CA2137238A1

CA2137238A1 - Immunoassay method

Info

Publication number: CA2137238A1
Application number: CA 2137238
Authority: CA
Inventors: Richard J. S. Duncan
Original assignee: Individual
Current assignee: International Murex Technologies Corp
Priority date: 1992-06-05
Filing date: 1993-06-07
Publication date: 1993-12-23
Also published as: DE643832T1; WO1993025905A1; JPH07507634A; GB9211980D0; EP0643832A1; ES2068790T1; AU4342693A

Abstract

A method is provided for determining false negative results in an immunoassay for an analyte of interest by determining the presence of sample in an immunoassay vessel in which the immunoassay is carried out. The method comprises using a sample monitor reagent or reagent system that is capable of producing a signal when sample is present, the sample monitor reagent or reagent system being different for the analyte-specific reagent or reagent system used in the immunoassay itself. A negative signal generated by the sample monitor reagent or reagent denotes a false negative result in the immunoassay itself.

Description

- 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.
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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 ~ .

.

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.

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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~;

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 ? ~

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 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;

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

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

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 ? ` '`

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.

.

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$

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 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.

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 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 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;

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 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 - `

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.

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 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.

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 .

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

Claims

1. A method for determining the presence of a sample of body fluid or of a sample derived from a human or non-human animal body in an immunossay 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, carrying out a sample addition step comprising admixing the sample and a liquid that comprises a sample monitor reagent or reagent system capable of producing a signal only when sample is present, or (i)(b) carrying out a sample addition step comprising 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 only when sample is present, and (ii) detecting any signal generated by the sample monitor reagent or reagent system;
the sample monitor reagent or reagent system capable or producing a signal only when sample is present being different from the analyte-specific reagent or reagent system used for detecting the analyte in the immunoassay or specific binding pair assay itself.

2. A method as claimed in claim 1, wherein the signal produced by the sample monitor reagent or reagent system is a change in colour, chemiluminescence or fluorescence.

3. A method as claimed in claim 1 or claim 2, wherein the sample monitor reagent or reagent system is capable or producing a signal in the presence of albumin, caeruloplasmin, transferrin, a serum or plasma enzyme, or another serum- or plasma-specific protein, or another high molecular weight component of serum or plasma.

4. A method as claimed in claim 3, wherein the sample monitor reagent or reagent system is capable of producing a signal in the presence of albumin, and the reagent for the detection of albumin is bromocresol purple, bromocresol green, cyano-di(4-nitrophenyl)-methane or Coomassie Brilliant Blue.

5. A method as claimed in claim 1 or claim 2, wherein the sample monitor reagent or reagent system is capable of producing a signal in the presence of a low molecular weight component of serum, plasma or any other body fluid.

6. A method as claimed in claim 5, wherein the low molecular weight component is glucose, calcium, magnesium, iron, urea or uric acid.

7. A method as claimed in claim 6, wherein a sample monitor reagent system for the detection of glucose comprises a glucose oxidase/peroxidase system.

8. A method as claimed in claim 6, wherein a sample monitor reagent system for the detection of urea comprises a urease/glutamate dehydrogenase system, and a sample monitor reagent system for the detection of uric acid comprises a uricase/peroxidase system.

9. A method as claimed in any one of claims 1 to 8, wherein the liquid comprising the sample monitor reagent or reagent system is a sample diluent.

10. A method as claimed in claim 1 or claim 2, wherein the sample addition step comprises admixing sample with a sample diluent that has little or no buffering capacity and the sample monitor reagent or reagent system comprises a component that generates a signal change as a result of the change in pH
when the sample and the diluent are admixed.

11. A method as claimed in claim 10, wherein the sample monitor reagent or reagent system comprises a pH indicator that changes colour when the pH changes from acid towards neutral or from alkaline towards neutral.

12. A method as claimed in any one of claims 1 to 11, where n the sample monitor reagent or reagent system is immobilized on the inner surface of the immunoassay vessel and/or on the surface of beads used in the immunoassay vessel.

13. A method as claimed in any one of claims 1 to 12, wherein the immunoassay vessel is a microwell and is present with a plurality of other microwells in the form of a microtitre plate.

14. A method as claimed in any one of claims 1 to 13, wherein the signal generated by the sample monitor reagent or reagent system is a colour change and the colour change is detectable by visual inspection and/or by spectrophotometric measurement.

15. A method as claimed in any one of claims 1 to 14, wherein the immunoassay or specific binding pair assay is a homogeneous phase assay.

16. A method as claimed in any one of claims 1 to 14, wherein the immunoassay or specific binding pair assay is a capture assay and wherein on the interior surface of the immunoassay vessel and/or on the surface of beads used in the immunoassay vessel is immobilized an antigen, antibody or one member of the specific binding pair.

17. A method as claimed in any one of claims 1 to 14, wherein the immunoassay is a latex agglutination assay, a haemagglut-ination assay or a flocculation assay.

18. 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, 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 of the sample in the immunoassay vessel is detected by means of a sample monitor reagent or reagent system that is capable of producing a signal only 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 assay itself.

19. An assay as claimed in claim 18, wherein the detection of sample is carried out as claimed in any one of claims 1 to 17.

20. An assay as claimed in claim 18 or claim 19, carried out simultaneously in a plurality of immunoassay vessels using a fully or partly automated system.

21. Use of a sample monitor reagent or reagent system that is capable of producing a signal only in the presence of a sample under investigation in an immunoassay or specific binding pair assay carried out on a sample of body fluid or of a sample derived from a human or animal body, to determine the presence in an immunoassay vessel of the sample under investigation and/or to to determine a false negative result in an immunoassay or specific binding pair assay.

22. A sample diluent for use in an immunoassay or specific binding pair assay, which comprises a sample monitor reagent or reagent system capable of producing a sample-specific signal in the presence of a sample under investigation in the assay.

23. A sample diluent as claimed in claim 22, wherein the sample monitor reagent or reagent system is as defined in any one of claims 2 to 8, claim 10 or claim 11.

24. An immunoassay vessel or a bead for use in an immunoassay vessel, on which bead or on the interior surface of which vessel is immobilized a sample monitor reagent or reagent system capable of producing a sample-specific signal in the presence o- a sample under investigation.

25. An immunoassay vessel or bead as claimed in claim 24, wherein the reagent or reagent system is as claimed in any one of claims 2 to 8, claim 10 or claim 11.

26. A plurality of immunoassay vessels as claimed in claim 2 or claim 25, in the form of an assembly or microwells.

27. A kit for carrying out an immunoassay or a specific binding pair assay on a sample under investigation, which kit comprises (a) a liquid comprising a sample monitor reagent or reagent system capable of producing a signal only in the presence of the sample and/or (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 only in the presence of the sample.

28. A kit as claimed in claim 27, wherein the liquid is a sample diluent as claimed in claim 22 or 23 and/or the immunoassay vessels and beads are as claimed in any one of claims 24 to 26.

29. A kit as claimed in claim 27 comprising a plurality of immunoassay vessels and beads for use in the vessel, wherein one of the components comprising the vessels and the beads has immobilized a sample monitor reagent or reagent system and the other component has immobilized a capture agent for the analyte of interest.

30. A method as claimed in any one of claims 1 to 17, an assay as claimed in any one of claims 18 to 20, or a kit as claimed in any one of claims 25 to 28, wherein the sample under investigation is donated blood.

31. A method for determining a false negative result in an immunoassay, which comprises determining the presence of a sample of body fluid or of a sample derived from a human or non-human animal body in an immunossay vessel in which an im-munoassay or a specific binding pair assay for an analyte in the sample is carried out, which method comprises (i)(a) in the immunoassay vessel, carrying out a sample addition step comprising admixing the sample and a liquid that comprises a sample monitor reagent or reagent system capable of producing a signal only when sample is present, or (i)(b) carrying out a sample addition step comprising 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 only when sample is present, detecting any signal generated by the sample monitor reagent or reagent system, the sample monitor reagent or reagent system capable of producing a signal only when sample is present being different from the analyte-specific reagent or reagent system used for detecting the analyte in the immunoassay or specific binding pair assay itself, and (iii) carrying out the immunoassay or specific binding assay for the analyte of interest, a negative signal generated by the sample monitor reagent or reagent system denoting a false negative result.

32. A method as claimed in claim 31, wherein the sample detection is carried out as claimed in any one of claims 1 to 17.