrate can be, but not limited to, a microtiter
plate. The ImCRACS of the antigen preparation is bound to the wells
of a microtiter plate so that the carrier-bound ImCRACS is brought
into contact with the biological sample under conditions that
permit the antibodies in the biological sample to bind with the
carrier-bound InCRACs to provide a pattern of carrier-bound
antibody-ImCRAC complexes. The non-binding antibodies can then be
removed.
[0036] The pattern of the carrier-bound antibody-ImCRAC complexes
and the substrate-bound antibody-ImCRAC complexes can be a banding
pattern consisting of, but not limited to, 29/33 KDa, 45/48 KDa, 64
KDa and combinations thereof.
[0037] The carrier-bound antibody-ImCRAC complexes and the
substrate-bound antibody-ImCRAC complexes can be made visualizable
by any method known in the art. Preferably, the carrier-bound
antibody-ImCRAC complexes can be made visualizable by using at
least one antibody-enzyme conjugate directed against at least one
antibody of an isotype selected from the group consisting of, but
not limited to, IgG, IgM, IgA and combinations thereof. The enzyme
of the antibody-enzyme conjugate can be any enzyme known in the art
which can make the carrier-bound antibody-ImCRAC complexes
visualizable, preferably peroxidase.
[0038] In another preferred embodiment of the present invention,
the carrier and the substrate can be, but not limited to, a
dip-stick. The mycobacterial in the antigen preparation is brought
into reaction with antibodies from a biological sample by dipping
the stick in the biological sample for testing. The carrier-bound
antibody-ImCRAC complexes are made visible by subsequently dipping
the stick in a solution with an antibody-enzyme conjugate and a
color substrate for the relevant enzyme.
[0039] The present invention is also a diagnostic kit which
includes (1) a test substrate having bound thereto an antigen
preparation from a culture of the standard Mycobacterium, wherein
the antigen preparation comprises at least two
immuno-cross-reactive antigen components (ImCRACs) that are
separable according to molecular weight, and wherein when the
ImCRACs of the test substrate are contacted with a biological
sample under conditions that permit antibodies in the biological
sample to bind with the substrate-bound ImCRACs, a pattern of
substrate-bound antibody-ImCRAC complexes is provided; and (2) a
means for visualizing the pattern substrate-bound antibody-ImCRAC
complexes.
[0040] The test substrate of the kit can be, but not limited to, a
dip-stick, a microtiter plate, and an immunoblot.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] Preferred embodiments of the invention have been chosen for
purposes of illustration and description, but are not intended in
any way to restrict the scope of the invention. The preferred
embodiments of certain aspects of the invention are shown in the
accompanying drawing, wherein:
[0042] FIG. 1 shows an example of Western blotting patterns which
are developed after incubation respectively with representative
negative and positive sera (positive for bovine tuberculosis).
[0043] FIG. 2a shows an example of different Western blotting
patterns developed after incubation with representative variable
sera of tuberculous patients.
[0044] FIG. 2b shows an example of different Western blotting
patterns developed after incubation with representative sera of
patients with Lepromatous Leprosy (LL) and Tuberculous Leprosy
(TT).
[0045] FIG. 2c shows an example of different Western blotting
patterns developed after incubation with representative sera of
patients with Crohn's Disease.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0046] The Im-CRAC comprise namely a number of antigens with
specific molecular weights which, as has now been found, after
immunoblotting, exhibit a binding pattern which correlates to the
disease or infection. The specific band pattern is characterized by
the presence or absence of four individual components, for
instance:
[0047] a region comprising different pronounced bands and/or
overlapping bands, which can be observed as a smear ("region");
[0048] sharp single bands which are strongly positive ("band");
[0049] sharp double bands which are strongly positive ("doublet");
and
[0050] other positive bands ("extra bands").
[0051] For a survey of the different antigens, their molecular
weights and binding characteristics, see Table 1.
1TABLE 1 Survey of characteristic bind patterns of mycobacterial
Immuno-Cross-reactive Antigen Components. MW range Binding Antigen
Diagnostic for (in KDa) characteristic A J <8 band B T, B, J,
10-16 band C B, J, T, 20-28 band or region D L 29/33 doublet E B,
J, T, 31-40 band or region F T 38-40 band or region G C, B, J,
45/48 doublet H T 58-60 band or region I L 64/65 doublet J J 66
band K T 68 band L L 30-64 region T: Human Tuberculosis L: Leprosy
C: Crohn's Disease B: Bovine Tuberculosis J: Johne's Disease
[0052] Each of the mycobacterioses is characterized by a specific
banding pattern which is formed when a blot having thereon an
antigen preparation of mycobacteria separated to size is incubated
with serum of an infected individual.
[0053] Tables 2a-2f below show a survey of the banding patterns of
a number of mycobacterial and immunological diseases.
2TABLE 2a Bovine tuberculosis Regions (MW in KDa) and/or and/or
and/or and Pattern 10-16 20-28 31-40 45-48 1. 14 KDa band 22 KDa
band 31 KDa band 45/48 KDa 20-28 KDa doublet region
[0054]
3TABLE 2b Johne's Disease Regions (MW in KDa) and/or and and/or
and/or and and Pattern >8 10-16 20-28 31-40 45-48 66 1. region
14 KDa and 22 31 KDa 45/48 66 KDa band (25) band KDa band - KDa
doublet band, and/or 27 KDa band
[0055]
4TABLE 2c Human Tuberculosis Regions (MW in KDa) Pattern 10-16
20-28 31-38 38-40 58-60 68 other* 1. 10 and / 33 KDa region band /
+/- 16 KDa band bands 2. 16 KDa region 33 KDa bands region / +/-
single band band 3. 10 or / / bands bands / +/- 16 KDa band 4. 16
KDa / 33 KDa region bands / +/- band band 5. 10 region 33 KDa bands
bands 68 +/- and/or band and/or KDa 16 KDa region band band *Extra
bands and/or regions can color but are not diagnostic for Human
Tuberculosis
[0056]
5TABLE 2d Leprosy Regions (MW in KDa) Pattern 29-33 30-65 64-65
other* LL pattern 1 29/33 KDa / / +/- doublet TT pattern 1 /
regions and/or / +/- bands TT pattern 2 / regions and/or 64/65 KDa
+/- bands doublet TT pattern 3 / / 64/65 KDa +/- doublet *Extra
bands and/or regions can color but are not diagnostic for
leprosy
[0057]
6TABLE 2e Crohn's Disease Regions (MW in KDa) Pattern 45-48 other*
1. 45/48 KDa doublet +/-
[0058]
7TABLE 2f Rheumatoid Arthritis Regions (MW in KDa) and/or and/or
and/or Pattern and 42 KDa 80-90 KDa 58-60 KDa 14-18 KDa 1. band
region region region
[0059] The method of the invention, such as an immunoblot, can be
used to answer two questions. First, the presence of any positive
band pattern will answer the question of whether a mycobacterial
infection is present. Second, the presence of specific banding
patterns indicates which mycobacterial species has caused the
infection, and therefore what the nature and etiology of the
disease will be. From the patterns in the immunoblotting it follows
which mycobacterial antigen preparations are suitable for diagnosis
of any particular disease.
[0060] The invention further relates to a heterogeneous enzyme
immunoassay. The antigens for a heterogeneous enzyme immunoassay is
preferably chosen from the group which consists of mycobacterial
immuno-cross-reactive antigen components with a molecular weight of
29/33 KDa, 45/48 KDa, 64/65 KDa and a fraction designated with the
term KP-100. These ImCRAC can be used separately or in combination
with each other for serological diagnosis of the correlating
diseases in a heterogeneous enzyme immunoassay (EIA).
[0061] In this form of assay, antibody-conjugates labeled with a
standard enzyme are used. An important detail is that the enzyme
activity does not change during the immunological reaction.
[0062] To test the immune response in patients to the selected
antigens, use is made for instance of microtiter plates ("Solid
Phase"). By means of standard published techniques the antigens are
irreversibly immobilized on the surface of the wells in such a
microtiter plate.
[0063] This binding takes place while retaining specific antigen
determinants on the used antigens. After incubation with serum, in
the wells of the microtiter plate, antibodies present therein can
specifically form a complex with the irreversibly bound
antigens.
[0064] After removal of non-binding serum components, binding
antibodies are detected using an anti-antibody antibody labeled
with an enzyme.
[0065] Binding of the enzyme is only possible when specific
antibodies have adhered to the immobilized antigens. Substrate
conversion by the binding enzyme to a visually or photometrically
observable signal is thereby directly related to the presence of
specific antibodies in the tested serum.
[0066] The choice of specificity of the enzyme-bound anti-antibody
antibody determines the type of reaction that takes place. For
instance, it may be desirable in some cases to demonstrate the
specifically binding immunoglobulins of the IgG type, while in
other cases immunoglobulins of the IgA and/or IgM type are just
demonstrated.
[0067] The combination of antigen and immunoglobulin type defines
the specificity of the test.
[0068] The said methods, that is, the immunoblot and the EIA, can
be used as mutual confirmation.
[0069] In addition, for the serological diagnosis based on the said
antigens, use can be made of a test stick as solid phase.
[0070] A particularly advantageous embodiment of the invention
relates to a test stick, the so-called "dip-stick", which is used
as solid phase in the heterogeneous enzyme immunoassay.
[0071] The said mycobacterial antigens can be irreversibly bound to
such a dip-stick.
[0072] The antigen is brought into reaction with antibody from
serum for testing by dipping the dip-stick in a serum sample for
testing. The formed antigen-antibody complex can be made visible by
subsequently dipping the dip-stick in an anti-antibody
antibody-enzyme conjugate solution.
[0073] With the binding enzyme a substrate can then be converted to
a visually or photometrically observable signal.
[0074] In another embodiment, the invention is a diagnostic kit
for:
[0075] an immunoblot assay; comprising IMCRAC antigens separated by
electrophoresis as described above, immobilized on a solid carrier,
in addition to an associated suitable detection system.
[0076] a heterogeneous enzyme immunological assay; comprising a
microtiter plate, the wells of which are coated with above
mentioned antigens or antigen preparations, in addition to an
associated suitable detection system.
[0077] a dip-stick assay; comprising test sticks coated with
antigen or antigen preparations, in addition to an associated
suitable detection system.
[0078] Detecting Protein with Antibodies
[0079] The probe may be an antibody, preferably a monoclonal
antibody. The antibodies may be prepared as described above.
[0080] Assays for detecting the presence of proteins with
antibodies have been previously described, and follow known
formats, such as standard blot and ELISA formats. These formats are
normally based on incubating an antibody with a sample suspected of
containing the protein and detecting the presence of a complex
between the antibody and the protein. The antibody is labeled
either before, during, or after the incubation step. The protein is
preferably immobilized prior to detection. Immobilization may be
accomplished by directly binding the protein to a solid surface,
such as a microtiter well, or by binding the protein to immobilized
antibodies. This and other immunoassays are described by David et
al., U.S. Pat. No. 4,376,110 which entirety is incorporated herein
by reference.
[0081] Immunoassays may involve one step or two steps. In a
one-step assay, the target molecule, if it is present, is
immobilized and incubated with a labeled antibody. The labeled
antibody binds to the immobilized target molecule. After washing to
remove unbound molecules, the sample is assayed for the presence of
the label.
[0082] In a two-step assay, immobilized target molecule is
incubated with an unlabeled first antibody. The target
molecule-antibody complex, if present, is then bound to a second,
labeled antibody that is specific for the unlabeled antibody. The
sample is washed and assayed for the presence of the label, as
described above.
[0083] The immunometric assays described above include simultaneous
sandwich, forward sandwich, and reverse sandwich immunoassays.
These terms are well known to those skilled in the art.
[0084] In a forward sandwich immunoassay, a sample is first
incubated with a solid phase immunosorbent containing antibody
against the protein. Incubation is continued for a period of time
sufficient to allow the protein in the sample to bind to the
immobilized antibody in the solid phase. After the first
incubation, the solid phase immunoabsorbent is separated from the
incubation mixture and washed to remove excess protein and other
interfering substances which also may be present in the sample.
Solid phase immunoabsorbent-containing protein bound to the
immobilized antibodies is subsequently incubated for a second time
with soluble labeled antibody cross-reactive with a different
domain on the protein. After the second incubation, another wash is
performed to remove the unbound labeled antibody from the solid
immunoabsorbent and to remove non-specifically bound labeled
antibody. Labeled antibody bound to the solid phase immunoabsorbent
is then detected and the amount of labeled antibody detected serves
as a direct measure of the amount of antigen present in the
original sample. Alternatively, labeled antibody that is not
associated with the immunoabsorbent complex can also be detected,
in which case the measure is in inverse proportion to the amount of
antigen present in the sample. Forward sandwich assays are
described, for example, in U.S. Pat. Nos. 3,867,517, 4,012,294, and
4,376,110.
[0085] In a reverse sandwich assay, the sample is initially
incubated with labeled antibody. The solid phase immunoabsorbent
containing immobilized antibody cross-reactive with a different
domain on the protein is added to the labeled antibody, and a
second incubation is carried out. The initial washing step required
by a forward sandwich assay is not required, although a wash is
performed after the second incubation. Reverse sandwich assays have
been described, for example, in U.S. Pat. Nos. 4,098,876 and
4,376,110.
[0086] In a simultaneous sandwich assay, the sample, the
immunoabsorbent with immobilized antibody, and labeled soluble
antibody specific to a different domain are incubated
simultaneously in one incubation step. The simultaneous assay
requires only a single incubation and does not require any washing
steps. The use of a simultaneous assay is a very useful technique,
providing ease of handling, homogeneity, reproducibility, linearity
of the assays, and high precision. See U.S. Pat. No.4,376,110 to
David et al.
[0087] In each of the above assays, the sample containing antigen,
solid phase immmunoabsorbent with immobilized antibody and labeled
soluble antibody are incubated under conditions and for a period of
time sufficient to allow antigen to bind to the immobilized
antibodies and to the soluble antibodies. In general, it is
desirable to provide incubation conditions sufficient to bind as
much antigen as possible, since this maximizes the binding of
labeled antibody to the solid phase, thereby increasing the signal.
The specific concentrations of labeled and immobilized antibodies,
the temperature and time of incubation, as well as other such assay
conditions, can be varied, depending upon various factors including
the concentration of antigen in the sample, the nature of the
sample and the like. Those skilled in the art will be able to
determine operative and optimal assay conditions for each
determination by employing routine experimentation.
[0088] There are many solid phase immunoabsorbents which have been
employed and which can be used in the present invention. Well known
immunoabsorbents include beads formed from glass, polystyrene,
polypropylene, dextran, nylon, and other material; and tubes formed
from or coated with such materials, and the like. The immobilized
antibodies may be covalently or physically bound to the solid phase
immunoabsorbent, by techniques such as covalent bonding via an
amide or ester linkage or by absorption.
[0089] Detecting Antibodies with Protein
[0090] The proteins may be labeled and used as probes in standard
immunoassays to detect antibodies against the proteins in samples,
such as in the sera or other bodily fluids of patients being tested
for. In general, a protein in accordance with the invention is
incubated with the sample suspected of containing antibodies to the
protein. The protein is labeled either before, during, or after
incubation. The detection of labeled protein bound to an antibody
in the sample indicates the presence of the antibody. The antibody
is preferably immobilized.
[0091] Suitable assays are known in the art, such as the standard
ELISA protocol described by R H Kenneth, "Enzyme-linked antibody
assay with cells attached to polyvinyl chloride plates" in Kenneth
et al., Monoclonal Antibodies, Plenum Press, New York, page 376
(1981).
[0092] Briefly, plates are coated with antigenic protein at a
concentration sufficient to bind detectable amounts of the
antibody. After incubating the plates with the protein, the plates
are blocked with a suitable blocking agent, such as, for example,
10% normal goat serum. The sample, such as patient sera, is added
and titered to determine the endpoint. Positive and negative
controls are added simultaneously to quantitate the amount of
relevant antibody present in the unknown samples. Following
incubation, the samples are probed with goat anti-human Ig
conjugated to a suitable label, such as an enzyme. The presence of
anti-protein antibodies in the sample is indicated by the presence
of the label.
[0093] For use in immunoassays, the probe may be the entire protein
or may be functional analogs thereof. Functional analogs of these
proteins include fragments and substitution, addition and deletion
mutations that do not destroy the ability of the proteins to bind
to their antibodies. As long as the proteins are able to detect
antibodies specific for the protein, they are useful in the present
invention.
[0094] The probes described above can be detectably labeled in
accordance with methods known in the art. In general, the probe can
be modified by attachment of a detectable label moiety to the
probe, or a detectable probe can be manufactured with a detectable
label moiety incorporated therein. The detectable label moiety can
be any detectable moiety, many of which are known in the art,
including radioactive atoms, electron dense atoms, enzymes,
chromogens and colored compounds, fluorogens and fluorescent
compounds, members of specific binding pairs, and the like.
[0095] Methods for labeling antibodies have been described, for
example, by Hunter et al. (1962) and by David et al., Biochemistry
13:1014-1021 (1974). Additional methods for labeling antibodies
have been described in U.S. Pat. Nos. 3,940,475 and 3,645,090, each
of which is incorporated herein by reference.
[0096] Methods for labeling oligonucleotide probes have been
described, for example, by Leary et al., Proc Natl Acad Sci USA
(1983) 80:4045; Renz and Kurz, Nucl Acids Res 12:3435 (1984);
Richardson and Gumport, Nucl Acids Res 11:6167 (1983); Smith et
al., Nucl Acids Res 13:2399 (1985); Meinkoth and Wahl, Anal Biochem
138:267 (1984). Other methods for labeling nucleic acids are
described, for example, in U.S. Pat. Nos. 4,711,955, 4,687,732,
5,241,060, 5,244,787, 5,328,824, 5,580,990, and 5,714,327, each of
which is incorporated herein by reference.
[0097] The label moiety may be radioactive. Some examples of useful
radioactive labels include .sup.32P, .sup.125I, .sup.131I, and
.sup.3H. Use of radioactive labels have been described in U.K.
patent document No. 2,034,323, U.S. Pat. Nos. 4,358,535, and
4,302,204, each incorporated herein by reference.
[0098] Some examples of non-radioactive labels include enzymes,
chromogens, atoms and molecules detectable by electron microscopy,
and metal ions detectable by their magnetic properties.
[0099] Some useful enzymatic labels include enzymes that cause a
detectable change in a substrate. Some useful enzymes (and their
substrates) include, for example, horseradish peroxidase
(pyrogallol and o-phenylenediamine), beta-galactosidase
(fluorescein beta-D-galactopyranoside), and alkaline phosphatase
(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The
use of enzymatic labels has been described, for example, in U.K.
2,019,404, EP 63,879, and by Rotman, Proc Natl Acad Sci USA
47:1981-91 (1961).
[0100] Useful reporter moieties include, for example, fluorescent,
phosphorescent, chemiluminescent, and bioluminescent molecules, as
well as dyes. Some specific colored or fluorescent compounds useful
in the present invention include, for example, fluoresceins,
coumarins, rhodamines, Texas red, phycoerythrins, umbelliferones,
LUMINOL.RTM., and the like. Chromogens or fluorogens, i.e.,
molecules that can be modified (e.g., oxidized) to become colored
or fluorescent or to change their color or emission spectra, are
also capable of being incorporated into probes to act as reporter
moieties under particular conditions.
[0101] The label moieties may be conjugated to the probe by methods
that are well known in the art. The label moieties may be directly
attached through a functional group on the probe. The probe either
contains or can be caused to contain such a functional group. Some
examples of suitable functional groups include, for example, amino,
carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.
[0102] Alternatively, label moieties such as enzymes and chromogens
may be conjugated to antibodies or nucleotides by means of coupling
agents, such as dialdehydes, carbodiimides, dimaleimides, and the
like.
[0103] The label moiety may also be conjugated to the probe by
means of a ligand attached to the probe by a method described above
and a receptor for that ligand attached to the label moiety. Any of
the known ligand-receptor binding pair combinations is suitable.
Some suitable ligand-receptor pairs include, for example,
biotin-avidin or -streptavidin, and antibody-antigen. The
biotin-avidin combination may be preferred.
[0104] The present invention will be further elucidated with
reference to a number of examples which are given herein by way of
illustration and are not intended to limit the invention.
EXAMPLE 1
Immunoblot
[0105] 1. Preparation of Crude Mycobacterial Mass ("Starting
Material")
[0106] The mycobacteria were cultured in commercially available
Sauton medium supplemented with 2 g MgSO.sub.4, 8 g citric acid, 2
g K.sub.2HPO.sub.4, 16 g asparagine, 2 g (Fe.sup.+) ammonium
citrate, and 240 mL glycerol. The bacteria were cultured under
standard conditions. The cells were harvested by filtration of the
culture medium with a 12 .mu.m filter. The cells were subsequently
resuspended in 20 mL PBS (phosphate-buffered salt solution) (pH
7.4) and the harvested cells were autoclaved under a pressure of 15
psi for 20 minutes in order to deactivate and sterilize the
bacteria. The thus obtained bacterial mass can be stored at
-80.degree. C.
[0107] To determine the quantity of starting material a 1/100
dilution of the harvested autoclaved suspension in PBS was made.
The optical density thereof, measured at 420 nm (O.D..sub.420) must
be 0.1. If necessary the concentrated bacterial mass is
supplemented with PBS (pH 7.4) until the correct O.D. is obtained.
An O.D..sub.420 of 0.1 indicates the presence of 7.times.10.sup.11
bacteria per 30 mL, which is equivalent to 12 g wet weight of the
bacterial mass.
[0108] For preparation of a crude mycobacterial extract, 5 g wet
weight of the bacterial mass was washed three times with PBS (pH
7.4). Centrifuging was then carried out at 3000.times.g until the
mass precipitated. The pellet was suspended in 50 mL PBS and
stirred carefully to reduce formation of lumps to a minimum. To
prevent lump formation, 0.05% Tween 80 was added. To avoid
bacterial contamination 3 mg penicillin/streptomycin was added to
this solution. This material was then diluted with PBS to a final
concentration of 2 g wet weight/mL.
[0109] The bacterial mass was subsequently broken open using an
automatic French-X-press or RIBI press (American Instruments
Company, Trevenollab. Inc. Maryland). The buckets were pre-cooled
overnight at -20.degree. C. Before use the buckets were held in a
mixture of ethanol and dry ice (-20.degree. C.). After the buckets
were filled with 1 g bacterial mass per bucket of 5 mL and cooled
at -80.degree. C. for 20 minutes, the buckets were placed in the
French-X-press and twelve tons of pressure was applied by pushing
the plunger of the press. The buckets were then removed and cooled
again at -80.degree. C. for 20 minutes. The buckets were inverted
and treated for the second time under the same conditions as the
first time with the exception of pressure being ten tons. The
sequence of cooling and breaking was then repeated five times. The
disrupted cells were eluted with a suitable volume of PBS and
subsequently centrifuged at 4.degree. C. at 300.times.g for 10
minutes in order to remove the unbroken bacteria with the sediment.
The collected supernatant was then centrifuged at 4.degree. C. and
145,000.times.g for 2 hours. The pellet was suspended in 0.1 M
Tris-HCl (pH 7.2), 0.01 M EDTA which contained 20 mM
MgSO.sub.4.multidot.7H.sub.2O in a concentration of about 1 g per
10 mL. 1 mg RNase and 1 mg DNase were added per 10 mL volume.
Samples were then incubated overnight at 4.degree. C. with careful
stirring, followed by incubation for 1 hour at 37.degree. C. The
lysate was centrifuged at 300.times.g and 4.degree. C. for 10
minutes in order to remove the last-remaining unbroken bacteria
(this material is referred to hereinafter as "starting
material").
[0110] 2. Manufacture of Membrane for Assays
[0111] A 12% polyacrylamide analytical gel of 1.5 mm thickness was
casted according to normal standard procedures. No comb was used in
the stacking gel. Five milligrams (5 mg) of the starting material,
KP-100, or SP-100 (see Example 2) were used for each gel. Forty
microlitres (40 .mu.L) of this material was diluted with 1.2 .mu.L
PBS. 300 .mu.L 5.times.loading mixture (0.3 g 250 mM Tris-HCl, 1.0
mL 10% SDS, 1.0 mL 10% dithioerythritol, 5 mg 0.05% bromophenol
blue) was then added.
[0112] Incubation was carried out for 20 minutes at 65.degree. C.
1.5 mL of total sample were subsequently applied to the gel and
electrophoresis performed under the following conditions: 150 V for
the run through the stacking gel for 30 minutes and 100 V through
the running gel for 6 hours.
[0113] To prepare a Western blot the proteins present in the gel
were transferred at 50 V for 3 hours to a nitrocellulose membrane.
After completion of the transfer the membrane was colored with 0.2%
amido black for 2 minutes to check the membrane for irregularities
and air bubbles. The membrane was the decolorized in 0.05% Tween 80
in PBS with 1% BSA (bovine serum albumin). The membrane was then
cut into strips and was ready for use.
[0114] 3. Immunodetection
[0115] The strips were incubated with human serum diluted 1:200
with PBS containing 3% BSA for 1 hour at room temperature. The
strips were subsequently washed three times (for 3 minutes at a
time) in PBS. The strips were then incubated with a goat anti-human
immunoglobulin-alkaline phosphatase conjugate in a dilution of 1 to
1000 in PBS with 3% BSA and 0.05% Tween 80. The strips were then
washed again three times in PBS, as before. The color was developed
with an NBT/BCIP (nitroblue tetrazolium/Bromo, Chloro Indolyl
phosphate) color solution (1 mg per 10 mL) to which 10 .mu.L
H.sub.2O.sub.2 were added. The strips were incubated for a maximum
of 2 hours in 1 mL of this solution per strip. The color reaction
was stopped by transferring the strips to 0.1 M Tris-HCl (pH 8.3),
0:01 M EDTA. The obtained patterns are interpreted by comparison
with a reference pattern.
[0116] The results are shown in FIGS. 1 and 2a-2c.
[0117] FIG. 1 shown in the blots A and B an example of Western
blotting patterns which are developed after incubation respectively
with representative negative and positive sera (positive for bovine
tuberculosis).
[0118] Blots C and D are exemplary Western blotting patterns which
are developed after incubation with a representative negative serum
sample (Blot C) or positive serum sample (Blot D) (positive for
Cattle Jones Disease).
[0119] Blots A and B: Lane 1: BCG (Bacillus Calmette-Gurin) crude
extract, Lane 2: crude extract of an M. tuberculosis strain, Lane
3: M. bovis crude extract.
[0120] Blots C and D: Lane 1: BCG derived KP-100, Lane 2: RIVM 7114
derived KP-100. RIVH is the abbreviation from the Netherlands
National Institute of Public Health and Environmental Protection
(Rijksinstituut voor Volksgezondheid en Milieuhgiene, Bilthoren,
the Netherlands).
[0121] Interpretation of Banding Patterns from Left to Right is as
Follows
[0122] Only the specific characteristics are stated herein.
[0123] Blot A: only the background bands can be observed in blots
incubated with negative serunm.
[0124] Blot B: region in the 10-16 KDa region in lane 3, 22 KDa
band in lane 2, 31 KDa bands in lane 1 and 2, 14 KDa band in lane
2.
[0125] Blot C: only background bands can be observed in blots
incubated with negative serum.
[0126] Blot D: 45/48 KDa doublet in lane 1 and 2, 22 and 25 KDa
band in lane 1 and 2, 66 KDa band in lane 1 and 2, 27 KDa band in
lane 1.
[0127] FIG. 2a is an example of different Western blotting patterns
developed after incubation with representative variable sera of
tuberculosis patients. Noticeable is the combination of different
patterns demonstrating the presence of different dominant bands, as
shown in table 1. These band patterns function as "hallmarks" for
TB patients as diagnosed serologically.
[0128] Applicable to all blots (from left to right): Lane 1=BCG
crude extract, Lane 2=crude extract of an M. tuberculosis
strain.
[0129] Interpretation of Banding Patterns is as Follows. Different
Blots (Originating from Different PAGE Gels) are Herein Compared
with Each Other
[0130] Blot A: Mycobacterium avium infected patient.
[0131] Blot B-F: Tuberculosis patients.
[0132] Blot G: non-endemic negative serum.
[0133] Blot H: endemic negative serum (known recent contact, blot
developed 2 weeks after patient returned to Netherlands from
endemic range). Only "hallmarks" are mentioned.
[0134] Blot A: Mycobacterium avium infected patients, sera, band at
68 KDa in lane 1 and 2, range in 10-16 KDa in lane 1, band in the
58-60 KDa region in lane 2. Patient shows low IgA titer in P-90
ELISA).
[0135] Blot B: 38-40 KDa band in lane 1 and 2, 10-16 KDa band in
lane 1 and 2, band in 58-60 KDa region in lane 2, smear in 22-28
KDa region in lane 1.
[0136] Blot C: 16 KDa band in lane 1 and 2, bands in 58-60* KDa
region in lane 1 and 2, bands in 38-40 KDa region in lane 1 and 2,
smear in 22-28 KDa region in lane 1, 33 KDa band in lane 1 and
2.
[0137] Blot D: 10 KDa band in 10-16 KDa region in lane 1, 16 KDa
band in 10-16 KDa region in lane 2, 68 KDa band in lane 1 and 2,
bands in 58-60* KDa region in lane 1 and 2.
[0138] Blot E: smear in 33-38 KDa region in lane 1 and 2, 16 KDa
bands in lane 1 and 2, bands in 58-60* KDa region in lane 2.
[0139] Blot F: bands in 10-16, 22-28, 38-40, 58-60 regions and 68
KDa band in both lanes 1 and 2.
[0140] Blot G: non-endemic negative serum.
[0141] Blot H: endemic negative serum (known contact).
[0142] FIG. 2b is an example of different Western blotting patterns
developed after incubation with representative sera of patients
with Lepromatous Leprosy (LL), Blot A and C, and Tuberculous
Leprosy (TT), Blot B and D.
[0143] The "hallmark" patterns are shown in table 1 and are for LL:
distinctive 29/33 KDa doublet, and for TT: distinctive 64/65 KDa
doublet (often observed as single band) or a very pronounced smear
in the 30-64 KDa region.
[0144] To Blot A and B are applied: Lane 1: BCG crude extract, Lane
2: crude extract of a M. tuberculosis strain, Blot C: Lane 1:
Molecular marker, Lane 2: not relevant, Lane 3: BCG crude extract,
Lane 4: crude extract of an M. tuberculosis strain, Blot D: Lane 1:
BCG crude extract, Lane 2: crude extract of an M. tuberculosis
strain, Lane 3: Molecular marker.
[0145] Interpretation of Banding Patterns, Wherein Only the
"Hallmarks" are Mentioned, is as Follows
[0146] Blot A/C: 29/33 KDa doublet in lane 1 and 2.
[0147] Blot B/D: 64/65 KDa doublet in lane 1 and 2.
[0148] The very intensive smear in the 30-64 KDa range on blot D is
distinct.
[0149] Finally, FIG. 2c is an example of different western blotting
patterns developed after incubation with representative sera of
patients with Crohn's Disease. The "hallmark" patterns are shown in
table 3 and are for Crohn's Disease a pronounced 45/48 KDa
doublet.
[0150] Applied to blot A is: BCG crude extract, blot B: crude
extract of an M. tuberculosis stain, blot C: Mycobacterium aviurn
crude extract, blot D: molecular marker.
[0151] Interpretation of Banding Patterns is as Follows
[0152] All lanes show a distinctive coloring of the 45/48 KDa
doublet positively, which indicates Crohn's Disease. The 45/48 KDa
doublet reacts positively in 65% of all Crohn patients.
EXAMPLE 2
Enzyme Immunoassay
[0153] 1. Preparation of Antigens
[0154] The starting material prepared according to Example 1 was,
depending on the chosen M. tuberculosis strain, centrifuged at
70,000.times.g to 120,000.times.g at 4.degree. C. for 2 hours. The
pellet was washed three times with PBS. Between the washing steps
centrifuging took place at 70,000.times.g to 120,000.times.g at
4.degree. C. for 2 hours. The pellet was collected and resuspended
in 10 mL PBS. In addition to the supernatant in Example 1, SP-100
can also be used for immunoblots (Example 1) and enzyme
immunoassays (this example). The suspension was subsequently
sonicated for 2 minutes at 80 watts at 4.degree. C. After the
protein concentration was determined, quantities of 100 .mu.L were
frozen at a concentration of 1 mg/mL and stored at -80.degree. C.
until time of use (this preparation is designated with the term
KP-100).
[0155] Thirty milligrams (30 mg) of the starting material was then
applied in the presence of loading buffer onto a preparative 12%
polyacrylamide gel of 0.5 cm thickness after 20 minutes of
incubation at 65.degree. C. Electrophoresis was carried out for 30
minutes at 150 V (stacking gel) and for 6 hours at 100 V (running
or separating gel). The electrophoresis was stopped after the blue
colorant band ("dry front") had ran off the gel. The gel was then
cut into horizontal strips of 2 mm thickness which in turn were
divided into pieces of 1 cm length. The gel pieces were each eluted
overnight at 4.degree. C. in a tube with 5 mL sterile distilled
water. Thorough mixing thereafter took place and the remaining gel
pieces were centrifuged to the bottom.
[0156] The elution was checked using a 12% polyacrylamide
analytical gel of 1.5 mm thickness. The gel was cast with a comb.
After 20 minutes incubation at 65.degree. C., 40 .mu.L of each tube
with gel pieces was placed in the slots in the presence of 10.mu.L
5.times.loading mixture. The electrophoresis was carried out for 30
minutes at 150 V in the "stacking gel" and for 6 hours at 100 V in
the "running or separating gel". The electrophoresis was stopped
and the gel made ready for preparation of a Western blot according
to the procedure described in Example 1. Similar results were
obtained using HPLC, FPLC, and other routine separating
procedures.
[0157] To establish which fractions contain the relevant antigens,
strips of the blot were incubated with sera of patients with
lepromatous leprosy, tuberculous leprosy and Crohn's Disease. Shown
herewith are respectively the 29/33 KDa antigens, the 64/65 KDa
antigen and the 45/48 KDa antigens. The complex formation was
visualized using anti-human IgG peroxidase conjugate and DAB. The
desired fractions were collected, combined and used to cast a
microtiter plate (see below).
[0158] 2. EIA
[0159] Microtiter plates are coated (via standard techniques) with
either KP-100, SP-100, starting material, whole bacteria, 29/33
KDa, 64/65 KDa or 45/48 KDa antigens.
[0160] After coating, the plates are blocked with a 3% BSA solution
in order to prevent a nonspecific binding of serum components.
Plates are then dried and stored at 4.degree. C.
[0161] 2.1. Tuberculosis EIA Test (Microtiter Plates Coated with
KP-100)
[0162] Test sera are pipetted in a 1:100 dilution into the coated
wells of a microtiter plate. The reaction takes place for 1 hour at
37.degree. C. Nonspecific serum components and non-binding serum
components are washed away with a washing buffer. A second
incubation with a suitable dilution of an anti-human IgA peroxidase
conjugate is carried out again for 1 hour at 37.degree. C., and
excess conjugate is then washed away.
[0163] Detection of human antibodies of the sub-type IgA binding
specifically to KP-100 takes place by adding TMB
(tetramethylbenzidine) to the wells.
[0164] Binding enzyme results in the occurrence of a blue color
which, after addition of a coloring stop solution, changes to
yellow. This yellow color has an absorption maximum of 450 nm.
[0165] The intensity of the resulting color is proportional to the
amount of bound KP-100specific IgA.
[0166] The results are shown in the tables below.
[0167] In the described test patient and control sera are used from
two different populations.
[0168] A=Endemic area (Africa, Ghana)
[0169] B=Non-endemic area (Europe, the Netherlands).
[0170] Each population is sub-divided into 4 sub-groups,
namely:
[0171] Group 1=culture confirmed TB patients
[0172] Group 2=negative control group (normal healthy
individuals)
[0173] Group 3=suspected positives (TB contacts)
[0174] Group 4=suspected negatives (no known data, but certainly no
TB, possibly leprosy or other nonspecific mycobacteriosis)
[0175] The test is performed with two kits having different lot
numbers and production dates.
[0176] Interpretation of the test results takes place on the basis
of the so-called calibration line which is made up of control sera
with a determined arbitrary unit definition which corresponds to a
known OD value (1 unit, 4 units, and 8 units).
[0177] Each time a test is carried out the units are included in
the assay. Found sample values can then be related to the unit
definition.
[0178] A test serum can be considered positive when the result
found in the test scores higher than 2.1 units.
[0179] A test serum can be considered negative when the result
found scores lower than 1.2 units.
[0180] Test sera with unit values between 2.1 and 1.2 units fall
into the set so-called reconfirmation zone. This means that in the
first instance positivity or negativity for tuberculosis cannot be
determined with this test.
[0181] Reconfirmation of these sera takes place using the described
Western blot strips with which, after serum incubation on the basis
of banding patterns and specific "hallmarks", an answer can be
given to the question of whether the test serum is positive (bands
present) or negative (bands absent).
8TABLE 3 Population A. Endemic range: Group 1: SERUM NO. # UNITS
TEST SCORE REMARKS 1 2.44 positive culture positive 2 3.43 positive
culture positive 3 1.78 reconfirmation culture positive 4 1.37
reconfirmation culture positive 5 5.74 positive culture positive 6
3.21 positive culture positive 7 1.66 reconfirmation culture
positive 8 2.00 reconfirmation culture positive 9 2.00
reconfirmation culture positive
[0182]
9TABLE 4 Population A. Group 2: SERUM NO. # UNITS TEST SCORE
REMARKS 1.16 negative healthy individual 11 0.86 negative healthy
individual 12 0.77 negative healthy individual 13 0.64 negative
healthy individual 14 0.74 negative healthy individual 15 0.79
negative healthy individual
[0183]
10TABLE 5 Population A. Group 3: SERUM NO. # UNITS TEST SCORE
REMARKS 16 3.13 positive sick individual 17 1.57 reconfirmation
sick individual 18 1.59 reconfirmation sick individual 19 5.39
positive sick individual 20 1.97 reconfirmation sick individual 21
2.29 positive sick individual 22 0.48 negative sick individual
[0184]
11TABLE 6 Population A. Group 4: SERUM NO. # UNITS TEST SCORE
REMARKS 23 1.54 reconfirmation normal control 24 1.76
reconfirmation normal control 25 0.58 negative culture negative 26
1.03 negative culture negative 27 0.79 negative culture negative 28
0.89 negative culture negative
[0185]
12TABLE 7 Population B. Non-Endemic range: Group 1: SERUM NO. #
UNITS TEST SCORE REMARKS 1 2.24 positive culture positive 2 2.53
positive culture positive 3 4.40 positive culture positive 4 14.95
positive culture positive 5 16.82 positive culture positive 6 10.54
positive culture positive 7 5.70 positive culture positive 8 6.72
positive culture positive 9 5.06 positive culture positive
[0186]
13TABLE 8 Population B. Group 2: SERUM NO. # UNITS TEST SCORE
REMARKS 10 1.04 negative healthy individual 11 0.92 negative
healthy individual 12 0.85 negative healthy individual 13 0.20
negative healthy individual 14 0.42 negative healthy individual 15
0.90 negative healthy individual 16 0.35 negative healthy
individual 17 0.73 negative healthy individual
[0187]
14TABLE 9 Population B. Group 3: SERUM NO. # UNITS TEST SCORE
REMARKS 18 2.23 positive sick individual 19 4.70 positive sick
individual 20 1.22 reconfirmation sick individual 21 1.43
reconfirmation sick individual 22 2.21 positive sick individual
with TB history 23 6.38 positive sick individual 24 1.59
reconfirmation sick individual
[0188]
15TABLE 10 Population B. Group 4: SERUM NO. # UNITS TEST SCORE
REMARKS 25 0.20 negative patient resistant to drug therapy
[0189] Thus, while there have been described what are presently
believed to be the preferred embodiments of the present invention,
those skilled in the art will realize that other and further
embodiments can be made without departing from the spirit of the
invention, and it is intended to include all such further
modifications and changes as come within the true scope of the
claims set forth herein.
* * * * *