OA12487A - Multiple target test useful for pre-donation screening of blood. - Google Patents

Abstract

Tests for establishing whether any of a plurality of different targets are present in a sample solution are described. If one or more of the targets are detected a positive result is obtained, but the different targets are not differentiated by the test, so there is no indication which targets have been detected. Such tests are useful for pre-donation screening of blood at public blood donation sessions to test whether the blood is safe for transfusion. The tests allow infected blood to be identified, but preserve the confidentiality of the reason for refusal of infected blood. This avoids the distress caused by public diagnosis of infection by diseases with which there is a social stigma attached, such as HIV. The tests are particularly suited for use in developing countries to screen for HIV, HBV, and HCV infection. Dipsticks and kits for performing the tests are also described.

Description

1 θ12487

Multiple Target Test üseful for Pre-Donation Screening of

Blood

This invention relates to tests, kits, dipsticks, and5 apparatus for détection of multiple targets in a samplesolution and allow, for example, détermination of whether aperson is infected with any of a number of disease-causingmicro-organisme. In particular, tests, kits, dipsticks andapparatus of the invention are for testing whether or not a îo person's blood is suitable for transfusion.

Transfusion or injection of unsafe blood accounts for 8-16million hepatitis B virus (HBV) infections, 2.3-4.7 millionhepatitis C virus (HCV) infections, and 80,000-160,000 HIVinfections each year. In most resource-poor countries of 15 Africa, Asia, and Latin America, post-donation screening byenzyme immunoassay (EIA) has revealed a cumulativeprevalence of 5-20% for hepatitis B surface antigen (HBsAg),anti-HIV antibody and anti-HCV antibody in blood donors.

Despite this high rate of infection, the blood supply in 20 many developing countries is incompletely screened or notscreened at ail for HIV antibody. An estimated 5-10% of HIVinfections in developing countries are due to‘ bloodtransfusion. The WHO estimate that 50% of blood donations in —sub-Saharan Africa are screened for HBsAg and only 5%^ for 25 anti-HCV. The following table gives an indication of theprevalence of HCV, HIV, and HBV in some countries of Africa: 012487 2

Country Prevalence in blood donors (%) Anti-HCV Anti-HXV HBsAg Total Ghana 0.6 0.3 15 15.9 Malawi 1 11.0 8 20 Botswana 1 10 5 16 South Africa 0.5 4.5 4 9 Egypt 12 1.0 5 18 Republic ofEquatorial Guinea 1.7 0.6 8.8 11.1 Somalia 0.6 0.8 19.1 20.5

In many cases, blood screening is carried out afterdonation. However, post-donation screening in developingcountries has several disadvantages. The high prevalence ofviral infection means that much of the blood donated cannotbe used. Thus, there is considérable wastage of- materialssuch as· blood bags and reagents. Tested and untested bloodis often stored in the only available refrigerator at theblood bank, thus allowing considérable risk of confusion ofsafe and unsafe blood. In many cases it is difficult toinform patients found to be infected because they hâve noaddress or because there is no adéquate means ofcommunication, or because they live long distances awaÿ andso are unlikely to return to receive the results once thepost-donation screening has been carried out.

In contrast, pre-donation screening allows considérablejsavings in materials and testing cost because the blood frompotential.donors found to be infected is not then drawn.Entry of infected blood to the blood bank facilities isthereby prevented. Patients found to be infected can beinformed of the infection at the donation session.Appropriate counselling, treatment, and prévention oftransmission can then be initiated immediately withouthaving to require donors to return at a later date.

In some areas, such as sub-Saharan Africa, Thailand andIndia, this pre-donation testing is carried out in publicblood collection sessions. Persons who are found to be 3 012487 infected with a disease-causing micro-organism, thus tnakingtheir blood unsuitable for transfusion, are advised of thisat the session. Because the session is public, the identityof these persons is not kept conf idential. This is of 5 particular concern if a person is found to be infected witha disease with which there is a social stigma attached, suchas HIV, as considérable distress can be caused to the personby such public diagnosis. In contrast, public diagnosis ofother infections, such as HBV, HCV or malarial infection is 10 much more readily accepted.

We hâve appreciated that there is a need to provide pre-donation tests which can be carried out in public toestablish whether or not a person's blood is suitable fortransfusion without disclosing whether a person has an 15 infection with which there is a social stigma attached.

According to the invention there is provided a test forestablishing whether any of a plurality of different targetsare présent in a sample solution which comprises contactingthe sample solution with a solid phase to allow capture of 20 target présent in the sample solution by the solid phase,and establishing whether or not any of the targets has beencaptured, wherein a positive resuit is obtained if one ormore of the targets are captured but does not indicate whichtarget or targets hâve been captured. —_ 25 _In preferred embodiments, tests of the invention are used totest whether a person is infected with any of a plurality ofdifferent micro-organisms or infectious agents therebymaking their blood unsuitable for transfusion. Thus,preferably the sample solution is, or is derived from, a 30 blood sample and the test is for establishing whether theblood is suitable for transfusion.

According to the invention a positive resuit shows that anyone or more of the different micro-organisms or infectiousagents are présent. However, the test does not differentiate 35 between the different micro-organisms or infectious agents. θ 1248γ

Thus, the person's blood is identified as unsuitable fortransfusion but the reason for this is not indicated (noteven the person carrying out the test is able to establishthis front the resuit) and therefore remains unknown. Theperson is then simply informed that their blood will not beaccepted for blood collection.

Subséquent testing, which can be carried out in aconfidential manner, can then establish the reason why theperson's blood is unsuitable for transfusion. If the personis then found to be infected with a disease causing micro-organism with which there is a social stigma attached, thedistress caused by public diagnosis of such infection isavoided.

The blood is préferably tested for any of the following:HIV, HBV, HCV, or malarial infection. Suitable markers ofsuch infection comprise or consist of : an antibody raisedagainst an HIV antigen; an antibody raised against an HCVantigen; an HBV antigen, preferably HBsAg; and a Plasmodiumantigen.

Most preferably, the blood is tested for HIV, HCV, and HBV.In areas where there is a high prevalence of infection (10%or more) by one or more of these viruses, such tests allowa considérable saving to be made of the materials and cqsts(which can constitute 1/3 to 1/2 of the blood bank budget)-which would otherwise be incurred by post-donationscreening..

Although blood screening tests of this type are primarilydesigned to meet the needs of developing countries, they canalso be useful in developed countries under circumstances inwhich a rapid test resuit is desired. An example is bloodscreening at inner city clinics. It has been found that upto 40% of the attendees of inner city clinics tested for HIVdo not return for their test results and are often nottraceable and therefore lost to the healthcare System. Testsof the invention allow pre-donation screening to be carried 5 ^12487 out so that infected attendees can be identified at their initial visit.

Tests of the invention may be used to test whether a patientis infected with other micro-organisms or infectious agents.Any marker of infection may be tested for. Examples includean antigen or nucleic acid of the micro-organism orinf ectious agent, or an antibody raised by the person to anantigen of the micro-organism or infectious agent.

Tests of the invention may also hâve wider application fortesting whether a sample solution contains any of aplurality of different targets in circumstances where it isnot desired or necessary to know which targets are présent.The different targets are not necessarily markers ofinfection by a micro-organism or inféctious agent. Othértargets may include hormones, métabolites (for example totest for metabolic disorders), illicit drugs, vitamins,steroids, or antibodies produced as part of an allergiereaction. ,

Tests of the invention are particularly suitable asscreening assays where it is desired to know whether any ofa number of different targets are présent in a plurality ofdifferent samples. Because several different targets aretested for, the time and cost of separately testing for~eachtarget is avoided. Once a test has been carried out, only _the positives need be re-tested to identify the particulartarget(s) présent.

The different targets may be captured by capture moietiesimmobilised to the solid phase, and détection moieties maybe used to detect the captured targets. Thus, according tothe invention there is further provided a test forestablishing whether any of a plurality of different targetsare présent in a sample solution which comprises: providing a solid phase having a plurality of differentcapture moieties immobilised thereto, each capture moietybeing capable of capturing a different target;

01248J providing a plurality of different détection moieties, eachdétection moiety allowing détection of a different targetcaptured by its capture moiety, détection of the differenttargets being such that there is no indication of whichtarget or targets hâve been detected; contacting the solid phase with the sample solution tothereby allow capture of target by the capture moieties; andusing the détection moieties to establish whether any of thedifferent targets are captured from the sample solution.

There is also provided according to the invention a kit forestablishing whether any of a plurality of different targetsare présent in a sample solution, the kit comprising: i) a solid phase having a plurality of different capturemoieties immobilised thereto, each capture moiety beingcapable of capturing a different target; and ii) a plurality of different détection moieties, eachdétection moiety allowing détection of a different targetcaptured by its capture moiety, détection of the differenttargets by the détection'moieties being such that there isno indication of which target or targets hâve been detected.

Capture of the different targets should be such that thereis no indication, for exaraple by the position on the solidphase at which a target is captured, of which target ortargets hâve been captured. " -Where the targets are markers of infection by mioro-organisms or infectious agents, the capture moieties arecapable of binding the markers. Depending on the target tobe captured, a capture moiety may be an antibody or antibodyfragment (capable of binding an antigen of the micro-organism or infectious agent) , a nucleic acid or nucleicacid analogue (capable of binding nucleic acid of the micro-organism or infectious agent), or an antigen (capable ofbinding an antibody raised against an antigen of the micro-organism or infectious agent). Where the targets are markersof HIV, HCV, HBV or Plasmodium infection, preferably thedifferent capture moieties comprise or consist of any of the 7 012487 following: an HIV antigen, an HCV antigen, an antibody toHBV, and an anti-Plasmodium antibody, and fragments ordérivatives thereof.

The solid phase may be any solid phase on which capture anddétection of the targets may be carried out. Examples ofsuitable solid phases include a dipstick (such as anitrocellulose dipstick), an Enzyme Linked Immuno-SorbentAssay (ELISA) plate, or micro-particles (for example,beads) . Whether or not any of the targets has been capturedmay be established by any suitable method including ELISA,other methods of antibody détection - for example usinglabelled antibodies (labelled for example with radioactivityor preferably a colour label) , or by agglutination ofmicroparticles.

In preferred embodiments of the invention, the solid phaseis a dipstick. Dipstick tests are quick and easy to perform,can be carried out with minimum training, and do not requireexpensive equipment. Dipstick tests are therefore relativelycheap to perform.

Dipstick tests are especially advantageous for screeningblood (particularly in developing countries) compared toother screening tests, such as microtiter plate enzymeimmunoassay (EIA) tests. EIA tests require expensiveequipment, are more complicated and slower to perform than -dipstick-tests, and need to be carried out by highly skilledtechnicians. In developing countries, many blood banks(which are mostly located in hospitals) screen too fewsamples to justify the expense of performing microtiter.plate EIAs. The cost of EIA tests is substantially higherwhere low numbers of samples are processed because there isa higher proportion of control wells in relation to testsamples.

Dipstick tests may be performed by immersing the dipstick ina sample solution or adding a fixed volume of sample with orwithout a diluent to allow binding of target in the sample 8 012487 solution to its capture moiety immobilised to the dipstick.However, preferably the dipstick comprises a contact end forcontacting the sample solution and is capable oftransporting the sample solution by capillary action to a 5 part of the dipstick remote from the contact end at whichthe different capture moieties are immobilised.

Preferably the different capture moieties are immobilised toa single capture zone of the dipstick. The different capturemoieties may be interspersed with each other at the singleîo capture zone. Preferably, however, the different capturemoieties are not interspersed with each other but areimmobilised at adjacent régions of the single capture zoneand are contiguous with each other (as shown in Figure 1) .

In such preferred embodiments, the capture moieties are not 15 ' diluted by one another so the sensitivity of target détection is thought to be higher than for embodiments inwhich the capture moieties are interspersed with oneanother. The adjacent régions of the single capture zoneshould be close together so that they cannot be 20 distinguished visually. The same visual resuit is thenobtained no matter which target or targets are captured.

Alternatively, each different capture moiety may be. immobilised at a different capture zone of the dipstick. Aslong , as there is no indication of which target binds_ to 25 ‘ which capture zone, and the tester is not informed of this,-then tests carried out using such dipsticks will._ notindicate which target or targets hâve been captured. Itwill, however, be possible to tell the number of targets which hâve been captured. 30 Détection according to the invention using dipsticks withmultiple capture zones is different to conventional dipstickdétection of multiple targets where the tester knows whichtarget binds to each capture zone. EP applicationno. 0301141 discloses a dipstick for simultaneously 35 detecting the presence of a variety of spécifie antibodiesin a clinical specimen. Different spécifie antigens used to 012487 capture the spécifie antibodies are separately immobilisedto different capture zones of the dipstick. The capturezones are labelled so that a tester can identify whichtarget binds to each capture zone.

In such conventional tests, it is desired to be able todistinguish the different targets and to identify -whichtargets are présent. Thus, a signal at a particular capturezone will indicate to the tester that the target for thatcapture zone is présent. This is in contrast to tests of theinvention in which it is desired to establish only whetherany of the targets are présent without establishing which ofthose targets are présent. A signal at one of the capturezones will indicate only that one of the targets is présent,and not which target is présent, because the tester does notknow which target is captured at that capture zone.

However, a disadvantage of using dipsticks with a pluralityof different capture zones is that it is possible to deducewhich target binds to each capture zone from the results ofsubséquent tests performed to identify which target(s) isprésent once a positive resuit using a test of the inventionhas been obtained. This could be avoided if the order of thecapture zones is changed for different dipsticks.

The different détection moieties are preferably sepâratefrom the dipstick, but may be releasably immobilised to thedipstick.- If the détection moieties are releasablyimmobilised to the dipstick, they should be released oncontact with the sample solution so that they can bind totarget in the sample solution.

Where a target is a marker of infection by an infectiousagent or micro-organism, a détection moiety is capable ofbinding the marker. The détection moiety may comprise anantigen, an antibody or fragment thereof, or a nucleic acidor nucleic acid analogue. 10 0 ί 24 8 7

Binding of a détection moiety to its target may allow directdétection of the target, for example if the détection moietycomprises a label. Alternatively, binding of a détectionmoiety to its target may allow indirect détection of the 5 target, for example, by a primary antibody capable ofbinding a détection ligand coupled to the détection moiety.The primary antibody may be labelled, or a labelledsecondary antibody may also be provided which is capable ofbinding the primary antibody. In other embodiments, indirect 10 détection may be achieved by conversion of a chromogenicsubstrate by a converting enzyme linked to the détectionmoiety (as in ELISA methods) .

If a détection moiety of a kit of the invention allowsindirect détection of its target, the other reagents 15 necessary for détection of the' target (such as primary andsecondary antibodies, chromogenic substrates) may beprovided with the kit.

Preferably the label is a-visually détectable label, such asa colour label, although other types of labels (such as 20 luminescent or radioactive labels) may be used. Détection of the different targets such that there is noindication of which target or targets hâve been detected maybe achieved by labelling the different détection moiefcieswith the same label. Capture of each target from the sample 25 -solution-will then be indicated by the presence of the_samelabel on the solid phase so that capture of one target isindistinguishable from capture of another target.

Alternatively, the different détection moieties may belabelled with different labels, but without informing the 30 person performing the test or the person being tested whichlabel représente détection of which target.

In some embodiments of the invention, it may be desired toprovide different capture agents capable of binding thedifferent targets. Each different capture agent comprises a 11 012487 capture ligand which can be bound by a capture moietyimmobilised at a capture zone of the solid phase. Theligands of the different capture agents are of the sanietype, so that on-ly a single type of capture moiety need beimmobilised to the solid phase. The different capture agentsare contacted with the sample solution so that target in thesample solution can bind to its capture agent. Eachdifferent target can then be captured on the solid phase bybinding of the capture agent for that target to the capturemoiety. Because the solid phase only comprises a single typeof capture moiety, préparation of the solid phase issimplified.

In other embodiments of the invention, the solid phase maycomprise a plurality of micro-particlés wherein each targetis capable of causing the micro-particles to agglutinatethereby indicating the presence of the target in the samplesolution. Thus, if any of the target s are présent in thesample solution, the micro-particles will agglutinate.Because a positive resuit' is indicated only by agglutinationof the micro-particles, it is not possible to tell whichtarget(s) has caused the agglutination.

This may be achieved by use of different capture moietiesimmobilised to the micro-particles. Different capturemoieties should be capable of binding to different sites oneach target, or to the same site présent in multiple copies -on the target, so that groups of micro-particles becomecross-linked via the target, thereby causing agglutinationof the micro-particles.

Preferred embodiments of the invention are now described, byway of example only, with reference to the accompanyingdrawings in which:

Figure 1 which shows a nitrocellulose dipstick of a firstpreferred embodiment of the invention;

Figure 2 shows a nitrocellulose dipstick of a secondpreferred embodiment of the invention; and 12 012487

Figure 3 shows a multi-sided dipstick of a third preferred embodiment of the invention.

In figure 1, the nitrocellulose dipstick 10 has a contactend 12 for contacting a sample solution, a single capturezone 14 remote from the contact end, and a conjugate zone 16between the contact end and the capture zone. A flow pad 18is at the other end of the dipstick, and a control zone 19is between the flow pad and the capture zone.

Four different capture moieties are immobilised at thecapture zone. The different capture moieties are : HIVantigen, HCV antigen, HBV antibody, and Plasmodium antibody.Each of the four capture moieties is immobilised to adifferent thin strip (14a, b, c, d) of the capture zone.Adjacent capture moieties are contiguous with one another.

The following détection moieties are releasably immobilisedto the conjugate zone 16 of the dipstick: i) colloïdal gold labelled anti-human antibody, orcolloïdal gold labelled HIV antigen (to detect anti-HIVantibody target); ii) colloïdal gold labelled anti-human antibody, orcolloïdal gold labelled HCV antigen (to detect anti-HCVantibody target); iii) . colloïdal gold labelled antibody to HBV (to detectlHBVtarget); and -iv) colloïdal gold labelled Plasmodium antibody (to de.tectPlasmodium target).

To detect whether a person is infected with HIV, HBV, HCV,or Plasmodium, a blood, or plasma, or sérum sample iscollected from the person and is contacted with the contactend and conjugate zone of the dipstick in an undiluted ordiluted form. The releasably immobilised détection moietiesare solubilised by contact with the sample and bind totarget (if présent) in the sample. The sample passes up thedipstick by capillary action to the capture zone. If targetis présent in the sample, it should be bound by the θ12487 13 appropriate détection moiety to form a complex which thenpasses up the dipstick to be captured at the capture zone.The presence of target in the sample is then indicated byaccumulation of colour label at the capture zone of the 5 dipstick. This is seen as a visible colour line.

The flow pad 18 absorbs sample solution which reaches it bycapillary action. The control zone has a control moietyimmobilised to it which is capable of binding directly toeach of the different détection moieties to provide aîo positive control for the test. If a visible line appears atthe control zone this confirms that sufficient amounts ofthe détection moieties hâve migrated through the dipstick membrane and are available for binding to the targets.

If a positive resuit is obtained at the'capture zone, thenis because each different détection moiety has the same colourlabel, it is not possible to tell whether the person from which the sample has been obtained is infected with HIV,HBV, HCV or Plasmodium. It is also not possible to tellwhich target(s) is présent in the sample from the position 20 in the capture zone at which the détection moietyaccumulâtes because the strips of the capture zone areextremely thin and close together and cannot bedistinguished visually. Thus, the person carrying out thetest,cannot identify to which strip(s) in the capture"zone 25' the détection moiety has bound.

The person can then be advised that their blood is notsuitable for collection and transfusion and referred forfurther testing ·· in the confidential environment of ahospital diagnostic or blood bank laboratory in order to 30 establish which infection (s) the person has. It will beappreciated that the distress of public diagnosis of HIVinfection is thereby avoided.

The further testing may be done for example by use of adipstick having four separate lanes, each with a single 35 capture zone, or by a single dipstick with four separate 14 012487 capture zones for spécifie détection of infection by HIV, HBV, HCV, and Plasmodium, respectively. A dipstick for performing a test according to a secondpreferred embodiment of the invention is shown in Figure 2.The test is a triple screening test for markers of HCV, HIVand HBV infection to establish whether blood collected froma patient is suitable for transfusion. The sample solutionis sérum from venous blood.

The · dipstick 20 has a contact end 22 for contacting thesérum, a single capture zone 24 (in the form of a lineacross the dipstick) remote from the contact end, a controlzone 26 further remote from the contact end, and a flow pad28 at the other end of the dipstick. The flow pad absorbssample solution which has passed through the dipstick by'capillary action from the contact end.

The markers of viral infection are anti-HIV antibody, anti-HCV antibody, and HBsAg. Different capture moieties capableof binding the different markers are immobilised to thecapture zone. The different capture moieties are immobilisedto adjacent régions of the capture zone and are contiguouswith each other.

To capture anti-HIV antibody, the capture moiéty“'_maycomprise any of the following antigens: HIV core protein~p24, the- extra-cellular portion of gp41, or the extra-cellular portion of p31. Preferably a mixture of theantigens is used. One or more of the antigens can also beused as a détection moiety to detect captured anti-HIVantibody. Alternatively, the détection moiety may comprisean anti-human antibody capable of binding the anti-HIVantibody. In a further alternative arrangement, the capturemoiety may comprise an anti-human antibody capable ofbinding the anti-HIV antibody and the détection moiety maycomprise one or more of the above antigens. 01248 7 - 15 -.

If it is desired to capture and detect anti-HIV 2 spécifieantibody this may be achieved using the extra-envelope,hydrophilic part of gp36. Although individuals infected withHIV-1 group 0 or group N are expected to cross-react with 5 the above antigens, spécifie peptides can be used to improveantigenic reactivity of the capture.

To capture anti-HCV antibody, the capture moiety maycomprise any of the following antigens: recombinant antigencorresponding to antigen of the NS3 or E2 région of HCV, aio core peptide, or a peptide of the NS4 or E2 région.

Preferably a mixture of the antigens is used. One or more ofthe antigens can also be used as a détection moiety todetect captured anti-HCV antibody. Alternatively, thédétection moiety may comprise an anti-human antibody capable 15 of binding the anti-HCV antibody. In a further alternativearrangement, the capture moiety may comprise an anti-humanantibody capable of binding the anti-HCV antibody and thedétection moiety may comprise one or more of the aboveantigens. 20 To capture HBsAg, the capture moiety may comprise amonoclonal antibody to the 'a' déterminant of the S protein.Captured HBsAg can also be detected by a monoclonal antibodyto the 'a' déterminant of the S protein because thisdéterminant is présent in multiple copies in HBsAg. 25 ’ Alternatively, the capture moiety may comprise polyclonal-antibody- to HBsAg and the détection moiety a monoclonalantibody or an appropriate mixture of monoclonal antibodies to HBsAg, or vice versa.

Where the détection moiety for detecting anti-HIV antibody 3σ or anti-HCV antibody comprises an antigen, this ischemically linked to a universal tail comprising one or moreligands. Where the détection moiety for detecting any of thetargets comprises an antibody, this is chemically coupled toone or more ligands. The ligand (s) of the détection moiety 35 can be bound by a colour-labelled anti-ligand antibody (as 16 012487 a labelling agent) . For example, if the ligand is biotin, ananti-biotin antibody conjugated to colloïdal gold orcoloured latex particles may be used. The ligands allow thesame labelling agent to be used to label each markerindirectly by binding to that marker's spécifie détectionmoiety.

To perform a test according to the second embodiment, thedifferent détection moieties and the anti-biotin antibodyconjugated to colloïdal gold are mixed with a sérum sample.The sample is then contacted with the contact end of thedipstick to allow sample to travel by capillary actionthrough the capture and control lines of the dipstick,Sample which reaches the other end of the dipstick isabsorbed by the flow pad.

If a viral marker is présent in the sample, the spécifiedétection moiety for that marker, bound to the anti-biotincolloïdal gold conjugate should bind to the marker and themarker, détection moiety, and anti-biotin colloïdal goldconjugate should be captured at the capture zone of thedipstick. Because there is only a single capture zone andthe same colour line appears at the capture zone no mat terwhich marker or markers are captured and detected, the testwill only reveal that one or more of the viral markers is inthe sample, but will not reveal which of the viral markersis présent. A moiety capable of binding directly to each of thedifferent détection moieties (for example an anti-biotin IgMantibody) is iromobilised to the control zone to provide apositive control for the test. If a visible line appears atthe control zone this confirms that sufficient amounts ofthe détection moieties and of the labelled anti-ligandantibody hâve migrated through the dipstick membrane and areavailable for binding to the markers.

The triple screening test of the second embodiment allowscollected blood to be tested without anyone présent, 17 012487 including the tester, being able to identify which viralmarker(s) is responsible for a positive resuit. Theconfidentiality of the reason for the positive resuit istherefore preserved. A third preferred embodiment of the invention is shown infigure 3. The figure shows a dipstick 30 comprising a closedtube of triangular cross-section. First 32, second 34, andthird 36 sides of the dipstick are for capture and détectionof a marker indicating infection by HIV, HCV, and HBV,respectively. The dipstick has a contact end 38 forcontacting sérum from venous blood and a flow pad 40 at theother end for absorbing sérum which has passed to that endby capillary action. Each side has a capture line remotefrom the contact end, and a control line between the captureline and the flow pad. The capture lines of the differentsides join to form a triangle around the dipstick, as do thecontrol lines.

The markers of viral infection are anti-HIV antibody, anti-HCV antibody, and HBsAg. A capture moiety capable of bindinganti-HIV antibody is immobilised to the capture zone of thefirst side of the dipstick. A capture moiety capable ofbinding anti-HCV antibody is immobilised to the capture zoneof the second side of the dipstick. A capture moiety capableof binding HBsAg is immobilised to the capture zone' of Ithethird side of the dipstick. However, there is no indication-of which -markers are captured by the different sides of_ thedipstick.

Three different détection moieties are used to detect theviral markers. Each détection moiety is chemically linked toa biotinylated tail and is capable of specifically bindingits viral marker. An anti-biotin antibody conjugated tocolloïdal gold is used to bind to the détection moieties tothereby allow indirect labelling of the targets.

To perform a test using the dipstick of the . thirdembodiment, the détection moieties and the anti-biotin 18 0124 8 7 antibody conjugated to colloïdal gold are mixed with a sérumsample or plasma-containing sairçple. The sample is t-hencontacted with the contact end of the dipstick to allowsample to travel by capillary action through the capture andcontrol lines of each side of the dipstick. Sample whichreaches the other end of the dipstick is absorbed by theflow pad.

If a viral marker is présent in the sample, the spécifiedétection moiety for that marker, bound to the anti-biotincolloïdal gold conjugate should bind to the marker and themarker, détection moiety, and anti-biotin colloïdal goldconjugate should be captured at the capture line of the sideof the dipstick for that marker. Because there is noindication of which side of the dipstick captures eachmarker, and there is no Visual différence' between thedifferent capture lines, the test will only reveal that oneof the viral markers is in the sample, but will not revealwhich of the viral markers is présent. A moiety similar to that described above for the secondembodiment may be immobilised to the control lines toprovide a check that the test is working properly.

Instead of being mixed with the sample solution, thedétection moieties and/or the anti-biotin colloïdal "goldconjugate may be releasably immobilised to a conjugate zone-of each s-ide of the dipstick between the contact end and. thecapture zone of each side.

In other embodiments, the dipstick may hâve a differentnumber of sides to allow capture and détection of adifferent number of targets. The dipstick may be ofgenerally circular or oval cross-section, or cône shaped.The dipstick may be pyramid shaped (with a base and three ormore sides, one side for each different target) . The tip ofthe cône or pyramid may form the contact end of thedipstick. -·»- 19 072487

In a further embodiment, the dipstick may comprise a diskwith a central contact zone for contacting the samplesolution, and different capture zones arranged radiallyaround the contact zone. The different capture zones are5 substantially équidistant from the contact zone andsubstantially equally spaced from, or contiguous with eachother. Different capture moieties capable of binding thedifferent targets are immobilised to the different capturezones. Again, there is no indication of which target iscaptured at which capture zone so that capture and détectionof a target using the dipstick does not reveal which targethas been captured and detected. 10

Claims (19)

θ T24 8 7 20 Clairns

1. A test for establishing whether any of a plurality ofdifferent targets are présent in a saraple solution whichcomprises contacting the sample solution with a solid phase 5 to allow capture of target présent in the sample solution bythe solid phase, and establishing whether or not any of thetargets has been captured, wherein a positive resuit isobtained if one or more of the targets are captured but doesnot indicate which target or targets hâve been captured. îo

2. A test according to claim 1 which comprises : providing a solid phase having a plurality of differentcapture moieties immobilised thereto, each capture moietybeing capable of capturing a different target; providing a plurality of different détection moieties, each 15 détection moiety allowing détection of a different targetcaptured by its capture moiety, détection of the differenttargets being such that there is no indication of whichtarget or targets hâve been detected; contacting the solid phase with the sample solution to 20 thereby allow capture of target by the capture moieties; andusing the détection moieties to establish whether any of thedifferent targets are captured from the sample solution.

3. .A test according to claim 1 in which the sol'i'd "phasecomprises micro-particles and each different target is 25 -capable -of causing the micro-particles to agglutinatethereby establishing that one or more targets hâve beencaptured without indicating which target or targets hâvebeen captured.

4. A test according to any preceding claim in which the 30 sample solution is or is derived from a blood sample taken from a person and the test is for establishing whether theperson's blood is suitable for transfusion withoutdisclosing why the blood is unsuitable for transfusion if apositive resuit is obtained. 21 012487

5. A dipstick for establishing whether any of a pluralityof different targets are présent in a sample solution whichcomprises a plurality of different capture moieties eachdifferent capture moiety being immobilised to a differentrégion of a capture zone of the dipstick and being capableof capturing a different target, wherein the differentrégions are not visually distinguishable.

6. A dipstick for establishing whether any of a pluralityof different targets are présent in a sample solution whichcomprises a capture zone to which a plurality of differentcapture moieties are immobilised, each different capturemoiety being capable of capturing a different target,wherein the different capture moieties are interspersed withone another.

7. A multi-sided dipstick for establishing whether any ofa plurality of different targets are présent in a samplesolution which comprises a different capture zone at eachdifferent side of the.dipstick, a different capture moietycapable of binding a different target being immobilised ateach different capture zone, wherein the different capturezones are at équivalent positions on each side of thedipstick so that if a target is captured and detected thereis no indication of which target of the different targetshas been captured and detected. ‘ "L

-8. A dipstick according to any of daims 5 to 7 in whichthe dipstick comprises a contact end for contacting thesample solution and the dipstick is capable of transportingthe sample solution by capillary action from the contact endto the capture zone or zones.

9. A dipstick for establishing whether any of a pluralityof different targets are présent in a sample solution whichcomprises a contact zone for contacting the sample solutionand a plurality of different capture zones arranged radiallyaround the contact zone substantially équidistant from thecontact zone and substantially equally spaced from each 22 θ T 24 8 7 other, a different capture moiety capable of binding adifferent target being itranobilised to each capture zone andthe dipstick being capable of transporting sample solutionfrom the contact zone to the capture zones. 5

10. A kit for testing whether any of a plurality of different targets are présent in a sample solution whichcomprises : i) a solid phase having a plurality of different capturemoieties immobilised thereto, each different capture moiety îo being capable of capturing a different target; and ii) a plurality of different détection moieties, eachdifferent détection moiety allowing détection of a differenttarget captured by its capture moiety; wherein a positive resuit is obtained if one or more of the15 different targets is captured and detected but there is noindication of which target or targets hâve been captured and detected.

11. A kit according to claim. 10 in which the solid phasecomprises a dipstick. 20

12. A kit according to claim 10 comprising a dipstick according to any of daims 5 to 9.

13. A kit according to claim 11 or 12 in which~Ltheplurality of different détection moieties are releasably -immobilised to a conjugate zone of the dipstick between. the25 contact end and the capture zone or capture zones.

14. A kit according to any of daims 11 to 13 in whicheach different détection moiety is provided with a ligandand the kit further comprises .a labelling agent capable ofbinding the ligands of the different détection moieties, the 30 labelling agent being labelled with a visible label.

15. A kit for testing whether any of a plurality ofdifferent targets are présent in a sample solution whichcomprises : 23 01248? i) a solid phase having a capture moiety immobilisedthereto; ii) a plurality of different capture agents, each differentcapture agent being capable of binding to a different 5 target, and each different capture agent being provided witha ligand which can be bound by the capture moiety; and iii) a plurality of different détection moieties, eachdifferent détection moiety allowing détection of a differenttarget captured via its capture agent by the capture moiety; îo wherein a positive resuit is obtained if one or more of thedifferent targets is captured and detected but there is noindication of which target or targets hâve been captured anddetected.

16. A test, dipstick or kit according to any preceding 15 claim in which the different capture moieties are capable of capturing an antigen or nucleic acid of a micro-organism orinfectious agent, or an antibody raised to an antigen of amicroorganism or infectious agent.

17. A test, dipstick or kit according to any preceding 20 claim in which the different capture moieties comprise or consist of any of the following: an HIV antigen; an HCVantigen; an antibody to HBV; and a Plasmodium antibody.

18. - A dipstick or kit according to any of daims 5 to 17for performing a test according to claim 4.

19. Apparatus for establishing whether any of a plurality of different targets are présent in a sample solution whichcomprises a plurality of micro-particles wherein eachdifferent target is capable of causing the micro-particlesto agglutinate so that agglutination occurs if one or more 30 of the different targets are présent in the sample solution.