FIELD OF THE INVENTION

The invention relates to a method and an apparatus for the determination of chorionic gonadotropin (CG), luteinizing hormone (LH) or CG-like material of a hormone from animals, preferably from husbandry animals.

BACKGROUND OF THE INVENTION

US 4,016,250 describes a method for the determination of the hormone human chorionic gonadotropin (HCG), luteinizing hormone (LH), prolactin (PRL) and HCG-like material in an aqueous sample comprising contacting a highly specific receptor for these hormones with an aqueous sample to be tested, providing a means of indicating whether binding has taken place between the receptor and hormone possibly contained in the sample and observing the indicating means to determine the presence of the hormone in the sample. PRL may be determined separately or simultaneously with the HCG and LH. The method has particular application to the determination to pregnancy in the human female.

US 3,817,239 describes a monitor for use in a catheterising tube system has a hollow closed chamber, preferably resilient, containing test viewing means, for example, a transparent portion of the chamber, and urine testing means disposed within the chamber. The urine specimen collection device is for use with a urinary catheter drainage system. The urine testing means includes a test medium, which is visually responsive to a pre- selected urine factor to be tested. Urine transferring means, such as a needle, communicate between the chamber interior and the exterior. Catheter attaching means may be provided for connecting the monitor to a urine catheter drainage system.

SUMMARY OF THE INVENTION

In accordance with the present invention, there has been discovered an efficient and a reliable and fast method and apparatus especially suited for determining the possible pregnancy of husbandry animals such as cows, but possibly also pigs, sheep and horses.

This object may be obtained by a method for the determination of chorionic gonadotropin (CG), luteinizing hormone (LH) or CG-like material of a hormone from animals, said method comprising the steps of contacting the animal urine with an agent capable of selectively binding said hormone, providing a means for indicating at least whether said binding has taken place and observing said indicating means to determine the presence of said hormone in the urine.

By addressing any chorionic gonadotropin (CG), luteinizing hormone (LH) or CG-like material in urine from animals, and contacting said urine with an agent capable of selectively binding said hormone, it has become possible to establish a very early determination of possible pregnancy of animals. Also, testing on the urine leads to the provision of a very easy and fast way of determination. Finally, testing on the urine makes it possibly for the test to be performed by an average person in contrast to persons such as veterinarians perhaps not being around when needed and perhaps performing pregnancy tests under a certain national, educational and/or ethical authorisation.

According to a first possible aspect of the method according to the invention, the husbandry animal to be tested is a cow. In case the husbandry animal is a cow, the CG- like material in the urine is denoted bovine chorionic gonadotropin (bCG). In the present application, the abbreviation bCG is not to be confused with the vaccine against tuberculosis, said vaccine prepared from weakened strain of tuberculosis bacteria named bacille Calmette-Guerin, for the French scientists who developed the vaccine, and being abbreviated BCG, or possibly also being abbreviated bCG, i.e. with the small letter b.

According to a second possible aspect of the method according to the invention, the husbandry animal to be tested is a sow. In case the husbandry animal is a sow, the CG- like material in the urine is denoted suidae chorionic gonadotropin (sCG).

According to a third possible aspect of the method according to the invention, the husbandry animal to be tested is a sheep. In case the husbandry animal is a sheep, the CG-like material in the urine is denoted ovine chorionic gonadotropin (oCG).

According to a fourth possible aspect of the method according to the invention, the husbandry animal to be tested is a goat. In case the husbandry animal is a goat, the CG- like material in the urine is denoted capra chorionic gonadotropin (cCG).

According to a fifth possible aspect of the method according to the invention, the husbandry animal to be tested is a mare. In case the husbandry animal is a mare, the CG- like material in the urine is denoted equine chorionic gonadotropin (eCG).

According to a sixth possible aspect of the method according to the invention, the animal to be tested is a ruminant. In case the animal is a ruminat, the CG-like material in the urine is denoted any suitable prefix to chorionic gonadotropin (CG), said prefix being a suitable denomination of the animal.

According to a seventh possible aspect of the method according to the invention, the animal to be tested is an animal of a zoological garden. In case the animal is an animal of a zoological garden, the CG-like material in the urine is denoted any suitable prefix to chorionic gonadotropin (CG), said prefix being a suitable denomination of the animal.

According to an eighth possible aspect of the method according to the invention, the animal to be tested is an endangered animal. In case the animal is an endangered animal, the CG-like material in the urine is denoted any suitable prefix to chorionic gonadotropin (CG), said prefix being a suitable denomination of the animal.

According to a preferred aspect of the method according to the invention, the steps of providing and observing said indicating means further comprise the steps of inserting a catheter into the urethra of the animal, extracting a sample of urine flowing through the catheter, and contacting said receptor and said sample in the catheter.

A further object of the present invention resides in the provision of a pregnancy testing method, which produces nearly 100% reliability in detecting pregnancy.

It is also an object of the invention to provide a method and means for simultaneously determining CG-like proteins, or LH and perhaps PRL in an urine sample.

By using a catheter as mentioned above, a very efficient and fast method is obtained of testing the animals. A catheter may be used in the manner described also by persons not having any veterinary background, because such insertion only demands the knowledge of the urethra properties of the animal, i.e. primarily the position of the urethra, and secondarily the length of the urethra, if not the catheter is provided with indication means of how far the catheter is to be inserted not the urethra of the animal.

According to an apparatus according to the invention, the objects of the invention may be obtained by an apparatus comprising a catheter divided into at least a first section and a second section, said first section having an inlet for the urine and said second section encompassing a testing means. The length of the catheter and the outer diameter of the catheter are adapted to the urethra of the animal which the catheter is intended for.

A catheter to be inserted directly into the urethra of the animal will ease and expedite the testing of husbandry animals in relation to determination of possible pregnancy. Also, insertion of a catheter directly into the urethra may be performed by a non-professional person in relation to testing of animals. Thus, in case the animal is a husbandry animal, the farmer himself or herself may perform the test without the need for a veterinary taking the test. Thus, the test may be performed independently on any time schedule of the veterinary, and the test may be performed any time during the day and without the cost involved when needing a veterinary performing the test. The possible pregnancy of the animal may thus bed established at a very early state of any possible pregnancy.

According to a preferred embodiment of the apparatus according to the invention, said first section also comprises an outlet for the urine, and said first section and said second section is divided by a transition wall, and a transition between the first section and the second section is established by a test strip extending from the first section to the second section.

The method according to the invention is not species specific, it may also be employed when detecting possible pregnancy in any other animal, which have been found to possess a specific receptor. Thus, even though the receptor is very specific for bCG in cows and LH in cows, it is also capable of selectively binding CG-like proteins, which play in animals a role corresponding to bCG in cows, i.e., they correspond to the chorionic gonadotropins in cows. The advantage to this is that standard catheters may be used when testing animals according to the method, whereas separate antibodies are required for each animal in accordance with immuno-chemical more clinical testing methods on blood from animals.

Another embodiment of the present invention is the determination of the hormone PRL. In some cases, only small samples are available, and therefore it is desired to have a very specific test, which requires only a small sample and only a short period of time to carry out.

Specificity is desired in order to differentiate from other hormone species which may be present in the sample, and this is particularly true in cases where it might be desired simultaneously determine or measure two or more different hormones. There exists at present no truly specific means for making such a determination.

The specific receptor employed in the testing procedure according to the present invention is preferably derived from ovarian tissue. As pointed out hereinabove, the receptor is significantly more selective for CG and CG-like materials or PRL than is an CG or PRL antibody, since the receptor requires the native configuration of the hormone in its biologically active form for binding. Hence, contrary to interactions between antibodies and antigenes as known from the prior art, there is less risk of non-specific immune response. Thus, the method according to the present invention is more reliable.

In all animals, which possess this specific receptor, it is readily detectable in the ovarian tissue, which represents the target tissue of the hormone. While the receptor may be isolated from any mammal and possible from certain non-mammals, it is preferred to obtain the receptor from the ovaries of relatively large animals, such as cows, sheep, pigs, horses and the like in view of the fact that these animals have proportionately a greater amount of ovarian tissue, and furthermore, a ready supply of such tissue exists by virtue of the fact that these animals are commercially slaughtered for their meat.

Additionally comprised in the present method is simultaneous determining two or more different hormones contained in the urine. In this embodiment, the steps are similar to those for measuring single hormones, except that the urine is contacted with one or more agents capable of selectively binding each of the hormones to be determined, providing a separate and distinguishable means for indicating whether binding has taken place for each hormone and observing each of the indicators to determine the presence of each hormone.

Possible hormones, the presence of which may be determined, comprise: oestrogen, progesteron, LH and placental lactogens. The list is not exhaustive, but merely constitutes possible and preferred hormones, the presence of which is readily determined.

Furthermore, in another embodiment of the invention there is provided a means for the determination of a plurality of hormones in urine, comprising (a) a first reagent and a number of second reagents equal to the number of hormones to be determined; (b) said first reagent comprising a substantially pure form of the specific fraction of plasma membrane extract from a body organ of a species having the specific receptor in said organ for selectively binding each of said hormones in biologically active form.

It is preferred that the method for determination of CG is initiated at a given time after conception, preferably when the throphoblast produces the CG, e.g. approximately 14 days after cover. In a presently preferred aspect, samples are tested once or possibly just twice within a one week interval. In some specific applications of the method according to the invention the samples may be tested within two days intervals, such as within 3 days interval, up to one week interval. The duration of the testing may be from 2-21 days, 4-14 days, 6-14 days, 8-12 days or 10-12 days after conception, or longer if needed. Preferably 1 testing is enough, but more than one test, such as 2-6 tests may be needed for almost one hundred percent reliability. The animals, e.g. the husbandry animals, are in early stage of pregnancy when the determination is performed. An early stage of pregnancy is preferably the stage from approximately two weeks after cover up to perhaps three months after cover.

Possible glycol-proteins from the trophoblasts, the presence of which may be determined includes: PAG (Pregnancy Associated Glycoprotein) including all subtypes, PSP (being the same as PAG in French literature), PSPB (being the same as PAG in American (USA) and French literature), bCG (Bovine Chorion Gonadotropin), ECF (Early Conception Factor), EPF (Early Pregnancy Factor), bTP (Bovine Trophoblast Protein) and boIFN-tau (Interferon Tau). The list is not exhaustive, but merely constitutes possible and preferred glyco¬ proteins, the presence of which is readily determined, or at least desirable to determine.

In the present context the agent capable of selectively binding said hormone being an extract from the corpus luteum of a mammal species with the receptor for animal chorionic gonadotropin. Preferably when at least two hormones are to be determined in the urine, the receptor is a plasma membrane extract of the corpus luteum of a mammal, which possesses receptor sites for both HCG and PRL.

Furthermore, the indicating means employed in this method may be any of those conventionally employed in a similar immunochemical method for determining proteins and/or polypeptides. For example, the indicator may be a material which has been treated with the hormone so that in the presence of the instant receptor, the indicator will discolour, become coloured, agglutinate, precipitate, fluoresce or give some other visible indication (for example, turning from bright to dull) of the presence or absence of the hormone in the urine being tested. Alternatively, the indicator comprises an electronic sensing device and the indicator will sound at a predetermined frequency or by a continuous sound or give other audible indication (for example, a selected tune) of the presence or absence of the hormone in the urine being tested.

In applying this method for the detection of pregnancy in animals, an urine sample is contacted with the above-described specific receptor and the presence or absence of CG in the sample is determined based upon the amount of binding evidenced by the indicator system. The indication may be provided as a detectable signal, which may be transmitted wire-bound or wireless to a remote signal processing means, perhaps for remote monitoring of the tests performed, or for storing of present test data, together with storing of any past test data and any future test data, of the animal being tested. In one embodiment the results of the present method provide an indication of non- pregnancy, only if the animal is not pregnant for thereby establishing the determination of the condition of pregnancy. Such embodiment is only applicable when testing at an advanced stage of early pregnancy, i.e. testing subsequent to a covering of the animal and at least 10-30 days thereafter, when possible pregnancy has been initiated.

In another embodiment the reagent for receptor determination of the hormone bovine chorionic gonadotropin (bCG), comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone bovine chorionic gonadotropin, said fraction being capable of selectively binding biologically active bovine chorionic gonadotropin.

In a further embodiment the reagent for receptor determination of the hormone suidae chorionic gonadotropin (sCG), comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone suidae chorionic gonadotropin, said fraction being capable of selectively binding biologically active suidae chorionic gonadotropin.

In yet another embodiment the reagent for receptor determination of the hormone ovine chorionic gonadotropin (oCG), comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone ovine chorionic gonadotropin, said fraction being capable of selectively binding biologically active ovine chorionic gonadotropin.

In a further embodiment the reagent for receptor determination of the hormone capra chorionic gonadotropin (cCG), comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone capra chorionic gonadotropin, said fraction being capable of selectively binding biologically active capra chorionic gonadotropin.

In yet another embodiment the reagent for receptor determination of the hormone equine chorionic gonadotropin (eCG), comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone equine chorionic gonadotropin, said fraction being capable of selectively binding biologically active equine chorionic gonadotropin.

In a different embodiment the reagent for receptor determination of the hormone chorionic gonadotropin (CG) from any animal being a ruminant, comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone chorionic gonadotropin of ruminants, said fraction being capable of selectively binding biologically active chorionic gonadotropin of ruminants.

In yet a different embodiment the reagent for receptor determination of the hormone chorionic gonadotropin (CG) from any animal situated in a zoological garden, comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone chorionic gonadotropin of zoological garden animals, said fraction being capable of selectively binding biologically active chorionic gonadotropin of zoological garden animals.

In yet a different embodiment the reagent for receptor determination of the hormone chorionic gonadotropin (CG) from any endangered animal, comprising in substantially pure form the specific fraction of plasma membrane extract from the corpus luteum of a species having the receptor for the hormone chorionic gonadotropin of endangered animals, said fraction being capable of selectively binding biologically active chorionic gonadotropin of endangered animals.

DETAILED DESCRIPTION OF THE INVENTION

The invention will hereafter be described with reference to the accompanying drawing, where

fig. 1 shows an embodiment of a catheter according to the invention for performing a method of testing an animal according to the invention by testing made on the urine of the animal, and fig. 2 shows a possible test strip to be used in a catheter according to the invention for performing a method of testing an animal and according to the invention by testing made on the urine of the animal, fig. 3A-3C shows a preferred test strip to be used in a catheter according to the invention for performing a method of testing "positive" the pregnancy of an animal and according to the invention by testing made on the urine of the animal, and fig. 4A-4C shows the preferred test strip to be used in a catheter according to the invention for performing a method of testing "negative" the pregnancy of an animal and according to the invention by testing made on the urine of the animal.

Fig. 1 shows a catheter consisting in an elongated tube, either being flexible or being rigid. The tube may be made of any suitable material, preferably a plastic material, alternatively a metal, more alternatively a combination of plastic and metal and even more alternatively being made by glass or a combination of metal and glass. The tube is divided into at least two sections, a first section 1 to be inserted into the urethra of the animal and a second section 2 encompassing a testing means 3. The first section 1 comprises an orifice constituting an inlet 4 for urine and an outlet 5 for the urine.

The second section 2 is substantially divided from the first section 1 so that a flow of urine along the first section cannot enter the second section, but will be let out through the outlet of the first section. However, a transition between the first section and the second section is established by a test strip 6 extending from a proximate end 7 of the test strip situated in the first section to a distant end 8 of the test strip situated in the second section.

An intersection between the first section and the second section is provided by a transition wall 9 having a narrow slit (not shown), through which the test strip 6 extends. Thus, the transition between the first section and the second section is established only along the test strip through the slit, the transition wall blocking any other transitional possibility between the first section and the second section.

In case the tube is made of a metal such as stainless steel the first section and the second section may be divided into two separate elements capable of being mutually jointed by a close fit or by any joining means such as a screw thread. Thus, the first section constitutes one separate element, which may be made of metal, and the second section constitutes another separate element, which may be made of plastic or of glass.

The first section made of metal can be manufactures very cheaply, and the second section is manufactured separately and may be separated from the first section after the testing has been performed. Also, the first section may be used more times (subsequent to proper intermediate cleaning), and the second section may be provided separately from the first section, the mutual joining taking place just before performing the test.

The test strip may be made of any material suitable for the purpose, but preferably a felt material is chosen due to the absorbent properties of a felt material. Alternatively or additionally, a material having adsorbent properties may be chosen. The test strip is provided with reactants. The function of the test strip is described in more detail with reference to fig. 2 and to fig. 3A-3C together with fig. 4A-4C

Preferably, the test strip is intended for one-time use. The test strip together with the catheter as such may be mutually dependent so that it is not possible to discard the test strip without also having to discard the catheter as such. Thus, both the test strip itself and the catheter itself will be manufactured from low-cost materials and the manufacture of the combined test strip and catheter will be conducted in a cost-effective manner.

The testing as such takes place by inserting into the urethra of a female cow, of a female pig, of a female sheep or of a female horse. Either the insertion is made during normal urination by the animal, or a urination is promoted when inserting the catheter. During urination, at least part of the urine flow into the first section via the inlet, through the first section and further out of the first section via the outlet.

During the flow of urine through the first section, the proximate end 7 of the test strip is moistened by the urine. Due to the absorbent properties, or alternatively the adsorbent properties, of the test strip and due to a propagation of urine in the test strip, the urine is transported through the slit in the wall and to the distant end of the test strip. In the distant end of the test strip, the reagent is present, and a reaction takes place between the urine and the reagent.

Due to the transparent properties of at least the second section of the catheter, the result of the reaction is immediately apparent, i.e. within a few minutes, and thus an immediate establishment of a possible pregnancy of the animal is provided, compared to prior art establishment of possible pregnancy of animals such as husbandry animals. Furthermore, the establishment of a possible pregnancy of the animal may be made by any non¬ professional person such as the farmer himself or herself, or a keeper at a zoological garden, or a shepherd of endangered animals or other wild animals.

Fig. 2, and fig. 3A-3C together with fig. 4A-4C shows two different kinds of test strips 10 consisting of paper or other pulp material capable of providing an absorptive effect to urine contacting the left side part of the test strip. At least two different antibodies, a first antibody 11 and a second antibody 12, but preferably three different antibodies, also a third antibody 13, are embedded within parts of the test strip 10. The antibodies must be non-dependent on widely variations in pH of animal urine, and in inorganic and organic concentrations of the animal urine, all being dependent on what and how much the animal is digesting. The anti-bodies are intended for binding to a specific antigen.

The antigen for the antibodies is the chorionic gonadotropin (CG), luteinizing hormone (LH) or CG-like material of a hormone from animals. Preferably, the antigen for the antibodies is the bCG, the sCG, the oCG, the cCG or the eCG that the placenta secretes into blood of the pregnant animal and from the blood into the pregnant animal's urine, and which chorionic gonadotropin (CG), luteinizing hormone (LH) or CG-like material of a hormone is intended for maintaining the pregnancy so that the pregnancy does not "slip" back into the menstrual cycle. As mentioned above, the bCG is the bovine chorionic gonadotropin, sCG is the suidae chorionic gonadotropin, oCG is the ovine chorionic gonadotropin, cCg is the capra chorionic gonadotropin and eCG is the equine chorionic gonadotropin. In the remainder of the description, bCG will be used as an example, but the example is not to be construed as limiting the possible other chorionic gonadotropines.

Thus, chorionic gonadotropin (CG), luteinizing hormone (LH) or CG-like material of a hormone from other animals than cows may be accepted as the antigens. However, exemplary speaking, the bCG is the antigene applied to the left side part of the test strip, and a complex situated on the right side of the test strip is one of anti-bCG, where "anti- bCG" is an "antibody that binds bCG". The test method according to the invention comprises the following steps and reactions for determining the presence of the CG of the animal, thus indicating the animal being pregnant:

Urine 14 is absorbed by the left side part 15 (see fig. 2) of the test strip 10, i.e. the test strip placed in the first section of the catheter (see fig. 1). The urine along with its dissolved components including bCG, move along the test strip from the left side to a neighbouring part 16 of the test strip, from the first section, past the slit in the intervening wall (see fig. 1) and to the second section of the catheter (see fig. 1).

When the urine reaches this part 16 of the test strip being applied the unbound first antibody 11, which is in great excess, the bCG begins reacting with the first antibody (see fig. 3B), but the complexes and the excess un-reacted first antibody flow along with the urine, by the influence of a capillary effect and/or the absorbent nature of the test strip 10, further to a central part 17 of the test strip 10.

When the urine together with the first antibody 11 reaches the horizontal line 18 , where the second antibody 12 is immobilized to the test strip 10, another portion of the bCG is "displayed" by the first antibody and binds with the second antibody 12 (see fig. 3C). When this happens, the distorted second antibody 12 may, in a possible embodiment of the test strip as shown in fig. 2, trigger an enzyme to start making an insoluble dye, which upon accumulating causes the vertically extending dotted line 19 of the "plus sign" (see fig. 2) to become visible. Alternatively, in a preferred embodiment of the test strip as shown in fig. 3A-3C, no enzymatic reaction takes place for triggering an insoluble dye, but the first antibody 11 itself is died with any suitable dye.

The urine continues moving along and eventually crossing the last right hand part 20 and reaching the horizontal line 21, where any excess of the first antibody 11 starts reacting with the third antibody 13. This triggers the formation of a dye that completes the vertically extending dotted line 22 of that right hand "plus sign" (see fig. 2). Alternatively, in a preferred embodiment of the test strip as shown in fig. 3A-3C, no enzymatic reaction takes place for triggering the formation of a dye, but the first antibody 11 itself is died with any suitable dye. The last right hand part 20 of the test strip constitutes a control part indicating that the antibodies in the test strip are alright and working. In the case, where the animal is pregnant, both the dotted line 19 in the central part 17 and the dotted line 22 in the right hand part 20 will show. The control part is optional and is provided to ensure that the device has not become overheated or otherwise become defective.

If the animal is not pregnant, then a reaction scheme as shown in fig. 4A-4C will take place. When the urine reaches the part 16 of the test strip being applied the unbound first antibody 11, which is in great excess, no reacting with the first antibody will take place due to the non-presence of bCG (see fig. 4AB). But the complexes and the un-reacted first antibody flow along with the urine, by the influence of a capillary effect and/or the absorbent nature of the test strip 10, further to a central part 17 of the test strip 10.

When the urine together with the first antibody 11 reaches the second antibody 12 being immobilized to the test strip 10, no binding with the second antibody will take place, because no other portion of the bCG can be "displayed" by the first antibody due to the non-presence of bCG (see fig. 4B). Thus, the second antibody 12 will not trigger an enzyme to start making an insoluble dye, or the first antibody 11 itself being died will not show along the dotted line 19, because no binding takes place in the central part 17.

The urine continues moving along and eventually crossing the last right hand part 20, where the first antibody 11 starts reacting with the third antibody 13. This triggers the formation of a dye that forms the vertically extending dotted line 22. Alternatively, in a preferred embodiment of the test strip as shown in fig. 4A-4C, no enzymatic reaction takes place for triggering the formation of a dye, but the first antibody 11 itself is died with any suitable dye. As mentioned above, the last right hand part 20 of the test strip constitutes a control part indicating that the antibodies in the test strip are alright and working. In the case, where the animal is not pregnant, only the dotted line 22 in the right hand part 20, and not the dotted line 19 in the central part 17, will show.

Accordingly, any further steps towards covering of the animal may be initiated at once if needed or if convenient. This may of course also be initiated by the farmer himself or herself, or a keeper at a zoological garden, or a shepherd of endangered animals or other wild animals, by simply allowing a male animal access to the female animal or by artificial insemination of the female animal. Especially in a stock of cattle, it will be possible at a very early state to have cows covered again after having given birth to a preceding number of calves.