Method of determining pregnanediol in female urine and test device for use therein

Abstract

A method is provided for determining the concentration of pregnanediol glucuronide (PG) in a woman's urine which is characterized by utilization of the reagent 20α-hydroxy-4-pregnen-3-one (20-α reagent) in a form in which it reacts with antibodies binding to PG. The method is adaptable to a visual color indication test which can be performed by the woman herself as well as by laboratory analysis. The method can be used to define the period in which conception can occur, to define a post-ovulation safe period in which conception is prevented, and as an early pregnancy indicator.

I claim: 
     
       1. The method of determining the concentration of pregnanediol glucuronide (PG) in female urine comprising: (a) adding to a measured sample of said urine a predetermined quantity of antibodies binding to PG, said antibodies being labeled with a substance capable of providing a visual color indication;   (b) contacting the reacted urine sample with a predetermined quantity of a 20-α reagent immobilized on a color indicator support, said reagent consisting essentially of 20α-hydroxy-4-pregnen-3-one, the unreacted labeled antibodies from said urine binding to said 20-α reagent on said support; and   (c) determining from the intensity of color developed the concentration of PG in the urine sample.   
     
     
       2. The method of claim 1 in which said PG-binding antibodies are conjugated to a color indicator dye. 
     
     
       3. The method of claim 1 in which said PG-binding antibodies are conjugated to rhodamine. 
     
     
       4. The method of claim 1 in which the antibodies are conjugated to an enzyme capable of providing a color indication reaction. 
     
     
       5. The method of claim 1 in which said color indicator support provides glass surfaces for binding to said 20-α reagent. 
     
     
       6. The method of claim 1 in which said PG-binding antibodies are conjugated to a color indicator dye, and in which said color indicator support provides glass surfaces for binding to said 20-α reagent. 
     
     
       7. The method for claim 1 in which said antibodies after adsorption on said support are stained with a protein-staining dye for more intense color development. 
     
     
       8. The method of determining the concentration of pregnanediol glucuronide (PG) in a woman's urine, comprising: (a) adding to a measured sample of said urine a predetermined quantity of a 20-α reagent, said reagent consisting essentially of 20α-hydroxy-4-pregnen-3-one and being labeled with a substance capable of providing a visual color indication;   (b) contacting the reacted urine with a predetermined quantity of antibodies binding to PG immobilized on a color indicator support, said labeled 20-α reagent and said PG reacting competitively with said antibodies; and   (c) determining from the intensity of color developed the concentration of PG in the urine sample.   
     
     
       9. The method of claim 8 in which said 20α reagent is conjugated to an enzyme capable of providing a color indication reaction. 
     
     
       10. A test device for use in determining the concentration of pregnanediol glucuronide (PG) in a sample of female urine, comprising a color indicator support having immobilized thereon a known quantity of a 20-α reagent consisting essentially of 20α-hydroxy-4-pregnen-3-one. 
     
     
       11. The test device of claim 10 in which said support provides glass surfaces for binding to said 20-α reagent. 
     
     
       12. A test strip for use in determining the concentration of pregnanediol glucuronide in a sample of a woman's urine, comprising an adsorbent fibrous strip having immobilized thereon a known quantity of a 20-α reagent consisting essentially of 20α-hydroxy-4-pregnen-3-one. 
     
     
       13. The test strip of claim 12 in which said strip is composed of a glass fiber paper. .Iadd. 
     
     
       14.  The method of determining the concentration of pregnanediol glucuronide (PG) in female urine, comprising: (a) adding to a measured sample of said urine a predetermined quantity of antibodies binding to PG for reacting said antibodies with the amount of PG in the urine, said antibodies being in excess of the amount of PG to leave unreacted antibodies;   (b) contacting the antibody-containing sample with a quantity of a 20-α reagent immobilized on a support for reacting the antibodies which have not reacted with said PG with said 20-α reagent on said support, said 20-α reagent consisting essentially of a 20-α-hydroxy-4-pregnen-3-one, the said quantity of 20-α reagent being at least sufficient to bind the PG-unreacted antibodies;   (c) separating the residual urine sample from said support with said PG-unreacted antibodies bound thereto; and, thereafter,   (d) determining the amount of PG-unreacted antibodies thus separated from the urine sample as a measure of the PG concentration in the urine sample by (i) a color change reaction indicating separated antibody quantity, or (ii) by means of an enzyme associated with said separated antibodies which enzyme is capable of providing a color reaction indicating antibody quantity, or (iii) by means of a protein-staining dye which dye is capable of providing a color reaction indicating antibody quantity, or (iv) by means of a radiolabel associated with said separated antibodies which radiolabel is capable of providing an indication of antibody quantity. .Iaddend. .Iadd.15. The method of claim 14 in which said support provides glass surfaces to which said 20-α reagent is bound. .Iaddend. .Iadd.16. A test device for use in determining the concentration of pregnanediol glucuronide (PG) in a sample of female urine comprising a support having immobilized thereon a quantity of a 20-α reagent consisting essentially of a 20-α hydroxy-4-pregnen-3-one, said immobilized 20-α reagent being capable of binding to antibodies   
     
     
        binding to PG. .Iaddend. .Iadd.17.  The test device of claim 16 in which said support provides glass surfaces to which said 20-α reagent is bound. .Iaddend.