Note: Descriptions are shown in the official language in which they were submitted.
- W093/25905 213 7 2 3 ~ PCT/GB93/0120
Immunoassay Method
The present invention relates to an improvement in methods of
carrying out immunoassays, especially on samples of body fluids
5 and other samples obtained from humans and non-human animals.
There are essentially two types of immunoassay format, one in
which an antibody or antigen is captured selectively onto a
solid phase from a sample under investigation, and another, a
lO homogeneous phase assay format, in which the antibody or antigen
under investigation is detected in solution. (unless stated
otherwise, the term "antigen~' as used herein includes haptens.)
It is particularly convenient to carry out immunoassays in small
-15 tubes or, especially, in the wells of microtitre plates
(microwells) because of the automated systems available for
handling such vessels. An automated system may simply be a
spectrophotometric microplate reader, or the system may be more
advanced and capable of performing such steps as washing,
20 dispensing various reagents and incubating, and then reading the
results of an assay, especially spectrophotometrically.
In an immunoassay in which an antigen or antibody is captured
from the sample under investigation and immobilised within an
2s immunoassay vessel, the solid phase on which the capture occurs
is, for example, beads of a polymeric substance, especially a
synthetic plastics matPrial, or particles, for example, so-
; called l~latex~ particles, stabilised blood or red blood cells,
bacterial or fungal cells, spores, gold or other metallic sols,
30 and proteinaceous colloids. The size of particles is generallyfrom about O.l to 5 microns, and beads generally larger, for
example, from about 2 mm to lO mm. Alternatively, the solid
phase on which the antibody or antigen is captured may be the
coated wells of a microtitre plate, or a slide or ~tile~ having
35 coated hollows. Slides, cards and tiles may be used as
immunoassay vessels for agglutination assays.
A further type of immunoassay is a flocculation assay, which
involves an immunological interaction between two components
W093/25905 ~ ~ PCT/GB93/01205
resulting in flocculation that may be determined by eye and/or
may be determined photometrically. Microparticles, f3r example,
microparticulate carbon, may be used to enhance the visual
difference between a positive and negative reaction.
s
Microtitre plates and beads of polymeric material are
particularly convenient for large-scale use since, as described
above, automated systems are available for handling such test
formats. The sample and a diluent, generally required in
10 immunoassays, may be dispensed manually when using such a
system, or an automated sample and diluent dispenser may be
used.
-The term ~immunoassay vessel~ is used herein to denote a vessel
15 in which an immunoassay may be carried out, for example, a micro
well, a tube for use with beads, a slide, card or tile. Assays
for specific binding pairs other than antibodies and antigens,
for example! for receptors and their ligands, may be carried out
analogously to i~munoassays in immunoassay vessels, and the use
20 of the term is not limited to use in immunoassays only.
In general, in homogeneous assays the antibody and the antigen
are both labelled so that, when the antibody and antigen
interact in solution, the two labels also interact, for example,
25 to allow non-radiative transfer of energy captured by one label
to the other label, with appropriate detection of the excited
second label or quenched first label, for example, by
fluorimetry, magnetic resonance or enzyme measurement. Addition `
of antigen or antibody in a sample under investigation results
30 in modification of the interaction of the labelled pair and so
to a different level of signal. In many cases, the signal is
preferably a colour change.
. ~.
In a solid phase assay, the captured antigen or antibody is
3S generally detected by any means that will give a signal, for
example, by the use of a labelled molecule or particle that will
react with the captured antibody or antigen. In the case of a
captured antibody, the labelled component may be protein A,
~1372?~ `
W093/25905 PCT/GBg3/01205
protein G, an anti-species or anti-immunoglobulin-sub-type,
rheumatoid factor, antibody to an antigen used to capture the
antibody ~that antibody being used in a competitive or blocking
manner), or any molecule containing an epitope of an ant`igen ',
5 that will interact with the antibody, for example, the antigen
used for capture. In the case of a captured antigen, a labelled 'i
antibody that will interact with the antigen is generally used.
The detectable signal may be optical, radio-active or physico-
10 chemical, and may be provided directly by labelling a molecule
or particie as described above, for example, with a dye, radio-
label, electroactive species, magnetically resonant species,
chemiluminescent species or fluorophore; or indirectly by
labelling a molecule or particle as described above with an
15 enzyme itself capable of giving rise to a detectable change of
any sor~. Alternatively, the signal may result from
flocculation, agglutination, a diffraction effect or a
birefringement effect occurring if any of the solid phase
comprises appropriately sized particles, for example, as
20 described above.
Numerous books and review articles describe the theory and
practice of immunoàssays. Advice is ~iven on the design of
immunoassays, for example, on the choice of homogeneous or
25 capture format, on the characteristics and choice of solid
substrate in the case of capture assays, on the nature and
choice of label (signal generating system), and on various other
practical matters such as the composition of diluents and
washing solutions. An example of a standard textbook is "ELISA
30 and Other Solid Phase Immunoassays, Theoretical and Practical
Aspects n, Editors D.M. Kemeny & S.J. Challacombe, published by
John Wiley, 1988.
As indicated above, homogeneous assays are conveniently carried
3~ out in small tubes or microwells. Coated microwells`and coated
beads are widely used for large scale solid phase assays. The
automated systems currently available for bead and microwell
formats are generally designed for enzyme immunoassays using
W 0 93/25905 ~ ~ 4 PC~r/GB93/01205
chemiluminescent, fluorescent or colour labels for detection.
Agglutination assays are often carried out on slides, cards, or
tiles with hollows for small-scale investigations. Agglutination
assays, for example, latex and haemagglutination assays on a
5 lar~er scale are often carried out in microtitre plates, the
aggregation being measured spectrophotometrically. Flocculation
assays may be carried out in a microwell or a small cup.
In all assays carried out in immunoassay vessels, whatever the
10 format of the assay or the nature of the analyte, the sample
under investigation is added to the immunoassay vessel, for
example, a tube containing coated beads, a coated or uncoated
well of a microtitre plate, a hollow in a slide or tile or a
circle on a card or tile. one test unit may comprise a
15 plurality of immunoassay vessels, for example, a microtitre
plate test unit generally comprises 96 microwells, each being an
immunoassay vessel.
`
The present invention is based on our appreciation that it is
20 important that users should be able to ascertain readily whether
sample has, in fact, been added to each immunoassay vessel to
which it~should be added because the absence of sample is one
cause of false negative results. with increasing use of
automation, technical staff only rarely carry out immunoassays
25 manually, and so become less adept at procedures, for example,
~ ~ when carrying out immunoassays manually they require assistance
;~- in defining which immunoassay ~essels have received the sample.
Automated systems are not infallible, and sometimes fail to
dispense sample into an immunoassay vessel. We believe that it
30 is most desirable that all immunoassays, including automated
immunoassays, are monitored for the presence of sample.
It might be thought that it would be rèlatively easy to detect
the presence of sample in an immunoassay vessel, but in practice s
~- 35 it is surprisingly difricult. Examination by eye of the volume -
in a microwell or other vessel is not a practical way of
determining if the sample has been added, because in many
commercial assays the sample has a very small volume and is
~ .
.
W093/25905 5 PCT/GB93/01205
admixed with a very much larger volume of a diluent or other
reagent.
The present invention provides a method for determining the
5 presence of a sample of body fluid or of a sample derived from a
human or non-human animal body in an immunoassay vessel in which
an immunoassay or a specific binding pair assay for an analyte
in the sample is carried out, which method comprises
(i)(a) in the immunoassay vessel, admixing the sample and a
lO liquid that comprises a sample monitor reagent or reagent system
capable of producing a signal when sample is present, or
(i)(b) adding the sample to an immunoassay vessel that
comprises, immobilized on a solid support, a sample monitor
- reagent or reagent system capable of producing a signal when
l5 sample is present, and
(ii) detecting the signal,
the sample monitor reagent or reagent system capable of
producing a signal when sample is present being different from
the analyte-specific reagent or reagent system used for
20 detecting the analyte in the immunoassay itself.
The present invention also provides an immunoassay or a specific
binding pair assay for an analyte in a sample, the analyte being
an antibody or antigen or a member of a specific .binding pair, ;
25 the assay comprising a specific interaction between the analyte
and a corresponding antigen or antibody or between the two
members of the specific binding pair in an immunoassay vessel
and the detection of that interaction, characterized in that the
presence or absence of the sample in the immunoassay vessel is
30 detected by means of a sample monitor reagent or reagent system
that is capable of producing a signal in the presence of sample,
the sample monitor reagent or reagent system being different
from the analyte-specific reagent or reagent system used in the
detection of the analyte in the immunoassay or specific binding
35 pair assay itself.
In thé method of the present invention for determining the
presence of sample and in the immunoassay and specific binding
S~ ~3~ ~3~
W093/2590~ ~ PCT/GB93/01205j-
pair assay of the present invention, the sample monitor reagent
or reagent system is present in the reaction mixture, which
ought to comprise the sample under investigation. The signal
generated by the sample monitor reagent or reagent system in the
s presence of sample may be determined qualitatively or
quantitatively. A positive or negative signal denotes the
presence or absence of sample, respectively. Quantitative
determination of the signal enables the sample to be determined
quantitatively. The immunoassay or specific binding pair assay
lO itself is generally carried out after the sample detection step
in the case of a capture assay. In the case of a homogeneous
phase assay, the sample monitor step is generally carried out as
a preliminary step before the addition of all of the various
-reagents required for the assay itself is completed. If
15 desired, however, the order of sample monitor step and the assay
proper may be varied.
If the sample monitor system reveals that sample is missing from
an immunoassay vessel, sample may be added at that stage and the
20 assay proper carried out. Alternatively, the whole assay
process may be repeated. In the case of small-scale assays it
may be more convenient to add sample to any vessel from which
it is missing and to continue the assay procedure. In the case
of large-scale assays, especially when carrying out a plurality
25 of assays simultaneously, it may be more convenient to note the
vessel from which sample is missing, to continue with the
assays, and to repeat the assay for that sample. In a system
where the results of the sample detection step and of the assay
proper are recorded, especially in a partly or fully automated
30 process, the whole assay process may be carried out without
noting separately the result of sample detection step. At the
end of the assay, any false negative result will be apparent
from the negative result of the sample detection step. The
assay for that sample can then be repeated.
Accordingly, the present invention provides a method for
determining a false negative result in an immunoassay, which
comprises determining the presence of a sample of body fluid or
- W093~259~5 21~ 7 2 3 ~ PCT/GB93/0120S
of a sample derived from a numan or non-human animal body in an
immunoassay vessel in which an immunoassay or a specific binding
pair assay for an analyte in the sample is carried out, which
method comprises
5 (i)(a) in the immunoassay vessel, admixing the sample and a
liquid that comprises a sample monitor reagent or reagent system
capable of producing a signal when sample is present, or
(i)(b) adding the sample to an immunoassay vessel that
comprises, immobilized on a solid support, a sample monitor
10 reagent or reagent system capable of producing a signal when
sampie is present,
(ii) detecting the signal generated by the sample monitor
reagent or reagent system,
-the sampie monitor reagent or reagent system capable of
15 producing a signal when sample is present being different from
the analyte-specific reagent or reagent system u~ed for
detecting the analyte in the immunoassay or specific binding
pair assay itself, and
(iii) carrying out the immunoassay or specific binding assay for
20 the analyte of interest,
~ a negative signal generated by the sample monitor reagent or
; reagent system denoting a false negative result.
The term "analyte~ is used herein to denote the antibody or ~;
25 antigen of interest or, in the case of a specific binding pair
other than an antigen and an antibody, the member of that pair
to be determined, in a sample under investigation. AS indicated
above, the sample monitor reagent or reagent system used for the
detection of the sample itself is dlfferent from the reagent or
30 reagent system used in the assay proper for the detection of the
analyte of interest. The terms "sample monitor reagent or
reagent system" and "non-analyte-speclfic reagent or reagent
system" are both used herein to denote the reagent or reagent
system used to detect the sample under investigation, that
3~ reagent or reagent system being different from the reagent or
reagent system used in the detection of the analyte of interest
(the ~'analyte-specificll reagent or reagent system)~ that is to
say, the respective signals generated are different.
W093/25905 2~3~ ~ V 8 PCT/GB93/0120
The present invention further provides the use of a sample
monitor reagent or reagent system that is capable of producing a
signal in the presence of a sample of interest to detect the
presence or absence in an immunoassay vessel of the sample when
S under investigation in an immunoassay or a specific binding pair
assay and/or to detect a false negative result.
The present invention also provides a liquid reagent for use in
an immunoassay or specific binding pair assay for an analyte,
10 which liquid reagent comprises a sample monitor (non-analyte-
specific) reagent or reage~t system capable of producing a
signal in the presence of a sample under investigation. The
liquid reagent is generally a diluent, especially a sample
-diluent.
The present invention further provides a bead or an immunoassay
vessel for use in an immunoassay or a specific binding pair
assay, to which bead or to-the interior surface of which
immunoassay vessel is immobilized a sample monitor (non-analyte-
20 specific) reagent or reagent system capable of producing asignal in the presence of a sample under investigation.
Alternatively or in addition, the bead or well may be coated
with a capture agent for the analyte. A further possibility is
that one of an immunoassay vessel and a bead is coated with a ;
25 sample monitor reagent or reagent system and the other is
coated with capture agent for the analyte of interest. Such a
two-component system is part of the present invention.
The invention also provides a plurality of such .immunoassay
30 vessels, especially in the form of an assembly of microwells, in
particular, a microtitre plate.
The present invention also provides a kit for carrying out an ¦~
immunoassay or a specific binding pair assay on a sample under
35 investigation, which kit comprises
(a) a liquid comprising a sample monitor reagent or reagent
system capable of producing a signal in the presence of the
sample and/or
W O 93/25905 ~ 13 7 2 ~ ~ PC~r/GB93/01205
(b) a plurality of immunoassay vessels or of beads for use in an
immunoassay vessel, each vessel or bead comprising an
immobilized sample monitor reagent or reagent system capable of
producing a signal in the presence of the sample.
S
A capture agent for the analyte may also be immobilized on the
beads or wells on which a sample monitor reagent or reagent
system is immobilized. If the kit does not comprise beads or
wells on which a sample monitor reagent or reagent system is
10 immobilized, it may comprise beads or wells coated with a
capture agent for the analyte under investigation or it may
comprise uncoated wells. The two-component system described
above where one of an immunoassay vessel and a bead is coated
~ with a sample monitor reagent or reagent system and the other is
15 coated with a capture agent for the analyte of interest may be
provided in a kit of the present invention. Slides or tiles for
agglutination or flocculation assays may be provided as an
alternative to wells and beads.
20 If desired, an immunoassay vessel used in the present invention
may comprise one component of the sample monitor reagent system
in immobilized form and a liquid reagent, generally a diluent,
especially a sample diluent for an immunoassay or specific
binding pair assay may comprise another component of the sample
25 monitor reagent system. Such a reagent system may be provided
in a kit of the present invention.
~,
The sample monitor system of the present invention enables the
elimination one source of false negative results, that is to -;
30 say, the absence of sample in the immunoassay vessel. For
example, in immunoassays or specific binding pair assays where
the level of an analyte is determined, the use of a sample
monitor reagent or reagent system provides confirmation that
sample was present in the case of results that are close to the
35 lower limit of detection. In immunoassays or specific binding
pair assays where the presence or absence of an analyte is to be
determined, the use of a sample monitor reagent or reagent
system provides clear and immediate proof of whether a negative
W093/2590~ ~3~ 2~ 3 1 o PCT/GBg3/01205 i
result is a true negative or is a false negative due to missing
sample.
The sample monitor system of the present invention is therefore
5 of value in all immunoassays and specific binding pair assays in
enabling detection of and hence elimination of false negative
results. It is especially valuable in immunoassays and specific
binding pair assays the results of which are used in clinical
diagnosis and prognosis. It is also of especial value in
10 immunoassays used in the screening of donated blood for
pathogenic organisms, for example, for HIV, hepatitis viruses
and syphilis. A false negative result in a blood screening
assay will enable contaminated blood to enter the blood supply.
-
15 The sample under investigation may be a sample of a human ornon-human animal body fluid, for example, blood (generally in
the form of serum or plasma)~ urine, saliva or cerebrospinal
~ fluid. The sample may be a sample derived from a human or non-
--~ human body, for example, it may be derived from tissue, for
20 example, from a biopsy specimen or may be derived from a body
product, for example, faeces, sputum, pus, abscess fluid or
other exudations.
Examples of analytes to be investigated by immunoassays or
25 specific binding pair assays are given below. One important
aspect of the present invention is the application of a sample
monitor reagent or reagent system of the present invention to
the ~detection of sample in immunoassays or specific binding pair
assays for the detection of a disease condition, for example, as
30 caused by a pathogenic organism or by a physiological or
biochemical change or malfunction within the body, for example,
cancer or an autoimmune disease. A further important aspect of
the present invention is the application of a sample monitor `
reagent or reagent system of the present in~ention to
35 immunoassays for screening donated blood.
The signal that is generated by the sample monitor reagent or
reagent system is, for example, a colour change or a change in a
; W093/25905 2 13 72 ~ ~ PCT/GB93/0120a
chemiluminescent or fluorescent signal. The change may be such
that it can be observed directly by eye, for example, the colour
of the diluent may change when sample is added, and/or the
colour or other change may be such that it can be measured, for
5 example, using a photometer or fluorimeter. If an automated or
semi-automated immunoassay system is used, an extra measuring
step after the sample addition step can be incorporated easily
in the assay protocol.
lO The method of the pres~nt invention may be used to detect if
sample is present or absent in an immunoassay vessel and/or it
may be used to detect how much sample has been added,
particularly if the colour or other change is measured using the
~appropriate equipment, for example, photometer or fluorimeter.
For those immunoassays and specific binding pair assays in which
the sample is serum or plasma and that require the use of a
diluent, which diluent does not comprise serum or plasma, it is
possible to detect the presence of sample by use of a sample
20 monitor reagent or reagent system that is capable of producing a
signal in the presence of any component that is present in serum
or plasma but not in the diluent.
It is particularly advantageous to detect albumin but any other
25 serum- or plasma-specific protein or other high-molecular weight
component, for example, icaerulopasmin, transferrin or any serum
or plasma enzyme, may be detected. As a further alternative, a
low molecular component of serum or plasma, for example, a metal
ion, amino acid or sugar, for example, glucose, calcium,
30 magnesium, iron, triglycerides, tyrosine or urea may be
detected, for example, as described below.
.
Whether it is a serum- or plasma-specific protein or a high or ~;
low molecular weight component of serum or plasma that is to be
35 detected, the sample monitor reagent or reagent system present
in the diluent should be such that, when the diluent and the
sample are admixed there is a detectable signal, for example, a
colour change.
W093t2~905 3~ ~ a 12 PCT/GB93/01205~
If the presence of a serum or plasma sample is to be detected by
the presence of albumin in the resulting mixture, a variety of
suitable indicator reagents are known, for example, bromocresol
purple, bromocresol green, bromocresol blue, and cyano-di(4-
s nitrophenyl)-methane, each of which exhibits a characteristic
colour change in the presence of albumin. Bromocresol purple,
for example, changes from yellow to purple in the presence of
albumin. Coomassie Brilliant Blue is a further suitable
indicator reagent. It changes from yellow to blue on binding to
10 albumin. In each case, the colour change may be determined by
eye andJor using a spectrophotometer.
Each of the above reagents has a particular pH range over which
it gives optimum performance, and the choice of reagent should
lS preferably take into account the pH of the particular assay
system in which it will be used. Bromocresol purple, for
example, functions best at pH values just below neutrality,
- cyano-di(4-nitrophenyl)-methane functions best at pH values
above neutrality, bromocresol green functions best near pH 6.
U.S. Patent Specification No. 5,182,214 describes anionic
cyanine dyes that are said to be specific for human serum
- albumin and to be particularly sensitive. They are fluorescent.
25 Although the trend in immunoassay design is away from the use of
serum or plasma in diluents, many immunoassays still require the
use of diluents that comprise serum or plasma. It is good
manufacturing practice and it is also preferable for the purpose
of the present invention to dialyse or gel-filter serum or
30 plasma before use in an immunoassay diluent in order to remove,
for example, relatively low molecular weight components, for
example, metal ions, sugars and amino acids.
~-;
Provided that either the diluent used does not contain any serum
35 or plasma, or any serum or plasma used in the diluent has been
treated as described above to remove low molecular weight
components, the prese~ce of sample may be detected by the
presence in the immunoassay vessel of a low molecular weight
! - WO 93/2sgoS ~313 7~3 8 Pcr/Gng3/ot2os
component of serum or plasma. Examples of detectable low
molecular components are, for example, glucose, calcium,
magnesium, iron, triglycerides, tyrosine and urea.
5 Glucose may be detected in a serum or plasma sample using a
colorimetric enzymic detection system, for example, glucose
oxidase~peroxidase. Such a system is well known; a colour
change at 505nm will occur in the presence of glucose. Reagents
and reagent systems suitable for the detection of other low
10 molecular weight components are also well known, for example,
calcium may be detected by o-cresolphthalein, magnesium by
eriochrome black, iron by ferrozine, and urea using a urease/-
glutamate dehydrogenase system and measuring the absorbance
-change at 340nm.
If, however, the diluent to be used does contain serum or
plasma, it is still possible under certain circumstances to
detect sample by the presence of albumin. The colour change
observed when an indicator reagent for albumin is used results
20 from binding of the indicator reagent to part~cular sites on the
albumin molecule~. Other compounds, for example, salicylates,
a~ino-naphthalene sulphonic acid, valproic acid and bile
pigments, can bind to those sites, generally more strongly than
indicator reagents bind. It is possible to block binding sites `
2s on albumin using such stronger binding compounds as blocking
compounds. By titration of a blocking compound into a diluent
containing albumin, it is possible to find a concentration of
the blocking compound such that a minimum amount of indicator
reagent is bound to the albumin present in the diluent, but that
30 when sample is admixed with the sample, the albumin in the
sample binds a maximum amount of indicator reagent and hence a
signal, preferably a colour change, results. For example, in a
diluent comprising 10% serum, and using equal volumes of sample ~,
and diluent in the final immunoassay mixture, the optimum
35 concentration of valproic acid used as bloc~ing agent is about
0.5 mM in the diluent.
If the sample to be detected is other than serum or plasma, the
~ .
W093/2s905 '~ ~31 ~ V PCT~GB93/0120~;
14
diluent for the assay should comprise a sample monitor reagent
or reagent system that will cause a detectable signal, for
example, a colour change, in the presence of a substance that is
a component of the sample but that is not present in the
5 diluent.
. i
If the sample is, for example, urine, the diluent may contain a
reagent or reagent system that will detect the presence of, for
example, urea, for example, using the urease/glutamate
lO dehydrogenase system described above, or uric acid, for ~xample,
using a uricase~peroxidase system and measuring the colour
change at 520nm.
If the sample is, for example, cerebrospinal fluid, the presence
15 of sample may be determined by the presence of glucose or
albumin. Similarly, if the sample is saliva, the presence of
glucose will indicate the presence of sample.
A further approach to the detection of a sample is to utilise
20 the pH effect that occurs when the sample and a diluent are
~; admixed. It is customary to buffer reagents used in
immunoassays ànd also in specific binding pair assays to ensure
that the pH is maintained at optimum values at various stages of
the assay. If, however, a reagent, for example, a sample `
25 diluent is buffered only weakly or even not at all, then when
sample is added the pH will change. For example, if a sample
diluent is initially slightly acid or slightly alkaline and has
weak or no buffering capacity, then when it is admixed with
serum or plasma the pH will move towards neutrality because of
30 the buffering effect of the proteins and inorganic constituents
of the serum or plasma. Such a change in pH can be exploited
for use in monitoring sample addition by the incorporation of a 7
pH indicator reagent (dye) in a reagent, for example, a sample ~`
diluent. The indicator may give a colour change that is visible t
3s by eye and/or that may be detected by spectrophotometric
measurement.
The pH for immunoassays is generally maintained within the range
2 1 3 7 2 ? ~
W093/25905 PCT/GB93/01205
of from pH5 to pHs~ Accordingly, the initial pH of the sample
monitor reagent should be such that, after addition of the
sample, the pH of the resulting mixture is within the range of
from 5 to 9, for example, from 6.S to 7.5. The chosen indicator
5 should change colour as the pH moves towards neutrality. For
example, whether the initial pH is acid or alkaline, the
indicator should change colour as the sample of plasma or serum
drives the pH towards neutrality, for example, it should change
colour at a pH around 5, preferably about 6.5 when moving from
10 acid) or at a pH of about 9, preferably about 7.5 when moving
from an alkaline pH to neutrality.
Examples of suitable indicators for pH changes from acid to
-neutral are ethyl red (pink to yellow), methyl red (red to
15 yellow), neutral red (plum red to orange), chlorophenol red
(yellow to purple) and phenol red (orange to red). Examples of
suitable indicators for pH changes from alkaline to neutral are
phthalein purple (purple to yellow)~ thymol blue (blue to
green), and thymolphthalein (blue to colourless).
The concentration of indicator may be adjusted according to
need. For example, if sample monitoring is to be carried out by
visual inspection it may be desirable to use a higher
concentration of indicator than if spectrophotometric methods '~
25 are to be used. Some indicators, for example, thymolphthalein,
chorophenol red and phenol red are more soluble in aqueous
solution than others, so may be more suitable for use in visual
assessment of sample addition than other indicators because a
higher concentration of indicator can be used.
The colour change of some indicators is more easy to detect by
eye than that of other indicators. We have found chlorophenol
red to be particularly useful for visual assessment as the
colour change of yellow to purple is very clear and dramatic. `!
35 Colour changes can be read by spectrophotometer as an
alternative to or in addition to visual assessment. Quantitative '~
determination of sample may be carried out using spectro-
photometric methods.
ç: ` '
w093/2590~ 3~ ~P3 l6 PCT/GB93/01205
As indicated above, the sample monitor reagent or reagent system
that is capable of producing a signal in the presence of sample
is generally incorporated in a diluent. The diluent ls
generally a sample diluent, but may alternatively be a diluent
5 used at a different stage of an immunoassay procedure. In the
case of an agglutination a~say, for example, a latex
agglutination assay or a haemagglutination assay, or a
flocculation assay, the diluent may be that in which the
particles are suspended. The reagent or reagent system that
lO causes a signal in the presence of sample may be incorporated in
another reagent to be used in an t~munoassay or specific binding
pair assay, particularly in the case of a homogeneous phase
assay. (It is to be understood that any reference in the
~pPcification to a diluent also applies to any other liquid
15 reagent used in the assay in question.)
As an alternative to providing the sample monitor reagent or
reagent system in liquid form, all or part of the sample monitor
reagent or reagent system may be immobilized on a solid support.
20 The support may be the walls of the immunoassay vessel itself,
particularly in the case of microwells, or the solid support may
be beads or smaller particles, generally the beads used in
commercially available systems. When immobilized on a solid
support, the sample monitor reagent or reagent system may be ~'"
- 25 coated on all or part of the walls of the vessel or on beads,
either alone or as one component of a multi-purpose coating, for
example, a coating that also comprises a capture agent for the
analyte of the immunoassay itself. An immunoassay vessel coated ~,
thus may be present in conjunction with a plurality of other
30 such vessels, for example, as coated microwells in the form of a `.
microtitre plate. IQ the case of the bead system, one of the
bead and the vessel may be coated with the sample monitor
reagent or reagent system and the other with an analyte capture ~:
agent. 1
If the sample monitor reagent is immobilized, it is generally
necessary for it to be a more stable reagent than is required
for use in a liquid, for example, for immobilization the reagent
W093/2590~ 2 1 3 7 2 3 8 PCT/GB93/01205
is preferably a dye or indicator reagent rather than an enzyme-
based system. Examples of dyes and indicator reagents that
interact with a component of the sample and examples of pH
indicator reagents are given above. The system may be used for
5 qualitative or quantitative detection of sample.
In many cases it is advantageous to use a sample monitor reagent
or reagent system at such a concentration that a technician
carrying out a number of immunoassays or specific binding pair
l0 assays can easily check for the presence of sample by a distinct
colour change that can easily be seen. Alternatively, or in
addition, th~ system may be such that the detectable signal can
be determined using appropriate e,quipment, for example, a colour
~ change that can be determined using a standard microplate
lS spectrophotometric reader with an appropriate choice of
wavelength. Such an additional step can be easily incorporated
in an automated system. The detection may be qualitative or, if
desired, may be quantitative, for example, if it is desirable to
know the volume of sample present.
The sample monitor reagent or reagent system chosen should be
such that the signal produced in the presence of sample does not
interfere adversely with the subsequent immunoassay or specific
binding pair assay. One way is to use a sample monitor (non-
25 analyte-specific) reagent or reagent system that is capable of
producing a different type of signal from that produced by the
analyte-specific reagent system in the assay proper. For
example, if the signal in the assay proper is a colour change,
the signal for sample detection may be a chemiluminescent or
30 fluorescent signal. Conversely, if the analyte-specific reagent
system utilises a`chemiluminescent or fluorescent signal, the
sample monitor reagent or reagent system may provide a colour
change.
- 35 The use of different types of signal for sample detection and
the assay proper may be preferred for homogeneous phase im-
munoassays and specific binding pair assays, and also for
immunoassays and specific binding pair assays that use
~ ~3~ 18 PCT/GB93tOl205i
chemiluminescent or fluorescent signals for the analyte detec-
tion. A particularly simple but effective sample detection
system is the use of a sample monitor reagent or reagent system
that gives a colour change detectable by eye when sample is
5 present. The presence of sample can be determined simply by
visual inspection, without the need for further monitoring
equipment.
Another way of avoiding interference is to use the same type of
10 signal for both the sample detection step and the assay proper
but to choose reagents or reagent systems for the two steps such
that the resulting changes in signal do not interfere with each
other, for example, the reagents should not absorb or transmit
~ight in the same spectral region.
15 ,
In the case of colour signals, for example, the reagents should
be chosen such that the wavelengths at which the two reagents
absorb light do not overlap to any significant extent. For
example, peroxidase is very commonly used in enzyme
20 immunoassays, with hydrogen peroxide and a chromogen, often
3,3',5,S'-tetramethylbenzidine. That system gives a signal that
may be measured at 450nm. Numerous sample monitor reagents and
reagent systems g ve characteristic colour changes at waveleng-
ths distinct from the wavelength of the peroxidase system. For
~; ~ 25 example, bromocresol purple gives a characteristic colour change
in the presence of albumin, and has peak absorbance at about
620nm and urease~glutamate dehydrogenase, which may be used to
detect urea gives a colour change at about 340nm. Similarly,
the pH indicators whose use as sample monitor reagents is
30 described above also have colour changes at characteristic
wavelengths, for example, the colour change of chorophenol red
from yellow (acid) to purple (neutral) may be read at 570nm. ~
- Appropriate sample monitor reagents or reagent systems may be ¦`
selected analogously for use with other colour analyte reagent
3S systems. The same considerations apply to systems using
chemiluminescent or fluorescent systems.
In the case of a capture immunoassay or specific binding pair
W093/25905 213 7 2 3 8 PCT/GB93/0120~
assay, sample is generally incubated with the immobilized
component that is to capture the analyte, and then there is a
washing step before detection of bound analyte. Accordingly~ ¦
the sample detection step will generally take place before the
5 analyte detection step. The choice of signal detection systems
may therefore be broader than 's the case with homogeneous phase
assays. Detection systems involving a colour change may be used
for both sample detection and for analyte detection. If the
signal for both sample and analyte detection is a colour change,
lO the colour change for sample detection is generally at a
wavelength different from that for the detection of the analyte.
In the case of an agglutination or flocculation assay, for
~ example, a latex agglutination assay, a haemagglutination assay
l5 or a flocculation assay, for example, using microparticles of
carbon to enhance the flocculation effect, the sample detection
signal may be a colour change or another signal, for example, a
fluorescent signal.
20 As indicated above, the present invention may be used to detect
the presence of sample in a homogeneous phase immunoassay or
specific binding pair assay, or in a solid phase capture
immunoassay or specific binding pair assay carried out in an
immunoassay vessel. It is particularly useful when a plurality
25 of assays are carried out simultaneously, with either manual or
automated sample dispensing. The signal may be a colour or
other change determined by eye and/or the change may be measured
- in a spectrophotometer or other measuring device. It is
particularly use~ul when the assays are carried out in fully or
30 partly automated systems, as are available for handling micro-
titre plates and also for carrying out assays using the bead
format. Sample detection in a homogeneous assay is generally
carried out when sample has been added to the immunoassay vessel
and before all the various reagents required for the immunoassay
35 or specific binding pair assay have been added.
In the case of capture assays, the sample detection step may be
carried out at any point after the sample addition step, for
~ i
W093~2sgos ~3~3~ 20 PCT/GB93/01205!-
example, at the beginning, during or at the end of theincubation step. An additional detection step, especially to
measure a colour change, can easily be incorporated in an assay
protocol, particularly in the case of automated systems.
The present invention is also particularly useful for larger
scale agglutination or flocculation assays carried out in
immunoassay vessels when a large number of samples are to be
dispensed manually or automatically, for example, when a
lO plurality of latex agglutination assays, haemagglutination
assays or flocculation assays are to be carried out.
In any of the types of assay, if the technician is able to
inspect the immunoassay vessels after the sample addition step
15 and before the assay proper, it is advantageous to use a sample
monitor reagent or reagent system that gives a colour change
detectable by eye. Such a sample monitor system is simple,
cheap and effective.
.:
20 As an alternative or in addition, an extra spectrophotometric or
other measuring step may be incorporated in an immunoassay or
specific binding pair assay protocol. To do so is easy, whether
a manual or partly or fully automated system is used. It is
also cheap, as no extra equipment is required. An advantage in
25 using an extra measuring and recording step in an assay protocol
is that the assay can be carried out from start to finish
without stopping to inspect at the sample detection stage. The
results of the sample detection step and the results of the
assay proper may be compared at the end of the assay. A
30 negative result at the sample detection stage indicates a false
negative result due to missing sample. The assay can then be
repeated on that sample.
. ~
A further option in the case of an assay using a chemi-
35 luminescent or fluorimetric method to determine the analyte is
to use a sample monitor reagent or reagent system that gives a
colour change, and to incorporate in the assay protocol a
spectrophotometric measuring step after the sample addition.
- W093/2~905 PCT/GB93/01205
21
Although extra eouipment is required~ spectrophotometers, for
example, microtitre plate readers, are standard equipment in
laboratories carrying out immunoassays and specific binding pair
assays.
Several different sample monitor reagents and reagent systems
have been described above. In some cases the choice of reagent
or reagent system may be constrained, for example, by the
equipment available, by the assay format, by the nature of the
lO analyte detection system, or by the nature of the sample under
investigation. It should be noted, however, that the sample
monitor reagents and reagent systems that use reagents or dyes
that give a colour change on interaction with a component of the
-sample, and the pH indicator reagents that give a colour change
lS on addition of sample, for example, as described above, are
generally cheaper than those using chemiluminescent, fluorescent
or enzyme systems. They are also generally more robust. They
are also quic~ and easy to use, particularly when visual
inspection is used to detect the presence of sample. There are
20 therefore practical advantages in choosins such a sample monitor
reagent or reagent system.
Immunoassays are well established and widely used in both
clinical diagnosis and prognosis and in screening of donated
25 blood. The analyte to be determined in an immunoassay may be an
antigen, an antibody or a hapten.
An antigen or hapten may be associated with disease, for
example, with infection, for example, by viruses, bacteria,
30 protozoa, fungi and other parasitic organisms. The antigen or
hapten to be detected may be associated with or derived from the
pathogenic organism itself, For example, one test for hepatitis
B virus involves the detection of hepatitis B surface antigen.
Many other assays for pathogenic antigens are known and are
3s available commercially. Also of interest are products of
infective organismst for example, toxins and toxoids. Antigens
produced by the body in response to disease or produced in
abnormal amounts as a result of disease and which are sometimes
W0~3/2s905 2~ C~a 22 PCT/GB93/01205
known as ~surrogate markers" may be determined in immunoassays.
An example is the enzyme alanine transaminase tALT), which is a
surrogate marker for liver disease.
,
5 Any hormone to which an antibody can be raised may be determined
in an immunoassay, for example, insulin, oestrogens, progestero-
nes and peptide hormones. Indeed, any substance to which an
antibody can be raised can be determined by an immunoassay.
Examples of haptens that can be determined by immunoassays are
lO therapeutic drugs and drugs of abuse, for example, digoxin,
thyroxine, anti-epileptic drugs, morphine and other opiates,
amphetamines, barbiturates, and cocaine metabolites.
I~munoassays may also be used to determine antibodies. An
15 antlbody may be of direct diagnostic or prognostic interest in
itself as, for example, in the case of autoimmune antibodies,
myeloma antibodies and antibodies to various allergens. In some
cases, it may be necessary as a practical matter to determine
antibodies to an antigen of interest because the amount of
20 antigen itself in a sample is too small to be determined by
immunoassays currently available. That is the case at present
with certain diseases caused by pathogenic organisms, for
example, certain blood-borne viruses, for example, HIV and ~
hepatitis C viruses. Antibody assays are therefore useful in ~``
25 the indirect detection of infection by pathogenic organisms of
many types, for example, viruses, bacteria, fungi, protozoa and
other parasitic organisms.
~,
The immunoassay techniques described herein in connection with
30 antibodies and antigens can be applied equally to the detection
and/or determination of members of specific binding pairs other
than antigens and antibodies, especially of receptors and their
ligands, and references in the herein to immunoassays also ~;~
relate to the corresponding specific binding pair assays.
35 Examples of ligands are hormones, for example, insulin and
glucagon, amino acid transport proteins, viral coat proteins,
molecules involved in neurotransmission, for example, adrenergic
and cholinergic transmitters, serotonin and dopamine, adrenalin,
- W093/25905 23 2~7~ PCT/GB93/01205
histamine and cytokines. In some cases, where an antibody to a
ligand or a receptor can be produced, it may it may preferable
- to determine a ligand or a receptor by a conventional
immunoassay. If it is difficult or impossible to produce an
5 antibody, or i~ it is desirable for any other reason, a ligand
or receptor may be determined in an assay analogous to an
immunoassay, for example, in a homogeneous phase or solid phase
assay. In a solid phase assay, either the ligand or the
receptor may be immobilised on the solid phase. Agonists and
l0 anta~onists of ligands may be determined in such a system, and
the term ~lanalyte'l includes such agonists and antagonists.
As pointed out above, the sample monitor system of the present
~invention is particularly useful in that it enables elimination
15 of one source of false negative results, that is to say, the
absence of sample in the assay vessel. For example, in assays
where the level of an analyte is determined, the use of a sample
monitor reagent or reagent system provides confirmation that
sample was present in the case of results that are close to the
20 lower limit of detection. In assays where the presence or
absence of an analyte is to be determined, the use of a sample
monitor reagent or reagent system provides clear and immediate
proof of whether a negative result is a true negative or a false
negative due to missing sample.
One example of the usefulness of the sample monitor reagent or
reagent system of the present invention is in the screening of
donated blood in order to maintain a supply of blood and blood
products free from pathogenic contamination. Blood screening is
30 often carried out on a large scale, and automated equipment has
been developed to deal with the large numbers of tests that must ~.
be carried out within a short time, as the shelf-life of blood
is short. The regulatory authorities in each country specify the
pathogens for which testing is to be carried out. In most
35 countries, screening is mandatory for HIV, hepatitis C viruses
(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 mandatory to test for both HIV-l and HIV-2.
: ;
W093~25905 ~ 24 PCT/GB93/0120
Blood to be used for transfusion to patients who are to or who
have received a transplant or are otherwise immuno-compromised
is generally tested for the presence of CMV (cytomegalovirus).
5 The most widely used tests for blood screening are immunoassays.
For most of the pathogenic oryanisms listed above it is more It`
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
lO and CMv are antibody tests. There are two tests for hepatitis
B: ar antigen assay for 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 antibodies is mandatory in some countries
l5 and may be introduced in more. There are both antibody and
antigen tests for syphilis. The syphilis tests currently in
use are haemagglutination tests, and also flocculation assays
using microparticulate carbon to enhance the flocculation effect
and hence the determination of a positive or negative result.
20 The other pathogens are generally tested using enzyme immunoas-
says.
The results obtained in blood screening assays are classified as
positive, negative or indeterminate. Positive and indeterminate ``
2S samples are investigated further using different assays.
It will be appreciated that a false negative result in any of
the screening assays will result in contamination of the blood !
supply and in the infection of the recipient of the blood. The
30 importance of the use of a sample monitor reagent or reagent
system of the present invention in eliminating ralse negative
results in the screening of donated blood should not be
underestimated.
35 Any of the sample monitor reagents and reagent systems described
above for the detection of samples of serum or plasma may be
used for blood screening. Provided the appropriate reagents,
especially the sample diluent, contain little or no plasma or
W093/25905 25 213 7 2 ? ~ PCT/GB93/01205
serum, it may be advantageous to detect the presence of sample
by detecting the presence of albumin, for example, using a
colour indicator reagent as described above. A further ad-
vantageous sample monitor reagent comprises a colour indicator
5 that is responsive to the pH change when the sample is added to
an unbuffered or slightly buffered diluent.
It may be advantageous to use a colour indicator that can be
detected by eye. Spectrophotometric measurement may be used in
lO addition to or in place or a visual inspection. The use of
bromocresol purple as sample monitor reagent in the sample
diluent for blood screening assays using microtitre plates, with
visual inspection of the plates after the sample addition step
_has proved very effective in use in blood screening
15 laboratories.
W093/25905 ~3~ ~ PCT/GB93/01205;
26
The following Examples illustraté the invention.
EXAMPLE l
Detection of a serum samPle by detection of albumin
5 The assay used was a commercial enzyme immunoassay tEIA) f~r
antibodies to HIv, Wellcozyme VK54 ("Wellcozyme" is a Trade
Mark). The reagents provided in the immunoassay kit were as
follows:
lO A 96-well microtitre plate coated with HIV-l and HIv-2 related
peptide epitopes. A sample diluent, which is a detergent and pH
buffering solution of pH8.0 and which does not contain serum. A
conjugate solution, which is a pH controlled solution of
~ntibody conjugates of HIv-l and HIV-2 peptide epitopes. A
15 substrate solution, comprising components of an amplification
system for the coniugate, and a stop solutlon, consisting of 2M
sulphuric acid.
~; The sample diluent was supplemented by the addition of bromo-
20 Gresol purple to a concentration of 0.125 mM.
.' - '.
The assay was carried out according to the directions provided
with the immunoassay kit, except that the sample dlluent was
added to all wells prior to the addition of the samples to the
25 wells. In outline, 25~1 of sample diluent was added to each
well in the microtitre plate, giving a yellow colour in each
well. 25~1 of individual serum or plasma samples under
investigation were then added to wells in the plate. Samples
were deliberately omitted from wells Fl2, Gl2 and Hl2. The
30 colour in the wells changed from yellow to purple immediately on
adding`the sample, and those wells from which sample had been
omitted could be distinguished by eye. In addition to a visual
inspection, the absorbance of each well was measured at 620nm ` ~^
using a Multiskan MCC/340P Version 2.20 plate reading spectro- ~,
35 photometer. The results of wells Al to H6 are show~ in Table lA ' -
~and of wells A7 to Hl2 are shown in Table lB.
W093/2~905 2~ ~ ~ 2 3 8 PCT/GB9~/0l20
TABLE lA
1 2 3 4 5 6
A 0.506 0.504 0.492 0.525 0.527 0.575
B 0.490 0.508 0.49S 0.512 0.530 0.493
5 C 0.483 0.511 0.471 0.485 0.479 0.536
D 0.465 0.497 0.445 0.463 0.504 0.527
E 0.468 0.494 0.486 0.526 0.521 0.441
F 0.503 0.507 0.451 0.510 0.5~3 0.519
G 0.506 0.490 0.454 0.492 0.489 0.516
10 H 0.490 0.514 0.489 0.472 0,460 0.476
TABLE lB
7 8 9 10 11 12
A 0.557 0.573 0.507 0.569 0.558 0.582
B 0.460 0.515 0.507~0.518 0.547 0.546
C 0.468 0.480 0.548 0.521 ~.477 0.499
D 0.515 0.509 0.593 0.477 0.536 0.509
20 E 0.534 0.491 0.499 0.502 0.477 0.515
F 0.524 0.500 0.499 0.563 0.472 0.056
G 0.508 0.613 0.526 0.517 0.505 0.058
H 0.515 0.523 0.585 0.548 0.477 0.058
'i `
25 (x = 0.508+ 0.03)
The results given in Table lA and Table lB show clearly that
wells F12, G12 and H12, from which sample had been omitted, have
an optical density that is conspicuously different from that of
30 the wells containing sample.
The overall results of the HIV assay were the same whether or
not the diluent had been supplemented with bromocresol blue: i?~
35 A second microwell plate was prepared as described above, except
that some of the samples added were known to be HIV-l antibody
positive. The HIV positive samples were added to duplicate
wells, one of each pair of wells containing diluent supplemented
:
W093/25905 ~ ~3~ ~ PCT/GB93/01205 ? ` '`
28
with bromocresol purple and the other containing the same
standard diluent without any additional agent. The plate was
examined as described above after sample had been added, and
then the HIV assay was completed by incubating the plate and its
5 contents for 30 minutes at 37C, washing adding 50~1 of
conjugate solution to each well and re-incubating at 37C for 30
minutes. After washing, the amplifying substrate system was
added to all the wells and finally the optical densities were
finaliy measured at 492nm in the Multiskan plate reader.
In wells Al to H6 the diluent used was that supplemented with
bromocresol purple, in wells A7 to H12 unsupplemented diluent
was used. Several weakly positive HIV samples were assayed.
-(Weakly positive samples were chosen because they are more
15 difficult to detect in the assay system than are strongly
positive samples.) Sample 843 was used in wells A5 and B5, All
and B11; sample 845 in wells C5 and D5, Cll and Dll; sample 951
in wells E5 and F5, Ell and F~l; sample 834 in wells G5 and H5,
Gll and Hll. HIV-l assay controls were in wells A6 and B6, A12
2Q and B12; HIV-2 controls in wells C6 and D6, C12 and D12. All
other wells contained HIV negative sera.
:
The results obtained in the Multiskan reader (1 Filter 620;
2 Filter 690) are shown in microplate format and as averaged
25 optical densities in Tables 2A (wells ~l to H6) and 2B (wells A7
to H12), and in Table 3.
TABLE 2A
.
1 2 3 4 S 6
A 0.049 0.048 O.OS2 0.535 0.519 1.092
B 0.043 0.051 0.049 0.048 0.4B4 1.138
C 0.049 0.039 0.043 0.049 0.253 0.799
D 0.038 0.047 0.047 0.050 0.264 0.778
35 E 0.037 0.036 0.039 0.049 0.177 0.061
F 0.037 0.039 0.042 0.050 0.175 0.061
G 0.033 0.036 0.037 0.066 0.386 0.060
H -0.03S 0.03S 0.038 0.059 0.390 0.056
W093/2S905 29 ~ 1 ~ 7 2 ~ 8 PCT/GB93/Ot20$
TABLE 2B
7 8 9 10 11 12
A 0.050 0.048 0.047 O .048 0.490 1.089
5 B0.095 0.053 0.052 0.048 0.472 1.157
C 0.047 0.048 0.045 0.049 0.246 0.772
D 0.051 0.048 0.050 0.048 Q .266 0.829
E 0.046 0.048 0.048 0.049 0.173 0.060
F 0.052 0.050 0.049 0.050 0.162 0.062
10 G0.050 0.049 0.049 0.051 0.356 0.063
H 0.049 0.049 0.049 0.050 0.366 0.060
TABLE 3
sample with bromocresol control
purple
843 0.502 0.481
845 0.259 0.256
951 0.176 0.168
834 0.388 0.361
HIV-l l . llS 1.123
HIV-2 0.789 0.800
negative control 0.060 0.061
25 From the Tables set out above, it can be seen that the overall
results of the HIV assay were the same whether or not the
diluent had been supplemented with the bromocresol purple.
This Example was carried out using bromocresol purple as sample
30 monitor reagent in the sample diluent of a commercially
available HIV assay kit for use with serum samples. The sample
monitor reagent gave a distinct and clearly visible colour
change in the presence of sample even though the pH of the
sample diluent is not the optimum for the reagent. The colour 3i
35 change was confirmed by spectrophotometric measurement. The
presence of the sample monitor reagent did not affect the
subsequent immunoassay for antibodies to HIV.
W093/25905 ~ ~3~ 30 PCT/GB93/0120~;
Instead of using the commercially available immunoassay Wellcoz-
yme VK54, the present Example may be carried out using another
commercially available enzyme immunoassay, for example~ another
assay for antibodies to HIV, provided the sample diluent for use
5 in the chosen assay contains no serum or plasma. Bromocresol ',
purple or one of the other albumin`indicator reagents described
above may be used. It is preferable to take into account the pH
of the sample diluent, for example, bromocresol purple functions
best at pH values just below neutrality, cyano-di(4-nitro-
10 phenyl)-methane at pH values above neutrality and bromocresol
green near pH6.
EXAMPLE 2
Ouantitative determination of_a serum sam~le bv measurement of
15 albumin r``~ The antibody assay system used was an assay system for non-A
non-B hepatitis (HCv). The reagents were as follows:
A-standard ~6 well microplate, coated by standard means with
20 antigenic peptides of non-A non-B hepatitis. A sample d`luent,
containing no serum, based on a pH buffered detergent in a salt
solution, supplemented with bromocresol purple at a
concentration of 0.125nM.
25 The assay was carried out as follows: 190yl of sample diluent
was added to all the wells of the plate, then 10~1 of individual
serum or plasma samples was added to the wells with the
exception that in wells A12, B12, C12, and D12 only 5ul of
sample were added, in wells E12, F12, G12 and H12 no sample was
30 added and in wells All, Bll, Cll and Dll 20~1 of sample were
added.
As in Example 1, there was an immediate colour change from `
yellow to purple, easily detectable by eye, when the sample was
35 added to the diluent. The absorbance at 620nm was m`easured `
using a Multiskan plate reader as described in Example 1. The
results obtained are shown in Tables 4A (wells Al to H6) and 4B
(wells A7 to H12):
W093/25905 ~13 7 2 ~ 8 PCT/GB93/01205
TABLE 4A t
1 2 3 4 5
A 0.477 0.473 0.471 0.405 0.468 0.472
5 B 0.420 0.480 0.440 0.436 0.491 0.431
C 0.441 0.504 0.487 0.413 0.488 0.433
D 0.432 0.450 0.505 0.434 0.475 0.447
E 0.444 0.45i 0.508 0.443 0.530 0.423
F 0.467 0.447 0.467 0.429 0.487 0.486
10 G 0.421 0.477 0.471 0.436 0.491 0.388
H 0.490 0.433 0.490 0.436 0.525 0.422
TABLE 4 B
- 7 8 9 10 11 12
15 A 0.443 0.476 0.472 0.492 0.732 0.275
B 0.459 0.443 0.472 0.433 0.716 0.251
C 0.461 0.366 0.471 0.492 0.703 0.276
D 0.469 0.443 0.430 0.475 0.684 0.258
E 0.445 0~442 0.438 0.433 0.409 0.100
20 F 0.423 0.445 0.445 0.482 0.421 0.106
G 0.423 0.428 0.414 0.446 0.486 0.105
H 0.492 0.430 0.491 0.472 0.425 0.108
As can be seen from Table 4, in the present case, the colour
25 change obtained using the microplate reader was quantifiable,
there being a clear difference between the absorbance values
obtained for the control wells having 10~1 of sample (average OD
0.455), the wells with no sample (average OD 00105), the wells
with 5~1 of sample (average OD 0.265), and those wells having
30 20~1 of sample (average OD 0.709).
As the amount of albumin in serum or plasma from normal blood
donors is relatively constant at about 4.Og/lOOml and has a
range of about +15%, any significant error in sample volume can
35 be detected readily.
The peak absorbance of the albumin-bromocresol purple complex is
at about 600nm, but in order to quantitate the amount of the
W093/25905 ~3 PCT/GB93/01205;
32
complex using a microplate reader having a limited optical
dynamic range, it may be necessary to read the plate off-peak, a
well-known technique.
S This Example may be carried out using another enzyme immunoas-
say, provided the sample diluent contains no serum or plasma.
As set out in Example 1, another albumin indicator reagent may
be used, the choice being influenced by the pH of the sample
diluent used.
EXA~PLE 3
Determination of serum and plasma samples usina unbuffered
~diluent and PH effect of serum or plasma
15 An assay system for HIVl/HIV2 was used. The reagents were as
follows:
A standard 96-well microtitre plate coated with polyclonal
anti-human IgG (DAKO). HIv-l recombinant protein comprising
20 core and envelope antigens from the CBL-l isolate of HIV-l
(Sattentau Q.J. et al, (1986) Science 234 1120) conjugated to
HRP (horse-radish peroxidase) ("HIV-l conjugate'~) HIV-2 peptide
comprising gp36 envelope protein antigen, conjugated to HRP
("HIV-2 conjugate"). Positive samples: HIV-l`positive, diluted
25 in HIV 1 negative serum; HIV-2 positive, diluted in HIV-2
negative serum. Controls: HIV-l~HIV-2 negative sera and plasma
obtained from donated blood. Wash solution (glycine borate
buffer containing Tween); conjugate buffer (HEPES buffer
containing bovine serum albumin and detergents); substrate
30 solution containing TMB (3,3'5,5'-tetramethylbenzidine) and
hydrogen peroxide).
SamPle diluents
Sample diluent A (pH effect diluent):
35 Saponin 2g; EDTA 2ml of 500 mM solution pH 8.0; Bronidox L (as
preservative) O.sml; Triton x-100 (non-ionic detergent) lml;
distilled water to lOOml, giving a pH of 4.7.
2~3~23~
W093/25905 PCT/GB93tOl20
33
To aliquots of sample diluent A was added chlorophenol red
(O.lmg/ml final concentration) or phenol red (0.33mg/ml final
concentration~.
5 Sample diluent B (albumin detection diluent): ;
To sample diluent A was added 0.2M citric acid 5.6ml;
0.2M trisodium citrate 4.4ml; 25% sodium azide 0.08ml;
bromocresol purple 6.8mg; f inal pH 6 . O .
10 Method:
An assay was carried out simultaneously on a single microtitre
- plate to compare the effect of the following four sample
diluents: sample diluent A containing chlorophenol red; sample
- diluent B; sample diluent A containing phenol red; sample
15 diluent A with no added dye.
50 ~1 aIiquots of the selected sample diluent were dispensed
into allocated wells in a microtitre plate according to the
protocol given below. Samples of various HIV-l and HIv-2
20 positive sera and samples of HIV-l~HIV-2 negative serum or
plasma were added to the wells according to the sample protocol
set out below. Any colour change was noted and the plate was
; read immediately at OD 570 nm with 6so nm as the reference
wavelength using 2 Multiscan plate reader. This was to monitor '~
25 the sample addition.
The assay was then completed as follows:
The microtitre plate was covered and incubated under humid
conditions at 37C for 60 minutes. The wells were then washed
30 thoroughly five times with a wash solution, each wash step ,
involving removal of the contents of each well by aspiration,
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
35 inverted and tapped dry on a paper towel or tissue.` 50~1 of a
working strength solution of the mixed conjugates in HEPES 3
buffer containing bovine serum albumin and detergents were added
to the wells and the plate was incubated at 37C for 30 minutes
W093/2S905 .~ ~3~ ?, ~ ~ PCT/GBg3/01205 1 - `
34
under humid conditions. After a further wash step as described
above, 100~1 of substrate solution containing TMB (3,3',5,5'-
` tetramethylbenzidine) and hydrogen peroxide was added to eachwell, the plate was incubated at 37C under humid conditions for
5 30 minutes and the reaction was then stopped us~ng 50~1 of 2M
sulphuric acid. The absorbance in the wells was recorded at
450nm with 690nm as the reference wavelength.
Sam~le diluent and samPle Protocol:
10 Sample diluent A containing chlorophenol red was used in rows 1-
3, diluent B (containing bromocresol purple) in rows 4-6, sample
diluent A with phenol red in rows 7-9 and sample diluent A with
no sample monitor reagent was used in rows 10-12.
-
15 The samples were added to the wells of the microtitre plate asfollows:
Al, Bl: blank
Cl: HIV-l positive sample (HIV-l+) diluted l/lQ in HIV-l
negative serum
20 Dl: HIV-l+ diluted 1/50 in HIV-l negative serum
El: HIV-l+ diluted 1/100 in HIV-l negative serum
Fl: HIV-l+ diluted 1/200 in HIV-l negative serum
Gl: HIV-2 positive sample (HIV-2+) diluted 1/2 in HIV-2 negative
serum
25 Hl: HIV-2+ diluted 1/4 in ~IV-2 negative serum
A2: HIV-2+ diluted 1/8 in HIV-2 negative serum
B2: HIV-2+ diluted 1/16 in HIV-2 negative serum
C2-H2: HIV-l/HIV-2 negative serum controls
A3-H3: HIV-l/HIV-2 negative plasma controls
The sample protocol of rows 1-3 was repeated for rows 4-6, 7-9
and 10-12.
. ~
Results
35 With the exception of the blank wells, on adding the samples to
the sample diluents in the plate, an immediate colour change was
observed in rows 1 to 9, which contain sample monitor diluents.
No colour change was observed in rows 10-12, where the control
; --
W093/25905 213 7 2 ~ 8 PCT/GBg3/01205
sample diluent contained no monitor reagent. In rows 1-3, the
colour change was from red to purple~ in rows 4-6 from lime
green to purple, and in rows 7-9 from orange to red. In all
cases the colour change was both immediate and clearly visible.
5 The most vivid colour change was from red to purple in the
chorophenol red-containing wells (rows 1-3)~ The results
obtained on reading the plate at S70 nm are presented in Tables
5A and 5B.
TABLE 5A
1 2 3 4 5 6
blank 0.975 0.740 blank 0.5720.508
B blank 1.030 0.814 blank 0.5570.573
- C 1.036 1.058 0.699 0.565 0.4300.478
D 1.109 1.087 0.788 Q.S80 0.5380.508
E I.167 1.198 0.996 0.614 0.6240.813
F 1.149 1.117 0.881 0.624 0.5150.571
G 1.148 1.178 0.894 0.694 0.6210.621
H 1.162 1.157 1.107 0.661 0.6080.585
TABLE 5B
7 8 9 10 11 12
A blank 0.669 0.478 blank 0.0830.035
B blank 0.821 0.543 blank 0.0680.005
C 0.750 0.430 0.415 0.072 0.0060.038
D 0.791 0.832 0.457 0.068 0.0210.018
E 0.844 0.901 0.621 0.070 0.084-0.054
F 0.902 0.689 0.510 0.079 0.021-0.038
G 0.524 0.976 0.448 0.152 0.~480.035
H 0.717 1.004 0.542 0.091 0.0320.039
The results confirm the observations made by eye that the
diluents containing pH effect monitor reagents (rows 1-3 and 7-
9) undergo a distinct change in OD when a serum or plasma sample
35 is added. The sample diluents having a pH effect monitor
reagent give results that are directly comparable to the sample
diluent containing a monitor reagent (bromocresol purple) that
interacts with albumin in a serum or plasma sample (rows 4-6).
W093/2590~ PCTtGB93tO1205~-`
~3~ 36
The results on completion of the assay are presented in Tables
- 6A and 6B. It is clear from those results that the presence of
a sample monitor reagent during the assay proper does not affect
5 the result of the assay proper, that is to say, the assay for
HIV-l and HIV-2.
TABLE 6A
1 2 3 4 5 6
A blank 0.312 0.129 blank 0.295 0.112
B blank 0.224 0.093 blank 0.207 0.091
C 1.669 0.079 0.102 1.616 0.081 0.084
D 0.750 0.096 0.094 0.593 0.082 0.082
~ E 0.489 0.117 0.099 0.370 0.106 0.086
F 0.267 0.085 0.109 0.230 0.073 0.083
G 0.766 0.086 0.090 0.745 0.075 0.083
H 0.441 0.089 0.093 0.445 0.079 0.081
.
TABLE 6B
7 8 9 10 11 12
A blank 0.295 0.120 blank 0.326 0.123
B blank 0.213 0.091 blank 0.214 0.096
C 1.686 0.083 0.096 1.693 0.079 0.094
:~ 25 D 0.750 0.097 0.094 0.765 0.097 0.094
~ E 0.448 0.118 0.091 0.456 0.115 0.093
- F 0.271 0.086 0.089 0.267 0.089 0.096
G 0.820 0.090 0.090 0.799 0.088 0.093
: H 0.457 0.092 0.092 0.453 0.084 0.101
