Systems and methods for detecting animal pregnancy

Abstract

Testing systems and methods are disclosed for detecting a pregnancy marker of an animal. A test kit may include a first standard with a first concentration of the marker, a second standard with a second concentration of the marker lower than the first concentration, and at least three test surfaces coated with a biomolecular recognition element selected to bind with the marker. The test may also include a reagent solution with a conjugated biomolecular recognition element that binds with the marker, and a visual indicator that produces a visually detectable change when reacting with the conjugated biomolecular recognition element bound to each test surface. A detectable change generated by the marker from the sample with an intensity greater than the first concentration yields a pregnant result, lower than the second concentration yields a not pregnant result, and between the first and second concentrations yields a retest result.

What is claimed is: 
     
         1 . A method of determining whether a ruminant is pregnant comprising:
 providing a test kit including at least three test surfaces, each surface being coated with a pregnancy-specific protein B (PSPB) specific antibody, a first reagent solution including a conjugated PSPB specific antibody, a first standard corresponding to a minimum amount of PSPB present when the ruminant is pregnant, and a second standard corresponding to a maximum amount of PSPB present when the ruminant is not pregnant,   obtaining a first blood sample from the ruminant after insemination of the ruminant,   introducing an amount of the first standard to a first one of the test surfaces,   introducing an amount of the second standard to a second one of the test surfaces,   introducing an amount of the sample to a third one of the test surfaces,   allowing the standards and sample to react with the PSPB specific antibody on each test surface under a substantially constant temperature for a first specified time period,   introducing equal amounts of the first reagent solution to each of the three test surfaces,   allowing the conjugated PSPB specific antibody to react with the PSPB bound to each test surface under a substantially constant temperature for a second specified time period,   removing any unbound materials from each test surface, including unbound sample, unbound PSPB, unbound first and second standards, unbound first reagent solution, and unbound conjugated PSPB specific antibody,   introducing under a substantially constant temperature for a specified period of time a visual indicator to undergo a visually detectable change, the magnitude of which is related to the amount of conjugated PSPB specific antibody bound to each surface, and   
       retesting a second blood sample received from the ruminant about 3 days to about 7 days after the first sample was received if the magnitude of the visual indicator on the third surface is between the magnitudes of the visual indicators on the first and second surfaces. 
     
     
         2 . The method of  claim 1 , wherein the first standard includes between about 0.1 nanograms per milliliter to about 1.5 nanograms per milliliter of PSPB in buffer, serum or plasma and the second standard includes between about 0.01 ng/ml to about 1.0 nanograms per milliliter of PSPB in buffer, serum or plasma. 
     
     
         3 . The method of  claim 1 , wherein the first standard includes about 0.4 nanograms per milliliter of PSPB in buffer, serum or plasma and the second standard includes about 0.2 nanograms per milliliter of PSPB in buffer, serum or plasma. 
     
     
         4 . The method of  claim 1 , further comprising between the first and second allowing steps, the step of removing any unbound material from each test surface, including unbound sample, unbound PSPB, and unbound first and second standards. 
     
     
         5 . The method of  claim 1 , wherein each test surface is an inner surface of a well that is part of an assay. 
     
     
         6 . The method of  claim 1 , wherein the ruminant is a cow, and the first sample is derived from blood obtained from the cow around 30 days after insemination. 
     
     
         7 . The method of  claim 6 , wherein the cow can be re-inseminated about 1 to about 7 days after a not-pregnant result indication from the first sample or the second sample. 
     
     
         8 . The method of  claim 1 , wherein the first and second PSPB specific antibodies are selected from the group consisting of polyclonal rabbit immunoglobulin, polyclonal goat immunoglobulin, polyclonal sheep immunoglobulin, polyclonal mouse immunoglobulin, monoclonal mouse immunoglobulin, and monoclonal rabbit immunoglobulin. 
     
     
         9 . The method of  claim 1 , wherein the PSPB being detected includes one or more variants of pregnancy associated glycoproteins (PAG). 
     
     
         10 . A test kit for detecting a selected marker of pregnancy from a sample of an animal, comprising:
 a first standard solution including a first concentration of the selected marker,   a second standard solution including a second concentration of the selected marker different than the first concentration,   at least three test surfaces, each test surface coated with a biomolecular recognition element selected to bind with the selected marker,   a first reagent solution including a conjugated biomolecular recognition element selected to bind with the selected marker, and   a visual indicator selected for its property of producing a visually detectable change under a substantially constant temperature and a specified reaction time when reacting with the conjugated biomolecular recognition element bound to each test surface, wherein the first and second concentrations of the marker are selected to generate the visually detectable change such that a visually detectable change generated by the marker from the sample with an intensity greater than the first concentration yields a first result, lower than the second concentration yields a second result, and between the first and second concentrations yields a retest result indicating to retest a second sample retrieved from the animal at a time period after the first sample.   
     
     
         11 . The kit of  claim 10 , wherein the first result is a pregnant result and the second result is a not pregnant result. 
     
     
         12 . The kit of  claim 11 , wherein a visually detectable change generated by the marker from the sample with an intensity equal to the first concentration yields a pregnant result, and equal to the second concentration yields a not pregnant result. 
     
     
         13 . The kit of  claim 10 , wherein the marker is pregnancy-specific protein B (PSPB), estrone sulfate, progesterone, or an interferon stimulated gene protein. 
     
     
         14 . The kit of  claim 10 , wherein the biomolecular recognition element is a detection antibody and the conjugated biomolecular recognition element is a conjugated detection antibody. 
     
     
         15 . The kit of  claim 14 , wherein the kit includes the first reagent solution including the conjugated detection antibody conjugated with a hapten, a second reagent solution with an agent selected to bind with the hapten-conjugated detection antibody, and a third reagent solution including the visual indicator selected to bind with the agent. 
     
     
         16 . The kit of  claim 14 , wherein the kit includes the first reagent solution with the conjugated detection antibody conjugated with the visual indicator. 
     
     
         17 . The kit of  claim 14 , wherein the kit includes the first reagent solution with the conjugated detection antibody conjugated with an enzyme, and a second reagent solution including the visual indicator selected to bind with the enzyme-conjugated second detection antibody. 
     
     
         18 . The kit of  claim 10 , wherein the biomolecular recognition element coated on each well is sealed with a blocking agent, and a preserving agent derived from a stabilizer block solution that includes a non-reducing sugar and a blocking material. 
     
     
         19 . The kit of  claim 18 , wherein the stabilizer block solution has a pH of about 7.4, the non-reducing sugar is about 0.25% to about 20% weight to volume, and the blocking material is bovine serum albumin. 
     
     
         20 . The kit of  claim 10 , wherein the test surfaces are inner surfaces of wells of an array or part of a test strip.