Note: Descriptions are shown in the official language in which they were submitted.
W0 9sl26503 r~ 4
21 3~742
DET~CTION OF ANALYTES
The present invention relates to a method for the
detection of analytes, in particular specific binding
molecules such as antibodies or antigens in saliva
samples. The invention is especially concerned with the
detection of analytes in fresh saliva samples which have
not been stored or frozen.
The detection of ~nt;hQrl;es in saliva is a convenient
method for the ~ n~R; R of various diseases and
conditions, in particular gut infections. Gut infections
in mammals, and in particular humans, stimulate an immune
response in mucous secretions, such as saliva, through
activation of the common mucosal immune system. This
response often initially parallels an antibody response
in serum although is generally characterised by the
presence of IgA antibodies. However, the immune response
in secretion, ;nc~ ;ng saliva, rapidly ~;Tn;n;Rl~a
following ol ;m;n~t;~n of the antigen (eg bacteria or
virus) from the body. Accordingly, the presence of
antibody in saliva reflects current, ie ~ nt~ rlri~ry~
infection. In the case of a microbial infection, for
example, ~nt;ho~;oR in saliva, hereinafter referred to as
secretious ~nt;hn~;~R, reflect the current status of
colonisation of the microbe, such as in the gut, and thus
is a useful monitor o~ rr~nt , ~ry infection. Serum
antibody, on the other hand, persists for some time after
the microbe is eliminated from the body. A positive
serum antibody test, therefore, reflects both past and
present exposure to antigen which is less helpful to the
clinician. A positive secretious antibody test indicates
present or contemporary infection by the microbe.
WO95126503 r~ /14
2186742
The provision of saliYa samples is greatly preferred by
patients to -the provision of samples of other body
fluids, partIcularly serum. Further, in nht~;n;n~ a
sample of saIiva, there is a very low risk of infection
of either the patient or the cli~ician since it is not
nf~r~cs~ry to~ uge a needle as is the ca6e with serum
samples .
Methods of ~iagnosis from saliva samples are known and,
lQ in particular, AU-A-9Q67676 is directed to the ~l~tect;nn
of IgG specific to Helicobacter pylori antigen in mucous
secretions such as saliva and thereby provides a means of
monitoring current, ie rnnt~ ,Inraryl infection by that
microorganism in mammals. The corresponding academic
1~ publication is Witt et al, ~rontiers in Mllcosal
Irranunology 1 6g3 - 696 ~1991 ) .
WO-A-9322682 relates to a saliva-based test for H. pylori
in which IgG aIltibodies to H. pylori are detected.
One disadvantage with currently available saliva-based
tests is that they may, in certain circumstances, be
unreliable; in particular, the number of false positives
obtained in the test can be unacceptably high. In
2~ experiments which were carried out in an attempt to
develop i ~~csays for salivary antibodies to H.
pylori, 78 subjects were asked to pool saliva flow into
a clean sterile f~r~nt~in~r_ When the samples were tested
for the presence of ~nt;ho~;~R to iY. pylori, it was found
that the results did not correlate well with the results
of serum tests because of a significant number of false
positive results.
A ~urther disadvantage which saliva based tests have in
~ W0 95/26503 P~~
2 1 ~6742
common with most other types of diagnostic test is that
in order to obtain the results, it is necessary to send
the sample to a laboratory in order f or it to be
analyzed. This may take several days and in part
destroys the advantage of being able to obtain an
indication of present infection by a microbe. In
addition, the patient may forget to collect the results
of the test, particularly if any symptoms have
disappeared in the interval between submitting the sample
and receiving the results of the test.
A test kit for the detection of an analyte in a sample of
saliva which would provide reliable results in a few
minutes would therefore be of great value as the test
could be carried out during a consultation with a general
medical practitioner. This would have the added
advantage of ensuring that the patient collects the
results and i8 prescribed treatment if n~CF.q8 ~ry .
However, the problems of false positive results which
exist with any saliva based test are greatly multiplied
when saliva samples are analyzed within a few minutes of
collection, 80 much 80 that any results from assays
carried out on fresh saliva samples have, in the past,
been almost ~ tPly r -n;ngl ess.
It is believed that this lack of r~ hi l; ty,
particularly in fresh saliva samples, may arise because
of the presence in the sample of agents which bind non-
specifically to the test reagent and thus give the false
positive results. It is, of course, possible to add to
the sample agents which reduce non-specific binding but
this has also proved problematic as many such agents also
reduce specif ic binding to such an extent that true
positive results are ~-1 ;min;~t~l along with the false
W095l26503 2 1 86742 ~ /14
positiveæ. The present invention makes it possible to
overcome this problem and to eliminate false positive
results whilst retaining the true positive ones.
In a first aspect of the present invention there is
provided a method for detecting the presence of an
analyte in a sample of saliva, the method comprising
~ nt;lCtin~ the saliva sample with a specific binding
agent capable of forming a specific binding complex with
the analyte and detecting the presence of qr~ f i ~~
binding complex; characterised in that the saliva sample
is initially c--nt~ctp~ with a so1ution comprising
polyoxyethylenesorbitan derivatives of palmitic and/or
stearic acids.
The advantage~ of the method of the present invention is
that the results of the test can be obtained quickly and
reliably by a non-invasive method and, in addition, it
overcomes the problems which arise when fresh saliva is
used and enables reliable results to be obtained rom "on
the spot" tests. In the context of the present invention
the term "fresh salivan refers to saliva which has been
stored for not longer than about thirty mimltes,
preferably fo-r not longer than ten minutes and often for
a shorter length of time than that.
It i5 essential to choose the surfactant extremely
careully and, indee~, one of the surprising features of
the present invention is that from the vast range o
surfactants available, the only ones which enabled us to
obtain reliable ~esults were those def ined above .
Suitable surfactants are available under the trade marks
TWEEN 4O, TWEEN 6O, TWEEN 61, TWEEN 6~ and TWEEN 8O .
W0 9Sl26~03 2 1 ~3 6 7 4 2 r~ J"7 ~ /14
It is particularly preferred that the surfactant contains
from 409s to 659~ stearic acid derivatives and m~WEEN 60
which ~r~ntRinA about 5596 stearic acid derivatives with
the balance being palmitic acid derivatives is
particularly preferred and provides si~nif;t-Antly more
reliable results than most other surfactants.
The amount of surfactant present will, when a solid
substrate is used as is discussed below, for preference
be chosen so as to maximise the flow of sample through
the substrate. The flow is usually m-~rim;~d when the
surfactant is present in an amount of from 0.196 to 1~ by
volume, typically about 0.5~. This amount of surfactant
give the best results for eliminating non-specific
binding without greatly af f ecting the specif ic binding
and thus the threshold of detection obt~in~hl~ using the
method of the invention.
The best results are obtained when the solution is
buffered to a pH of from about 6.8 to 7.8 but a solution
buffered to pH 7.4 has been found to be optimal. Any
buffer may be used provided that it results in the pH of
the solution being r-intA;n,od in the preferred range.
Phosphate is a particularly preferred buffer for use in
the present invention but other examples of buffers which
could be used to ensure that the pH of the solution is
within the preferred range are familiar to those skilled
in the art.
Any water soluble salt such as a sodium, potassium or
ammonium salt may be used for the preparation of the
buffer solution although sodium salts often give the best
results. It has been found that effective buffering is
obtained using a 0.001 - 0.05 M, preferably about 0.02 M,
Wo95/26503 r~ b '~ /14
2~ 86742
solution of s--odium phosphate.
Other agents may be present in the solution in order to
minimise the non-specific binding of mucins and
particulate material in the test sample to the test
reagents. Such agents include inorganic salts such as
sodium chloride and protqins such as boYine serum albumin
( BSA) .
lQ Sodium chloride may be present in a ~ c~ tion of from
about O .1 to O . 2 M. It is greatly pr~ferred that the
upper rnnt-Pntr~tion limit of 0.2 M is not exceeded since
this would tend to discourage specific binding.
Typically, the concentration of sodium chloride present
in the solution i9 about Q.125 M.
BSA, if present will typicaIly be included in an amount
of about 0.05% to 0.5% by weight, preferably of 0.1%.
2 0 The analyte may be any specif ic binding molecule capable
of reacting with the specific binding agent to form a
specific binding complex. Examples of ~ specific binding
complexes include antibody-a~tigen complexes and thus the
analyte may be either an antibody or an antigen.
In the case where the analyte is an antibody, it may be
of any isotype and may be an antibody against any
r~h~ n Analysis of saliva samples is particularly
useful in the r7; ~gn~s; c of gut infqctions caused by
3~ p~hr~ nc such as ~P7 i~ h~rter pylori (formerly known as
Campylobacter~pylori). As discussed above, H. pylori
infection is indicated by the presence in saliva of IgG
and therefor~, if the aim o~ the test is to detect ~.
pylori infection, the analyte may be Ig~ specific to ~.
Wo95/26503 1~ ,S,. /14
21 86742
pylori antigen .
The expres6ion "antigen" is used in its broadest sense
and includes whole pathogen cells or homogeneous, near
homogeneous or heterogeneous extracts from a pathogen,
all of which are capable of binding to specif ic antibody
in saliva.
When the specif ic binding agent is an antigen, it may be
a protein, polysaccharide or lipid or any combination
thereof. Preferred ~p~;f;c binding agents which are
antigens include protein, lipopolysaccharide or cell
extract of pathogen prepared by, for example, sonication,
pressure .1; ei nt~ation, detergent e~traction or
fractionation.
When the method of the invention is used to detect
infection with N. pylori, the specific binding agent may
be an antigen derived from H. pylori. Antigens derived
from H. pylori suitable for use as specific binding
agents in the method of the present invention are
disclosed in WO-A-9322682. ~owever, any H. pylori
derived antigen could be used as a specific binding
agent .
The specific binding agent will for convenience and
preference be bound to a solid support. Suitable solid
supports include a nitrocellulose membrane, glass or
polymer solid supports. The most commonly used polymers
for this purpose are cellulose, polyacrylamide, nylon,
polystyrene, polyvinyl chloride or polypropylene, but the
invention is not limited to these. The solid supports
may be in the form of strips, tubes, beads, discs or
microplates, or any other surface suitable for conducting
WO 95/26503 F~
21 86742
an i ~m-lnn~ qq~y .
A particularly useful solid support may comprise a
nitrocellulose membrane backed by an i~h~rhPnt pad so
that, on adding the sample to the solid support, the
analyte will be i hi l; ce~l by the specific binding agent
on the top surface of the nitrocellulose membrane whilst
the rP~in~lPr of the sample passes through the membrane
and is absæbed on the pad. This ensures that any
lln-~ntPri material is removed from the area in which the
specif ic binding complex is detected .
The nitrorp~ rqp membrane may have a pore size of from
- about 0.5 to ~ llm with from about l to 2 llm being
preferæd. ~_
The pad may be formed from any absorbent material but
absorbent paper will often be the material of choice,
generally because of considerations of cost.
The sperifir hin~lin~ molecules useful in this invention
may be either covalently or non-covalently (npassively")
bound to the solid surface. Suitable binding processes
are well knoDm in the art and generally consist of cross-
linking, covalently binding or physically i3Aqnrhinr~ the
antigen to the solid support.
The presence o~ the analyte is diagnosed by means of the
present invention by detecting the formation of a complex
between the analyte and the specific binding agent. Some
form of niPtPrt;ng means is therefore necessary to
identify the presence (or, if rer~uired, amount) of the
specif ic binding complex .
WO 9S/26503 2 1 ~ 6 7 4 2 1~ bS' ~ ~ 14
The detection means may be an antibody, conjugated with
a reporter molecule, and which is capable of binding
specifically to the specific binding complex.
In the case where an antibody is to be detected, the
detection means may comprise a labelled second antibody
specific i~or all antibodies of the isotype of the analyte
antibody. In tests for H. pylori in humans, the analyte
antibody will often be of the IgG isotype and in that
io case the second antibody may be anti-human IgG.
A "reporter molecule~ is a molecule or group which, by
it3 chemical nature, has an analytically identifiable
characteristic or provides an analytically identifiable
signal which allows the tl~tenti nn of antigen-bound
antibody. Detection may be either qualitative or
quantitat~re. Reporter molecules used in this type of
assay may be either enzymes, fluorophores or radinnl-rl;~l~
nnnt~;n;n~ molecules (ie radioisotopes). In the case of
an enzyme; ~nn~qpi3y, an enzyme is conjugated to the
second antibody, generally by means of glutaraldehyde or
periodate. As will be readily recognised, however, a
wide variety of differer,t co~jugation technis~ues exist,
which are readily available to those skilled in the art.
Commonly used enzymes include horseradish peroxidase,
glucose oxidase, ~-galactosidase and Alk~l ;n~
rhnSph~t~qe, among others. The chromophores to be used
with the specific enzymes are generally chosen for the
production, upon hydrolysis by the corr~qponfl;ng enzyme,
of a detectable colour change. Chromophores can be
soluble or insoluble, depending upon the chosen
application. For example, 5-bromo-4-chloro-3-~ndolyl
rhn~rh~te/nitroblue tetrazolium is suitable ~or use with
;~lk~l;n~ phosphatase conjugates; for peroxidase
W0 95/26503 ~ J7i~ 4
21 86742
conjugates, l, 2-phenylPnp~ m;np-5-aminosalicylic acid,
3, 3, 5, 5-tetramethylbellzidine, tolidi~e or dianisidine are
commonly used. It is also possible to employ
fluororl~rrPc, which yield a fluorescent product, rather
than the chromophores noted above. I~xample6 of
fluorophores are fluorescein and rlln~l~m;np When
activated by illumination with light of a particular
wavelength, the fluorochrome-labelled antibody absorbs
the light energy, inducing a state of excitability in the
molecule, followed by emission of the light at a
characteristic colour which is usually visually
~PtPrt~hl f' w~h a light microscope.
However, the present inve~tion is particularly we~l
adapted for use as an 'instant diagnosis' test from which
the results will be available in a few minutes and which
may be carried out by a general practitioner during a
consultation. For this reason, it is greatly preferred
that the method of detection is as simple as possible and
rer~uires no--specialised er~uipment . Therefore, the
reporter molecules preferred in the present invention are
colour reagents such as rr)l l r~ l gold or carbon,
polystyrene or latex particles.
When this type of rPrnrtPr substance is used, it is
important that unbound second antibody be removed 80 that
the bound se~o~d a~tibody with the attached colour agent
is clearly visible. This can be achieved by the
provisioIl of the ~rPr;f;r h;nr~;nrJ agent immobilised o~ a
3 0 nitrocellulose membrane backed with an absorbent pad as
described above. Any surplus seco~d antibody will pass
through the~membrane and be absorbed in the pad leaving
only bound second antibody and ascnri ~tP~ colour agent on
the surface of the nitrocellulose membrane.
WOgs/26s03 2~G742 P~
11
Further investigations into the problems of obtaining
reliable results from saliva based assays suggested that
one possible cause of the false positive results was the
presence of mucins and particulate material in the saliva
sample which bind non-specifically to the test reagents
thus producing false positive results. Therefore, it is
advantageous to include in the method of the present
invention the step of f iltering the sample bef ore
attempting to detect the presence of a specific binding
complex.
Typically, the filter will have an effective pore size of
from about 1 to 15 ~Lm, preferably from 3 to 8 ~m. This
may be achieved using any type of filter but a preferred
type is a frit made from a plastics material and having
a typical actual pore size of about 5 to lO~Lm. In this
type of frit, the effective pore size is smaller than the
actual pore size because the frit is relatively deep and
the pores are out of alignment.
The remoyal from the saliva sample of particulate
material helps to ensure that a substrate to which the
specif ic binding agent is bound does not become
contAm; nAted~ This is particularly important when the
substrate is a membrane since particulate material may
block the pores of the membrane preventing unbound sample
from passing through the membrane and being removed from
the test surface and thus increasing the flow through
time for the sample.
A further step which may be included before the
filtration step is a primary separation step in which the
sample is passed through a coarse filter such as a cotton
or cotton wool pad. This reduces the viscosity of the
Wo 95l26503 .~ ~IA.D~. /l4
21 86742
12
saliva sample, possibly by the removal of high lPrlllAr
weight mucopolysaccharides. A sample of reduced
viscosity is helpful when the specific binding molecule
is ; ,h; 1 i ~P-l on a membrane substrate since a viscous
saliva sample may pass through the pores of the membrane
only with dii:i~iculty and thus the test takes much longer
when this step is not present. In addition, unless this
step is ;nrl~ , a residue is often let on the test
surface which may interfere with either the specific
binding react~ion or with the detection step.
I~ven these preliminary filtration steps do not appear to
be completely ef f ective in removing mucins and
particulate material from saliva and ~he flow through
time is slow and a number o f alse pos~ltive results are
often obtained even when the sample has been pre-
f iltered .
However, it has now been shown that it is possible to
reduce the ~mber of false positive results obtained in
the test o the present invention simply by wiping the
surf ace of .~}he substrate af ter the sample has been
~ orhP-l onto it. This was most unexpected because it
had been thought that all rnntAm;n:~ntPI had been removed
by the f iltration step which had been attempted
previously whereas it now seems that this was not the
case .
Therefore, in cases when the spPr;f;r binding agent is
il~enrhPIl on a substrate, it is preferred that the method
of the invention includes the step of wiping the
substrate aii:er the sample has been added to it and
before attempting to detect the specific binding complex.
The wiping step both dramatically tlPrrPi~Pq the flow
_ _ _
Wo9S/26503 21 86742 ~ ,, C ll4
13
through time of the sample and reduces the occurence of
false positive results.
The wiping may be carried out manually or, alternatively,
the process may be i~llt tP~l. Generally an absorbent
material will be used to wipe the substrate and examples
of suitable materials are cotton wool and absorbent
paper .
The wiping step should be carried out sufficiently
vigorously to remove from the surface of the substrate
any material which is not bound to a specific binding
molecule. In order to make it clear that all of the
unwanted material has been removed, a colouring agent may
be added to the sample on the substrate. For
convenience, the colouring agent may be included in the
surfactant solution but this will not necessarily be the
case. The surfactant solution may contain from about
0 . 005~ to 0 . 05~ (w/w) of a particulate colouring agent
and preferably from about 0 . 0196 to 0 . 029~ but other types
of colouring agents may be pre3ent in greater amounts.
The colouring agent may be an agent which is specific for
the mucins and other rrnt~m;n;lntc which remain on the
substrate and are a cause of many of the problems of
false positives which occur with assays of saliva.
However, it is often simpler to provide as a colouring
agent a coloured particulate material such as latex,
agarose polystyrene or another polymer. The particles
should of course be larger than the pore size of the
~ubstrate but must also be smaller than the pore size of
any pre-filters which may be used. It has been found
appropriate in many cases to use particles of diameter of
about 3 ILm since, as discussed above, the filters used in
Wo 95l26503 ~ ~ ~ 6 7 4 2
14
any initial ~urif ication steps can be chosen to have a
pore size larger than this. The coloured particles will
remain on the surface o_ the substrate along with the
mucins and particulate impurities which appear to be
causing the ~ problem of false positives when smaller
particles pass through the substrate.
Thus, when the colouring agent is used, it will be a
simple matter for a practitioner to wipe the colouring
agent from the surface of the substrate and thus to
ensure that all surface debris has been removed from the
substrate.
A further rf~f;n t of the method which assists in the
elimination of false positive results is the provision on
the substrate of a rontrol reagent which is capable of
reacting with the detection reagent. The control reagent
will be present in a different location from the specific
binding molecule and will be capable of specifically
binding the detection agent. Thus, for example, if the
detection reagent is anti-human IgG, the control reagent
will be human IgG. The presence of the control reagent
is a means of monitoring the viability of the method of
the invention since if its presence is not detected, then
clearly, the detection method is not working correctly.
Thus the present invention provides a simple and
effective method for assaying saliva samples even when
the saliva samples are fresh.
The surfactant solution is an important part of the
invention since it has been found that only the
surfactants discussed above in relation to the method are
.
WO95/26503 21 86742 P~l, " 0~/14
at all effective in preventing false positive results in
the assay.
Theref ore, in a second aspect of the invention there is
provided a surfactant solution for use in the method of
the invention, the solution comprising from 0.1% to 1% by
volume polyoxyethylenesorbitan derivatives of palmitic
and/or stearic acids; a buffer capable of m~inti:;n;n~ the
pH of the solution at a level of from 6 . 8 to 7 . 8; and
optionally, a water soluble salt, a protein such as BSA
and a particulate colouring agent.
The preferred buffers and cr~n~pnt~;~t;ons of salt, BSA and
colouring agent are as discussed above in relation to the
first aspect of the invention.
The method may be carried out using a kit which itself
forms a further aspect of the invention.
In this aspect of the invention there is provided a kit
comprising:
i. a solution comprising polyoxyethylenesorbitan
derivatives of palmitic and stearic acids;
ii. a specific binding agent immobilised on a
substrate and capable of forming a specific binding
complex with the analyte; and
iii. a detection reagent for detecting the presence
3 0 of specif ic binding complex
The present invention is particularly useful for the
detection of antibodies against H. pylori which may be
present in the saliva of H. pylori infected patients. As
WO9S/26s03 2 1 8 6742 ~ 4
16
discussed above, ~. pylori is unusual in that infection
gives rise to antibodies of the IgG isotype present in
the saliva.
Therefore in a fourth aspect of the invention there is
provided a kit for the detection of IgG specific for H.
pylori, the kit comprising:
i. a solution comprising polyoxyethylenesorbitan
derivatives o palmitic and stearic acids;
ii . an antigen derived from H. pylori; hi 1 i ~ed
on a substrate; and
iii. a solution of a labelled antibody capable of
binding spf~c;f;r~lly to human IgG.
The preferre~ t~t , t~nt~ntq of the solution, and preferred
substrates a~d detection reagents are those which are
2 o described f or the method of the f irst aspect of the
invention . .
The kit may also c~ntain a saliva collection device. A
coarse filter such as a cotton or cotton wool pad for
rrt-l;m;nAry =iltration o~the sample may also be included
and may optionally form a part of the saliva collection
device. Furthermore, the kit may include a filter or
removing particulate material rom the sample. As
discussed above in relation to the method of the irst
3~ aspect, the ilter may have an efective pore size of
from about 1 to 15 ~m, preferAbly from 3 to 8 ILm. This
may be achieYed using any type o ilter but a preferred
type is a frit made from a plastics material and having
a typical actual pore size o about 5 to lQ~Lm. In this
¦ WO 95/26503 2 18 6 7 4 2 r~ 714
17
type of frit, the effective pore 6ize is smaller than the
actual pore size because the f rit is relatively deep and
the pores are out of alignment.
S A colouring agent capable of n~-~;n;n~ on the surface of
the substrate may also be included if the method of the
invention is to include the wiping step mentioned above.
The colouring agent may be an agent which i8 capable of
staining the mucins and particulate impurities but will
preferably consist of coloured particles of latex,
agarose, polystyrene or some other polymer in which the
particle size is chosen so that the particles will not be
removed in any preliminary filtration steps which are
carried out but will be too large to pass through the
pores of the substrate.
The invention will now be described in detail with
ref erence to the f ollowing examples .
2 0 EXP MPLE
Preparation of Surfacta~t Solution
A surfactant solution was prepared from the following
ingredients:
0.02M sodium rhncrh~te solution 100 m~
60dium chloride o . 73 g
Bovine serum albumin 0.1 g
T~EEN 6 oY O 5 g
dark ~lue latex particles (3.0 llm diameter) 0.1 g
by mixing at room temperature. The dark blue latex
particles are available from Polymer ~aboratories, UK.
W095/t6~03 P~I~B ' /14
21 86742
18
EXAMPLE 2 ~
Preparation of ~. pylori derived antigen
An antigen derived from H. pylori was prepared according
to the method set out in Example 1 of W0-A-9322682. In
summary, a crude sonicate o H. pylori was prepared and
fractionated. A 440 kDa protein was removed leaving a
mixture ~nntAin;n~ 265 and 340 kDa proteins.
EX~MPT .T' 3
Preparation of Test Device
Between 1 and 5 ~lL of a 0 . 059~ (w/w) solution of the
antigen of Example 2 was spotted onto a substrate to ~orm
a test area . The substrate was a 1. 2 ~m
nitrocellulo6e membrane supported upon a backing layer of
Schleicher & Schuell chromatography paper No 3469
(available from Anderman & Co, Kinston upon Thames, ~TK)
which acts as a wicking material.
From 1 to 5 . I~L of a 0 . 005~ (w/w) solution of purified
normal human IgG (Sigma Chemical Company Ltd, Poole,
Dorset, UK) was spotted onto the substrate in a co~trol
area distinct from the test area.
~MPT,T~ 4
Preparation o~ Di~closing Agent
A disclosing agent ~as T~repared by diluting colloidal
gold conjugated to goat anti-human IgG (heavy and light
chains) (Biocell Research T AhnrA~n~ies, Cardiff, ~JK) in
~I WOgs/~6s03 21 86742 p~l 1 "~
19
phosphate buffered saline (PBS) cr~ntR;n;n~ 0.0596 by
volume of the surfactant available under the trade mark
TWEEN 20 and 0.1~ by weight BSA to an absorbance at 520
nm, 1 cm path length of 0.5 optical density units.
MPT ,~ 5
Assay of Saliva Sample
Saliva (lmL) was collected using the collection device
available under the trade mark OMNISAL (Saliva Diagnostic
Systems, Vancouver, Washington, USA) in which the sample
is collected in a pad which also acts as a coarse filter.
The collection device c(~nt~in1ng the sample was then
transferred to a tube ~fmt~;n;n~ 1.0 m~ of the solution
of Example 1. The collected saliva was f iltered using a
Porex Ultrafine serum separator having an approximate
exclusion of 5 ~Lm and was then added to the test device
prepared in Example 3.
After the diluted saliva 3ample had flowed through the
nitrocellulose membrane into the chromatography paper
backing layer, the blue latex particles formed a layer on
the surface of the substrate. This layer was removed by
wiping firmly but gently with cotton wool until no blue
colour L- i n.o~l .
O . 5 mL of the disclosing agent of Example 4 was then
added to the test device. The disclosing agent was
allowed to drain through the nitrocellulose membrane and
then the test was read.
.
A single pink spot in the control area indicates a viable
but negative test result whereas a test which results in
Wo95/26503 r~J~ 4
2186742
a spot in the test area and a spot in the control area
indicates a positive result.
The test was capable of detecting levels of anti-~.
pylori IgG o~ as low as 0 . 8 units ~on a scale of from 0
to 10) and did not give false positive results.
