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WO2000051520A2 - Test de grossesse pour bovins - Google Patents

Test de grossesse pour bovins Download PDF

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Publication number
WO2000051520A2
WO2000051520A2 PCT/US2000/005616 US0005616W WO0051520A2 WO 2000051520 A2 WO2000051520 A2 WO 2000051520A2 US 0005616 W US0005616 W US 0005616W WO 0051520 A2 WO0051520 A2 WO 0051520A2
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WO
WIPO (PCT)
Prior art keywords
animal
bovine
antibody
liquid
pregnancy
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2000/005616
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English (en)
Other versions
WO2000051520A9 (fr
WO2000051520A3 (fr
Inventor
Susan L. M. Frushour
Martin Pearson
Edward Slowikowski
Kevin D. Jones
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KEMS BIO-TEST Ltd
Original Assignee
KEMS BIO-TEST Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by KEMS BIO-TEST Ltd filed Critical KEMS BIO-TEST Ltd
Priority to AU35119/00A priority Critical patent/AU3511900A/en
Publication of WO2000051520A2 publication Critical patent/WO2000051520A2/fr
Publication of WO2000051520A3 publication Critical patent/WO2000051520A3/fr
Anticipated expiration legal-status Critical
Publication of WO2000051520A9 publication Critical patent/WO2000051520A9/fr
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour

Definitions

  • the present invention relates to a bovine early pregnancy factor antibody, process for producing and isolating the same, and its use in a diagnostic assay for the detection of pregnancy in cattle.
  • the length of gestation in the cow, from the time of fertilization until actual birthing, of a calf ranges between 279- 285 days with an average of 283 days. Therefore, 60 percent of the reduction in calf may could be attributed to failure to mate, fertilization failure and/or embryonic mortality. This is a very conservative estimate because beef cows could be mated two to three times during the 45 to 60 day exposure to bulls. Dairy cattle or artificially inseminated cattle can be "short cycled" to allow for maximum breeding opportunities.
  • Maternal recognition must take place about day 16 to 17, or the uterus will produce prostaglandin F2a which will regress the corpus luteum and allow progesterone levels to drop with the result that the embryo can not implant. Significant embryo losses can occur during this period due to either failure of the embryo to produce the signal or failure of the mother to recognize the signal from the embryo. In either one of these situations, the cow can be short cycled or allowed to naturally return to estrous in about 21 days.
  • a current method for determining pregnancy in cow involves a rectal palpitation which requires an experienced veterinarian or technician to detect pregnancy as early as 30 days after breeding or insemination. This method requires great care, much practice and a well-developed sense of touch. Some experienced vets can detect a pregnancy by 30 days but very few persons can reliably determine pregnancy using this method. The risk factor of Bovine abortions is extremely high until day 20. Rectal palpitation is an invasive manual veterinary diagnostic method that is costly, requires a specialist and carries risk of peritonitis and death of the cow and calf.
  • the present inventors have developed an early immunochromatographic assay which detects specific markers (i.e., factors) present in saliva, sera or blood which indicate whether the conception (i.e., pregnancy), has occurred.
  • pregancy detection as early as 30 to 48 hours (the fertile life span of bovine sperm) after insemination (as opposed to the currently forty days) is feasible.
  • the present invention provides a method for producing an antibody to a factor indicating conception and pregnancy (i.e., early pregnancy factor) and its use in detecting pregnancy in any animal producing the early pregnancy factor after pregnancy.
  • an antibody refers to an immunoglobulin which binds to a specific antigen or hapten. Specifically, the term “antibody” refers to an antibody to a bovine' s early pregnancy factor, unless otherwise stated.
  • an early pregnancy factor refers to a compound, for example, a protein or a hormone, that is present in the blood stream and is produced by a pregnant cow in the early stages of the pregnancy and is not produced in any significant amount, if at all, by a non- pregnant cow. It should be understood that the early pregnancy factor may include more than one type of compound. However, for the sake of brevity and clarity, both plural and singular forms of the early pregnancy factor and the corresponding antibodies will be referred to in a singular form. Thus, the antibody of the present invention can be a polyclonal antibody or a monoclonal antibody, unless otherwise stated.
  • the early pregnancy factor is produced in cow within about 5 days after becoming pregnant, preferably within about 2 days, and more preferably within about 1 day. Moreover, the early pregnancy factor is believed to decrease to undetectable level after about 40 days.
  • the antibody of the present invention is produced by obtaining a biological fluid of a cow in its early pregnancy, injecting the biological fluid to another animal which is capable of producing antibody to the biological fluid, isolating antibodies raised against the biological fluid and substantially removing antibodies that are negative to the early pregnancy factor.
  • Figure 1 is a schematic representation of a test device for use in an assay test in accordance with the invention
  • FIG. 1 is an illustration of the test device of Figure 1 showing the direction of liquid sample flow in accordance with the invention
  • Figure 3 illustrates a variety of detection zone markers for a possible positive pregnancy test indication
  • Figure 4 shows one embodiment of the top portion of a liquid sampling tube of the present invention for use with a test device
  • Figure 5 shows one embodiment of a body portion of a liquid sampling tube of the present invention
  • Figure 6 shows one embodiment of a bottom portion of a liquid sampling tube of the present invention.
  • the production of an antibody in response to a given antigen or hapten is common occurrence in humans and animals.
  • the antibody is polyclonal in mammals; however, monoclonal antibodies can also be produced, for example, by using carcinoma cell lines.
  • the antibody in the present invention can be prepared by taking a biological fluid sample from a bovine that is less than 100 days pregnant, preferably from about 20 days to about 40 days pregnant.
  • biological fluid refers to any fluid that can be obtained from the bovine. Such fluids include blood, saliva, urine, milk, . ⁇ perspiration and chorionic fluid.
  • the biological fluid is blood.
  • acellular fraction is separated from the cellular fraction by any known method such as by centrifuge, settling, or filtration.
  • the acellular fraction, i.e., serum is then injected into a non-bovine animal following standard immunization procedures.
  • non- bovine animals useful in generating antibodies to the early pregnancy factor include mammals such as sheep, goats, equines, swine, mice, rabbits and poultry.
  • an egg can also be used to produce an antibody, e.g., IgY, for the early pregnancy factor.
  • the antibody is isolated from the non-bovine animal.
  • the incubation period is from about 10 days to about 45 days, preferably from about 20 days to about 30 days, and more preferably from about 25 days to about 30 days.
  • Isolation of the antibody of the present invention generally involves obtaining antibodies present in the blood or the lymph node system of immunized animal and isolating the antibody to the bovine early pregnancy factor. For example, in cases where a goat is used to generate the antibody, the goat is incubated with the serum containing bovine early pregnancy factor for about 6 weeks to about 2 months. The blood of the goat is then
  • immunoglobulin ⁇ i.e., Ig ⁇
  • the purity of the antibody of the present is at least about 30 % by weight (by wt.), preferably at least about 60 % by wt . , and more preferably at least about 90 % by wt.
  • Monoclonal antibody to the bovine early pregnancy factor can also be prepared using a process similar to that discussed by Milstein and Kohler as reported in Nature, 1975, 256, 495-497.
  • the preparation of a monoclonal antibody involves injecting a mouse (or other suitable animal) with partially or completely purified bovine early pregnancy factor.
  • the immunized animals are sacrificed and the cells from their spleens are fused, e.g., with mouse myeloma cells.
  • the result is a hybrid cell, known as a "hybridoma" that is capable of reproducing in vitro .
  • the population of hybridomas is then screened for immunoglobulin production using any of the known methods, for example, as described in U.S. Patent No. 4,016,043.
  • the immunoglobulins present in the cell culture fluids are then further examined for their ability to react with the bovine early pregnancy factor used for immunization.
  • the antibody of the present invention may be used in a variety of manners, e . g. , in a solid phase or in a solution, to determine pregnancy in a female bovine.
  • a test device includes a dry porous solid phase with immobilized antibody in a detection zone.
  • a use of solid phase assay with visual readout to determine the presence or the absence of a given substrate is generally described in U.S. Patent Nos. 4,703,017, and 5,656,503 which are incorporated herein by reference in their entirety.
  • the porous solid phase comprises any material or combination of materials having a high permeability to liquids.
  • exemplary porous solid phase materials include porous plastics material, such as polypropylene, polyethylene (preferably of very high molecular weight) , polyvinylidene fluoride, ethylene vinyl acetate, acrylonitrile and polytetrafluoroethylene; glass fibers; paper; cellulosic materials such as nitrocellulose; cellulose esters such as cellulose acetate; Nylon 0 ; Rayon 15 ; polyester; polyeth ⁇ rsulfone; and other suitable materials known in the art or combinations thereof. It can be advantageous to pre-treat the porous solid phase material with a surface-active agent during manufacture, as this can reduce any inherent hydrophobicity in the porous solid phase material and therefore enhance its ability to take up and deliver a moist sample efficiently. '
  • the porous solid phase material includes nitrocellulose sheet having a pore size of between 0.1 micron and 20 micron, more preferably between 3 micron and 12 micron, and most preferably 5 micron and 8 micron.
  • the antibody may be bound to the matrix of the detection zone by any one of a number of methods known to the art.
  • the antibody may be absorbed onto various water insoluble matrices such as microtiter plates, Dextran beads, nylon web, glass, cellulose, polyacrylamide, charcoal, urethane, ceramic, or mixtures thereof; chemically bonded to the porous solid phase material, i.e., by the formation of ionic or covalent bonds, including disulfide bond formation, hydrogen bonding, Van der Waals bonding and charge attraction; or physically attached to the porous solid phase material, i.e., by absorption, entrapment in an insoluble matrix; and the like.
  • the bound antibody can then be provided in a kit wherein body fluids from female bovine animals would be added and activity of the antibody with the fluids could be measured.
  • the antibody in the detection zone is relatively permanently immobilized in the detection zone on the porous solid phase material and is therefore not mobile in the moist state.
  • the relative positioning of the labelled antibody, if present within the test device, and the detection zone are such that a liquid sample which is applied to the device can pick up a labelled antibody (either from the liquid sample or the labelled antibody zone) and thereafter permeate into the detection zone.
  • the antibody in the detection zone can be immobilized firmly with or without prior chemical treatment. For example, if the material comprising the porous solid phase is nitrocellulose, which is preferred, then the antibody can be immobilized firmly by applying a liquid solution containing the antibody and allowing it to dry.
  • nitrocellulose refers to nitric acid esters of cellulose, which may be nitrocellulose alone, or a mixed ester of nitric acid and other acids, and in particular aliphatic carboxylic acids having from one to seven carbon atoms, with acetic acid being preferred.
  • Such solid supports which are formed from cellulose esterified with nitric acid alone, or a mixture of nitric acid, are often referred to as nitrocellulose paper.
  • nitrocellulose is a preferred porous solid phase material, it is to be understood that other materials, having a surface area sufficient for supporting the antibody in a concentration as hereinafter described may also be employed for producing such porous solid phase.
  • the porous solid phase which is used in the assay has a surface area such that it is capable of supporting antibody in a concentration of at least about 0.2 ⁇ M/cm 2 , and preferably in
  • a concentration of at least about 1 ⁇ M/cm 2 a concentration of at least about 1 ⁇ M/cm 2 .
  • the immobilized antibody in the detection zone is impregnated throughout the thickness of the porous solid phase material in the detection zone (e.g., throughout the thickness of the sheet or strip if the porous solid phase material is in this form) .
  • Such impregnation can enhance the extent to which the immobilized antibody can capture any labelled antibody-early pregnancy factor complex present in the migrating sample.
  • the liquid solution useful in applying the antibody to the detection zone for immobilization can include a buffer solution.
  • Useful buffer solutions include any buffer solution having a pH from about pH 6.5 to about pH 9.5.
  • Exemplary useful buffer solutions include phosphate buffers with a phosphate level in the range of from about 10 mM to about 100 mM, phosphate buffered saline buffers, TRIS buffer pH 8.2 in the range of from about 10 mM to about 50 mM, TRIS buffered saline pH 8.2, acetate buffer, carbonate buffer, bicarbonate buffer, MOPS piperazine buffer, BIS- TRIS buffer, tricine buffer, HEPES buffer and the like.
  • the liquid solution can also include a surfactant.
  • a surfactant Any general surfactant can be used.
  • Exemplary surfactants useful for the present invention include Tween 20 ® , Triton X-IOO 0 .
  • the amount of surfactant can be from about 0.01% to about 10%, preferably about 0.5%.
  • the immobilization of the antibody in the detection zone needs to be performed by covalent linkage which can be achieved through chemical coupling using, for example, aldehydes, azo compounds, carboxylic acids, isothiocyanates, cyano compounds, CNBr, carbonyldiimidazole, or tresyl chloride.
  • the remainder of the porous solid phase material may be treated to block any remaining binding sites. Blocking can be achieved by treatment with protein (e.g., bovine serum albumin or milk protein) , with polyvinyl alcohol or ethanolamine, surfactant, polymers (such as PVA, PVP, PEG), or combinations thereof.
  • the test device can also include a sample receiving zone for applying a sample, e.g., a liquid biological sample such as urine, serum, or saliva.
  • a sample e.g., a liquid biological sample such as urine, serum, or saliva.
  • the porous solid phase material in the sample receiving zone can include any bibulous, porous or fibrous material capable of absorbing liquid rapidly.
  • the porosity of the material can be unidirectional (i.e., with pores or fibers running wholly or predominantly parallel to an axis of the member) or multidirectional (omnidirectional, so that the member has an amorphous sponge-like structure) .
  • the porous solid phase material of the sample receiving zone should be chosen such that the sample receiving zone can be saturated with aqueous liquid within a matter of seconds.
  • the material remains robust when moist.
  • the test device can also include a labelled bovine early pregnancy factor antibody zone ("labelled antibody zone") within the porous solid phase located downstream relative to the sample receiving zone but upstream relative to the detection zone.
  • labelled antibody zone refers to antibody which is bound to a label.
  • the label can be any entity the presence of which can be readily detected by any of the known methods in the art including by visual inspection, using an instrument, conducting a chemical reaction and combinations thereof.
  • the label is a direct label, i.e., an entity which, in its natural state, is readily visible either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g., UV light to promote fluorescence.
  • minute colored particles such as dye sols; metallic sols such as gold, platinum, palladium, iron, copper; colored latex particles; and carbon sols are all suitable. Of these metallic sol particles are preferred with gold sol particles being particularly preferred.
  • sol particle refers to particles of a sol.
  • metal sol particle refers to particle of a sol, consisting of a metal, a metal compound or polymer nuclei coated with a metal or metal compound.
  • the useful size of a sol particle depends on the particular sol particle being used, for example, for gold sols useful size ranges from about 15 nm to about 100 nm, with about 40 nm being particularly preferred.
  • Using metallic and non-metallic sol particles for immunoassay is generally described in U.S. Patent Nos. 4,313,734 and 4,954,452, respectively, which are incorporated by reference herein in their entirety. Concentration of the label into a small zone or volume should give rise to a readily detectable signal, e . g. a strongly-colored area. This can be evaluated by eye, or by instruments if desired.
  • Indirect labels such as enzymes (e.g., alkaline phosphatase and horseradish peroxidase) , radio isotopes, fluorescent compounds or other compound, which can be detected using appropriate instruments, can also be used but these usually require an instrument or the addition of one or more developing reagents such as substrates before a visible signal can be detected.
  • additional reagents can be incorporated in the porous solid phase material or in the sample receiving zone, such that they dissolve or disperse in the aqueous liquid sample.
  • the developing reagents can be added to the sample before contact with the porous solid phase material or the porous solid phase material can be exposed to the developing reagents after the binding reaction has taken place.
  • Coupling of the label to the specific binding reagent can be by covalent bonding, if desired, or by hydrophobic or electrostatic bonding. Such techniques are well known to one of ordinary skill in the art.
  • the porous solid phase material which is used in the assay has a surface area such that it is capable of supporting labelled antibody in a concentration of at least about 0.2 ⁇ M/cm 2 , and preferably in a concentration of at least about 0.5
  • the labelled antibody is applied to the porous solid phase as a surface layer, rather than being impregnated in the thickness of the porous solid phase material. This can minimize interaction between the porous solid phase material and the labelled antibody.
  • the porous solid phase material is pre-treated with a glazing material in the region to which the labelled antibody is to be applied. Glazing can be achieved, for example, by depositing an aqueous sugar or cellulose solution, e.g., of sucrose or lactose, on the porous solid phase at the relevant portion, and drying. The labelled antibody can then be applied to the glazed portion. The remainder of the porous solid phase should not be glazed.
  • the labelled antibody can be provided separately from the test device and admixed with the sample prior to being applied to the sample receiving zone of the test device. In this manner, any bovine early pregnancy factor which may be present in the sample is allowed to bind with the labelled antibody prior to or during application of the sample to the sample receiving zone.
  • Such labelled antibody can be provided as a solution or a solid which is then admixed with the sample.
  • the solution generally comprises a buffer solution which stabilizes the labelled antibody, i.e., prevents a significant decrease in the antibody activity by, e.g., preventing decomposition or denaturing of the antibody.
  • Useful buffer solutions include those described above.
  • the solution may also include a surfactant described above.
  • the labelled antigen is an integral part of the test device or is provided separately, it is essential that the labelled antibody migrates with the liquid sample as this progresses to the detection zone.
  • the flow of the sample continues beyond the detection zone and sufficient sample is applied to the porous solid phase material in order that this may occur and that any excess labelled antibody which does not participate in any binding reaction in the detection zone is flushed away from the detection zone by this continuing flow.
  • an absorbent is applied to the porous solid phase material in order that this may occur and that any excess labelled antibody which does not participate in any binding reaction in the detection zone is flushed away from the detection zone by this continuing flow.
  • the absorbent sink can be provided at the distal end of the porous solid phase material.
  • the absorbent sink may comprise, for example, Whatman
  • 3MM chromatography paper should provide sufficient absorptive capacity to allow any unbound labelled antibody, i.e, conjugate, to wash out of the detection zone.
  • a sink it can be sufficient to have a length of porous solid phase material which extends beyond the detection zone.
  • test device employed in the assay is preferably in sheet form, generally being in the form of a card, a test strip, a flow through device or dipstick, etc. It should be appreciated, however, that other forms of test devices are also within the spirit and scope of the invention.
  • the antibody and the labelled antibody, if present, are applied in spatially distinct zones of the test device, and the liquid sample is allowed to permeate through the test device from one side or end to another.
  • the porous solid phase of the test device is capable of conveying a liquid sample in a fluid flow direction parallel to the length of the test device.
  • the spatial separation between the zones (i.e., sample receiving zone, labelled antibody zone, if used, and the detection zone) , and the flow rate characteristics of the porous solid phase material can be selected to allow adequate reaction times during which the necessary specific binding can occur, and to allow the labelled antibody in the labelled antibody zone to dissolve or disperse in the liquid sample and migrate through the porous solid phase material. Further control over these parameters can be achieved by the incorporation of viscosity modifiers (e.g., sugars and modified celluloses) in the sample to slow down the reagent migration.
  • viscosity modifiers e.g., sugars and modified celluloses
  • the porous solid phase material is "backed", e.g., with plastic sheet, to increase its handling strength.
  • This can be manufactured easily by forming a thin layer of porous solid phase material such as nitrocellulose on a sheet of backing material.
  • the actual pore size of the porous solid phase material when backed in this manner may be lower than that of the corresponding unbacked material.
  • a pre-for ed sheet of porous solid phase material can be tightly sandwiched between two supporting sheets of solid materials, e.g., between two supporting plastic sheets.
  • the antibody and/or the labelled antibody can be applied to the porous solid phase material in a variety of ways.
  • porous solid phase materials e.g., micro-syringes, pens using metered pumps, direct printing and ink-jet printing, and any of these techniques can be used in the present invention.
  • the porous solid phase material e.g., sheet
  • the porous solid phase material can be treated with these reagents and then subdivided into smaller portions (e.g., small narrow strips each embodying the required reagent containing zones) to provide a plurality of identical porous solid phase material units.
  • the sensitivity of the assay i.e., bovine pregnancy testing, can be increased by increasing the concentration of the labelled antibody and/or the antibodies on the detection zone.
  • porous solid phase materials having high surface areas are particularly preferred in that the labelled antibody and/or the antibodies in the detection zone may be supported on such material in a high concentration. It should be appreciated, however, that the concentration of the labelled antibody and/or the antibodies in detection zone which is actually used is dependent in part on the binding affinity of the labelled antibody and/or the antibodies in the detection zone. Therefore, the scope of the present invention is not limited to a particular concentration of labelled antibody and/or the antibodies on the detection zone.
  • Pregnancy testing of a female bovine generally involves contacting the test device as set forth above with an aqueous liquid sample (e.g., liquid biological sample such as serum, saliva, urine or chorionic fluid) of the female bovine.
  • aqueous liquid sample e.g., liquid biological sample such as serum, saliva, urine or chorionic fluid
  • the sample is allowed to permeate by capillary action through the porous solid phase material via the labelled antibody zone, if present, migrates from the labelled antibody zone to the detection zone and the labelled antibody migrates therewith from the solution or from the labelled antibody zone, depending on how the labelled antibody is presented in .the test.
  • the labelled antibodies are carried downstream as the liquid sample front is moved along the test device by a capillary action.
  • any early pregnancy factor that may present in the liquid sample binds to the labelled antibody and the resulting complex is carried along the length of the test device to the detection zone.
  • the presence of an early pregnancy factor in the sample can be determined by observing the extent (if any) to which the complex becomes bound in the detection zone.
  • the labelled antibody zone if present in the test device, is located at a height sufficiently high enough in the test device such that when the liquid sample is applied, the labelled antibody zone is above the liquid sample level to avoid having the labelled antibodies diffusing into the liquid sample itself.
  • the distance between these two zones should be sufficiently large enough to allow the formation of labelled antibody-early pregnancy factor complex prior to the capture of the complex by the antibodies present in the detection zone.
  • a distance is at least about 1 cm, and more preferably at least about 2 cm.
  • the detection zone can be any shape which aids in visualizing the bovine pregnancy test result.
  • the detection zone can be a strip of line across the partial or entire width ⁇ of the test device, an "X", "+", a check mark, a circle, or a ring.
  • the observation is made visually by the presence of a particular color within the detection zone where the antibody has been immobilized. For example, with gold sols as labels the color red indicates the positive result, with carbon or palladium sols the positive pregnancy is indicated by black color, and a wide variety of color can be used as indicator for latex particles depending on the dye that is used.
  • liquid sampling tube which may be used in conjunction with the test device discussed above.
  • the term "tube” refers to any conduit with a hollow interior.
  • the cross section of the hollow interior of the tube can be a circle, rectangle, hexagon, triangle, oval or any similarly shaped symmetrical or non- symmetrical area.
  • the liquid sampling tube includes a transparent region for reading the result of a female bovine pregnancy test using the test device described above. Preferably the entire length of the tube is transparent.
  • Such a liquid sampling tube can be made from any variety of materials which is transparent or allows incorporation of a transparent material • on or near the detection zone of the above test device. Exemplary materials suitable for, manufacturing the liquid sampling tube include polycarbonate, tetrafluoroethylene polymer, polyacetate, plastics, glass and combinations thereof.
  • the tube includes a bottom portion having a means for allowing a liquid to enter into the tube.
  • a means includes any device which allows a liquid sample to enter into the tube.
  • Exemplary devices include an opening in the bottom portion of the tube, a filter which may or may not be removable, a porous membrane, and combinations thereof.
  • the porosity of the filter should be sufficiently large enough to allow any dissolved material, in particular the early pregnancy factor, within the liquid to also enter into the tube.
  • the filter should have porosity of no greater than 200 micron with a flow rate greater than 10 liters per minute per square meter of material, at a differential pressure of 1 bar.
  • the liquid sampling tube of the present invention also includes a body portion having at least one opening or pressure equalizing means.
  • a "pressure equalizing means” refers to a device or a means for allowing the pressure within the tube to equalize with the pressure outside the tube. Such means includes an opening (e.g., a hole), and a porous membrane. The presence of means for equalizing pressure allows a relative control as to how much the liquid is sampled by the tube.
  • the liquid sampling tube of the present invention also includes a top portion for enclosing the tube. In this manner, the gas that is displaced within the tube by the liquid sample is released primarily through the pressure equalizing means.
  • the amount of liquid sampling by the tube is substantially proportional to the height of the opening on the body portion of the tube relative to the bottom of the tube.
  • the liquid sampling tube of the present invention eliminates the requirement for a small sample to be removed from the body of a liquid in order to be tested. The process of correct use of the liquid sampling tube also ensures that sufficient liquid material is presented to the testing area, provided that sufficient material is available. The requirement for accurate placement of the sample is also eliminated as the design of the liquid sampling device ensures that with correct operation, material is only presented to the correct sampling location.

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  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un test de grossesse pour bovins dans lequel on utilise un anticorps au facteur de grossesse bovine précoce préalablement produit et isolé. Le test s'effectue au moyen d'un dispositif de test renfermant l'anticorps immobilisé au facteur de grossesse bovine précoce. En outre, il est aussi prévu un dispositif d'échantillonnage de liquide biologique permettant seulement une quantité prédéterminée d'un échantillon de liquide biologique.
PCT/US2000/005616 1999-03-02 2000-03-02 Test de grossesse pour bovins Ceased WO2000051520A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU35119/00A AU3511900A (en) 1999-03-02 2000-03-02 Bovine pregnancy testing

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US12240099P 1999-03-02 1999-03-02
US60/122,400 1999-03-02

Publications (3)

Publication Number Publication Date
WO2000051520A2 true WO2000051520A2 (fr) 2000-09-08
WO2000051520A3 WO2000051520A3 (fr) 2001-01-11
WO2000051520A9 WO2000051520A9 (fr) 2002-03-28

Family

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Family Applications (1)

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PCT/US2000/005616 Ceased WO2000051520A2 (fr) 1999-03-02 2000-03-02 Test de grossesse pour bovins

Country Status (2)

Country Link
AU (1) AU3511900A (fr)
WO (1) WO2000051520A2 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031513A1 (fr) * 2000-10-09 2002-04-18 Icpbio Limited Detection de grossesse
WO2003028582A3 (fr) * 2001-09-28 2003-06-19 Aspenbio Inc Test de grossesse pour bovins
WO2003093493A3 (fr) * 2002-05-02 2005-04-28 Aspenbio Inc Detection de la gravidite
US7125728B2 (en) 2001-06-19 2006-10-24 Idaho Research Foundation Determination of pregnancy status in cattle and sheep
US20150335902A1 (en) * 2012-12-26 2015-11-26 Koninklijke Philips N.V. Intuitive Overlaid Readiness Indicator for Defibrillators
CN108314732A (zh) * 2018-01-15 2018-07-24 妊达（北京）生物技术有限公司 一种抗牛早孕因子单克隆抗体细胞株单克隆抗体的制备方法
CN118962159A (zh) * 2024-08-07 2024-11-15 吉林农业大学 一种基于体液诊断绵羊早期妊娠的方法

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1986005498A1 (fr) * 1985-03-12 1986-09-25 University Of Queensland Procede de detection de facteur de determination precoce de grossesse (epf) ches des mammiferes, procedes de purification d'epf et de production d'anticorps monoclonaux
DE3603053A1 (de) * 1986-01-30 1987-08-06 Schering Ag Schwangerschaftsnachweis mit epf
WO1988004779A1 (fr) * 1986-12-22 1988-06-30 University Of Queensland Procede de traitement des mammiferes utilisant des anticorps contre le facteur de grossesse precoce (epf)
DE3856542T2 (de) * 1987-04-27 2003-10-30 Inverness Medical Switzerland Testgerät zur Durchführung von spezifischen Bindungsprüfungen
US5646003A (en) * 1994-03-23 1997-07-08 Barnea; Eytan R. Preimplantation factor

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002031513A1 (fr) * 2000-10-09 2002-04-18 Icpbio Limited Detection de grossesse
US7125728B2 (en) 2001-06-19 2006-10-24 Idaho Research Foundation Determination of pregnancy status in cattle and sheep
US8658431B2 (en) 2001-06-19 2014-02-25 Idaho Research Foundation Determination of pregnancy status in ungulate ruminants
WO2003028582A3 (fr) * 2001-09-28 2003-06-19 Aspenbio Inc Test de grossesse pour bovins
US7393696B2 (en) 2001-09-28 2008-07-01 Aspenbio Pharma, Inc. Bovine pregnancy test
US7687281B2 (en) 2001-09-28 2010-03-30 Aspenbio Pharma, Inc. Bovine pregnancy test
WO2003093493A3 (fr) * 2002-05-02 2005-04-28 Aspenbio Inc Detection de la gravidite
US7842513B2 (en) 2002-05-02 2010-11-30 Aspenbio Pharma, Inc. Pregnancy detection
US20150335902A1 (en) * 2012-12-26 2015-11-26 Koninklijke Philips N.V. Intuitive Overlaid Readiness Indicator for Defibrillators
US9808639B2 (en) 2012-12-26 2017-11-07 Koninklijke Philips N.V. Intuitive overlaid readiness indicator for defibrillators
CN108314732A (zh) * 2018-01-15 2018-07-24 妊达（北京）生物技术有限公司 一种抗牛早孕因子单克隆抗体细胞株单克隆抗体的制备方法
CN118962159A (zh) * 2024-08-07 2024-11-15 吉林农业大学 一种基于体液诊断绵羊早期妊娠的方法

Also Published As

Publication number Publication date
AU3511900A (en) 2000-09-21
WO2000051520A9 (fr) 2002-03-28
WO2000051520A3 (fr) 2001-01-11

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