WO2014012558A2

WO2014012558A2 - A dry stick device and a method for determining an analyte in a sample

Info

Publication number: WO2014012558A2
Application number: PCT/DK2013/050245
Authority: WO
Inventors: Kim Clausen
Original assignee: Lattec IS
Current assignee: Lattec IS
Priority date: 2012-07-20
Filing date: 2013-07-19
Publication date: 2014-01-23
Anticipated expiration: 2015-01-20
Also published as: JP2015528114A; CN104704363A; KR20150034732A; WO2014012558A3; EA201590169A1

Description

A DRY STICK DEVICE AND A METHOD FOR DETERMINING AN ANALYTE IN A SAMPLE

Field of the invention

The present invention relates to improved dry stick constructions, methods for producing such dry sticks and their use in analysing an analyte in a sample. The present invention also relates to an automated or semi-automated system comprising the improved dry stick constructions. Additionally the present invention relates to an automated or semi-automated system for optimising the production performance of a milk producing animal herd comprising the improved dry stick constructions.

Background of the invention

The demand for rapid and reliable diagnostic tests is increasing. Today most of the diagnostic tests employed comprise a single layer of porous material (such as a filter pad) impregnated with one or more reagent(s) capable of generating a detectable signal. Such diagnostic tests are non-partitioned as the one or more reagent(s) are present in the single layer of porous material. When using such a non-partitioned dry stick, the porous material is contacted with a fluid sample thereby creating a colour change or a change in colour intensity which is used to verify whether or not a particular effect is achieved or to quantitatively determine the amount of an analyte present in the sample. Dry stick devices comprising non-partitioned environments may compromise both the storage stability and the performance of the impregnated reagent(s) as optimal storage stability and optimal performance is hardly ever achieved at the same environmental conditions (e.g. pH and/or salt content). Thus, by employing non-partitioned dry stick devices the environment of the test paper is created in such a way that the dry stick device displays acceptable (but not optimal) storage stability and acceptable (but not optimal) performance. In order to overcome these problems partitioned dry sticks have been developed. The partitioned dry sticks of the prior art have been created in such a manner as to favour the (i) storage stability of the reagent(s), (ii) performance of the reagent(s) and (iii) the rate of reaction between the analyte and the reagent(s). The partitioned dry sticks of the prior art comprises different layers of pads made from porous materials impregnated with different reagents.

The use of pads however makes the manufacturing process of such partitioned dry sticks time consuming and costly.

When preparing partitioned dry sticks the porous materials making up the pads are impregnated with the respective reagents. The porous materials are subsequently laminated, then sliced and cut into pads before being mounted onto a solid support. Alternately the porous material when sliced is mounted onto a solid support before being cut into sticks. All these process steps are time consuming, costly and increase the risk of manufacturing failures. Additionally the dust generated during the slitting and cutting steps may adversely affect the working environment. Yield losses may be encountered because the porous material must be trimmed in order to avoid using the material edges, where impregnation inconsistencies are frequent.

Except for the lamination step the same process steps have to be employed in the manufacturing of non-partitioned dry sticks comprising a single pad of a porous material.

Clearly there is a need in the art for improved non-partitioned as well as partitioned dry sticks that are reproducible, cheap and which are easy and fast to produce. Moreover there is a need for a production method which does not adversely affect the working environment. Summary of the invention

An object of the present invention relates to improved dry stick constructions for the determination of an analyte in a sample. The improved dry stick constructions may be non-partioned or partioned dry stick constructions.

In particular, it may be seen as an object of the present invention to provide improved dry stick constructions that solve the above mentioned problems of the prior art by avoiding the use of porous materials.

Thus, the present invention relates in one aspect to a dry stick for the

determination of an analyte in a sample, said device comprises a solid support comprising a cavity and at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent.

Another aspect of the present invention relates to a dry stick for determining an analyte in a sample, said device comprising a solid support comprising a cavity and at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity. An aspect of the present invention relates to a dry stick for determining an analyte in a sample, said device comprising a solid support comprising a cavity and at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity by drying.

A further aspect of the present invention relates to a method for the

determination of an analyte in a sample, said method comprising the steps of (a) applying the sample to a cavity of a dry stick device comprising a solid support comprising the cavity, at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent and (b) permitting the at least one dry reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte to react and provide a detectable signal.

Yet a further aspect of the present invention relates to a method for the determination of an analyte in a sample, said method comprising the steps of (a) applying the sample to a cavity of a dry stick device comprising a solid support comprising the cavity, at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity and (b) permitting the at least one dry reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte to react and provide a detectable signal.

Yet another aspect of the present invention relates to a method for preparing a dry stick device, said method comprises the steps of providing a support comprising a cavity, providing at least one dry reagent or at least one reagent, applying the at least one dry reagent or the at least one reagent to the cavity and obtaining the dry stick device.

Still another aspect of the present invention relates to an automated or semi- automated system comprising (a) a dry stick device for the determination of an analyte in a sample, said device comprises a solid support comprising a cavity, at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent and (b) sampling means for obtaining a sample to be analysed, said sampling means being controlled by means for storing data, said sampling means is only activated at pre-selected points in time or at pre-selected time intervals.

An aspect of the present invention relates to an automated or semi-automated system comprising (a) a dry stick device for the determination of an analyte in a sample, said device comprises a solid support comprising a cavity, at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity and (b) sampling means for obtaining a sample to be analysed, said sampling means being controlled by means for storing data, said sampling means is only activated at pre-selected points in time or at pre-selected time intervals.

A further aspect of the present invention relates to an automated or semi- automated system for optimising the production performance of a milk producing animal herd comprising a plurality of individual herd members each assigned a unique identification code that is recognisable by the system, the system comprising analysing means for analysing at least one analyte in a milk sample of an individual member of the milk producing animal, said analysing means comprising (a) a dry stick device for the determination of an analyte in a sample, said device comprises a solid support comprising a cavity, at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent and (b) means for directing a part of the milk sample to each separate analysing means, said directing means being controlled by means for storing data for the physiological and nutritional state of each individual herd member, including data indicating point in time in the reproduction and lactation cycles of said herd member, such that the directing means is only activated at pre-selected points in time or at pre- selected time intervals in the production or lactation cycles of the individual herd member.

A further aspect of the present invention relates to an automated or semi- automated system for optimising the production performance of a milk producing animal herd comprising a plurality of individual herd members each assigned a unique identification code that is recognisable by the system, the system comprising analysing means for analysing at least one analyte in a milk sample of an individual member of the milk producing animal, said analysing means comprising (a) a dry stick device for the determination of an analyte in a sample, said device comprises a solid support comprising a cavity, at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity and (b) means for directing a part of the milk sample to each separate analysing means, said directing means being controlled by means for storing data for the physiological and nutritional state of each individual herd member, including data indicating point in time in the reproduction and lactation cycles of said herd member, such that the directing means is only activated at pre-selected points in time or at pre-selected time intervals in the production or lactation cycles of the indivi- dual herd member.

Brief description of the figures

Figure 1A-B shows examples of a dry stick device for the determination of an analyte in a sample.

Figure 2A-2C shows examples of a compartmented dry stick device for the determination of an analyte in a sample.

Figure 3 shows an example of a tiered dry stick device for the determination of an analyte in a sample.

Figure 4 shows examples of a dry stick for the determination of an analyte in a sample comprising lines of one or more dry reagent(s). Figure 5 shows examples of a dry stick for the determination of an analyte in a sample comprising dots of one or more dry reagent(s).

Figur 6 shows an example of a dry stick device for the determination of an analyte in a sample comprising separate layers of different dry reagent(s).

Figure 7 shows a comparison between (i) a method for producing a traditional non-partitioned dry stick where the reagents are present in the same pad and (ii) the method of the present invention. As an example of such a traditional non- partitioned dry stick is used a BHB dry stick. Figure 8 shows a comparison between (i) a method for producing a traditional partitioned dry stick where the reagents are separated into different pads and (ii) the method of the present invention. As an example of such a traditional partitioned dry stick is used an LDH dry stick.

Figure 9 shows the performance curve of (i) a traditional LDH stick vs. (ii) the new LDH dry stick. The present invention will now be described in more detail in the following. Detailed description of the invention

The inventor of the present invention has developed new and improved dry stick constructions comprising no porous materials. The dry stick constructions of the present invention may be non-partitioned or partitioned with respect to the reagents involved. In the present context the term "partitioned" relates to a dry stick device having different environments (such as different reagents, pH-values and/or salt concentrations) in the same dry stick. The term "non-partitioned" relates on the other hand to a dry stick device comprising only one environment in which the reagent(s) are present.

In an embodiment the dry stick constructions of the present invention

simultaneously complies with the requirements of good stability during storage and good performance during testing or assaying as required by the industry and/or end user.

Dry stick construction and methods for determining an analyte in a sample

The improved dry stick construction of the present invention comprises (i) a solid support comprising a cavity and (ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent. In the present context the term "cavity" is to be understood as a hollow space such as a well or any similar indentation on the solid support. In an embodiment of the present invention the volume of the cavity is in the range from 0.1-1000 μΙ_, such as in the range from 1-100 μΙ_, e.g. in the range from 25-750 μΙ_ , such as in the range from 100-500 μΙ_, preferably in the range from 2-50 μΙ_.

As the detectable signal generated using the dry stick of the present invention does not need to pass through a porous material before being detected the detectable signal generated is not diluted by the presence of the porous material and less susceptible to variations. In traditional dry sticks such variations may be caused by the porous material. Such variations are difficult to avoid since porous materials notoriously vary both in terms of quality and colour. Using a porous material with decreased quality or with a different or uneven colour may lead to a differentiation in the detectable signal otherwise generated, thus, compromising performance of the dry stick. Additionally mixing of analyte and dry reagent(s) may be difficult in porous materials. It is therefore a great advantage that the detectable signal can be obtained directly from the sample in the dry stick of the present invention in a one-step procedure. Thus, in an embodiment of the present invention the detectable signal generated is obtained from the sample. In a further embodiment of the present invention the detectable signal generated is directly obtained from the sample

In an embodiment the present invention therefore relates to a dry stick device for the determination of an analyte in an aqueous sample, said device comprises a solid support comprising a cavity and at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent and wherein the detectable signal generated is obtained from the sample.

In the present context the term "traditional dry stick" is to be understood as a dry stick comprising a porous material. The traditional dry stick may be a non- partitioned or a partitioned dry stick. In the present context the term "porous material" relates to a material which adsorbs the sample and thereby permits it to spread out more or less evenly throughout the porous material.

Additionally the amount of reagent(s) and/or dry reagent(s) used in the dry stick constructions of the present invention may be reduced compared to the amount used in traditional dry sticks. The reason being that a much stronger signal is generated when no porous material is to be passed before detection. Likewise the amount of sample needed to generate a detectable signal using the dry stick of the present invention is reduced (compared to traditional dry sticks) as adsorption in the porous material is avoided.

Thus, in an embodiment of the present invention the dry stick does not comprise a porous material. The at least one dry reagent may be present in the dry stick as a solid (e.g. a powder or a compacted powder). If two or more dry reagents are present in the dry stick such dry reagents may be present as seperate powders or separated compacted powders in the cavity of the dry stick. In order to keep the solid in the cavity during handling of the dry stick the cavity may be sealed with a membrane. In one embodiment the sample is applied to the cavity by perforating the membrane thus bringing the at least one reagent in contact with the sample applied. In a preferred embodiment the membrane may be selected from the group consisting of polyethylene, polypropylene,

polystyrene, polyvinyl chloride, polytetrafluoroethylene, bio plastics and combinations thereof .

It may be contemplated to use a transparent membrane. Alternatively the membrane may be made from a material which is dissolvable by the sample applied thus bringing the at least one dry reagent in contact with the sample.

Alternatively, and in order to avoid the use of a membrane, the at least one dry reagent may be fixed to at least part of the cavity. Thus, "a dry reagent fixed to at least part of the cavity" is to be understood as a dry reagent substantially impeded to move until contacted with the sample.

Thus, in one embodiment the present invention relates to a dry stick for the determination of an analyte in a sample, said device comprises: (i) a solid support comprising a cavity and (ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compround for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity.

In an embodiment of the present invention such fixing comprises coating, adhesion and drying.

Immobilizing the at least one dry reagent would prohibit the movability of the molecules of the at least one dry reagent when the sample is applied. Thus, such immobilization may negatively affect the performance during testing or assaying. In order to compensate for this lack of performance during testing the amount of immobilized dry reagents needed to achieve optimal performance may need to be increased thus, leading to an increased cost per dry stick produced.

Thus, in an embodiment of the present invention the at least one dry reagent is not immobilized to the cavity of the dry stick.

Immobilizing is especially known from immunoassays (such as ELISA) where the presence or concentration of a macromolecule is measured in a solution through the use of an immobilized antibody and/or immobilized immunoglobulin. The mode of operation of an immunoassay is different from the mode of operation of the dry stick of the present invention. In the dry stick of the present invention the sample is applied and the detectable signal is obtained directly from the applied sample. In immunoassays several washing steps are needed before a detectable signal can be obtained making such an assay more expensive and time consuming compared to the dry stick of the present invention. Moreover one may need to add a liquid reaction mixture to the immunoassay after applying the sample in order obtain a detectable signal. In another embodiment of the present invention the dry stick does not comprise an antibody and/or immunoglobulin. In yet another embodiment of the present invention the dry stick is not an immunoassay. Depending on the form of the at least one reagent(s) and/or at least one dry reagent(s) the at least one reagent(s) and/or at least one dry reagent(s) may automatically adher to at least part of the cavity once applied - this may be the case where the at least one dry reagent(s) and/or the at least one dry reagent(s) are in the form of a "sticky" substance (comprising substantially no water).

In a particular preferred embodiment the at least one reagent is dried onto at least part of the cavity. Thus, such a dry stick device may comprise: (i) a solid support comprising a cavity and (ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compround for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity by drying.

In the present context the term "dried" is to be understood as a process where water and/or organic solvent(s) are removed (e.g. by evaporation) from a reagent thus obtaining a dry reagent.

The inventor of the present invention has also provided a new and improved method for the determination of an analyte in a sample. The method comprising the steps of:

(a) applying the sample to a cavity of a dry stick device comprising :

(i) a solid support comprising the cavity (ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent, and (b) permitting the at least one dry reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte to react and provide a detectable signal. The detectable signal may be generated by any substance which directly or indirectly is capable of being observed by any kind of visual or instrumental means. The instrumental means may be e.g. a magno(magne)tometer, spectrophotometer, ELISA-reader and/or CCD camera. Various suitable

compounds may be suitable as a signal producing compound. In the present invention the signal producing compound may be selected from the group consisting of chromogens, catalysts, fluorescent compounds, chemiluminescent compounds, radioactive labels, metals, magnetic particles, dye particles, organic polymer latex particles, liposomes or other vesicles containing signal producing substances and the like.

Thus, in an embodiment of the present invention the method comprising the steps of:

(a) applying the sample to a cavity of a dry stick device comprising :

(i) a solid support comprising the cavity

(ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent,

(b) permitting the at least one dry reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte to react and generate a detectable signal, and

(c) obtaining the detectable signal from the sample.

As mentioned above no washing step is needed in order to obtain a detectable signal using the dry stick of the present invention. Thus, in an embodiment of the present invention the method does not comprise a washing step.

Before the detectable signal can be obtained a short incubation may be needed. Thus, in an embodiment of the present invention the at least one dry reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte is permitting to react in at least 1 minute, such as at least 2 minutes, e.g. in at least 3 minutes, such as in at least 4 minutes, e.g. in at least 5 minutes, such as in at least 6 minutes, e.g. in at least 7 minutes, such as in at least 8 minutes, e.g. in at least 9 minutes, such as in at least 10 minutes, e.g. from 1-10 minutes, such as from 2-9 minutes, e.g. from 3-8 minutes, such as from 4-7 minutes, e.g. from 5-6 minutes, preferably from 1-5 minutes.

In the present context the term "in a moistened state" relates to the contact between the at least one dry reagent and the sample whereby the at least one dry reagent becomes wet or slightly wet. The effect of the moistened state is that the at least one dry reagent is liberated and dissolved (mobilised) and the reaction in the dry stick device commences and a detectable signal is produced, which is dependent on the amount of analyte present in the sample.

In an embodiment the detectable signal is obtained from the top, bottom or sides of the cavity directly after application of the sample. This means that no washing step is required in order to obtain a detectable signal - thus, the detectable signal can be obtained from the cavity in a one-step procedure. Accordingly, there is also no need to sequentially add liquids to the cavity besides the addition of the sample in order to obtain a detectable signal. Therefore the dry stick constructions of the present invention is not ELISA.

In a further embodiment the present invention relates to a method for the determination of an analyte in a sample, said method comprising the steps of:

(a) applying the sample to a cavity of a dry stick device comprising :

(i) a solid support comprising the cavity (ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the at least one dry reagent is fixed onto at least part of the cavity, and

(b) permitting the at least one dry reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte to react and provide a detectable signal.

It may be preferred that such fixing is drying. Solid support

The dry stick of the present invention comprises a solid support. In the present context, the term "solid support" refers to a material, which has no influence on the migration or on the reaction of the sample or on reagent(s). In other words the solid support is a fixture to keep the reagents in place.

The solid support provides a stabilising basis for the dry stick and provides sufficient strength to maintain the desired physical shape and has substantially no interference with the production of a detectable signal.

In an embodiment of the present invention, the material for the solid support may be selected from the group consisting of membranes, plastic, glas, metal, porcelain and combinations thereof. If the detectable signal is obtained from the side and/or the bottom of the cavity it may be preferred that the solid support (or at least the cavity) is made from a transparent material.

The plastic may be a synthetic organic solid, a semi-synthetic organic solid and a combination thereof. Likewise the plastic may be a thermoplastic polymer, a thermosetting polymer and combinations thereof. In an embodiment the plastic may be selected from the group consisting of polyethylene, polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, bio plastics and combinations thereof . The reaqent(s) and/or dry reaqent(s)

The "reagent" and/or "reagents" may be in the form of a solid, a semi-solid or a liquid. Prior to being applied to the solid support the "reagent" and/or "reagents" may be dissolved in water and/or organic solvent(s) - alternatively they may be applied to the solid support in their original form (i.e. the form of a solid, a semisolid or a liquid).

Thus, in the present context the terms "reagent", "reagents", "dry reagent" and/or "dry reagents" relates to the chemical and/or enzymatic substances that react with, participate in or are necessary for the determination of an analyte, a derivative of said analyte or an indicator compound for said analyte present in a sample to provide a detectable signal. A similar definition of the combination of reagents may be provided which relates more specifically to 2 or more reagents, such as 3 or more reagents, e.g. 4 or more reagents, such as 5 or more reagents, e.g. 6 or more reagents.

In the present context the term "dry reagent" is to be understood as a reagent comprising substantially no water. Thus, in an embodiment of the present invention the dry reagent comprises less than 5% w/w water, such as less than 4% w/w water, e.g. less than 3% w/w water, such as less than 2% w/w water, e.g. less than 1% w/w water, such as less than 0.5% w/w water, e.g. less than 0.01% w/w water, such as in the range from 0.01% w/w - 5% w/w water, such as in the range from 1% w/w - 4% w/w water, e.g. in the range from 2% w/w - 3% w/w water, such as in the range from 0.5% w/w - 1% w/w water, e.g. in the range from 0.01% w/w - 0.5% w/w water . In a preferred embodiment the dry reagent comprises no water. In an embodiment of the present invention the dry reagent may be in the form of a solid (e.g. a powder), a semi-solid or a liquid in the obtained dry stick. Analvtes

A dry stick or a method based on the above principles can be used to determine a wide range of analytes by choice of appropriate signal producing compounds known to the person skilled in the art. Thus, the invention need not be limited to the examples mentioned herein. In an embodiment of the present invention the analyte to be assayed may be selected from the group consisting of a protein, an enzyme, a lipid, a

carbohydrate, an antibiotic, a hormone, a vitamin and a chemical compound.

In an embodiment of the present invention the hormone is a steroid. The steroid may be selected from the group of pregnenolone, progesterone, testosterone, dihydrotestosterone, estrone, estradiol, Cortisol, cortisone, aldosterone, corticosterone, androstenedione, 17a-OH- pregnenolone, 17a-OH- progesterone, 11-desoxy-corticosterone, 11-desoxycortisol, dehydroepiandrosterone and luteinising hormone.

The enzyme may be selected from the group consisting of catalase, lactate dehydrogenase (LDH), alkaline phosphatase, acid phosphatase, carboxylesterase, arylesterase, β-glucuronidase, lactoperoxidase, lipase, lysozyme, xanthine oxidase, plasmin and beta-N-acetylhexosaminidase (NAGase) and prostaglandin D synthase (PGDS).

In a preferred embodiment the enzyme is selected from the group consisting of LDH and NAGase.

The chemical compound may be selected from the group consisting of urea, triglyceride and ketone bodies. The ketone body mad be selected from the group consisting of acetoacetate, beta-hydroxybutyrate (BHB), citric acid, lactic acid, acetone, ascorbic acid, nitrates, urobilinogen and cholesterol.

In a preferred embodiment the chemical compound is selected from the group consisting of urea and BHB. The carbohydrate may a monosaccharide and/or a disaccharide. The

monosaccharide may be glucose or galactose. The disaccharide may be lactose.

In an even more preferred embodiment the analyte is selected from the group consisting of LDH, urea and BHB. In another embodiment the analyte may be an enzyme irrecpective of whether an enzyme is present in the dry stick and participate in the derermination of the analyte or not. Sample

In the present context, the term "a sample" relates to any sample found in the form of liquid or solution. The term also relates to a gas which may be liquefied, dissolved and/or suspended in a liquid at the time of assaying. The term also relates to a solid sample once it is modified to form a liquid sample, for instance in the form of a solution, a suspension or an emulsion.

It is preferred that a minimum number of handling steps, of the liquid sample is necessary before applying it to the dry stick test. In the present context, the term "handling steps" relates to any kind of pre-treatment of the liquid sample before application to the assay device. This pre-treatment comprises separation, filtration, dilution, distillation, concentration, inactivation of interfering

compounds, centrifugation, heating, fixation, addition of reagents, or chemical treatment. In an embodiment of the present invention the sample may be collected from a mammal, preferably the mammal is selected from the group consisting of herd animals, cows, camels, buffaloes, pigs, horses, deer, sheep, goats, pets and humans. The sample may also be collected from other types of animals such as snakes, fish and birds.

In a preferred embodiment of the present invention, the sample can be derived from any desirable source, however, it is preferred that the sample is selected from the group consisting of milk, blood, serum, plasma, saliva, urine, sweat, ocular lens fluid, cerebral spinal fluid, ascites fluid, mucous fluid, synovial fluid, peritoneal fluid, amniotic fluid, hair and the like.

Samples may also be obtained from a bacteria or a bacterial derived source, a plant or plant derived source, a fungi or a fungal derived source, a production process, an aqueous source, a food source, a feed source, an oil source and/or a mineral source. The production process may be any kind of production process such as but not limited to fermentation processes, biochemical processes and a chemical synthesis processes.

Likewise the aqueous source may be any kind of aqueous source such as but not limited to lakes, streams, rivers, open waters and purification plants.

The mineral source may be a mining stream.

Further embodiments of the dry stick construction

In an embodiment of the present invention the at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound of said analyte may be present in separate parts of the cavity.

Such separation of dry reagent(s) provides a faster and more uniform detectable signal during assaying. If two or more dry reagents are present in the dry stick such separation of dry reagents may improve the storage stability of the dry stick as any kind of reaction between the dry reagents is avoided. When in a moistened state the dry reagent(s) react with the analyte to provide a detectable signal.

Thus, a dry stick comprising no separation of dry reagents may be seen as a non- partitioned dry stick whereas a dry stick comprising two or more separated dry reagent(s) may be seen as a partitioned dry stick - as each part of the cavity in principle is a different environment. When in a moistened state a new

environment is generated.

In an embodiment the dry reagent(s) may be separated in dry lines or in dry dots present on the surface of the cavity (please see Figures 4 and 5 as examples).

To provide an easy separation of the dry reagents and improve the storage stability of the dry stick the cavity may be compartmented (please see Figures 2A-2C as examples). Thus, in an embodiment of the present invention the cavity comprises at least 2 compartments, such as at least 3 compartments, e.g. at least 4 compartments, such as at least 5 compartments, e.g. at least 10 compartments, such at least 20 compartments, e.g. at least 30 compartments.

It thus follows that 2 or more dry reagents may be fixed in separate

compartments, such as 3 or more dry reagents, e.g. 4 or more dry reagents, such as 5 or more dry reagents, e.g. 10 or more dry reagents, such as 20 or more dry reagents, e.g. 30 or more dry reagents, such as 40 or more dry reagents, e.g. 50 or more dry reagents. The dry reagents may be the same or different.

In a preferred embodiment 2 or more dry reagents are dried onto separate compartments, such as 3 or more dry reagents, e.g. 4 or more dry reagents, such as 5 or more dry reagents, e.g. 10 or more dry reagents, such as 20 or more dry reagents, e.g. 30 or more dry reagents, such as 40 or more dry reagents, e.g. 50 or more dry reagents. Again the dry reagents may be the same or different.

The compartmented cavity can be seen as an alternative to the embodiment where the dry reagent(s) are present in different parts of the cavity. The results are believed to be the same - the provision of a faster and more uniform signal and/or an improved storage stability of the dry stick.

In another embodiment the cavity is tiered (please see Figure 3 as an example). Thus, if a certain chain of reactions are desired the dry stick may be designed with a specific sequence of dry reagents on the tiered cavity. Such a dry stick comprising a separation of dry reagents may show improved storage stability compared to traditional dry sticks as any reaction between the dry reagent(s) are avoided prior to sample application. Moreover the sequence of dry reagents may be arranged to limit and/or avoid precipitation of sample components thus improving the detectable signal obtained. In that respect please see the parts below regarding a dry stick comprising separate layers as the same considerations regarding precipitation are applicable to a dry stick comprising a tiered cavity.

In a further embodiment of the present invention the dry reagents may be present in at least 2 separate layers on at least part of the cavity, such as in at least 3 layers, e.g. at least 4 layers, such as at least 5 layers, e.g. at least 6 layers on at least part of the cavity (please see Figure 6 as example).

In an embodiment one of the dry reagents may be a controlling compound capable of providing a condition required for the dry reagent or the combination of dry reagents to react with the analyte to provide a detectable signal. Additionally the controlling compound may be capable of increasing the rate of the reaction between the analyte a derivative of said analyte or an indicator compound for said analyte present in the sample and the dry reagent(s). In an embodiment of the present invention the controlling compound may be an acid, a base or a salt.

Preferably, the controlling compound is an acidic compound capable of providing a pH-value of the sample in the dry stick test device, when in a moistened state, below pH 6, such as below pH 5, e.g. below pH 4, such as below pH 3, e.g. below pH 2, such as below pH 1, e.g. below pH 0, such as in the range of pH 0-6, e.g. in the range of pH 0-5, such as in the range of pH 0-4, e.g. in the range of pH 0-3, such as in the range of pH 0-2, e.g. in the range of pH 0-1, such as in the range of pH 1-6, e.g. in the range of pH 2-6, such as in the range of pH 3-6, e.g. in the range of pH 4-6, such as in the range of pH 5-6.

In another embodiment of the present invention the controlling compound may be an alkaline compound capable of providing a pH-value of the sample in the dry stick test device, when in a moistened state, of pH 8 or above, such as in the range of pH 8-14, e.g. in the range of pH 8-13, such as in the range of pH 8-12, e.g. in the range of pH 8-11, such as in the range of pH 8-10, e.g. in the range of pH 8-9, such as in the range of pH 9-12, e.g. in the range of pH 10-13, such as in the range of pH 10-11.

In one embodiment of the present invention the controlling compound may be separated from the at least one dry reagent. Such separation may for example be in different parts of the cavity or in separate layers on at least part of the cavity.

Such a separation of dry reagents (including any controlling compound(s)) may improve stability, storage properties and applicability of the dry stick because non-compatible reagents can be included in different layers. Accordingly any reaction between the reagents is avoided prior to sample application.

Moreover the sequence of layers may be arranged to limit and/or avoid

precipitation. By precipitation of sample components are meant sample

components of the fluid sample or part of the fluid sample that change into a solid or semisolid mass, often caused by the action of e.g. heat or chemical substances such as a strong acid, a strong base or a high salt concentration (such a chemical substance may be a controlling compound).

Coagulated sample components may interfere and reduce the detectable signal. Thus, if a strong acid, a strong base or a high salt concentration is needed in the determination of a specific analyte it may be preferred that such a compound is placed in the layer next to the solid support in order to prevent precipitation of sample components.

The sample component may be selected from the group consisting of proteins, carbohydrate, fat, cells, or other components present in the sample. In a preferred embodiment the sample is milk.

In a preferred embodiment at least two reagents are present in at least 2 separate layers dried onto at least part of the cavity, such as in at least 3 layers, e.g. at least 4 layers, such as at least 5 layers, e.g. at least 6 layers.

Obviously the various dry stick constructions outlined above may be used for assaying an analyte in a sample.

Preparation of the dry stick

A comparison between a method for the preparation of a traditional dry stick and methods for the preparation of the dry stick constructions of the present invention can be seen in Figures 7 and 8. The dry stick device according to the present invention may be prepared by any conventional methods provided for the preparation of the dry stick devices. In a preferred embodiment the method for providing a dry stick device according to the present invention comprises the steps of:

(i) providing a support comprising a cavity;

(ii) providing at least one dry reagent,

(iii) applying the at least one dry reagent to the cavity and obtaining the dry stick device.

Comparing the traditional method for the preparation of a non-partitioned dry stick where reagent(s) are present in the same pad (i.e. the same porous material) with the above method of the present invention, the following process steps are made superfluous:

I. Sourcing of the porous material

II. Impregnation of one or more reagents into the porous material

III. Cutting the porous material into strips,

IV. Cutting the strips into pads,

V. Mounting the pad in a cartridge

In respect of methods for the preparation of immunoassays such a production requires several steps of incubation and washing to immobilize antibody and/or immunoglobulin - such steps are made superfluous by the method of the present invention.

In a further embodiment the dry stick of the present invention may be prepared by a method comprising the steps of: (i) providing a support comprising a cavity;

(ii) providing at least one solution comprising at least one reagent,

(iii) applying the at least one solution to at least part of the cavity.

(iv) fixing the solution, and obtaining the dry stick device. In one embodiment said fixing may be drying.

Comparing the traditional method for the preparation of a partitioned dry stick where reagents are present in separate pads (i.e. separate porous materials) with the above method of the present invention, the following process steps are made superfluous:

I. Sourcing of the porous material

II. Impregnation of reagents onto at least two separate porous materials, III. Lamination

IV. Cutting the impregnated porous materials into strips,

V. Cutting the strips into pads,

VI. Mounting the pads in a cartridge Clearly the number of method steps is significantly reduced using the methods of the present invention thus, improving process economy. Additionally the reduced number of method steps decreases the risk of process failures thus, limiting the amount of sticks discarded. In the traditional methods several intermediate products are generated (such as the one or more impregnated porous material(s)). As such intermediate products are avoided using the methods of the present invention the need for dry and cool storage is reduced. The methods of the present invention do not comprise the steps of cutting and/or slitting pads. Therefore, no cutting and/or slitting equipment for pads are needed in the methods of the present invention thus, providing improved manufacturing economy. Accordingly, the amount of dust generated during such a process step is avoided. The dust generated in the traditional methods for preparing dry sticks may affect subsequent process steps and may adversely affect the working environment. Thus, normally such dust is actively removed adding further costs to the manufacturing process. As no pads are needed in the dry stick constructions of the present invention the method for the preparation of these sticks can easily be adjusted to the size of the dry stick. Methods for the preparation of traditional dry sticks need new cutting and/or slitting equipment or at least an adjustment of the cutting and/or slitting equipment if the size of the dry stick is changed thus, adding time and costs to the manufacturing process.

Additionally, the pads used in traditional dry sticks are mounted using glue, tape or a similar adhesive. Glue remainders may affect performance of the dry stick. Glue remainders especially on the sides of the pads may cause the applied sample to move towards such side causing an oblique signal. Such oblique signal affects the read out thus, increasing the risk of false results (both false negative and false positive). In an embodiment the at least one solution comprises at least one reagent.

The solution comprising the at least one reagent may be selected from the group consisting of an aqueous solution and/or an organic solvent. If the solution comprises water it is preferred that such water is removed (e.g. by evaporation) by the end of the manufacturing process.

By using the method of the present invention it is possible to obtain a partitioned dry stick comprising separate dry reagents. Thus, if such a dry stick is desired the method comprises applying at least 2 different solutions comprising different reagents, such as 3 or more different solutions, e.g. 4 or more different solutions, such as 5 or more different solutions, e.g. 6 or more different solutions, such as 7 or more different solutions, e.g. 8 or more different solutions, such as 9 or more different solutions, e.g. 10 or more different solutions.

As mentioned above such a separation of reagents and/or dry reagents may be obtained by separating the reagents and/or dry reagents (i) in separate parts of the cavity, (ii) in different compartments of the cavity, (iii) on different parts of a tiered cavity and/or (iv) in different layers on the cavity. The separation of dry reagents (including any controlling compound(s)) in separate parts of the cavity may be achieved by non-contact dispensing or contact dispensing where lines and/or dots of a solution comprising the at least one reagent are dispensed on separate parts of the cavity. The reagents may be fixed by any one of the methods previously mentioned. In a preferred embodiment the lines and/or dots may be dried following application of the at least one solution comprising the at least one reagent.

The provision of different layers comprising separate dry reagents (including any controlling compound(s)) may be achieved by continuously repeating the steps of providing at least one solution comprising at least one reagent and applying such solution to at least part of the cavity. Such repetition may be conducted in order to obtain 2 or more separate layers, such as 3 more separate layers, e.g. 4 or more separate layers, such as 5 or more separate layers, e.g. 6 or more separate layers. Each layer comprises a dry reagent or a combination of dry reagent(s) which is distinct from each other.

Depending on the solutions used it may be advantageous to coat each layer with a layer preventing the dissolution of the layer previously applied. Such a coating may comprise a wax. It may be contemplated to use a coating which is

dissolvable by the sample applied in order for the at least one dry reagent to be able to react with the analyte once the sample is applied.

The above mentioned methods can be used to provide the various dry stick constructions disclosed above. Thus, the present invention pertains to dry stick devices obtainable by the above mentioned methods.

Additional embodiments

In an embodiment the dry stick test device according the present invention is used for the determination of an analyte in a sample. Preferably, the analyte is selected from the group consisting of a protein, a fat, a carbohydrate, an antibiotic, a steroid, a vitamin and a chemical compound. Preferably, the chemical compound is selected from the group consisting of urea, triglyceride and ketone bodies, such as acetoacetate, beta-hydroxybutyrate (BHB), ascorbic acid (citric acid) and acetone. If the analyte is a carbohydrate the carbohydrate may be selected from the group consisting of glucose and lactose.

Determination of LDH

As mentioned above, the inventor of the present invention has developed new dry stick constructions for the determination of an analyte in a sample, such analyte could be lactate dehydrogenase (LDH) in body fluids. The dry stick device according to the present invention may be useful for the qualitative detection and the quantitative determination of LDH in a sample wherein the test means comprises at least one dry reagent composition.

The quantitative determination of LDH may be important for the determination of mammary inflammation which affects the integrity of mammary gland structure and concurrently damages the secretary epithelia and the blood-milk barriers. Consequently, many milk components are influenced by mastitis. Major

components such as fat, protein and lactose are reduced and a number of enzymes are altered. LDH and/or N-acetyl glucosaminidase may be used as suitable indicators for the inflammation caused by mastitis. N-acetyl-β-Ο- glucosaminidase, also called N-acetyl glucosaminidase (NAGase), has been claimed to be one of the better markers for mammary inflammation. Furthermore, it has been shown that other enzymes in milk, like LDH may be of similar value and act as a suitable indicator of mastitis as does NAGase.

Alternatively, the quantitative determination of LDH may be extremely important in the detection of heart diseases, especially heart attacks, in that, following heart attacks, the concentration of LDH in e.g. blood rises noticeably over its normal concentration. The early detection of such an abnormal rise in LDH concentration can therefore obviously lead to a more accurate and rapid diagnosis of heart maladies.

Because early diagnosis of abnormal heart conditions is so important, a test for the detection of variables in the concentration of LDH in the blood must be rapid and simple enough for the clinician to carry out but accurate enough to enable the diagnosis to be made without extreme changes of error or false readings. Such a mechanism is represented by the novel dry stick device of the present invention. Utilizing this novel dry stick device, no instrumentation is necessary and no mixing or reconstitution of reagents is needed. Testing can therefore be conducted in the patient's home or in a doctor's office without any special equipment.

In an embodiment of the present invention a method for assaying LDH in a sample may comprise the steps of:

(i) applying the sample suspected of containing LDH to the dry stick device of the present invention

(ii) permitting the sample to dissolve a a first dry reagent or a combination of dry reagents capable of reacting with LDH, a derivative of LDH or an indicator compound for LDH to provide a detectable signal when in a moistened state, the first dry reagent or a combination of dry reagents providing a first environment for said reagent(s), first environment permitting an improved storage stability of the reagent(s) and dry stick device when in a non- moistened state

(iii) permitting the sample to react further with a second dry reagent composition or a combination of dry reagents, the regulating reagent composition, said regulating reagent composition creating a second environment for said reagent(s), when in a moistened state, said second environment permitting an increased rate of reaction between LDH and the reagent(s), and

(iv) permitting the reagent and LDH, the derivative of LDH or the indicator compound for LDH to provide a detectable signal.

In a further embodiment of the present invention the determination of LDH is based on an enzyme-based determination.

The novel dry stick device for the determination of the concentration of LDH, in a sample may comprise at least one dry reagent composition . The dry reagent compositon may comprise a dry reagent, a combination of dry reagents or a series of dry reagents.

One example of such a dry reagent may be a tetrazolium salt. This reagent is capable of imparting to the dry stick device contacted with sample a colour of such varying intensity as to be representative of the concentration of the LDH in the sample which is added to the indicator. These dyes are well known in the art and generally have the formula :

wherein R¹, R² and R³, individually, are the same or different aryl or substituted aryl radicals and X is an anion such as a halide etc. Examples of useful salts of this configuration include 2,3,5-Triphenyl-2H- tetrazolium chloride; 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT); nitroblue tetrazolium; blue tetrazolium; and the like. These salts may be incorporated into the novel dry stick device in concentrations ranging from about 0.05 part to about 0.35 part, preferably, from about 0.1 part to about 0.2 part, based on 100 parts of solution used, in a manner set forth hereinbelow.

The dry stick may further comprise an anti-oxidant which is employed in order to prevent premature coloration of the tetrazolium salt. Examples of suitable antioxidants include the alkylated phenols such as 2,6-ditertiary butyl-p-cresol; butylated hydroxytoluene, 4-t-butyl catechol, octadecyl-3,5-di-t-butyl-4-hydroxy hydrocinnamate; alkylidene bisphenols such as 2,2'-methylenebis (6-t-butyl-4- methyl phenol), 4,4'-butylidenebis (6-t-butyl-3-methyl phenol); thiobisphenols such as 4,4'-thiobis (6-t-butyl-3-methylphenol), 2,2'-thiobis (6-t-butyl-4-methyl phenol); polyphenols such as tetrakis [methylene (3,5-di-t-butyl-4- hydroxyhydrocinnamate)] methane, l,3,5-trimethyl-2,4,6-tris (3,5-di-t-butyl-4- hydroxybenzyl) benzene; esters such as ditridecyl thiodipropionate,

distearylthiodipropionate, dilaurylthiodipropionate; amines such as diaryl or dialkyl substituted p-phenylene diamines, diphenylamine, N-phenyl-. alpha. - naphthylamine; organic phosphites such as dibutyl phosphite, didecyl phosphite, dioctyl phosphite, diphenyldecyl phosphite, ditetradecyl phosphite, phenyldidecyl phosphite, phenylneopentyl phosphite, tridecyl phosphite, trilauryl

trithiophosphite, triphenyl phosphite, trisnonyl phosphite and various other well known anti-oxidants such as the quinones including hydroquinone, hydroquinone monomethyl ether, mono-t-butylhydroquinone, 2,5-di-t-butyl hydroquinone, toluhydroquinone, 2,5-di-t-amyl hydroquinone and the like. I may also use phenothiazine, hydroxybenzophenone, p-dimethylaminonitrosobenzene, thiodipropionic acid etc.

These anti-oxidant materials may be used in amounts ranging from about 0.01 part to 2.0 part, preferably from about 0.02 part to 1.0 part based on 100 parts of solution and may be used in conjunction with the tetrazolium salt or before or after deposition thereof.

Another example of a dry reagent may be diaphorase which is used to catalyze the reduction of the tetrazolium salt with NADH. This enzyme is well known in the art and should be employed in concentrations ranging from about 0.02 part to 0.2 part by weight and is perferably used from 0.03 part to 0.10 part based on 100 parts of solution used. Nicotinamide-adenine-dinucleotide, hereinafter sometimes referred to as NAD, in admixture with an alkali lactate salt such as lithium lactate, sodium lactate, potassium lactate and the like, comprises a further component. The use of NAD is well known in the art and should be employed in concentrations ranging from about 0.01 part to about 0.20 part and is preferably used from 0.015 part to 0.08 part by weight based on 100 parts of solution. The lactate salt is employed in amounts ranging from 0.03 part to about 1.5 parts and is preferably used from 0.02 part to 0.09 part based on 100 parts of solution used. It is believed that the following reaction of the reagents present in the

determination of LDH in a sample using the above described mechanism may be used :

LDH

L-lactate + NAD⁺ ► Pyruvate + NADH + H⁺

Diaphorase

NADH + NTB ^► NAD⁺ + Formazan dye

The above reaction scheme illustrates that once the sample is added to the dry stick device, the LDH therein may cause a reaction which results in the reduction of the tetrazolium salt and the formation of a colored indicator, the intensity of which is directly proportional to the concentration of the LDH. The clinician then merely compares the color which results to a standard color chart to ascertain the LDH concentration of the sample being tested.

In order to achieve optimum results utilizing the novel dry stick device of the present invention, it is also advantageous, although not necessary, to incorporate as a further component a suitable non-ionic wetting agent any of those which are well-known to being applicable to the skilled artisan. For instance, fatty

alkanolamides may be used, i.e. the alkanolamine reaction products with fatty acids such as lauric acid or stripped coconut fatty acid, suitable alkanolamines being diethanolamine, monoethanolamine, amonisopropanolamine and the like; ethylene oxide derived materials, i.e. those derived from the reaction of ethylene oxide with alkylphenols wherein the alkyl group is octyl, nonyl or higher, long chain fatty alcohols such as tridecyl alcohol, lanolin, lecethin alcohol etc., long chain fatty acids such as tall oil, oleic acid, abietic acid etc., long chain fatty mercaptans, long chain fatty amines, polyoxypropylene glycol, fatty sorbitan ester; sugar esters i.e. the alcoholysis reaction products of the methyl ester of a fatty acid and sucrose or raffinose; polysorbitol; polyvinyl alcohol; methyl cellulose; ethoxylated phenol/formaldehyde resins and the like. Concentrations of from about 0.01 part to about 1.0 part of wetting agent per 100 parts of solution are employed, the wetting agents preferably being added with each component, if the components are added singly or in admixture with the components if they are added as a complete admixed system. In an embodiment of the present invention an aqueous solution of the tetrazolium salt may be prepared and added in a separate part, in a separate compartment, as separate stripes, or as separate dots of the dry stick device. A buffer solution of diaphorase and optionally a carbohydrate stabilizer may then be prepared and added in a separate part, in a separate compartment, as separate stripes or as separate dots of the dry stick device. A buffer solution of the NAD and alkali lactate may be prepared and added in a separate part, in a separate

compartment, as separate stripes or as separate dots of the dry stick device. A drying step completes the preparation of the test indicator.

In an embodiment of the present invention the method for determining LDH in a sample may be performed using a dry stick device having at least one dry reagent composition comprising : (a) a colouring compound,

(b) diaphorase, and

(c) a nicotinamide-dinucleotide, and

(d) a lactate salt. The reagents (a), (b), (c) and (d) may be in one single dry reagent composition or in individual dry reagent compositions, such as in 2 different dry reagent compositions , e.g. in 3 different dry reagent compositions or such as in 4 different dry reagent compositions . In another embodiment of the present invention the dry stick device may further comprises at least one dry reagent composition comprising :

(e) an anti-oxidant The reagent (e) may be in one single dry reagent composition together with reagents (a), (b), (c) and (d) or in an individual dry reagent composition .

The reagents may be separated into at least 2 dry reagent compositions , such as at least 3 dry reagent compositions , e.g. at least 4 dry reagent compositions , such as at least 5 dry reagent compositions , e.g. at least 6 dry reagent compositions .

In an embodiment of the present invention the colouring compound is selected from the group consisting of tetrazolium salt or any derivative hereof

If wetting agents etc. are to be incorporated, they are added to any or all of the dry reagent compositions to obtain uniform dry reagent deposits. Materials suitable as the carbohydrate stabilizer include maltose and sorbitol as well as water soluble polymeric ethylene oxides both high and low molecular weight, diethylene glycol and the like in concentrations ranging from about 10.0 parts to about 25.0 parts, preferably about 15.0 parts to about 20.0 parts based on 100 parts of solution used. In an embodiment of the present invention a water-soluble anti-oxidant may be employed, and then all the reagents may be admixed together in the buffer solution the concentrations of each ingredient being as set forth above except that each is based on the same 100 parts of water, and a one dissolution -one dry cycle can be employed to produce the desired test indicator.

Example of buffers useful in either procedure include, phosphate buffer, phthalate buffer, tris buffer, citratephosphate buffer, borate-succinate buffer etc. The preferred buffer is tris buffer i.e. 2-amino-2-(hydroxymethyl)-l,3-propanediol in a 0.05 to 0.2M concentrations.

It is obvious for the person skilled in the art that the first- and the second environment may be changed if a different assay for the determination of LDH is being provided. Furthermore, it is also obvious for the skilled person how to optimise the first- and the second environment based on the knowledge provided by the concept of the present invention, namely, having a first environment which may be selected in such a manner as to favour the storage of the reagent(s) capable of reacting with the analyte and providing a detectable signal and having a second environment which may be created in such a manner as to favour the performance of the reagent(s) capable of reacting with the analyte and providing a detectable signal or as to favour the rate of reaction between the analyte and the reagent(s) capable of reacting with the analyte providing a detectable signal.

In a preferred embodiment of the present invention the dry stick device is developed to measure LDH in accordance with the above mentioned reaction scheme for detecting LDH. In this construction it may be preferred that the at least one dry reagent composition is provided with a pH-value of approximately pH 6.8 and the regulation dry reagent composition is provided with a pH- regulating agent capable of providing a second environment for the reagent or the combination of reagents of approximately pH 8.3.

Determination of β-hydroxybutyrate (BHB)

BHB is being formed when fat is mobilised for energy. The level of BHB, with other ketone bodies, increases during hunger or with underfeeding, of e.g. animals. The level is closely related to energy status when there is a high demand for glucose, i.e. during late pregnancy and lactation of herd animals, such as cows.

In an embodiment of the present invention the determination of BHB may be performed using the same reaction scheme as provided for the determination of LDH as provided above.

In a preferred embodiment of the present invention the dry stick device is developed to measure BHB in accordance with the above mentioned reaction scheme for detecting BHB. In this construction it may be preferred that the at least one dry reagent composition is provided with a pH-value of approximately pH 6.8 and the regulation dry reagent composition is provided with a pH- regulating agent capable of providing a second environment for the reagent or the combination of reagents of approximately pH 8.3. Determination of urea

Determination of protein utilisation may be an important parameter. In cattle farming, it is highly important that the animals (e.g. cows) optimally utilize the protein contained in the feed, because protein is one of the most expensive feed components. The utilization depends, inter alia, on the amount of energy and protein simultaneously present in the animal.

In an embodiment of the present invention the determination of urea may be performed using the following reaction scheme:

Urease

Urea + 2H₂0 ► 2NH₄ ⁺ + C0₃ ^2~

NH₄ ⁺ + Base ^► NH₃

NH₃ + Indicator ► Dye

In a preferred embodiment of the present invention the dry stick device is developed to measure urea in accordance with the above mentioned reaction scheme for detecting urea . In this construction, and when using urease obtained from Jack Beans, it may be preferred that the at least one dry reagent

composition is provided with a pH-value of approximately pH 8.0 and the regulation reagent composition is provided with a pH-regulating agent capable of providing a second environment for the reagent or the combination of reagents of approximately pH 6.0.

Determination of N-acetyl glucosaminidase (NAGase)

The quantitative determination of NAGase may in the same way as LDH be important for the determination of mammary inflammation which affects the integrity of mammary gland structure and concurrently damages the secretary epithelia and the blood-milk barriers.

In an embodiment of the present invention the determination of NAGase may be performed using the following reaction scheme:

NAGase

4-MU-NAG ► 4-MU-acid

4-MU-acid + Base +^■ 4-MU-phenolate dye Here 4-MU-NAG relates to 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide, 4-MU-acid relates to 4-methylumbelliferone and 4-MU-phenolate dye relates to a 4-methylumbelliferone salt. In a preferred embodiment of the present invention the dry stick device is developed to measure NAGase in accordance with the above mentioned reaction scheme for detecting NAGase. In this construction it may be preferred that the at least one dry reagent composition is provided with a pH-value of approximately pH 7.0 and the regulation reagent composition is provided with a pH-regulating agent capable of providing a second environment for the reagent or the combination of reagents of approximately pH 4.6.

It is obvious for the person skilled in the art that the first- and the second environment may be changed if a different assay for the determination of LDH, BHB, urea, NAGase or any other analyte is being provided. Furthermore, it is also obvious for the skilled person how to optimise the first- and the second

environment based on the knowledge provided by the concept of the present invention, namely, having a first environment which may be created in such a manner as to favour the storage of the reagent(s) capable of reacting with the analyte and providing a detectable signal and having a second environment which may be created in such a manner as to favour the performance of the reagent(s) capable of reacting with the analyte and providing a detectable signal or as to favour the rate of reaction between the analyte and the reagent(s) capable of reacting with the analyte providing a detectable signal.

An automated or semi-automated system

In an embodiment of the present invention it may be a significant feature that a system is provided that is capable of handling and analysing, e.g. simultaneously, a number of analytes in a sample obtained from a mammal, such as a herd member, out of those analytes possible at any given point in time. Preferably, this point in time is dependent e.g. on the reproduction or lactation cycle state of an individual animal. This analysis of a number of analytes in a sample is made possible by

operationally linking the analytical means to a database containing information on the reproduction and lactation state of each herd member and/or any other information that may be used to determine whether or not a particular analyte should be analysed at a particular point in time. In this manner the system operates in a "dynamic" mode.

Thus, the present invention relates to an automated or semi-automated system comprising :

(a) a dry stick device for the determination of an analyte in a sample, said device comprises:

(i) a solid support comprising a cavity

(ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, and wherein the cavity comprises the at least one dry reagent,

(b) sampling means for obtaining a sample to be analysed, said

sampling means being controlled by means for storing data, said sampling means is only activated at pre-selected points in time or at pre-selected time intervals.

Thus, in a further embodiment the present invention relates to an automated or semi-automated system comprising : (a) a dry stick device for the determination of an analyte in a sample, said device comprises:

(i) a solid support comprising a cavity (ii) at least one dry reagent capable of reacting with the analyte derivative of said analyte or an indicator compound for said ana to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent, and wherein the detectable signal generated is obtained from the sample.

(b) sampling means for obtaining a sample to be analysed, said

The at least one dry reagent may be fixed onto at least part of the cavity drying, coating or adhesion.

Furthermore, in an embodiment the present invention relates to an automated or semi-automated system for optimising the production performance of a milk producing animal herd comprising a plurality of individual herd members each assigned a unique identification code that is recognisable by the system, the system comprising analysing means for analysing at least one analyte in a milk sample of an individual member of the milk producing animal, said analysing means comprising :

(a) a dry stick device for the determination of an analyte in a sample, said device comprises: solid support comprising a cavity

(ii) at least one dry reagent capable of reacting with the analyte derivative of said analyte or an indicator compound for said ana to provide a detectable signal when in a moistened state, and wherein the cavity comprises the at lease one dry reagent, (b) means for directing a part of the milk sample to each separate analysing means, said directing means being controlled by means for storing data for the physiological and nutritional state of each individual herd member, including data indicating point in time in the reproduction and lactation cycles of said herd member, such that the directing means is only activated at pre-selected points in time or at pre-selected time intervals in the production or lactation cycles of the individual herd member. In a further embodiment the present invention relates to an automated or semi- automated system for optimising the production performance of a milk producing animal herd comprising a plurality of individual herd members each assigned a unique identification code that is recognisable by the system, the system comprising analysing means for analysing at least one analyte in a milk sample of an individual member of the milk producing animal, said analysing means comprising :

(i) a solid support comprising a cavity

(ii) at least one dry reagent capable of reacting with the analyte derivative of said analyte or an indicator compound for said ana to provide a detectable signal when in a moistened state, and wherein the cavity comprises the at lease one dry reagent, and wherein the detectable signal generated is obtained from the sample. (b) means for directing a part of the milk sample to each separate analysing means, said directing means being controlled by means for storing data for the physiological and nutritional state of each individual herd member, including data indicating point in time in the reproduction and lactation cycles of said herd member, such that the directing means is only activated at pre-selected points in time or at pre-selected time intervals in the production or lactation cycles of the individual herd member.

Again the at least one dry reagent may be fixed onto at least part of the cavity - e.g. by drying, coating or adhesion.

Obviously, the dry stick device may comprise the features explicitly stated on page 3-35. As used herein, the term "automated" implies that the system can be operated substantially without manual operations. Thus, the term indicates that milk samples are automatically collected on-line at the milking site from the milking system and automatically transported to analytical means which in turn automatically generate analytical data that are processed automatically to update the system and to provide instructions to the farm management for corrective measures. The milking site may a milking site of an automatic milking system for freely moving milking animal or one of several milking sites in a conventional milking system such as a herringbone milking system. The milking site may also be at rotating or parallel milking parlours.

The term "semi-automated" as used herein refers to a system where at least part of the operations of the system involves some manual operation, e.g. manual transport of samples to the analytical means. The term "production performance" as used herein, is intended to mean the production performance in its broadest aspect. Thus, included in this term is milk production, including milk quantity and quality, reproductive performance of herd members, e.g. the number of offspring per milking animal and optimum utilisation of feed rations

The pre-selected points in time or at pre-selected time intervals for obtaining a sample, preferably for obtaining a subsequent sample, may be determined in a dynamic mode or in accordance with a pre-fixed schedule for obtaining samples. An interesting feature of the present invention may be that the means for storing data for the physiological and nutritional state of each individual herd member is continuously updated with new data, so that the selection of the range of compounds/parameters that are analysed in a given sample at a given point in time is based on a constantly updated set of data for the particular herd member or for a particular group of the herds.

Thus, the pre-selected points in time or at pre-selected time intervals for obtaining a sample, preferably for obtaining a subsequent sample, may preferably be determined in a dynamic mode. The term "dynamic mode" relates to the determination of the point in time for providing a subsequent sample and performing a subsequent analysis of said subsequent sample is on the basis of the result obtained from a previously analysed sample. Further details on the features and interconnections of the automated or semi- automated system described above may be found in WO 02/069697 or WO 2004/017066 which are hereby incorporated by reference.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples and figures.

Detailed description of the figures

Figure 1A shows a dry stick device for the determination of an analyte in a sample. The dry stick as shown in Fig. 1A a) comprises a solid support (1) and a cavity (2) . The dry stick as shown in Fig. 1A b) comprises a solid support (1) and a dry reagent (3). The dry stick as shown in Fig. 1A c) comprises a solid support (1) and sample (4), wherein the dry reagent (3) is dissolved. Figure IB shows a dry stick device for the determination of an analyte in a sample. The dry stick as shown in Fig. IB a) comprises a solid support (1) and a cavity (2). The dry stick as shown in Fig. IB b) comprises a solid support (1) and a dry reagent (3). The dry stick as shown in Fig. IB c) comprises a solid support (1) and sample (4), wherin the dry reagent (3) is dissolved.

Figure 2A shows a compartmented dry stick device for the determination of an analyte in a sample. The cross section of the dry stick as shown in Fig. 2A a) comprises a solid support (1), a cavity (2) and 2 separate compartments (3). The cross section of the dry stick as shown in Fig. 2A b) comprises 2 separate compartments (3) comprising 2 different dry reagents (4) and (5). The dry stick as shown in Fig. 2A c) comprises a solid support (1) and sample (6), wherein the dry reagents (4) and (5) are dissolved. Figure 2B shows a compartmented dry stick device for the determination of an analyte in a sample. The dry stick shown in Fig. 2B a) comprises a solid support (1), a cavity (2) and 4 separate compartments (3). Accordingly the dry stick may comprise 4 different dry reagents. The cross section of the dry stick as shown in Fig. 2B b) comprises 2 separate compartments (3) comprising 2 different dry reagents (4) and (5). The dry stick as shown in Fig. 2B c) comprises a solid support (1) and sample (6), wherein the dry reagents (4)-(7) are dissolved.

Figure 2C shows a compartmented dry stick device for the determination of an analyte in a sample. The dry stick shown in Fig. 2B a) comprises a solid support (1) and 2 separate compartments (5). The cross section of the dry stick as shown in Fig. 2B b) comprises a solid support (1) and 2 different compartments comprising 2 different dry reagents (2) and (3). The cross section of the dry stick as shown in Fig. 2B c) comprises a solid support (1) and sample (4), wherein the dry reagents (2) and (3) are dissolved.

Figure 3 shows a tiered dry stick device for the determination of an analyte in a sample. The tiered dry stick as shown in Fig. 3 a) comprises a solid support (1) and 2 different dry reagents (2) and (3). The tiered dry stick as shown in Fig. 3 b) comprises a solid support (1) and a sample (4), wherein the dry reagents (2) and (3) are dissolved. Figure 4 shows a dry stick for the determination of an analyte in a sample comprising lines of one or more dry reagents. The dry stick as shown in Fig. 4 a) comprises a solid support (1), a cavity (2) and lines of different dry reagents (3), (4) and (5). The dry stick as shown in Fig. 4 b) comprises a solid support (1) lines of different dry reagents (3), (4) and (5). The dry stick as shown in Fig. 4 c) comprises a solid support (1) and a sample (6), wherin the dry reagents are dissolved. The dry stick as shown in Fig. 4 d) comprises a solid support (1) a cavity (2) and thinner lines of a dry reagent (3).

Figure 5 shows a dry stick for the determination of an analyte in a sample, comprising dots of one or more dry reagents. The dry stick as shown in Fig. 5 a) comprises a solid support (1), a cavity (2) and dots of different dry reagents (3), (4), (5) and (6). The dry stick as shown in Fig. 5 b) comprises dots of different dry reagents (3) and (4). The dry stick as shown in Fig. 5 c) comprises a solid support (1), a cavity (2) and smaller dots of the same dry reagent (3). The dry stick as shown in Fig. 5 d) comprises a solid support (1) and a sample (7), wherein the dry reagents are dissolved. Figur 6 shows a dry stick device for the determination of an analyte in a sample comprising a solid support (1) and layers of different dry reagents (2) and (3).

Figure 7 shows a comparison between (i) a method for producing a traditional non-partitioned dry stick where the reagents are present in the same pad and (ii) the method of the present invention. As an example of such a traditional non- partitioned dry stick is used a BHB dry stick.

Figure 8 shows a comparison between (i) a method for producing a traditional partitioned dry stick where the reagents are separated into different pads and (ii) the method of the present invention. As an example of such a traditional partitioned dry stick is used an LDH dry stick.

Figure 9 shows the performance curve of (i) a traditional LDH dry stick and (ii) the new LDH dry stick of the present invention. Examples

Example 1. Preparation and test of BHB dry sticks

Preparation of impregnation solution:

The following reagents are dissolved one at a time in 3400 mL 0.1 M phosphate buffer pH 8.0:

• 20.0 g of β-nicotinamide adenine dinucleotide (β-ΝΑϋ⁺)

• 9.0 g nitro blue tetrazolium (NTB)

• 32 mL of a 5% solution of Triton X-100

• 20.0 g bovine serum albumin (BSA)

· 400 KU of ascorbic acid oxidase (ASO-3 from Amano)

• 100 KU of diaphorase (Unitika)

• 50 KU of hydroxy butyrate dehydrogenase (HBDH from Amano)

to yield a slight yellow clear solution. Prepare tion of dry sticks :

In the cavitys of separate sticks were filled 10 μί of the above freshly prepared solution in each cavity. The sticks were then dried in a ventilated oven at 40 °C for 1 hour to yield in each cavity a dry residue (about 250 pg), which was stuck to the bottom of the cavity. Each separate dry stick obtained was then placed in a foil bag with drying agent, and the foil bag was sealed. The sealed bag with the dry stick was then stored in the refrigerator at 4 °C until use.

Preparation of standard series of BHB in UHT milk:

A stock solution of 1.05 mM of BHB in UHT milk was prepared by dissolving 34.7 mg BHB-Na salt in 275 mL of UHT milk. The quality of UHT milk employed here contained about 0.05 mM BHB.

A standard series of BHB in UHT milk was prepared as follows:

Test of BHB dry sticks by standard series:

To eight cavities of 8 separate dry sticks (as described above) were added at about 25 °C 10 μί of each of the BHB standards in UHT milk. The dry material in each well dissolved and after a short time the liquid in the 8 separate cavities started turning bluish. After 5 minutes incubation time the intensity of the blue color was compared visually, and it was noticed that the color intensity was proportional to the BHB concentration.

Example 2. Preparation and test of LDH dry sticks

Preparation of impregnation solution A:

The following reagents are dissolved one at a time in 2600 mL 0.1 M phosphate buffer pH 8.0:

· 60.0 g of β-nicotinamide adenine dinucleotide (β-ΝΑϋ⁺)

• 30.0 g Li-acetate

• 3.0 g PEG 20.000

• 30.0 g sucrose

• 30 mL of a 5% solution of Triton X-100

· 30.0 g bovine serum albumin (BSA)

• 300 KU of ascorbic acid oxidase (ASO-3 from Amano)

• 600 KU of diaphorase (Unitika)

• 3.0 g nitro blue tetrazolium (NTB)

to yield a slight yellow clear solution.

Preparation of impregnation solution B:

450 g TRIS were dissolved in 1080 mL DI water to yield a clear colorless solution of pH about 12. Preparation of dry sticks: In the cavities of a first stick were filled 8.4 μΙ_ of the above freshly prepared solution A. The stick was then dried in a ventilated oven at 40 °C for 1 hour to yield in the well a dry residue (about 770 μς), which was stuck to the bottom of the cavity. The obtained dry stick was then placed in a foil bag with a drying agent, and the foil bag was sealed. The sealed bag with the dry stick was then stored in the refrigerator at 4 °C until use.

In the cavity of a second stick was filled 3.4 μΙ_ of the above freshly prepared solution B. The stick was then dried in a ventilated oven at 40 °C for 1 hour to yield in each well a dry residue (about 1 mg), which was stuck as a small pellet to the bottom of the cavity. The dry stick was then placed in a foil bag with drying agent, and the foil bag was sealed. The sealed bag with the dry stick was then stored in the refrigerator at 4 °C until use.

A LDH dry stick of the partioned type was prepared by adding to the cavity of the first stick the pellet from the second dry stick cavity.

Preparation of standard series of LDH in UHT milk:

A stock solution of 1000 U/L of LDH (sigma L1378) in UHT milk was prepared. A standard series of LDH in UHT milk was prepared as follows:

Test of LDH dry sticks by standard series:

To the cavities of eight seperate LDH dry sticks as described above were added at about 25 °C 10 μί of each of the LDH standards in UHT milk. The dry material in each well dissolved and after a short time the liquid in the 8 cavities started turning bluish. After 5 minutes incubation time the intensity of the blue color was compared visually, and it was noticed that the color intensity was proportional to the LDH concentration. Example 3. New LDH dry stick vs traditional LDH dry stick

Phosphate buffer solution : 85.9 g Na2HP04.12H20 and 2.1 g NaH2P04 in 2520 mL DI water, pH 8.0.

Solution A: 2.6 mL phosphate buffer solution, 60.0 mg β-ΝΑϋ+, 30.0 mg Li- lactate, 3.0 mg PEG 20.000, 30.0 mg sucrose, 30.0 mg BSA, 300 U ASO-3, 600 U diaphorase, 3.0 mg NTB. Solution B: 450 mg TRIS with 1.08 mL DI water (totally 1.45 mL solution).

Solution C: 1.5 mg Triton X-100 in 300 μί DI water.

Standard series of LDH in milk: UHT milk. LDH Sigma L1378 added to give standard series 0, 50, 100, 200, 300, 400 U/L.

Stick frame: White polystyrene frame with a 19 μί box formed well (5.2mm x 5.2mm x 0.7mm height).

(i) Traditional LDH dry stick preparation :

Layer 1 : An 8.5 x 10 cm piece of Whatman 3MMChr filterpaper was impregnated with 2.8 mL solution A, and dried in a dry air ventilated oven at 40 °C for 20 minutes.

Layer 2: An 8.5 x 10 cm piece of Asahi PS-2 wiper was impregnated with 1.36 mL solution B, and dried in a dry air ventilated oven at 40 °C for 10 minutes.

Lamination : The dry layers 1 and 2 were laminated together by using a hot melt glue transfer tape with an open structure, and passing through a set of rollers. Cutting and mounting : The laminated material was cut into squares of 5 x 5 mm and mounted into stick frames.

Storage: The LDH dry sticks were stored in foil bags with desiccant at 4 °C. (ii) New LDH dry stick preparation :

In the bottom of a well of a dry stick were placed separately two drops of 2.0 μΙ_ of solution A plus one drop of 2.0 μΙ_ of solution B. The dry stick was placed in a dry air ventilated oven at 40 °C for 10 minutes, leaving a dry stick with three dry spots. A total of 25 sticks were prepared simultaneously. The dry stick was stored at 4 °C in a foil bag with desiccant.

Table 10. Composition comparison between the new LDH dry stick and a traditional dry stick:

*added via LDH standard (to 10 parts LDH standard in milk were added 1 part solution C).

Therefore, the new dry stick contains only about V2 chemistry compared to standard dry stick. Table 11. Test of dry sticks: 10 \ L of LDH standard in milk, 25 °C, 5 minutes incubation, reading at 525 nm

The results in Table 11 and figure 9 shows that the new LDH dry stick construction gives a performance curve of approximately the same shape as a traditional LDH dry stick, which is surprising since only about V2 chemistry was employed in the new dry stick construction. The results with 15 μΙ_ sample addition shows that the shape of the performance curve can be influenced in a desired direction by changing the sample volume from e.g. 10 to 15 μΙ_.

References

WO 02/069697 WO 2004/017066

Claims

1. A dry stick device for the determination of an analyte in a aqueous sample, said device comprises:

(i) a solid support comprising a cavity, and

(ii) at least one dry reagent capable of reacting with the analyte, a derivative of said analyte or an indicator compound for said analyte to provide a detectable signal when in a moistened state, wherein the cavity comprises the at least one dry reagent and wherein the detectable signal generated is obtained from the sample.

2. A dry stick device according to claim 1, wherein the dry stick does not comprise a porous material.

3. A dry stick device according to claim 1 or 2, wherein the at least one dry reagent is not immobilized.

4. A dry stick device according to any one of the preceding claims, wherein the dry stick does not compris an antibody and/or immunoglobulin.

5. A dry stick device according to any one of the preceding claims,wherein the dry stick is not an immunoassay.

6. A dry stick device according to any one of the preceding claims, wherein the at least one dry reagent is fixed onto at least part of the cavity by drying, coating or adhesion.

7. A dry stick device according to any one of the preceding claims, wherein the sample is selected from the group consisting of milk, blood, serum, plasma, saliva, urine, sweat, ocular lens fluid, cerebral spinal fluid, ascites fluid, mucous fluid, synovial fluid, peritoneal fluid, amniotic fluid, hair and the like.

8. A dry stick device according to any one of the preceding claims wherein the one or more dry reagents are fixed onto separate parts of the cavity.

9. A dry stick device according to any one of the preceding claims wherein the cavity is compartmented.

10. A dry stick device according to any one of the preceding claims wherein the at least one dry reagent is present in at least 2 separate layers fixed onto at least part of the cavity such as in at least 3 layers, e.g. at least 4 layers, such as at least 5 layers, e.g. at least 6 layers.

11. A method for the determination of an analyte in an aqueous sample, said method comprising the steps of:

(a) applying the sample to a cavity of a dry stick device according to any one of claims 1-10,

(b) permitting the at least one reagent and the analyte, the derivative of said analyte or the indicator compound for said analyte to react and generate a detectable signal, and

(c) obtaining the detectable signal from the sample.

12. The method according to claim 11, wherein the method does not comprise a washing step.

13. A method for preparing a dry stick device according to any one of claims 1-10, said method comprises the steps of:

(i) providing a support comprising a cavity;

(ii) providing at least one dry reagent

(ii) applying the at least one dry reagent to the cavity, and

obtaining the dry stick device.

14. An automated or semi-automated system comprising :

(a) a dry stick device for the determination of an analyte in a

sampleaccording to any one of claims 1-10 and

(b) sampling means for obtaining a sample to be analysed, said sampling means being controlled by means for storing data, said sampling means is only activated at pre-selected points in time or at pre-selected time intervals.

15. An automated or semi-automated system for optimising the production performance of a milk producing animal herd comprising a plurality of individual herd members each assigned a unique identification code that is recognisable by the system, the system comprising analysing means for analysing at least one analyte in a milk sample of an individual member of the milk producing animal, said analysing means comprising :

(a) a dry stick device for the determination of an analyte in a sample according to any one of claims 1-10, and

(b) means for directing a part of the milk sample to each separate analysing means, said directing means being controlled by means for storing data for the physiological and nutritional state of each individual herd member, including data indicating point in time in the reproduction and lactation cycles of said herd member, such that the directing means is only activated at pre-selected points in time or at pre-selected time intervals in the production or lactation cycles of the individual herd member.