Note: Descriptions are shown in the official language in which they were submitted.
~",4,~ o 21 3 7 7 8 6 PCT/GB94/00788
IMMUNOI~SSAY
The present invention relates to immunoassays.
Testing of samples of body fluids and of solid samples, for
example, of cells or tissue, obtained from a body for the
5 presence of various organisms, in particular bacteria, viruses,
parasites and other pathogenic (infectious) organisms, is carried
out routinely. Testing is carried out in two main contexts. One
is testing of samples of body fluids and of solid samples for the
purpose of diagnosing disease, monitoring the course of disease
10 and/or monitoring treatment in an individual. This type of
testing is often called "clinical" testing. A clinical
laboratory typically carries out tests for a large number of
different organisms. The term "pathogen" is used herein to
denote a ~ice~ce-causing organism.
15 Another major type of testing is the screening of donated blood
in order to maintain a supply of blood and blood products free
from pathogenic contamination. The regulatory authorities in
each country specify the pathogens for which testing is to be
carried out. In most ~o~.LLies, screening is currently mandatory
20 for HIV, hepatitis C (non-A non-B hepatitis), hepatitis B and
syphilis. In some countries there is a further requirement to
test for HTLV. In most countries it is currently mandatory to
test for both HIV-1 and HIV-2. Blood to be used for transfusion
to patients who are to or who have received a transplant or are
25 otherwise immuno-compromised is generally tested for the presence
of CMV (cytomegalovirus). As new pathogens and new sub-types of
known pathogens that pose a threat to the blood supply are
discovered, the mandatory requirements for testing donated blood
are extended to those pathogens.
30 The most widely used tests for blood screening are immunoassays.
For most blood viral pathogens, it is more difficult to obtain
the required sensitivity when testing blood for the presence of -
antigens than it is when testing for the presence of antibodies.
Generally the tests for HIV, HTLV, HCV and CMV are antibody
35 tests. There are two tests for hepatitis B: an antigen assay for
W094/2~ PCTIGB94/007~
2137~8~ -
-- 2
hepatitis B surface antigen (HBsAg), and a test for antibodies to
hepatitis B core antigen (HBc). At present, HBsAg testing is
mandatory in most countries; testing for hepatitis B core anti-
bodies is mandatory in some countries and may be introduced in
5 more. There are both antibody and antigen tests for syphilis.
Blood screening is carried out on a large scale, and it is par-
ticularly important that the results are obtained quickly, since
blood has a relatively short shelf-life. In order to save both
time and money, assays capable of detecting more than one
lO pathogen in a single test sample have been proposed.
Antibody assays for determining two sub-types of the same
pathogen, in particular, for HIV-l and HIV-2 (HIV-1+2), and also
for HTLV-I and HTLV-II (HTLV-I+II) are on the market. It is to
be noted, however, that there is often considerable cross-
15 reactivity between sub-types of the same species, for example,
the degree of immunological cross-reactivity between HIV-l and
HIV-2 is such that the HIV-l tests initially on the market did,
in fact, detect a very large proportion of HIV-2-containing
blood. Accordingly, as is also explained in EP-A-O 484 787,
20 there is a very considerable technical difference between
providing an antibody assay for two sub-types of the same virus
and an antibody assay for two completely different pathogens
having essentially no immunological cross-reactivity. The term
"combination assay" is used herein to denote an assay for the
25 detection or determination, in a single test on a sample, of two
or more pathogenic organisms that have substantially no
immunological cross-reactivity. The term "different pathogens"
is used herein to denote pathogens that have substantially no
immunological cross-reactivity.
30 EP-A-0 484 787 proposes combination antibody assays for viral
pathogens that may occur in donated blood (blood viral
pathogens), in particular, a combination assay for HIV and HCV
(hepatitis C virus). The proposed heterogeneous phase assay
involves the use of multiple peptides coated on a solid surface
WO ~AS~ 213 7 7 8 6 PCT/GB94/~7~
to capture the relevant antibodies. The resulting antigen-anti-
body complexes may then be detected.
A major disadvantage of such a combination assay, and also of the
HIV-1+2 and HTLV-I+II combination blood assays currently on the
5 market, is the requirement for co-coating a plurality of antigens
on a solid surface. Not only must the antigens (peptides and/or
proteins) be coated evenly, they must also be coated in such a
manner that the epitopes are available for antibody binding.
Furthermore, there must be no interaction between the various
10 antigens. The quality control testing that must be carried out
to ensure that the antigens have been coated in a satisfactory
manner adds to the ~ypence~ both in the cost of the testing
itself and in the cost of the rejects. The more antigens there
are to be coated, the worse the problems and the greater the
15 expense. Each additional pathogen to be detected requires the
presence of at ieast one additional immobilized antigen. ~or
some pathogens it is neo~cc~ry to enable the detection of
multiple an~iho~ies. In the case of HCV, for example, current
commercially available assays comprise four different antigens.
20 For a combination HIV-1+2/HCV assay at least six antigens are
required.
In addition to the practical manufacturing problems of co-
coating, it can be difficult to obtain the required specificity.
Very high purity antigens having strong antigenicity are
25 required, see EP-A-0 484 787.
Antibody/antigen combination assays have been proposed for HIV
and hepatitis B surface antigen. EP-A-0 286 264, for example,
proposes coating a solid phase with a peptide capable of binding
to an HIV antibody and coating a solid phase with an antibody
30 specific to HBsAg. The two solid phases may be different, or
both the peptide and the antibody may be coated on the same solid
phase. W091/10747 proposes improvements to the type of assay
described in EP-A-0 286 264, the improvements being in the nature
of the peptides used for coating and for detection of the HIV
wo ~n~s~ PCT/GB94/00788
Z 1 37 7 8 6 _ 4 _
antibody, and in the nature of the detection system.
It is noted, however, that in the only commercially available
combination assay for HIV and HBsAg based on the principle dis-
closed in EP-A-O 286 264, the various problems associated with
5 co-coating are circumvented by coating the HIV peptides on the
inner surface of small tubes and the anti-HBsAg antibody on beads
to be used in conjunction with the tubes. Furthermore, that assay
is not a true combination assay in the present sense because,
after bringing the sample into contact with the beads in the
10 tubes, the beads are removed and the detection steps are carried
out separately on the beads and the tubes. Accordingly, any
potential problems regarding interactions between the labelled
antibody and the labelled antigen used for detection, and any
potential problems between the detection systems (labels) during
15 the detection of any immune complexes formed are avoided.
According to the present invention, the problems of and resulting
from co-coating multiple antigens on a solid surface for a
combination antibody assay are overcome in a simple and elegant
manner by using an immunoglobulin capture format.
20 The immunoglobulin capture format has been known as such for many
years; it was described in 1979 by Duermeyer W. et al in Journal
of Medical Virology 4:25-32 for the detection of specific IgM
antiho~;es in an ELISA for hepatitis A. The principle of the
described assay for a sample obtained from a human is that a
25 solid phase, for example, the wells of a microtitre plate, are
coated with anti-human IgM, sample is added to the coated wells
and incubated, the wells are washed and then antigen is added to
the wells and incubated, and finally any bound antigen is
detected using enzyme-labelled antibody. The authors of that
30 paper ~o~o~ed the use of the assay for any individual IgM
antiho~ies, for example, against rubella, herpes simplex, CMV,
EBV, Toxoplasma gondii, hepatitis B core antigen or other
antigens.
W094~4~ 213 7 7 8 6 PCT/GB94/00788
Assays using the immunoglobulin capture tech~i que for the detec-
tion of single pathogens in blood, for example, a rubella assay
and a hepatitis A assay have been available for several years.
W088/07680 discloses the application of the immunoglobulin
5 capture technique to the determination of ant; h~ ies in body
fluids other than blood, in particular saliva and urine. A
commercially available HIV-1+2 assay for saliva and urine uses
the IgG capture technique described.
It is believed that there is a general prejudice in the art
10 against the immunoglobulin capture technique, as demonstrated by
the fact that although the technique has been available since
1979 only a very limited number of assays using the technique are
available commercially compared to the large number of
commercially available assays using antigen to capture anti-
15 bodies. In particular, it is believed that there is a generalperception that, in practice, the techn; que would not work
effectively, either with regard to specificity or with regard to
sensitivity.
With the immunoglobulin capture techni que, a considerable level
20 of undesired interactions would be expected, leading to lack of
specificity. WO89/12231, for example, which describes an anti-
body capture assay for multiple antibody detection, expresses
concern regarding lack of specificity caused by non-immunological
protein-protein interactions between antigens and the Fc region
25 of immunoglobulins, and proposes an immunoglobulin capture assay
in which the antihs~ies coated on the solid surface are anti-Fc
antibodies. Immunoglobulins are therefore captured specifically
at the Fc region, which is thereby made unavailable for non-
specific protein-protein interactlons.
30 Sensitivity is also regarded as a potential problem with
immunoglobulin capture assays, for example, the immunoglobulins
are not ca~Lu~ed selectively, so there is no enrichment of the
specific antibody species under investigation. If that specific
WO 94~24560 PCT/GB94/00788
2137786 6 -
antibody species is present in very small amounts, both in
absolute terms and relative to the other species of antibody
present in the serum sample, it would be expected that the
detection of the antibody under investigation would be difficult
5 and unreliable and, in particular, that the assay would lack
sensitivity.
Contrary to expectation and surprisingly, the present inventors
have found that, using the immunoglobulin capture technique,
antibodies to two or more different pathogens in samples under
10 investigation can be detected simultanëously with excellent
sensitivity and specificity.
The present invention provides the use of the immunoglobulin
capture technique in the determination of antibodies to two or
more different pathogens in a single test sample.
15 Accordingly, the present invention provides a method for
determining specific an~ihoAies to two or more different
pathogens in a liquid test sample, which comprises
(i) contacting the sample with a solid phase on which are immo-
bilized antibodies to one or more classes of immunoglobulin,
20 whereby immunoglobulins of the respective class or classes
present in the sample are captured on the solid phase,
(ii) simultaneously or sequentially contacting the solid phase,
on which immunoglobulins from the sample have been captured, with
two or more different antigens, each antigen being capable of
25 binding selectively to an antibody specific for one of the
pathogens under investigation, each antigen being provided with
means capable of providing, directly or indirectly, a detectable
signal, and
(iii) determining any resulting immunoglobulin-antigen complex
30 formed on the solid phase.
2137786
WO ~n4~ ~ PCT/GB94/007~
The immobilized antiho~;es may be directed against IgG, IgM or
IgA, or a mixture of antiho~;es against different classes of
immunoglobulin may be used. A mixture of anti-IgG and anti-IgM
antibodies is particularly preferred.
5 The assay of the present invention, a "combination assay" has
unlimited versatility because of the use of a universal solid
phase for the capture of an~;ho~ies. The user can chose which
antibodies and hence which pathogens are to be detected in a
sample under investigation simply by using the a~o~iate
10 antigen reagents for detection. This is in complete contrast to
the conventional assays that use antigens to capture ant; hoA; es,
where a different antigen-coated solid phase is required for each
combination of antibodies under investigation.
The versatility of the use of a universal solid phase is a major
15 advantage both to the manufacturer and to the user of assays for
multiple pathogens, particularly blood screening assays and
clinical assays. As pointed out above, a universal antibody-
coated solid phase, for example, coated beads or coated wells of
microtitre plates, can be used for antibody testing for any and
20 all combinations of pathogens because the selectivity of any
particular antibody assay is determined by the combination of
antigen reagents used for detection.
This versatility is an advantage for the manufacturer of assays
because a universal antibody-coated solid phase may be used for
25 all co~bination antibody assays, whether they are for blood
screening or for clinical testing. At present, the mandatory
requirements for antibody testing of donated blood vary from
country to oo~.LL~. Different antigen-coated solid phases are
therefore required for different territories. The provision of a
30 universal antibody-coated solid phase for all territories reduces
manufacturing costs significantly. For clinical testing, a
different antigen-coated solid phase is required for each and
every different combination antibody assay. Again,- provision of
a universal antibody-coated solid phase reduces manufacturing
WO ~nA5~ PCT/GB94/00788
2 131 ~ 8 ~ - 8 -
costs significantly.
A further advantage to the manufacturer is the reduction in the
number of components to be coated on the solid phase and hence a
reduction in manufacturing costs. Assays using antigens to
5 capture antiho~ies require multiple antigens for combination
antibody assays for as few as two pathogens. For example, at
least six different antigens are required for a combination
HCV/HIV-1+2 assay. The more pathogens to be detected, the more
antigens must be coated on the solid phase. According to the
10 present invention, however, it is not necescAry to immobilize
additional antibodies on the solid phase in order to detect
additional pathogens; the presence of anti-IgG alone enables the
detection of multiple pathogens. Furthermore, the antibodies
used for coating are generally cheaper than the high purity
15 antigens required for successful coating, especially for multiple
antigen coating.
The versatility of the combination assay of the present invention
is also an advantage for the user because, for example, testing
for any particular combination of pathogens may be done simply by
20 using the appropriate combination of antigen reagents. This is
particularly useful in the clinical laboratory since only one,
universal antibody-coated solid phase need be purchased and
stored to enable testing for any desired combination of
pathogens.
25 For screening ~oses, it is not neces~Ary to know the nature of
a contaminating organism. In the case of blood screening, if a
sample of blood is positive for an~iho~ies to any of the
proscribed pathogens, that blood is unsuitable for use for trans-
fusion or for the production of blood products, for example,
30 plasma or Factor VIII, and should be discarded.
The ability to test a sample of donated blood in one assay for
all the blood viral antibodies laid down by any particular
national regulation is a major advance, providing blood banks
wo ~4~ 213 7 7 8 6 PCTIGB94/007~
with very substantial savings in both time and money. The time
saving is important not just in saving the time of technicians
and hence being a further cost saving, it is also important in
view of the relatively short shelf-life of whole blood.
5 The immunoglobulin capture assay of the present invention has
excellent specificity and sensitivity. We have obtained results
for samples containing a mixture of antiho~ies of interest that
are substantially the sum of the results obtained for the
corresponding serum or plasma samples each cont~ining only one of
lO the antibody species. It could not have been predicted that, in
the combination assay of the present invention, each of a
plurality of antibodies, directed to different organisms, reacts
as if it were the only antibody species present. The
observation of an additive effect with regard to the number of
15 antibody species detected is an unexpected but major practical
advantage. It is also unexpected that the assay of the present
invention, which can be conducted using a conventional assay
format, for example, a microtitre plate, is so sensitive.
An assay of the present invention may be an IgG assay, an IgM
20 assay or, for special purposes, an IgA assay. Alternatively, a
mixture of antiho~ies to immunoglobulins of different classes may
be used for coating the solid phase. It is particularly
preferred to use a mixture of anti-IgG and anti-IgM antiho~ies.
A further important advantage of the assay of the present
25 invention compared with assays using coated antigens for antibody
capture is that, by using immobilized anti-IgM, early infection
may be detected reliably. This is important both for blood
screening and for clinical investigations.
IgM is the first class of immunoglobulin to be produced in
30 response to infection. Specific IgM antibody levels rise in the
first few weeks after infection, then specific IgG is produced a
little later. The IgG antibody response is persistent and may
last for many years, even for life. In theory, IgM can be
WO ~AS~ PCT/GB94100788
213~86 - 10 -
detected in conventional assays where an antibody of interest is
ca~u~d onto an antigen-coated surface and the presence of
ca~L~d antibody is detected using, for example, labelled anti-
human-IgM antibodies. In practice, however, such IgM assays
5 generally lack specificity because IgM is "sticky", that is to
say, IgM tends to bind non-specifically to the capturing antigen.
The labelled anti-IgM antibody used for detection of antigen-IgM
complex cannot distinguish between specific antigen-IgM complexes
and non-specifically bound IgM. For this reason, conventional
10 assays using immobilized antigens generally use anti-IgG anti-
bodies for detection i.e. the assays are for IgG only.
In the assay of the present invention, anti-IgM ant;ho~ies may be
coated on the solid phase. Those antiho~ies then capture IgM
immunoglobulins specifically from the sample, and the captured
15 IgM is detected specifically by means of the labelled antigen.
We have found, surprisingly, that the system is not "sticky" and
that specificity is not a problem as it is with the conventional
antigen-capture assays for the detection of IgM antiho~ies. As
indicated above, it is particularly preferable to use a mixture
20 of anti-IgG antibodies and anti-IgM antiho~ies for coating the
solid phase. The presence of anti-IgM antibodies on the solid
phase will ensure that any early antiho~ies present in a sample
are detected, and the presence of anti-IgG antibodies will ensure
that persistent antiho~ies are detected.
25 The antihoAies immobilized on the solid phase may be polyclonal
antiho~ies, for example, polyclonal anti-human antibodies, which
will contain antiho~ies to all immunoglobulin classes.
Alternatively polyclonal antihoAies directed against a particular
class of immunoglubulin may be used, for example, polyclonal
30 anti-IgG and polyclonal anti-IgM antihoAies. Polyclonal
antibodies may be purified, for example, by affinity chromato-
graphy. If desired, monoclonal antihoAies may be used. Anti-
immunoglobulins may be specific for the immunoglobulin gamma, mu
or alpha chains of IgG, IgM and IgA, respectively.
wo ~n~s~ 213 7 7 8 6 PCT/GB941~788
-- 11 --
Polyclonal and monoclonal antibodies used for coating the solid
phase may be prepared by methods known per se. Various anti-
immunoglobulins are available commercially, for example, rabbit,
sheep and goat polyclonal anti-IgG and anti-IgM immunoglobulins,
S and mouse monoclonal anti-IgG and anti-IgM immunoglobulins. If a
mixture of different antibodies is used for coating, the
proportions of the different antiho~ies in the mixture may be
varied as desired. The present invention is not limited to the
investigation of samples from humans and also finds veterinary
lO applications. Accordingly, the antibodies used for coating
should be directed against immunoglobulins of the species from
which the sample under investigation is obtained. In the case of
samples from humans, for example, a coating antibody should be an
anti-human antibody.
lS As mentioned above, a particularly advantageous feature of the
assay of the present invention is that a universal antibody-
coated solid phase means is used regardless of the number or
nature of pathogens under investigation. The antihoAies used as
coating antihoAies are directed against one or more classes of
20 immunoglobulins, especially against IgG and IgM, and capture a
representative proportion of immunoglobulins of the respective
classes from the sample. By choosing any particular combination
of specific antigens, the user of the assay can test for any
combination of pathogens. The antigen reagents can be provided
25 ready mixed, or the user can use separate antigen reagents in any
desired combination. If used individually, the antigen reagents
may be contacted simultaneously or sequentially in any order with
the solid surface carrying the ca~-~Led immunoglobulins. It is
generally more convenient to add the desired mixture of reagents
30 in one step.
An antigen to be used in an assay of the invention may be any
antigenic entity that interacts selectively with the antibody
under investigation. For example, an antigen may be a peptide or
polypeptide, and may be a synthetic or recombinant antigen, or a
35 purified antigen from cell culture, for example, from a viral
WO94nA~ PCT/GB94/00788
2137786 - 12 -
lysate. It may be particularly useful in certain cases, for
example, HCV, to use a recombinant fusion polypeptide that com-
prises more than one antigenic region of a particular organism.
An antigen may itself be labelled with means capable directly or
5 indirectly of providing a detectable signal to enable any
immunoglobulin-antigen complex to be detected. The labelled
antigen is called an "antigen ~bnjugate".
The detectable signal may be optical, radio-active or physico-
chemical, and may be provided directly by labelling the antigen,
lO for example, with a dye, coloured particle, radiolabel,
electroactive species, magnetically resonant species or
fluorophore; or indirectly by labelling the antigen with an
enzyme itself capable of giving rise to a measurable change of
any sort. Alternatively, the signal may result from
15 agglutination, a diffraction effect or a birefringement effect
involving the antigen conjugate.
In a further embodiment, the means capable indirectly of
providing a detectable signal comprises an antibody that binds to
the antigen. That antibody (Abl) may be provided with direct or
20 indirect label means as described above for the antigen (single
antibody detection system), or it may be labelled by means of yet
another antibody (Ab2), which binds to Abl and is itself labelled
directly or indirectly as described above for the antigen (dual
antibody detection system). A single or dual antibody system for
25 detection of captured antibody-antigen complex is particularly
useful if it is difficult to produce a suitable antigen
conjugate. Some antigen conjugates, for example, tend to be
insufficiently stable for commercial use, and some antigens lose
antigenicity on conjugation.
30 The single antibody detection system has the advantage of
involving fewer washing and incubation steps, but the
disadvantage that it is nece~c~ry to produce an antibody
conjugate (labelled antibody Abl) for each antigen to be
WO94nA5~ 21~ 7 7 8 6 PCT/GB94/007~
- 13 -
detected. The dual antibody detection system has the advantage
that, with appropriate choice of Abl and Ab2, it is possible to
use one antibody conjugate (labelled antibody Ab2) for the
detection of all captured antibodies as follows: when each anti-
5 body (Abl) is raised in the same animal species, the secondantibody (Ab2) can be an anti-species antibody. For example, if
each antibody Abl is a sheep antibody, then a labelled anti-sheep
antibody can be used as Ab2.--The advantage to the user of
requiring only one antibody conjugate for detection of all
10 analytes generally outweighs the disadvantage of the need for an
extra incubation and extra washing steps, particularly when an
automated system is used. The advantage of requiring only one
antibody conjugate is another significant advantage to the
manufacturer.
15 A labelling system that is particularly preferred for an antigen
or antibody conjugate is an enzyme labelling system, particularly
one in which the antigen or antibody is conjugated to an enzyme
that catalyses a detectable colour change in the presence of a
suitable substrate. This format, the enzyme-linked immunoassay
20 or ELISA, is widely used commercially, and automated instruments
are available for carrying out such assays using, for example,
microtitre plates, the proprietary "bead" or "IMX" (Trade Mark)
systems, or using hollow rods or pipette tips, and computer soft-
ware is available to process the results obtained.
25 The enzyme systems used are well known per se, see for example,
"ELISA and Other Solid Phase ImmunoA~cAys, Theoretical and Prac-
tical Aspects" Eds. Kemeny D.M. & Challacombe S.J.,
Examples of typical enzyme systems are those using alkaline
phosphatase, B-galactosidase, urease or peroxidase, for example,
30 horse-radish peroxidase.
The solid phase on which the antibodies are captured is, for
example, beads or the wells or cups of microtitre plates, or may
be in other forms, for example, as solid or hollow rods or
W094/~ PCT/GB94/~7
~ ~ 31 1 8 6 - 14 -
pipettes, or particles, for example, from 0.1~m to 5mm in
diameter. (Such particles are often called "latex" particles,
regardless of the material of which they are made.)
A solid phase may be of a plastics or polymeric material, for
5 example, of nitrocellulose, polyvinyl chloride, polystyrene,
polyamide, polyvinylidine fluoride or other synthetic polymers.
Particles may additionally be of natural polymers, for example,
latex or protein. Microtitre plates and beads are used
extensively for both blood screening and clinical testing and are
10 widely available commercially.
Other solid phases that may be used include membranes, sheets and
strips, for example, of a porous, fibrous or bibulous material,
for example, of nylon, polyvinyl chloride or another synthetic
polymer, of a natural polymer, for example, cellulose, of a
15 derivatized natural polymer, for example, cellulose acetate or
nitrocellulose, or of glass fibres. Paper, for example, diazo-
tized paper may be used. Films and coatings, for example, of
fibrous or bibulous material, for example, as described above,
may be used as the solid phase.
20 It is to be understood that the above examples of solid phases
are given by way of illustration only, and the invention is not
limited to the use of such solid phases. The invention may be
practised on any solid phase suitable for use in immunoassays.
A solid phase, for example, a membrane, sheet, strip, film or
25 coating, may be incorporated in a device for the determination of
multiple or, more generally, single samples.
The term "assay device" is used herein to denote means for
carrying out an immunoassay comprising a solid phase, generally a
laminar solid phase, for example, a membrane, sheet, strip,
30 coating, film or other laminar means, on which are immobilized
antibodies to one or more classes of immunoglobulin. The
immobilized antibodies are preferably present in a defined zone,
wo ~nAs~ ~13 7 7 8 6 PCT/GB94100788
-- 15 --
called herein the "antigen capture zone".
An assay device may incorporate the solid phase within a rigid
support or a housing, which may also comprise some or all of the
reagents required for carrying out an assay. Sample is generally
5 applied to an assay device at a predetermined sample application
zone, for example, by pouring or dripping the sample on the zone,
or by dipping the relevant part of the device into the sample.
If the sample application zone is at a different site from the
antibody capture zone, the arrangement of device is generally
10 such that antihs~ies in the sample migrate to the antibody cap-
ture zone. The required reagents are then applied in the app-
ropriate order at designated application zones, which may or may
not be the same as the sample application zone. Again, if the or
any reagent application zone is at a different site from the
15 antibody capture zone, the arrangement of a device is generally
such that the reagent(s) migrate to the antibody capture zone,
where any antigen-antibody complex formed is detected. All or
some of the reagents required for an immunoassay may be
incorporated within a device, in liquid or dry form. If so, a
20 device is generally arranged such that interactions between
different parts of the device, which interactions may occur
automatically during the operation of the device or may be
brought about by the user of the device, bring the various
reagents into contact with one another in the correct sequence
25 for the immuno~ss~y to be carried out.
A wide variety of assay devices are described in the literature
of immunoA-ss~ys. Examples of membrane devices are described in
U.S. Patents Nos. 4,623,461 and 4,693,984. Depending on their
design and their speed of action, some assay devices are called
30 "dipsticks" and some are called "rapid assay" devices. A "rapid
assayN device generally provides a result within ten minutes of
the application of sample. (A typical microtitre plate or bead
assay requires incubation steps, and generally takes at least an
hour to provide a result.) Accordingly, although assay devices
35 are generally more expensive than microtitre or bead format
W094nA~ PCT/GB94/007
2 ~ 3~ ~ 8 6 - 16 -
assays, they have particular uses in clinical testing, for
example, when a result is required rapidly, for example, in the
case of emergency surgery.
Assay devices have the particular advantage that they can be used
5 without the need for sophisticated laboratory facilities or even
without the need for any laboratory facilities. They may there-
fore be used for "on the spot" testing, for example, in an
emergency room, in a doctor's surgery, in a pharmacy or, in
certain cases, for home testing. They are particularly useful in
10 territories where laboratory facilities are few and far between.
As indicated above, the combination assay of the invention is
particularly useful for screening donated blood. The assay may
therefore be used to test for an~;hoAies to at least two viruses
selected from HIV, for example, HIV-1, HIV-2 and HIV subtypes,
15 for example, HIV-l subtype o; HCV; hepatitis B (antibodies to
core antigen); HTLV, for example, HTLV-I and HTLV-II; CMV; EBV
(Epstein Barr virus); and optionally also for syphilis.
Particularly preferred are combinations of two or more selected
from HIV, for example, HIV-1+2; HCV; HTLV, for example, HTLV-
20 I+II; and hepatitis B core (HBc); for example: HIV and HCV; HIVand HTLV; HTLV and HCV; HIV, HCV and HTLV; HIV, HCV and HBc;
HIV, HTLV and HBc; HTLV, HCV and HBc; HIV, HCV, HTLV and HBc.
Syphilis may-be included in any of the above combinations. The
combination of choice will be influenced by specific national
25 regulations. In some cases, donated blood may also be tested for
further pathogens, for example, for rubella.
Furthermore, the requirements for blood screening are reviewed as
new pathogenic agents (new organisms and new subtypes of known
organisms) are discovered, and the mandatory assays are extended
30 to cover those pathogens. For example, it is currently required
in most countries to test for both HIV-1 and HIV-2. The recent
discovery of HIV-1 subtype 0 has led to the requirement that
assays must also detect that subtype. Accordingly, the
combinations of pathogens to be detected will almost certainly
2137786
WO ~nAS~ PCT/GB94/007
enlarge with time. The present invention includes assays for
such combinations of pathogens.
If the tube/bead two component system is used, further
versatility can be introduced, for example, after contacting the
5 tube and bead with the sample, the bead may be removed and con-
tacted with antigen(s) to one or more different species of
pathogen and the tube contacted with a different antigen or
combination of antigens.
A further variant of the tube/bead two component assay format is
10 to immobilize one or more classes of anti-immunoglobulin on one
of the components and to immobilize anti-hepatitis B surface
antigen (anti-HBsAg) on the other component, for example, anti-
IgG and optionally also anti-IgM may be coated on the inner
surface of the small tubes and anti-HBsAg on the beads, or vice
15 versa. The beads and the tubes may then be used together in a
combined HBsAg/multiple antibody assay, especially an assay for
HBsAg, HIV, HCV and optionally one or more further analytes
selected from hepatitis B (core), HTLV, and syphilis.
Peptides and polypeptides that interact specifically with HIV-l
20 and HIV-2 are well known and are described, for example, in EP-A-
0 347 148. We have found that it is particularly useful with
synthetic HIV peptides to block the sulphydryl groups of the
cysteine ~o~s with blocking agents, see for example, EP-A-O 307
149, resulting in a labelled peptide, in particular enzyme-
25 labelled peptide, having greater immunological activity in theassay than a corresponding non-blocked peptide.
The antigen to be used for detection of hepatitis B core anti-
bodies is a core antigen.-DNA and predicted protein sequences for
various hepatitis B serotypes have been published, for example,
30 by Ono et al (1983) Nuc. Acids Res 11, 1747, and suitable peptide
and polypeptide core antigens, both synthetic and recombinant,
may be derived from published sequences.
21~7 8 PCT/GB94/~7~
- 18 -
Hepatitis C does not, at present, appear to have one
immunodominant epitope, and it is preferable to test for anti-
bodies to more than one region. For example, it may be advan-
tageous to use as antigen a fusion protein that comprises more
5 than one epitope, for example, an amino acid sequence from at
least one structural region and from at least one non-structural
region, for example, as described in GB-A-2,239,245. Antigens
from the core and envelope regions are preferred structural
antigens. Non-structural antigens may be selected, for example,
10 from the NS3, NS4 and NS5 regions.
An HTLV antigen may be, for example, a p21e or gp46 recombinant
protein or a peptide derived from p21e or gp46 (see, for example,
US Patent No. 4,743,678). Purified p21e is available from
Cambridge Biotech Corporation, 1600 East Gude Drive, Rockville,
15 Md 20850-5300, U.S.A and gp41 from Repligen Corporation, 1 Kendal
Square, Building 700, Cambridge, Ma 02139, U.S.A.
For detection of CMV antiho~ies~ there may be used purified
cultured CMV core proteins, for example, p66, or recombinant
pplS0, which is dominant in Western blots. For detection of EBV
20 antiho~ies, there may be used capsid or early antigen. Purified
T. pallidum antigen may be used for the detection of syphilis
antihs~ies.
A combination assay according to the present invention may be
used in clinical diagnosis as a preliminary, screening test for
25 several pathogens. If the test is positive, then a separate test
for each pathogen may be carried out. If the result is negative,
no further action is required. Overall, time and money is saved.
An example of such a combination is that used for the so-called
"TORCH" screening of pregnant women in many countries. The
30 combination of pathogens under investigation varies from country
to co~,LLy, in analogy to blood screening requirements, but is
generally selected from rubella, toxoplasmosis, CMV and herpes.
It is particularly advantageous to use a mixture of immobilized
anti-IgG and anti-IgM antibodies to ensure that recent infection
wo 94~ 21 3 7 7 8 6 PCrIGB94/00788
-- 19 --
is detected.
A combination assay of the present invention presented in an
assay device format, especially "rapid assay" device format, is
particularly useful when a screening result is required urgently,
5 for example, in the case of emergency surgery. For example, a
rapid assay for two or more different blood viral pathogens, for
example, as described above, for example, for HIV-1+2 and HBc
will inform a surgeon if special precautions are required.
An assay device for combinations of pathogens of interest is of
10 use, for example, for "on the spot" testing in doctors' surgeries
to assist with diagnosis and can, overall, save time and money in
both clinical laboratory testing and in the avoidance of repeat
visits to the surgery. Furthermore, assay devices are useful for
testing in situations where laboratory facilities are not readily
15 available, for example, in rural areas and in developing
countries. Examples of combinations of pathogens are CMV and
HIV; and TB and HIV.
The versatility of the use of a universal antibody-coated solid
phase for the detection of a plurality of antigens can be
20 exploited in a further manner in clinical testing. In clinical
testing, the object is generally to identify the pathogen(s) in a
sample. At present, if a number of samples have to be tested for
the presence of various pathogens using the conventional micro-
titre plate or bead format, it is customary to prepare aliquots
25 of the various samples and to run a series of assays, each for a
different pathogen. This is because the conventional assays use
antigen-coated means to capture pathogen-specific antibodies.
Accordingly, it is nec~c~Ary to use a different antigen-coated
means, generally either beads or a microtitre plate, for each
30 pathogen. This increases the time taken to obtain results on a
particular sample, and also creates a risk that aliquots of
samples will be mislaid or confused while all the various tests
are being carried out.
W094~5~ PCT/GB94/007
2 13 1 ~ 8 6 - 20 -
Furthermore, the antigen-coated means is generally presented with
other reagents, for example, positive and negative controls, wash
solutions and diluents, in the form of a kit. The user therefore
has to have a large number of different kits.
5 Using the assay of the present invention, a universal antibody-
coated means, for example, microtitre plate, can be used for the
simultaneous but separate determination of each of a plurality of
pathogens simply by the use of the appropriate antigen reagents.
A series of different samples may be tested at the same time,
lO some for one pathogen, some for another. It is particularly
advantageous to test aliquots of a single sample simultaneously
but separately for different pathogens, for example, to test each
aliquot in a separate well of a microtitre plate for a different
pathogen. This results in savings of time and money, and also
15 reduces the risk of mistakes through loss or confusion of
samples.
Accordingly, the present invention also provides a method for
determining simultaneously but separately, antihoA ies specific to
two or more different pathogens in liquid test samples, which
20 comprises
(i) bringing each sample into contact with one of a plurality of
units, for example, in a single module, each unit comprising a
solid phase having immobilized antibodies to one or more classes
of immuno-globulin, whereby immunoglobulins of the respective
25 class or classes present in each sample are captured on the solid
phase, the immobilized antibodies being of the same class or
classes in all units of the module,
(ii) contacting each unit that has previously been contacted with
sample with an antigen capable of binding selectively to an
30 antibody specific for one of the pathogens under investigation,
each antigen being provided with means capable of directly or
indirectly providing a detectable signal, and
W094~4~ 21 3 7 7 8 6 PCT/GB94/~788
- 21 -
(iii) determining any resulting immunoglobulin-antigen complex on
the solid phase.
The number of different units used and hence antigens used will
depend on the number of pathogens under investigation. Each unit
5 may be, for example, one of the wells of a microtitre plate, or a
vessel contAining beads.
Alternatively, each unit may be a defined region on a membrane,
strip, sheet, film or coating, especially when presented in an
assay device. Devices having predetermined regions for capture
10 of components from a sample under investigation are known per se.
At its simplest, an assay strip device for a simultaneous but
separate assay according to the present invention may comprise,
for example, a strip of bibulous material having a band of
capture immunoglobulin across the width of the strip, called
15 herein the antibody capture zone. A band is preferably divided
into sections by areas not coated with capture immunoglobulin.
Each section of immobilized immunoglobulins may be considered to
be a unit antibody capture zone.
An assay device for a simultaneous but separate assay is, for
20 example, as descibed above in relation to combination assays.
The versatility of an assay device of the present invention for a
simultaneous but separate assay for different pathogens provides
particular advantages for both manufacturer and user. A major
advantage is that one device, provided with a number of defined
25 antibody capture zones, can be supplied together with a plurality
of antigen reagents for simultaneous testing for a number of
different antibodies in a sample. As before, the manufacturer
has the advantage that only one device need be made for all
analytes. The user, too, has the advantage of requiring only one
30 device, and has the further advantage of free choice of the
combination of analytes to be determined.
As indicated above, a simultaneous but separate assay of the
WO ~n4~ PCT/GB94/007
2 1 3~ 7 8 6 - 22 -
present invention for different pathogens may be carried out for
the investigation of any particular combination of pathogens in a
sample. ~or example, a sample from a patient suffering from
hepatitis may be tested simultaneously for hepatitis A, hepatitis
5 B anti-core an~;ho~;es and HCV: a sample from a patient with non-
specific urethritis may be tested for gonorrhoeae, chlamydia and
candida.
In a variant of the simultaneous but separate assay of the
present invention, instead of each unit having immobilized
10 ant;ho~ies of the same class or classes, one or more of the units
may have a different class of immunoglobulin immobilized thereon.
For example, there may be provided a set of two units, one unit
having immobilized anti-IgG and the other immobilized anti-IgM.
A series of simultaneous assays for a particular antibody carried
15 out over a period of time using such a two-unit set on samples
obtained from the same individual is particularly useful for
detecting early infection and for following the course of a
disease, especially for following se~o~o.-version, for example, of
HIV or HCV. A set of three units, one having immobilized anti-
20 IgG, one having immobilized anti-IgM and the third having
immobilized anti-IgA is particularly useful for determining if a
baby has HIV: a baby's blood has both the baby's own antibodies
and maternal antibodies, so using conventional tests it is
difficult to determine if the baby does have its own HIV anti-
25 bodies, which are indicative of infection.
A further example of the use of more than one class of antibodyis in the so-called "TORCH" test carried out in many countries on
pregnant women. The pathogens are selected from rubella,
toxoplasmosis, CMV and herpes. The combination of choice varies
30 from ooullLL~ to C~I~L~. The rubella test should include an IgM
assay in order to detect a recent infection, which is potentially
dangerous. Accordingly, for a "TORCH" assay involving rubella,
at least one of the units in the set used should have an anti-IgM
coating.
wo ~4~ 213 7 7 8 6 PCT/GB941~7~
- 23 -
A module comprising two or more units having immobilized
antibodies against different classes of immunoglobulins is itself
part of the present invention. Accordingly, the present
invention provides a module comprising two or more units for use
5 in a simultaneous but separate assay for different pathogens, the
units comprising immobilized~an~i hoA; es against immunoglobulins,
at least one unit having having immobilized an~iho~;es directed
against a different class of immunoglobulins from the immobilized
immunoglobulins in the other units, for example, one unit may
lO have immobilized anti-IgM and one or more units may have
immobilized IgG. In another em~o~iment, a module comprises three
units, one unit having immobilized anti-IgG, one unit having
immobilized anti-IgM and the third unit having immobilized anti-
IgA.
l5 A module comprising two or more units may be present in an assay
device, for example, as an arrangement of two or more antibody
capture zones, for example, an IgG capture zone and an IgM
capture zone. Such an assay device may be used for a
simultaneous but separate assay for different pathogens, for
20 example, for any of the particular uses described above.
Alternatively, a module may comprise two or more sets of units
(each set comprising identical units). A series of sets of
different units may be presented as a module. For example, a set
of units may be presented as a strip of microwells. Such strips
25 may be assembled as desired into modules comprising two or more
sets of units, for example, a module may comprise two strips of
microwells, one coated with anti-human IgG and one coated with
anti-human IgM. A third strip, having immobilized anti-human IgA
may be added. Strips of microwells i.e. sets of units, may be
30 assembled as desired to form a conventional microtitre plate.
For example, a plate may have alternate rows or columns of anti-
IgM and anti-IgG coated microwells, for example, for following
seroconversion. Again, there are manufacturing cost advantages
and also the advantage of versatility of use, particularly for
35 strips of microwells or analogous sets of units.
WO ~AS~ PCTIGB94/~7
2 137 ~ 8 6 - 24 -
Unless otherwise specified, the following description applies to
both the combination and the simultaneous but separate assays of
the present invention:
For clinical testing, the liquid test sample may be any body
5 fluid, for example, blood, plasma, serum, saliva, urine, cerebro-
spinal fluid, milk, lymph fluid or tears. Alternatively, a solid
sample, for example, of cells or tissue may be brought into
liquid form for testing, for example, as tissue exudate.
For blood screening (which is advantageously carried out as a
10 combination assay) the pathogens of interest are so-called blood
viral pathogens (HIV, hepatitis B and hepatitis C, optionally
also HTLV, CMV and EBV), and also syphilis. For clinical
testing, a very much wider range of pathogens are of interest,
including the above organisms and also other viruses, bacteria
15 and other types of pathogenic organisms. By way of example, but
non-limiting, are the following:
Rubella, measles, Herpes (simplex and genitalis), Chlamydia,
Gonorrhoeae, hepatitis A, chickenpox, mumps, human parvovirus,
Mycobacteria tuberculosis, Mycobacteria leprae, Mycobacteria
20 avium, Staphylococcus aureus, Listeria monocytogenes, Bacillus
anthracis (antigen/toxins), Actinomycetes (for example,
Streptomyces, Nocardia, Rhodococcus), Salmonella typhi, Yersinia
enterocolifica, Helicobacter pylori, Campylobacter jejuni,
Pseudomonas mallei and pseudomallei, Pseudomonas aeruginosa,
25 Legionella pneumophila and spp, ~rancisella tolarensis, Brucella
melitenis, Mycoplasma pneumoniae, Leptospira interrogans, Borelia
spp, Treponema pallidum, Candida albicans, and diseases caused by
protozoal pathogens, for example, amoebiasis, babesiosis, Chagas'
disease, leishmaniasis, malaria and toxoplasmosis.
30 The immobilized anti-immunoglobulins may be of one or more
classes, that is to say, of the IgG, IgM and IgA classes. As
mentioned above, IgG is the most abundant immunoglobulin in
serum. IgM is the first immunoglobulin produced in response to
WO 94/24560 21 3 7 7 8 6 PCrIGB94100788
infection, so provides an earlier indication of infection than
does IgG. For testing blood (plasma or serum), generally either
IgG or IgM may be used alone, according to the ~uL~ose of the
assay, or à mixture of anti-IgG and anti-IgM may be used in one
5 unit for a combination assay or anti-IgG and anti-IgM may be
presented in separate units for a simultaneous but separate
assay. For assaying saliva, it is also preferable to capture IgG
and/or IgM. For assaying urine, anti-IgG is preferably used,
optionally in combination with IgM and/or IgA.
10 For those forms of assay of the present invention which utilise
the two-component bead/tube format, it is possible to increase
even further the adaptability of the assay of the present
invention by immobilising one or two classes of anti-
immunoglobulins on the inner surface of a plastics tube and
15 immobilizing one or two different classes of anti-immunoglobulins
on beads, the beads to be used in the tube. For example, anti-
IgG may be coated on one of the two components, and anti-IgM on
the other.
Furthermore, a combination bead/tube format may be used and,
20 after contacting the tube and bead with the sample, the bead may
be removed and contacted with one antigen and the tube contacted
with another antigen, enabling two antibody assays to be carried
out on a single sample.
Although both blood screening and clinical testing have been
25 described above, there is no difference in the principle of the
assays used, or in the type of materials used. For blood
screening a particular range of analytes are specified by the
specific national regulatory authorities, and the testing tends
to be more fully automated than clinical testing simply because
30 of the large numbers of identical tests. However, it is to be
understood that, throughout the specification, unless specified
otherwise, all descriptions relate to assays of the present
invention in general, and include assays carried out on assay
devices as well as those carried out on, for example, microtitre
W094/~ PCTlGB94/~7
2 1 3~ 7 8 6 - 26 -
plates or beads.
The assays of the present invention may be carried in a conven-
tional manner see, for example, "ELISA and Other Solid Phase
Immunoassays Theoretical and Practical Aspect", Eds. Kemeny D.M.
5 & Challacombe S.J. Immunoglobulins may be immobilized on a solid
phase, for example, by contacting the solid phase with a solution
of the immunoglobulins at an appropriate concentration and pH,
for example, at a pH within the range of from 7 to 11, especially
from 9 to 10. A buffer is preferably used, for example, a sodium
10 carbonate/sodium bicarbonate buffer. AS indicated above,
suitable immunoglobulin preparations are available commercially,
and a suitable dilution of a commercial antibody solution is, for
example, 1:200 to 1:4000 v/v.
A labelled antigen reagent (conjugate) may be produced by any of
15 a variety of methods see for example, Kemeny & Challacombe, loc
cit. Antigen-enzyme conjugates are generally used.
Samples may be diluted, for example, blood or plasma samples may
be diluted 1 in 1 v/v, for example, 50~1 of sample is added to
50~1 of diluent in a microwell. It should be noted that any
20 sample diluent used should not contain human immunoglobulins of
the class or classes that are to be captured. In a microtitre
plate or bead format assay, the sample is then generally
incubated with the solid phase. The temperature and time of
incubation are interdependent, a longer time being required at a
25 lower temperature. Typical incubation conditions are one hour at
37 C. After incubation, the plate or beads should be washed
thoroughly prior to incubation with the antigen reagent
(conjugate). Typical incubation conditions for the conjugate
stage are 30 minutes at 37 C. After incubation with the
30 conjugate, there is a further washing step, followed by
incubation with the substrate for the enzyme in the case of an
ELISA. Again, 30 minutes at 37 C are typical incubation
conditions. A stop solution is generally added at the end of the
incubation. In the case of an ELISA, results are generally
wo ~s~ 213 7 7 8 6 PCTIGB94/007~
- 27 -
obtained by reading the absorbence of each unit in a
spectrophotometer. If another labelling system is used, the
method is modified accordingly, for example, in the case of a
radioimmunoassay or a fluorescence assay, results are obtained
5 after incubation with a radiolabelled or fluorescent antigen
conjugate. In the case of coloured particles, it may be possible
to determine positive and negative results by eye.
In the case of an assay device for use outside a laboratory,
coloured particles are particularly useful as label, since a
10 positive or negative result can be determined by eye with a
minimum number of reaction steps. For a more sensitive assay, an
ELISA system may be used.
The present invention also provides a kit comprising
(a) a solid phase, especially plastics beads or a microtitre
15 plate, carrying immobilized antibodies to one or more classes of
immunoglobulin, especially a mixture of anti-IgG and anti-IgM
ant; hoA ies~
(b) two or more antigen reagents each capable of binding
selectively to an antibody specific for one of the pathogens
20 under investigation, and each antigen being provided with means
capable directly or indirectly of providing a detectable signal,
and optionally, one or more of the following:
(c) positive and negative control reagents, washing solutions and
diluents.
25 The kit may be provided as such, or the antibody-coated component
and the antigen reagent component may be provided separately.
The present invention also provides a solid phase suitable for
use in an immunoassay, on which is immobilized a mixture of anti-
IgG and anti-Ig~ antibodies. The mixture of antibodies may
30 additionally comprise anti-IgA antibodies. The antibodies are
especially anti-human antibodies. The solid phase is, for
example, any of the solid phases described above, for example,
microtitre plates and beads, and includes those suitable for use
W094~S~ PCTIGB94/~7
- 28 -
assay devices.
The terms "detection" and "determination~" are both used herein
to denote qualitative, quantitative and semi-quantitative assays
for pathogens.
5 The use of the immunoglobulin capture format for the
determination of an~ihoAies to a plurality of pathogens in
different samples ("combination assay") or for the simultaneous
but separate determination of a plurality of different antibodies
in aliquots of the same sample ("simultaneous assay") gives
10 advantages both in time and cost. The potential versatility of
the format, in that multiple pathogens can be determined
simultaneously either in one unit or in a series of parallel
units simply by choice of antigen reagent, had not been
appreciated previously. The very real practical and economic
15 advantages of being able to use a universal antibody-coated
means, for example, antibody-coated microtitre plates, antibody-
coated beads, and immobilized antihoAies in an assay device, for
any antibody assay should not be underestimated.
It will, of course, be appreciated that the present invention is
20 not limited to the detection of pathogens. The present invention
may be applied to the detection of any antibody of interest,
whatever the nature of the antigen that gives rise to the anti-
body, for example, non-pathogen-associated antibodies. Examples
of conditions giving rise to such antihoAies include autoimmune
25 dis~A~c and allergies, for example, non-organ-specific
autoimmune diseases, for example, rheumatoid arthritis, lupus
erythematosus and rheumatic fever, and organ-specific autoimmune
diseases and diseases considered to have some autoimmune
involvement, for example, autoimmune diseases of the thyroid,
30 myasthenia gravis, autoimmune haemolytic anaemias, multiple
disseminated sclerosis, aphthous ulcer, pernicious anaemia and
ulcerative colitis. All that is required is an entity capable of
binding specifically to any antibody of interest.
wo ~5~ 213 7 7 8 6 PCT/GB941~7~
- 29 -
Accordingly, it is to be understood that the teachings of present
specification are as relevant to the detection of non-pathogen
related antibodies as they are to antiho~;es to pathogens, and
that the present invention includes all such embodiments.
5 The present invention may be used in the detection of
determination of human or animal pathogens, and finds both human
and veterinary applications, including applications in the meat
trade.
The following non-limiting Examples illustrate the invention.
10 F~ ~T.~:
Reagents
The following reagents were used for the assay described below:
1. Solid phase: 96-well microtitre plate (Nunc) coated with a
mixture of polyclonal anti-human IgG (DAK0) and polyclonal anti-
15 human IgM (DAK0). (Nunc products are available from LifeTechnologies, P0 Box 35, ~ hington Road, Abbotts Inch Industrial
Estate, Paisley, Renfrewshire, TA3 4EF, Scotland, and DAK0
products from DAKO, 16 Manor Courtyard, Hughenden Avenue, High
Wycombe, Bucks HP3 5RE, England).
20 2. HIV-1 recombinant protein comprising core and envelope
antigens from the CBL-1 isolate of HIV-l (Sattentau Q.J. et al.
(1986) Science 234 1120) conjugated to HRP (horse-radish
peroxidase) ("HIV-1 conjugate").
3. HIV-2 peptide comprising gp36 envelope antigen, conjugated
25 to HRP ("HIV-2 conjugate").
4. Hepatitis core antigen conjugated to HRP
5. Positive samples:
a) HIV-l, internal reference No. 4034, diluted in HIV-1
negative serum
b) HIV-2, internal reference No. 91/174 diluted in HIV-2
negative serum
c) anti-HBc core positive samples diluted in HBc negative
W094~5~ PCT/GB94/00788
2 1 37 7 ~ 6 _ 30 _
serum
6. Negative samples: normal human serum obtained from donated
blood.
Method
S 100~1 of diluted samples (10 ~1 sample and 90~1 sample diluent)
were added to wells of a microtitre plate and incubated under
humid conditions at 37 C for 60 minutes. The wells were then
washed thoroughly five times with a wash solution, each wash step
involving removal of the contents of each well by aspiration,
10 filling the well with wash solution ((glycine borate buffer
containing Tween), and soaking for 30 seconds. After the final
wash step the contents of the well are removed and the wells are
inverted and tapped dry on a paper towel or tissue. 50~1 of a
working ~e.l~Lh solution of the relevant conjugate(s) in HEPES
15 buffer containing bovine serum albumin and detergents were added
to the wells either singly or in combination as described below
and the plate was incubated at 37 C for 30 minutes under humid
conditions. After a further wash step as described above, 100~1
of substrate solution containing TMB (3,3',5,5'-tetramethyl-
20 benzidine) and hydrogen peroxide was added to each well, theplate was incubated at 37 C under humid conditions for 30 minutes
and the reaction was then stopped using 50~1 of 2M sulphuric
acid. The absorbance in the wells was recorded at 4SOnm with
690nm as the reference wavelength.
25 EXAMPT~ 1
A series of positive samples each containing one species of
antibody of interest (anti-HIV-l, anti-HIV-2 and anti-HBc) were
diluted and tested according to the above protocol using the HIV-
1, HIV-2 and HBc conjugates described above, either alone or in
30 various combinations as shown in the Tables below. The HIV-l
sample was serially diluted 1/40; 1/80; 1/160 and 1/320. The
HIV-2 sample was diluted 1/32; 1/64; 1/128 and 1/256. A number
of different HBc-positive samples were diluted 1/2.
W094~S~ 21 3 7 7 8 6 PCTIGB94/00788
- 31 -
(i) HIV-l positive samples at increasing dilutions were tested
each with the following conjugates: HIV-1; HIV-l+HIV-2; HIV-
l+HBc; HIV-l+HIV-2+ HBc. The results are presented in Table 1.
TABLE I
5 Sample -> HIV-l HIV-l HIV-l HIV-l
increasing >3 2.897 >3 >3
dilutions 2.015 1.905 2.011 1.847
1.282 1.148 1.214 1.129
0.694 0.663 0.695 0.672
0.41 0.378 0.412 0.378
0.232 0.230 0.282 0.235
negative 0.058 0.061 0.068 0.076
negative 0.060 0.064 0.075 0.075
Conjugate HIV-l HIV-l+ HIV-l+ HIV-1+
HIV-2 HIV-2 HIV-2+
HBc
(ii) HIV-2-positive samples at increasing dilutions were each
tested with the following conjugates: HIV-2; HIV-2+HIV-l; HIV-
15 2+HBc; HIV-l+HIV-2+HBc. The results are presented in Table 2.
TABLE 2
Sample HIV-2 HIV-2 HIV-2 HIV-2
increasing 0.099 1.045 1.009 1.002
dilutions 0.588 0.635 . 0.619 0.619
0.386 0.397 0.386 0.383
0.232 0.252 0.249 0.251
0.158 0.167 0.171 0.183
0.111 0.124 0.126 0.135
negative 0.053 0.060 0.061 0.072
negative 0.053 0.062 0.063 0.075
Conjugate HIV-2 HIV-2+ HIV-2+ HIV-2+
HIV-1 HIV-l HIV-l+
HBc
W094~A5~ PCTIGB94/00788
2 ~3~ 1 ~ G - 32 -
(iii) Six different HBc-positive samples were each tested with
the following conjugates: HBc; HBc+HIV-l; HBc+HIV-2;
HBc+HIV-l+HIV-2. The results are presented in Table 3.
TABLE 3
5 Sample HBc HBc HBc HBc
0.956 1.072 0.970 0.926
0.980 1.073 0.986 0.913
0.994 1.053 0.966 0.032
1.072 1.189 1.058 1.030
1.062 1.177 1.066 1.025
1.058 1.156 1.062 1.005
negative 0.056 0.070 0.061 0.074
negative 0.060 0.069 0.063 0.073
Conjugate HBc HBc+ HBc+ HBc+
HIV-l HIV-2 HIV-l+
HIV-2
Discus$ion
10 The results set out in Tables 1 to 3 above show clearly that the
various antigen conjugates can detect the respective antibodies
as effectively in combination as when used alone. There is
little interference when two or even three conjugates are used.
There is only a small increase in the background levels observed
15 for the negative samples when two or more conjugates are used.
EXAMPT.~ 2
A series of samples A to D containing HIV-l, HIV-2 and HBc
antibodies were prepared, with dilutions of the original single
samples as shown in Table 4.
TABLE 4
HIV-1 HIV-2 HBc
A 1/40 1/32 1/2
B 1/80 1/64 1/2
C 1/160 1/128 1/2
D 1/320 1/256 1/2
W094~4~ 21 3 7 7 8 6 PCT/GB94100788
- 33 -
Samples A to D were then each tested with the following antigen
conjugates: HIV-l; HIV-2: HBc; HIV-l+HIV-2; HIV-l+HBc; HIV-2+HBc;
HIV-l+HIV-2+HBc. The results are given in Table 5.
TABT-~ 5
5 A 1.314 0.961 0.361 1.602 1.415 1.185 1.689
B 0.840 0.619 0.386 1.232 1.075 0.848 1.482
C 0.519 0.368 0.401 0.804 0.803 0.650 1.102
D 0.298 0.216 0.402 0.465 0.614 0.526 0.828
- ve 0.062 0.049 0.052 0.059 0.059 0.054 0.068
- ve 0.055 0.049 0.050 0.054 0.057 0.052 0.064
- ve 0.054 0.047 0.049 0.054 0.057 0.051 0.062
- ve 0.053 0.046 0.050 0.052 0.057 0.052 0.063
conjg HIV-1 HIV-2 HBc HIV-1 HIV-1 HIV-2 HIV-l
HIV-2 +HBc +HBc HIV-2
HBc
Discussion
15 These results show clearly that each of the antibodies can be
detected selectiveally in the presence of the other antibodies,
that is to say, the presence of the non-relevant antibodies does
not affect the detection of the relevant antibodies. Further-
more, the results obtained at lower dilutions illustrate quali-
20 tatively the presence of the three specific antibodies, and atgreater dilutions the results obtained are substantially
additive.
The results obtained show that specificity is not compromised
when antibodies to two different pathogens are detected, and also
25 show that the sensitivity of the combination assay is remarkably
high.
