WO2013037887A1

WO2013037887A1 - Competitive immunoassay for the detection of antibiotics

Info

Publication number: WO2013037887A1
Application number: PCT/EP2012/067946
Authority: WO
Inventors: Mylène Patrice Dominique CAUSSETTE; Maartje Maria Franse; Stéphen L. A. HENNART
Original assignee: Dsm Ip Assets B.V.
Priority date: 2011-09-16
Filing date: 2012-09-13
Publication date: 2013-03-21

Abstract

The present invention relates to a device and method for detecting analytes in a sample.

Description

COMPETITIVE IMMUNOASSAY FOR THE DETECTION OF ANTIBIOTICS

Field of the invention

The present invention discloses a device and method for detecting analytes in a sample.

Background of the invention

Antibiotics are used for combating infectious diseases, both in humans and in animals. It is well known that misuse of antibiotics such as administration of antibiotics whenever this is not required from a medical point of view or incomplete courses of treatment is the most important cause of the development of antibiotic resistance. Thus, methods for detecting the presence of antibiotics in samples such as e.g. milk, blood, fish, feed, meat, serum, urine, water and the like are of utmost importance in the prevention of the unwanted spread of antibiotics. In many areas, this process of detection can only be performed adequately, if a fast and simple test is available.

In general, there are two types of tests suitable for routinely monitoring the presence of antibiotics in samples. Firstly, there are microbial inhibition tests, wherein a test microorganism is contacted with the sample to be tested and the growth (or inhibition of growth) of the microorganism is observed, for instance with the use of an indicator. An example of such a test is described in EP 0 755 456 B1. The major drawback of microbial inhibition tests is that it takes a relatively long time to obtain results.

Secondly, there are competitive immunoassays, wherein the antibiotic to be tested and a reference antibiotic present in the test compete for binding with binding proteins and/or antibodies with affinity for the antibiotics. Visualization is usually done by means of labeling. One of many examples of such a test is described in EP 0 593 1 12 B1 . Although these types of tests are in general faster than microbial inhibition tests, they still require extensive handling by the end user and are therefore not user friendly. In view of the above, it is clear that there exists considerable room for improvement in the area of antibiotic testing, particularly where it concerns to ease-of- use, speed and convenience.

Description of the figures

Figure 1 is a side view of an embodiment of the test device according to the present invention. The test device comprises a solid support (f) comprising a sample receiving region (a), a reaction region (b), a detection region (c), an absorbing region (d) and a handling region (e).

Description of the invention

There is a wide range of applications for the methods, test devices and kits according to the present invention across the entire field of diagnostics and analysis. They can be used to detect any type of analyte in a sample, including antibiotics, carbohydrates, dietary substances, microorganisms, (poly)nucleotides, (poly)peptides, steroids, hormones, toxins, (agro)chemical agents such as fungicides, herbicides and pesticides, vitamins, drugs, metabolites, receptors, antibodies, allergens, to name just a few. In a preferred embodiment the methods, test devices and kits according to the present invention are used to detect antibiotics in a sample.

The term 'antibiotic' as used herein refers to one or more substances or chemical constituents (or metabolites of such substances or chemical constituents) of a sample that display activity against bacteria. In an embodiment of the invention the antibiotic to be detected by the method, test device and/or kit according to the present invention is selected from the group consisting of the family of beta-lactam antibiotics, the family of tetracycline antibiotics, the family of sulfonamide antibiotics, the family of aminoglycoside antibiotics, and the family of quinolone antibiotics. In a preferred embodiment of the invention the antibiotic to be detected by the method, test device and/or kit according to the present invention is a beta-lactam antibiotic. The term 'beta-lactam antibiotic' refers to compounds (or metabolites thereof) that comprise a beta-lactam substructure within their chemical structure and display activity against bacteria. Two important subclasses of the beta-lactam antibiotics are the cephalosporin-derived antibiotics and the penicillin- derived antibiotics. Examples of the cephalosporin-derived antibiotics are cefaclor, cefadroxil, ceftiofur, cephalexin, cephapirin and cephradine. Examples of the penicillin- derived antibiotics are amoxicillin, ampicillin, cloxacillin, dicloxacillin, flucloxacillin, oxacillin, penicillin G, penicillin V and ticarcillin.

The present invention relates to a method for detecting an antibiotic in a sample, said method comprising the steps of:

a) contacting a liquid sample with an antibiotic binding protein and an antibiotic to form a liquid composition, said antibiotic binding protein comprising a ligand 1 and a ligand 2, said ligand 1 capable of binding to a target molecule 1 and said ligand 2 capable of binding to a target molecule 2, said antibiotic comprising a ligand 3, said ligand 3 capable of binding to a target molecule 3,

b) providing a test device having a proximal and a distal end, said test device configured to allow lateral flow from the proximal to the distal end, said test device comprising a solid support comprising the following regions in sequence from the proximal to the distal end:

i. a sample receiving region,

ii. a reaction region, said reaction region comprising a mobilisable labeled target molecule 1 ,

iii. a detection region, said detection region comprising at least two zones:

A. a control zone comprising an immobilized target molecule 3, and

B. a detection zone comprising an immobilized target molecule 2, iv. an absorbing region, and

v. optionally, a handling region,

c) contacting the liquid composition with the sample receiving region of the test device,

d) allowing the liquid composition to move from the sample receiving region through the reaction region and the detection region to the absorbing region, so as to allow:

i. the liquid composition to mix with the labeled target molecule 1 , ii. ligand 1 to bind to target molecule 1 to provide a labeled antibiotic binding protein, and

iii. the liquid composition comprising the labeled antibiotic binding protein and the antibiotic comprising ligand 3 to move to and contact the control zone and the detection zone,

e) detecting a signal at the detection zone and a signal at the control zone, wherein i. the absence of antibiotic in the sample is indicated by the absence of a signal at the detection zone or the presence of a signal at the detection zone that is less intense than the signal at the control zone, and ii. the presence of antibiotic in the sample is indicated by the presence of a signal at the detection zone that is more intense than the signal at the control zone.

In a first step the sample is contacted with at least one antibiotic binding protein and at least one antibiotic. In an embodiment the sample may be contacted with more than one antibiotic binding protein and/or more than one antibiotic. The antibiotic binding protein and the antibiotic can be present in liquid or solid form before they are contacted with the sample. Both agents may be in liquid form, or both agents may be in solid form, or one agent may be in solid form and the other agent may be in liquid form. Preferably, both agents are present in solid form, preferably in powder form. The solid form can be made by drying or lyophilizing the compounds. The powder can be resuspended in the sample. If necessary, the obtained liquid composition can be mixed (by e.g. applying vortices) to improve and/or fasten resuspension of the powder in the sample. If desired, compounds facilitating resuspension and/or dissolution and/or mixing of the antibiotic binding protein, the antibiotic and the sample can be added. In a preferred embodiment these compounds are present with the labeled antibiotic binding protein and the antibiotic before they are contacted with the sample. Preferably, these compounds are also in solid form e.g. in powder form. Suitable compounds include, but are not limited to, a buffer e.g. an organic buffer such as a Tris buffer, a surfactant such as Triton X-100, a protein such as bovine serum albumin, a polyol such as glycerin, and a sugar e.g. a disaccharide such as saccharose. In an embodiment the amount of the liquid sample added to the antibiotic binding protein and the antibiotic is between 50 and 1000 μΙ, preferably between 75 and 7500 μΙ, more preferably between 100 and 500 μΙ, in particular between 125 and 250 μΙ. After the sample is contacted with the antibiotic binding protein and the antibiotic, the obtained liquid composition may be shaken. Shaking is generally done for 1 to 20 seconds, preferably 5 to 15 seconds with about 10 seconds being preferred.

In an embodiment the antibiotic binding protein and the antibiotic are present in a container. Preferably, the above compounds are also present in the container. The labeled antibiotic binding protein and the antibiotic may be present in different containers, but preferably they are present in one container. Containers that can be used in the present invention may be tubes of any shape and size and from any suitable material available. Containers may also be the wells such as those incorporated in microtiter plates. In a preferred embodiment the container comprises the labeled antibiotic binding protein, the antibiotic, a buffer e.g. an organic buffer such as a Tris buffer, a surfactant such as Triton X-100 (preferably in a concentration of between 0 and 0.1 % w/v), a protein such as bovine serum albumin, a polyol such as glycerin, and a sugar e.g. a disaccharide such as saccharose.

Alternatively, the antibiotic binding protein and/or the antibiotic may be part of the test device, for example they can be present in liquid or solid form in the sample receiving region or in a separate region located in front of the sample receiving region or between the sample receiving region and the reaction region. In this embodiment rather than add a pre-measured volume into the container, the test device might be arranged to be dipped into the fluid sample to absorb the selected amount of sample.

In an embodiment the antibiotic binding protein comprises at least one ligand. In a preferred embodiment it comprises at least two ligands. In a preferred embodiment, the ligands are different and each ligand is capable of binding a different target molecule. Each ligand is capable of specifically binding to a target molecule. In an embodiment the antibiotic binding protein comprises a ligand 1 and a ligand 2, said ligand 1 being capable of binding to a target molecule 1 and said ligand 2 being capable of binding to a target molecule 2. In a preferred embodiment the antibiotic also comprises at least one ligand. In a preferred embodiment, the ligand is different from the ligand(s) of the antibiotic binding protein and the ligand is capable of binding to a different target molecule than the ligand(s) of the antibiotic binding protein are capable of binding to.

In an embodiment the ligands are directly bound to the antibiotic binding protein and/or the antibiotic. The ligands may however also be bound through a spacer molecule to the antibiotic binding protein and/or the antibiotic. The type of conjugation is dependent on the ligand, the concentration of the ligand, the molecule the ligand needs to bind to, to name just a few.

In an embodiment the ligand bound to the antibiotic binding protein and the ligand bound to the antibiotic is an antigen, an antibody, a substrate, an agonist, an antagonist, an activator, a nucleic acid molecule, a hapten, to name just a few. In a preferred embodiment the ligand(s) bound to the antibiotic binding protein is a hapten such as for example biotin, streptavidin, avidin, and digoxigenin. In a preferred embodiment ligand 1 is biotin and ligand 2 is digoxigenin. In a preferred embodiment the ligand bound to the antibiotic is an antibody. In a preferred embodiment ligand 3 is an IgG molecule.

In an embodiment the sample might be solid and liquid comprising the antibiotic(s) needs to be extracted from the sample. Methods for extracting liquids from samples are dependent on the type of sample. Suitable extraction methods for different types of samples are known to the person skilled in the art and include disintegration of the solid sample by homogenization, vortexing with beads, grinding or sonication and/or solvent extraction. In a preferred embodiment the sample that is contacted with the antibiotic binding protein and antibiotic is liquid. In an embodiment of the invention the sample may be derived from a body liquid, an organ, meat or eggs. Antibiotics might also be present in food products in which these animal products are added as an ingredient. Examples of food products are milk; honey; meat of cow, pig, poultry and fish; sea food such as shrimps; processed meat products such as sausages; ready-to-eat meals; feed; and baby food. Antibiotics might also be present in body liquids or animal tissues, which are suitable for examination by for example food inspection authorities. Examples are blood, liver tissue, muscle tissue, heart tissue, kidney tissue or pre-urine obtained from the kidney and urine. Urine and blood are suitable for examination prior to slaughtering of an animal. Antibiotics may also be present in water such as waste water. In a preferred embodiment the sample is milk. The milk may be obtained from cattle (e.g. cows), horses, sheep, goats, yaks, water buffalo, humans, donkeys, reindeers, bison and camels. Antibiotics may also be present in semi-processed or processed food such as pasteurized products, UHT-products, skimmed or partially skimmed milk, whey, fresh or ripened cheese, yoghurt, cream, butter, sour cream, buttermilk, to name just a few.

In an embodiment the liquid composition (i.e. the liquid composition obtained in step a) is incubated for 30 seconds to 5 minutes, preferably 45 seconds to 4 minutes, more preferably 50 seconds to 3 minutes, most preferably 55 seconds to 2.5 minutes and in particular 1 to 2 minutes before the liquid composition is contacted with the test device. The liquid composition is incubated at a temperature of between 40 and 70°C, preferably a temperature of between 50 and 65°C, more preferably a temperature of between 60 and 64°C. In an embodiment, the liquid composition obtained in step a is incubated after shaking. In another embodiment, shaking is done after incubation of the liquid composition obtained in step a. In yet another embodiment, shaking is done before and after incubation. Shaking time before and after incubation may be equal, but may also differ. In a preferred embodiment incubation continues after the liquid composition is contacted with the test device. In an embodiment the test device is contacted with the liquid composition for 1 to 5 minutes, preferably 1.5 to 4 minutes, more preferably 2 to 3 minutes at a temperature of 40 to 70°C, preferably a temperature of between 50 and 65°C, more preferably a temperature of between 60 and 64°C. Incubation can be carried out with the aid of a thermostatic device such as a water bath or an incubator. In a preferred embodiment the temperature before and after the liquid composition is contacted with the test device is identical. Incubation may be stopped as soon as a signal is detected at the detection zone and/or the control zone.

The antibiotic binding protein may be any protein capable of binding to the antibiotic to be detected. The binding protein may bind a family of antibiotics which have similar structural binding sites. Suitable binding proteins include, but are not limited to, antibodies (monoclonal, polyclonal or recombinant), antibody fragments, enzymes, aptamers, and receptors such as penicillin binding protein. Preferably, the antibiotic binding protein is a protein obtained from a microorganism. In an embodiment the antibiotic binding protein is a protein obtained from an antibiotic-sensitive microorganism. In an embodiment of the invention the organism is selected from the group consisting of a Bacillus species, an Escherichia species and a Streptococcus species. In a preferred embodiment of the invention the organism is thermophilic. Examples are Bacillus stearothermophilus or Streptococcus thermophilus, with Bacillus stearothermophilus being preferred.

The antibiotic that is contacted with the antibiotic binding protein and the liquid sample may be an antibiotic or an analogue thereof. Examples of suitable antibiotics or analogues thereof are beta-lactam antibiotics, e.g. cephalosporins such as for instance 7-amino-cephalosporanic acid (7ACA).

When the liquid composition is contacted with the test device, it will flow from the sample receiving region to the reaction region, from the reaction region to the detection region and from the detection region to the absorbing region. The liquid composition will flow by capillary action. When the liquid composition comes in contact with the reaction region, it will mix with the reagents deposited in or on the reaction region. In addition, ligand 1 will bind to target molecule 1 providing a labeled antibiotic binding protein. In other words, the reaction region comprises a mobilisable labeled target molecule 1 , which is deposited on or in the reaction region and remains fixed at the reaction region prior to addition of the liquid composition to the test device. On addition of the liquid composition, the mobilisable reagent at the reaction region become mobile and is carried by the liquid composition through the remainder of the test device. On addition of the liquid composition, the mobilisable reagent at the reaction region will be capable of binding to a binding partner present in the liquid composition. For instance, ligand 1 will bind to labeled target molecule 1 providing a labeled antibiotic binding protein that for example together with the antibiotic comprising ligand 3 can move to and contact the detection region and absorbing region. The reagents at the reaction region may be diffusibly bound to the reaction region and application of the liquid composition to the test device and contact of the liquid composition with the reaction region will allow the diffusibly bound reagents to migrate along the test device to the detection region and absorbing region. The term "diffusibly bound" as referred to herein means that a reagent is attached or impregnated, but capable of dispersing with the liquid composition and being carried by the liquid composition in the lateral flow.

Mobilisable target molecule 1 comprises a label. A visible as well as a non-visible label can be used. Suitable labels include, but are not limited to, fluorescent compounds, chromogenic compounds, chemiluminescent compounds, radioactive compounds, colorimetric compounds, magnetic compounds (e.g. beads or particles), enzymes, catalytic compounds, substrates, vesicles with labels and particles such as dye particles, colored latex particles, carbon particles, metallic particles, non-metallic particles, colloidal metallic particles. In a preferred embodiment the label is a visible label with colloidal metallic particles being preferred and gold particles being most preferred. The label may be bound to the mobilisable target molecule 1 by any suitable means including conjugation, covalent bonding or non-covalent bonding. The label may be directly bound to the mobilisable target molecule 1 or the label may be bound through a conjugate or spacer. In a preferred embodiment the label is directly bound.

The antibiotic in the sample and the antibiotic comprising ligand 3 are generally the same, but they may also be different. For example, the antibiotic comprising ligand 3 may be an analogue of the antibiotic in the sample. If the antibiotic in the sample and the antibiotic comprising ligand 3 are different, they should both be able to bind to the antibiotic binding protein. In other words, they compete for binding to the antibiotic binding protein.

When the sample comprises antibiotic, this antibiotic will bind to the antibiotic binding protein and antibiotic comprising ligand 3 will stay (to a large extent) unbound. The unbound antibiotic comprising ligand 3 will flow to the detection region and bind to the control zone, as this zone comprises immobilized target molecule 3, i.e. a molecule specifically binding to ligand 3. The labeled antibiotic binding protein comprising the bound antibiotic from the sample will flow to the detection region and bind to the detection zone through the specific interaction between ligand 2 and the immobilized target molecule 2. When the sample does not comprise antibiotic (or only a low amount), antibiotic comprising ligand 3 will bind to the antibiotic binding protein and the labeled antibiotic binding protein comprising antibiotic comprising ligand 3 will flow to the detection region and bind to the control zone through the specific interaction between ligand 3 and the immobilized target molecule 3.

In an embodiment the test device is a test strip. In view of the small volumes of liquid sample added to the antibiotic binding protein and the antibiotic, it is recommended that the test device is placed such that it rests in the angle between bottom and wall of the container.

In the method of the present invention, when the label density (i.e. signal) in the detection zone is higher than that in the control zone, antibiotic is present in the sample at a concentration above a given threshold (in other words, the antibiotic is present in an amount in excess of allowable levels and the test is considered 'positive'). Ergo, when in the present application is referred to "the presence of antibiotic in the sample", it is meant that the sample contains antibiotic at a concentration above a given threshold. When the label density (i.e. signal) in the detection zone is equally intense as the label density (i.e. signal) in the control zone, antibiotic is present in the sample at a concentration above, at or below a given threshold. This depends on the chosen threshold. However, when the label density in the detection zone is less than the label density in the control zone, the sample contains no antibiotic or antibiotic at a concentration below a given threshold (in other words, the antibiotic is not present in a sufficient amount and the test is considered 'negative'). Ergo, when in the present application is referred to "the absence of antibiotic in the sample", it is meant that the sample contains no antibiotic or antibiotic at a concentration below a given threshold. 'Threshold' as used herein refers to the concentration value above which a given antibiotic is to be regarded as present and below which said antibiotic is to be regarded as absent. Generally, a threshold value is given for particular antibiotics in particular samples by local, regional or interregional authorities, but it can also be pre-set for certain research purposes. The signals may be detected visually by eye, but also by means of a signal reading device such as e.g. a spectrophotometer, a reflectance reader, a fluorometer, a camera, a magnetic detector, a scintillation counter, to name just a few.

The intensity of the detectable label at the detection zone can be measured to determine the result of the method of the present invention. The method of the present invention may provide a yes or no result (i.e. antibiotic present or absent) or may determine the presence or absence of an antibiotic above or below a certain threshold value (which is in fact also a yes or no result). The intensity of the signal can be inversely related to the concentration of antibiotic in the sample. Furthermore, the intensity of the signal at the detection zone can be compared to the intensity of the signal at the control zone to determine a result of the method of the present invention. The difference between the intensities of the various zones can even be analyzed by a signal reading device and used to calculate the concentration of antibiotic in the sample, for example by comparing the result to a predetermined value.

The invention further relates to a test device for detecting an antibiotic in a sample, said test device having a proximal and a distal end, said device comprising a solid support comprising the following regions in sequence from the proximal to the distal end:

a sample receiving region,

a reaction region, said reaction region comprising a mobilisable labeled target molecule 1 ,

a detection region, said detection region comprising at least two zones:

A. a control zone comprising an immobilized target molecule 3, and

B. a detection zone comprising an immobilized target molecule 2, an absorbing region, and

optionally, a handling region,

wherein said test device is configured to allow lateral flow from the proximal to the distal end, so as to allow a liquid composition comprising a liquid sample, an antibiotic binding protein and an antibiotic, said antibiotic binding protein comprising a ligand 1 and a ligand 2, said ligand 1 capable of binding to a target molecule 1 and said ligand 2 capable of binding to a target molecule 2, said antibiotic comprising ligand 3, said ligand 3 capable of binding to a target molecule 3, to move from the sample receiving region through the reaction region and the detection region to the absorbing region, so as to allow

the liquid composition to mix with the labeled target molecule 1 , ligand 1 to bind to target molecule 1 to provide a labeled antibiotic binding protein, and

the liquid composition comprising the labeled antibiotic binding protein and the antibiotic comprising ligand 3 to move to and contact the control zone and the detection zone.

'Solid support' as used herein refers to material that is used to provide support for the various regions of the test device. When used for a test device according to the present invention, a solid support usually is made from material that is inert with respect to the application for which the test device is to be used. Suitable materials are glass, metals, and various types of plastics such as for instance polystyrene. The solid support may have a thickness of between 0.1 and 1 mm. In an embodiment the test device may be housed within a non-absorbent or laminate casing. In other words, the test device includes a housing defining an elongated cavity for receiving and holding the test device. Suitable housings are known to a person skilled in the art.

Attachment of the regions to the backing can be performed following known techniques such as gluing, thermo compression and the like.

The test devices of the present invention usually has a length varying between 10 and 200 mm, preferably between 20 and 150 mm, more preferably between 30 and 100 mm, and in particular between 50 and 75 mm, a width varying between 1 and 20 mm, preferably between 2 and 15 mm, more preferably between 3 and 10 mm, and a thickness varying between 0.05 and 2 mm, preferably between 0.075 and 1.5 mm, more preferably between 0.1 and 1 mm.

The test devices of the present invention have a shelf-life of at least 6 months, preferably of at least 9 months when stored at 4°C. In another embodiment, the test devices of the present invention have a shelf-life of up to 10 days, preferably up to 20 days and more preferably up to 28 days when stored at -20°C. In yet another embodiment, the test devices of the present invention have a shelf-life of up to 1 day, preferably up to 4 days and more preferably up to 7 days when stored at 30°C. "Shelf- life" as used herein means that the sensitivity of the stored test device does not decrease. Ergo, the sensitivity of a stored test device is equal to the sensitivity of a freshly prepared test device.

In an embodiment the test device can be stored at a temperature of between - 20°C and 30°C. Preferably, the test device is stored at a temperature of between 4°C and 8°C. 'Sample receiving region' as used herein refers to the portion of the test device which is brought into direct contact with the liquid composition. In other words, the sample receiving region is the portion of the test device which is brought into direct contact with the liquid sample after the liquid sample has been contacted with the antibiotic binding protein and the antibiotic comprising ligand 3. The sample receiving section is made of porous material. In a preferred embodiment the sample receiving region is made of a material having a pore size of 3-8 μηη. Preferably, the sample receiving region is a polyvinyl alcohol-bound glass fibre membrane such as a VF2 membrane.

'Reaction region' as used herein refers to the portion of the test device which is in lateral flow contact with the sample receiving region and the detection region. The contact can be an end-to-end connection, but preferably there is overlap between the reaction region and the sample receiving region and between the reaction region and the detection region. The overlap may be between 1 and 2 mm. The reaction region is made of porous material. In an embodiment the reaction region and the sample receiving region may be combined into one region. The reaction region comprises a mobilisable labeled target molecule 1. Target molecule 1 may be any suitable molecule including an antigen, an antibody, an enzyme, a receptor, to name just a few. Since the ligand is preferably a hapten such as for example biotin, streptavidin, avidin, and digoxigenin, the target molecule is preferably a compound capable of binding a hapten. In a preferred embodiment ligand 1 is biotin and target molecule 1 is a compound capable of binding to biotin such as streptavidin, avidin or neutravidin.

'Detection region' as used herein refers to the portion of the test device which is in lateral flow contact with the reaction region and the absorbing region. The contact can be an end-to-end connection, but preferably there is overlap between the detection region and the reaction region and between the detection region and the absorbing region. The overlap may be between 1 and 2 mm. The detection region is made of porous material. Preferably, the detection region is a HF90 membrane. The detection region usually comprises one or more zones, for instance, a detection zone and a control zone for detecting the presence or absence of the antibiotic. The control zone might also function as a control site. The detection region may also have two or more detection zones and/or two or more control zones. The detection zones may have the same functionality or may have different functionalities. The same holds true for the control zones. The one or more zones may be made of a porous material different than that of the detection region. The separate zones may be made of a different porous material. Preferably, they are made however of the same material. Preferably, the one or more zones are made of the same material as the detection region. When the liquid composition moves through the detection region, it may first be contacted with the control zone and then be contacted with the detection zone or vice versa. In case the detection region has several detection zones and/or control zones, any sequence of zones may be provided. The zones can be in a variety of configurations including lines, dots or other configurations. In a preferred embodiment the zones are lines.

'Lateral flow' as used herein refers to liquid flow of a sample in a material in which all of the dissolved and/or dispersed components of the sample are transported at essentially equal velocities and with relatively unimpaired flow laterally through the material.

The detection zone and the control zone each may comprise at least one capture reagent. Preferably, the detection zone and the control zone are made by applying the appropriate capture reagent or mixture of capture reagents to the detection region, either by means of covalent linkages or other bonding processes. 'Capture reagent' as used herein refers to any reagent that can be used to create the required functionality in the detection zone and/or the control zone. The application of the capture reagent to the detection region can be done by known methods such as spraying, dispensing, painting, drawing, printing, striping and the like. The zones are capable of generating a signal, for instance a visual color signal, upon presence or absence of the complex between the capture reagent and its binding partner.

A capture reagent may be any natural or non-natural compound. Examples of suitable capture reagents are antibiotics, antibodies, antigens, ligands, proteins, to name just a few.

In a preferred embodiment the capture reagent at the detection zone of the test device according to the present invention is target molecule 2. Target molecule 2 may be any suitable molecule including an antigen, an antibody, an enzyme, a receptor, to name just a few. Since the ligand is preferably a hapten such as for example biotin, streptavidin, avidin, and digoxigenin, the target molecule is preferably a compound capable of binding a hapten. In a preferred embodiment ligand 2 is digoxigenin and target molecule 2 is a compound capable of binding to digoxigenin such as an antibody against digoxigenin or a fragment of such an antibody. In a preferred embodiment the capture reagent at the control zone of the test device according to the present invention is target molecule 3. Target molecule 3 may be any suitable molecule including an antigen, an antibody, an enzyme, a receptor, to name just a few. As ligand 3 is an antibody, e.g. an IgG molecule, target molecule 3 may be a member of a binding pair e.g. a specific binding pair such as an antigen/antibody pair (e.g. an IgG/anti-lgG antibody). It may however also be an antibody binding protein such as e.g. protein A. The capture agent may be present in a solution comprising an additional protein e.g. bovine serum albumin, a sugar e.g. a disaccharide such as saccharose and a salt such as NaCI when applied to the control zone.

Immobilization of the capture reagent to the detection region may be carried out in a manner known per se, for example by covalent or non-covalent adsorption to the detection region. The capture reagent may also be covalently conjugated to the detection region through a carrier such as for instance bovine serum albumin (BSA). Optionally, the capture reagent may be coupled to the carrier via a spacer. Many bifunctional compounds are suitable as a spacer. All methods available to construct bonds, e.g. coupling techniques for instance those known from peptide chemistry, could be applied, unless they are detrimental to the capture reagent. Suitable spacers, carriers and coupling techniques are well known to the person skilled in the art.

The test device according to the present invention may also comprise an additional control zone that produces a signal irrespective of whether or not an antibiotic is present in the sample and gives an indication that the test device functions as required. Such a control zone provides a consistent signal that does not vary with the concentration of antibiotic in the sample. Such a control zone can also be used to inform the user that the liquid composition has flowed through the test device. In that sense, such a control zone can be used as a flow control. Furthermore, such a control zone can be used for comparison to the detection zone and/or other control zone.

'Absorbing region' as used herein refers to the part of the test device, which is in lateral flow contact with the detection region and functions to promote lateral flow through the detection region and is capable of absorbing excess liquid sample. The contact an end-to-end connection, but preferably is an overlap between the detection region and the absorbing region. The overlap may be between 1 and 2 mm. The absorbing region is made of porous material. In a preferred embodiment the absorbing region is at least 1 cm. 'Handling region' as used herein relates to a region of the test device that can be used to hold and manipulate the test device without interfering with the test result. The handling region may be a separate region attached to the solid support, but the handling region can also be a part of the solid support itself. The handling region and absorbing region may also be combined into one region, if desired.

In yet another embodiment of the present invention, the test device comprises a member that covers one or more of the sample receiving region, the reaction region, the detection region and the absorbing region. Said member, which can be made of any material, preferably a clear plastic material, advantageously provides protection for said regions with regard to fingerprints and/or mechanical destruction and/or fumes and the like. One or more regions may be covered with a single member, however also multiple members optionally of different materials may be used.

'Porous material' as used herein refers to any material capable of providing lateral flow. Examples of suitable porous materials are polymeric materials such as polyvinyl or polyester, cotton, glass fiber, nitrocellulose, blends of nitrocellulose with polymeric materials, nylon, paper, rayon and the like.

In an embodiment the test device comprises a detection region that is longer than the absorbing region, an absorbing region that is longer than the handling region, and a handling region that is longer than the sample receiving region and/or the reaction region.

The test devices according to the present invention are manufactured by the methods known to the skilled in the art. Solid supports can have the form of cards. These can be prepared, for example, using commercially available laminators. The cards can be laminated. Onto the cards the various regions can be attached. The reagents used (capture reagents, mobilisable reagents, etc) are deposited on the respective regions in the form of solutions, before or after the assembly of the cards. These solutions can be deposited very precisely using commercially available apparatus such as dispensers from BioDot, Inc. Before and/or after application of the detection and/or control zone, the detection region can be blocked by for instance spraying a blocking solution. Preferred blocking solutions comprise a buffer e.g. an organic buffer such as a Tris buffer, a surfactant e.g. Tween such as Tween-20, and a protein such as bovine serum albumin. Preferably, the blocking solution is not sprayed directly onto the detection zone and/or control zone. The deposited solutions can be immediately evaporated, for example, by placing the card under a stream of hot air. After all regions and zones have been applied, the cards are preferably dried in a dry atmosphere, i.e. an atmosphere having a relative humidity of < 50%, < 40%, < 30%, < 20%, < 10%, preferably 0%. For large-scale production it is also possible to prepare rolls. Subsequently, the cards and rolls bearing the desired reagents can be cut into strips, each of these strips constituting a test device according to the invention. Alternatively, the reagents (capture reagents, mobilisable reagent, etc) can also be deposited on the respective regions, prior to the assembly of the cards or rolls by simply immersing the respective regions in a solution containing the reagents.

The current invention is also directed to a kit comprising an antibiotic binding protein, an antibiotic and a test device according to the present invention. The test device is preferably stored in a package comprising a desiccant. Preferably, the antibiotic binding protein and the antibiotic are present in a container. Preferably, the antibiotic binding protein comprises a ligand 1 and a ligand 2, said ligand 1 being capable of binding to a target molecule 1 and said ligand 2 being capable of binding to a target molecule 2. Preferably, the antibiotic comprises a ligand 3, said ligand 3 being capable of binding to a target molecule 3. Preferably, the kit comprises more than one container and more than one test device, e.g. 10, 20, 30, 40, 50 or even 100 containers and/or test devices. The kit according to the present invention may also comprise a sampling device. This is a device with the aid of which sample, e.g. liquid sample, can be added to the antibiotic binding protein. Examples include, but are not limited to, a container (optionally with volume markings), a syringe, a pipette or an automated pipetting system. Such a syringe or pipette may be designed in such a fashion that with only one mode of operation a predetermined volume can be withdrawn from the liquid sample to be analyzed. Optionally, systems known in the art with which more than one syringe or pipette can be operated with one single handling may be applied. In case, the kit comprises a sampling device such as a pipette, it may additionally comprise pipette tips. Preferably, the amount of pipette tips is equal to the amount of containers (i.e. the containers wherein the antibiotic binding protein and the antibiotic are present) and test devices. This way, with only one pipette different samples can be applied to different containers. In case the kit comprises disposable pipettes as sampling device, the amount of disposable pipettes is equal to the amount of containers (i.e. the containers wherein the antibiotic binding protein and ligand comprising antibiotic are present) and test devices. Optionally, the kit further comprises an insert with instructions for use and/or a means for setting the time needed for incubation. Optionally, the kit further comprises a thermostatic device such as an incubator or a water bath, with the aid of which samples can be kept at a pre-set temperature, such as the temperature at which the liquid sample, the antibiotic binding protein and optionally the test device should be incubated. Preferably, said thermostatic device is designed in such a fashion that it can hold the containers filled with the antibiotic binding protein, the antibiotic and the liquid sample and optionally the test device. Optionally, the thermostatic device is coupled to a means for setting the time needed for incubation such that heating is stopped after lapse of a pre-set period. Optionally, the kit also comprises a sample-reading device, a data carrier loaded with a computer program suitable for instructing a computer to analyze digital data obtained from the sample-reading device.

The embodiments and features disclosed above for the method of the present invention also pertain to the test device and kit according to the present invention. The embodiments and features disclosed above for the test device of the present invention also pertain to the method and kit according to the present invention. The embodiments and features disclosed above for the kit of the present invention also pertain to the test device and method according to the present invention.

EXAMPLES

Example 1

Preparation of the test device

A nitrocellulose HF90 membrane (Millipore; length 25 mm) is used as a detection region. The membrane is glued to a polystyrene laminated card (thickness 0.254 mm) 8 mm from the proximal end of the card. Next, the detection zone and the control zone are applied onto the nitrocellulose membrane. The detection zone is applied by dispensing 0.15 mg/ml of an anti-lgG antibody in 20 mM KP0₄-buffer (pH 7.5) containing 0.675 mg/ml bovine serum albumin (BSA), 5% w/v saccharose and 20 mM NaCI with a Biodot Dispense workstation XYZ 3050 with frontline at 0.8 μΙ/cm. The control zone is applied by dispensing 0.15 mg/ml of an anti-digoxigenin antibody in 20 mM KP0₄-buffer (pH 7.5) comprising 0.675 mg/ml BSA, 5% w/v saccharose and 20 mM NaCI with a Biodot Dispense workstation XYZ 3050 with frontline at 0.8 μΙ/cm. After drying of the zones, the nitrocellulose membrane is blocked by spraying a solution comprising 10 mM Tris buffer (pH 8) comprising 2% w/v BSA and 0.05% w/v Tween-20 using a Biodot Dispense workstation XYZ 3050 with airjet 3x. The solution is sprayed onto the membrane at a distance of 2 mm below the detection zone.

Streptavidin-coated gold particles are synthesized by reacting colloidal gold (diameter 40 nm; 1 OD/ml) with 10 μg/ml streptavidin. Next, the obtained solution is concentrated by tangential flow filtration, washed and stored in a Tris buffer (pH 8) including NaCN.

Thereafter, 140 mOD of the streptavidin-gold conjugate is solubilized in 100 μΙ buffer (80 mM Tris buffer comprising 0.01 % w/v Triton-X-100, 0.4% w/v BSA, 5% v/v glycerin and 2% w/v saccharose (pH 8.5)) and 1.44 μΙ/cm of the solution is dispensed on the sample receiving region (VF2 membrane (pore size 3-8 μηη); Millipore; length 10 mm) and allowed to dry.

Thereafter, the sample receiving region (a VF2 membrane) is glued at the proximal end of the card overlapping 2 mm with the nitrocellulose membrane. The absorbing region (10038 membrane from Millipore; length 20 mm) is glued at the distal end of the card with an overlap of 2 mm on the nitrocellulose membrane. The obtained cards are dried at 37°C in a dry atmosphere (0% relative humidity). The card is cut in strips of 5.2 mm width and kept at a dry atmosphere (0% relative humidity).

Example 2

Preparation of the antibiotic binding protein and conjugated antibiotic

Penicillin binding protein purified from Bacillus stearothermophilus was biotinylated (1 :4) with D-biotinyl-epsilon-aminocaproic acid-N-hydroxysuccinimide ester. The biotinylated penicillin binding protein was conjugated to digoxigenin giving a penicillin binding protein having a biotin ligand and a digoxigenin ligand.

Antibiotic (7-amino-cephalosporanic acid (7ACA)) was conjugated to digoxigenin in a molar ratio of 1 :1.2 in 100% DMSO.

Thereafter, penicillin binding protein having the biotin ligand and digoxigenin ligand is solubilized to a final concentration of 0.1 μΜ in 100 μΙ buffer (80 mM Tris buffer comprising 0.01 % w/v Triton-X-100, 0.4% w/v BSA, 5% v/v glycerin and 2% w/v saccharose (pH 8.5)) and 0.75 μΙ of the solution is dispensed per tube and dried for 12 hours at 40°C. Next, antibiotic (7-amino-cephalosporanic acid (7ACA)) conjugated to digoxigenin ligand is solubilized to a final concentration of 0.01 to 10 μΜ in 100 μΙ buffer (80 mM Tris buffer comprising 0.01 % w/v Triton-X-100, 0.4% w/v BSA, 5% v/v glycerin and 2% w/v saccharose (pH 8.5)) and 0.75 μΙ of the solution is dispensed per tube and dried for 12 hours at 40°C. The dried material is combined in tubes and the obtained tubes are sealed.

Example 3

Detection of antibiotics with the test device

150 μΙ of spiked milk is added per tube with the dried conjugates prepared as described in Example 2. The concentration of penicillin G in the added milk varies from 0 to 4 ng/g (tube 1 : 0 ng/g; tube 1 : 1 ng/g; tube 2: 2 ng/g; tube 3: 3 ng/g; and tube 4: 4 ng/g). The obtained liquid composition is incubated for 2 minutes at 64°C in an incubator. A test device is put into the tube and incubated for 3 minutes at 64°C in the incubator. The result is read by eye.

The results show that the intensity of the control zone decreases with an increase of the concentration penicillin G in the milk. The signal at the detection zone increases with an increase of the concentration penicillin G. At concentrations of 0 ng/g and 1 ng/g the intensity of the detection zone is clearly lower than the intensity of the control zone. At concentrations of 2 ng/g and 3 ng/g the signal intensity of the detection zone is similar to the signal intensity of the control zone. At a concentration of 4 ng/g the intensity of the detection zone is significantly more intense than the intensity of the control zone.

Example 4

Determination of the signal intensity as a function of the concentration of the conjugated penicillin binding protein

Anti-digoxigenin antibody was diluted with phosphate buffered saline (PBS) to a final concentration of 50 μg/ml. Next, a slot blot assay was performed, wherein anti- digoxigenin antibody was applied to slots containing nitrocellulose membrane (10 μg anti-digoxigenin antibody per slot). The membranes were washed three times with 200 μΙ PBS and blocked with 5% w/v BSA in PBST (PBS with 0.05% w/v Tween-20) under agitation for 1 hour at room temperature. The membranes were washed three times with PBST and then 500 μΙ penicillin binding protein having the biotin ligand and digoxigenin ligand prepared as described in Example 2 was added to each slot in a different concentration. After one hour incubation under agitation at room temperature, the membranes were washed three times with 750 μΙ PBST. Each slot was incubated with 500 μΙ streptavidin-gold conjugates (1000 mOD/slot) under agitation for 1 hour at room temperature. The results are shown in Table 1 and demonstrate that the signal intensity is a function of the concentration of the binding protein.

Example 5

The example was done as described in Example 4 with the proviso that streptavidin-horseradish peroxidase conjugate instead of streptavidin-gold conjugate was used. Each slot was incubated with 500 μΙ streptavidin-horseradish peroxidase conjugate (20 mU/ml) under agitation for 1 hour at room temperature. The membranes were washed three times with 750 μΙ PBST under agitation for 5 minutes at room temperature. Next, 500 μΙ tetramethylbenzidine (TMB) was added to each slot and incubated under agitation for 3 minutes at room temperature. Then, 1 ml water was added to each slot to stop the reaction. The results are shown in Table 2 and demonstrate that the signal intensity decreases when the concentration of the binding protein increases.

Table 1 : Signal intensity as a function of the concentration of the conjugated penicillin binding protein

intensity.

Table 2: Signal intensity as a function of the concentration of the conjugated penicillin binding protein

intensity.

Claims

A method for detecting an antibiotic in a sample, said method comprising the of:

contacting a liquid sample with an antibiotic binding protein and an antibiotic to form a liquid composition, said antibiotic binding protein comprising a ligand 1 and a ligand 2, said ligand 1 capable of binding to a target molecule 1 and said ligand 2 capable of binding to a target molecule 2, said antibiotic comprising a ligand 3, said ligand 3 capable of binding to a target molecule 3,

providing a test device having a proximal and a distal end, said test device configured to allow lateral flow from the proximal to the distal end, said test device comprising a solid support comprising the following regions in sequence from the proximal to the distal end:

ii. a sample receiving region,

iii. a detection region, said detection region comprising at least two zones:

A. a control zone comprising an immobilized target molecule 3, and

v. optionally, a handling region,

contacting the liquid composition with the sample receiving region of the test device,

allowing the liquid composition to move from the sample receiving region through the reaction region and the detection region to the absorbing region, so as to allow:

detecting a signal at the detection zone and a signal at the control zone, wherein i. the absence of antibiotic in the sample is indicated by the absence of a signal at the detection zone or the presence of a signal at the detection zone that is less intense than the signal at the control zone, and ii. the presence of antibiotic in the sample is indicated by the presence of a signal at the detection zone that is more intense than the signal at the control zone.

2. A method according to claim 1 , wherein the liquid composition is incubated for 30 seconds to 5 minutes before it is contacted with the test device.

3. A method according to claim 1 or 2, wherein the liquid composition is incubated at 40 to 70°C before it is contacted with the test device.

4. A method according to any one of the claims 1 to 3, wherein the antibiotic binding protein and the antibiotic are present in powder form before they are contacted with the liquid sample.

5. A method according to any one of the claims 1 to 4, wherein the antibiotic binding protein and the antibiotic are present in a container.

6. A method according to any one of the claims 1 to 5, wherein the antibiotic binding protein is obtained from an antibiotic-sensitive microorganism.

7. A method according to any one of the claims 1 to 6, wherein ligand 1 , ligand 2 and ligand 3 are different and are capable of binding a different target molecule.

8. A method according to claim 7, wherein the label of target molecule 1 is a gold particle.

9. A method according to any one of the claims 1 to 8, wherein the antibiotic comprising ligand 3 and the antibiotic in the sample compete for binding to the antibiotic binding protein.

10. A method according to any one of the claims 1 to 9, wherein the test device is a test strip.

1 1 . A method according to any one of the claims 1 to 10, wherein the test device is contacted with the liquid composition for 1 to 5 minutes at a temperature of 40 to 70°C.

12. A method according to any one of the claims 1 to 1 1 , wherein the sample is milk.

13. A test device for detecting an antibiotic in a sample, said test device having a proximal and a distal end, said device comprising a solid support comprising the following regions in sequence from the proximal to the distal end:

a sample receiving region,

a detection region, said detection region comprising at least two zones:

A. a control zone comprising an immobilized target molecule 3, and

optionally, a handling region,

the liquid composition comprising the labeled antibiotic binding protein and the antibiotic comprising ligand 3 to move to and contact the control zone and the detection zone. comprising:

a container comprising an antibiotic binding protein comprising a ligand 1 and a ligand 2 and an antibiotic comprising ligand 3, said ligand 1 capable of binding to a target molecule 1 , said ligand 2 capable of binding to a target molecule 2, and said ligand 3 capable of binding to a target molecule 3, and

a test device according to claim 13.

15. A kit according to claim 14, further comprising a pipette.