Method for determining vitamin B12 and reagent therefor

Abstract

Method for determining vitamin B 12 in aqueous samples by contacting said sample with radioactivity labelled vitamin B 12 and with particles of water insoluble polymers to which substances capable of binding vitamin B 12 have been bound, thereafter separating said particles and determining the radioactivity.

What we claim is: 
     
       1. A method for determining vitamin B 12  in an aqueous sample, which comprises contacting particles of water-insoluble polymers to which a substance capable of binding vitamin B 12  has been bound, by covalent bonds, with the sample and with a certain amount of vitamin B 12  labelled with a radioisotope, and subsequent to the reaction between vitamin B 12  and the substance capable of binding vitamin B 12  attached to the particles having taken place, separating the particles from the sample liquid and determining the radioactivity of the particle material. 
     
     
       2. A method according to claim 1, wherein the radioactivity is also determined in the fluid. 
     
     
       3. A method according to claim 1, wherein vitamin B 12  labelled with a radioisotope of cobalt is used in the determination. 
     
     
       4. A method according to claim 1, wherein the substance capable of binding vitamin B 12  which has been bound to the polymer particles by covalent bonds, is intrinsic factor. 
     
     
       5. A method according to claim 1, wherein the determination is effected quantitively. 
     
     
       6. A method for determining vitamin B 12  in an aqueous sample, which comprises contacting particles of water-insoluble polymers to which a substance capable of binding vitamin B 12  has been bound, by covalent bonds, with the sample and with a certain amount of vitamin B 12  labelled with a radioisotope, and subsequent to the reaction between vitamin B 12  and the substance capable of binding vitamin B 12  attached to the particles having taken place, separating the particles from the sample liquid and determining the radioactivity in the fluid. 
     
     
       7. A method according to claim 6, wherein vitamin B 12  labelled wih a radioisotope of cobalt is used in the determination. 
     
     
       8. A method according to claim 6, wherein the substance capable of binding vitamin B 12  which has been bound to the polymer particles by covalent bonds, is intrinsic factor. 
     
     
       9. A method according to claim 6, wherein the determination is effected quantitively. 
     
     
       10. A reagent for use in the determination of vitamin B 12 , containing particles of water insoluble polymers to which have been bound, by covalent bonds, a substance capable of binding vitamin B 12 , in dried form. 
     
     
       11. A reagent according to claim 10, wherein the reagent is in lyophilized form. 
     
     
       12. Sealed ampoules containing the reagent as claimed in claim 11. 
     
     
       13. Test pack for the determination of vitamin B 12  chiefly comprising one or more sealed ampoules containing particles of water insoluble polymers to which have been bound, by covalent bonds, a substance capable of binding vitamin B 12  in dried form and another ampoule with vitamin B 12  labelled with a radioisotope in dried form. 
     
     
       14. Test pack according to claim 13, wherein the particles of water-insoluble polymers to which have been bound by covalent bonds a substance capable of binding vitamin B 12  is in lyophilized form. 
     
     
       15. Test pack according to claim 14, wherein the vitamin B 12  labelled with a radioisotope is in lyophilized form. .Iadd. 16. A method according to claim 1, wherein the substance capable of binding vitamin B 12  is a protein fraction from blood plasma. .Iaddend..Iadd. 17. A method according to claim 1, wherein said covalent bonds are formed through a group of the formula --NH·CS·NH--, --NH·CO·NH--, or --N=N--. .Iaddend..Iadd. 18. A method according to claim 17, wherein said group is --NH·CS·NH--. .Iaddend..Iadd. 19. A method according to claim 1, wherein said water-insoluble polymers contain at least one reactive group selected from the group consisting of amino, hydroxyl and carboxyl. .Iaddend..Iadd. 20. A method according to claim 19, wherein said reactive group is hydroxyl and wherein said substance capable of binding vitamin B 12  is covalently bound to said polymer through reaction with a cyanogen halide. .Iaddend..Iadd. 21. A method according to claim 19, wherein said reactive group is amino and wherein said substance capable of binding vitamin B 12  is covalently bound to said polymer through reaction with a cyanogen halide. .Iaddend..Iadd. 22. A method according to claim 1, wherein said polymer is obtained by cross-linking a material selected from the group consisting of carbohydrates, sugar alcohols, and polyvinyl alcohol, with a bifunctional compound of the formula X--R--Z, wherein X and Z are each independently halogen or epoxy, and R is an aliphatic radical containing from 3 to 10 carbon atoms. .Iaddend. .Iadd. 23. A method according to claim 22, wherein said material is dextran and said bifunctional compound is epichlorohydrin. .Iaddend..Iadd. 24. A reagent according to claim 10, wherein the substance capable of binding vitamin B 12  is a protein fraction from blood plasma. .Iaddend..Iadd. 25. A reagent according to claim 10, wherein the substance capable of binding vitamin B 12  is intrinsic factor. .Iaddend..Iadd. 26. A reagent according to claim 10, wherein said covalent bonds are formed through a group of the formula --NH·CS·NH--, --NH·CO·NH--, or --N=N--. .Iaddend..Iadd. 27. A reagent according to claim 26, wherein said group is --NH·CS·NH--. .Iaddend..Iadd. 28. A reagent according to claim 10, wherein said water-insoluble polymers contain at least one reactive group selected from the group consisting of amino, hyroxyl and carboxyl. .Iaddend..Iadd. 29. A reagent according to claim 28, wherein said reactive group is hydroxyl and wherein said substance capable of binding vitamin B 12  is covalently bound to said polymer through reaction with a cyanogen halide. .Iaddend..Iadd. 30. A reagent according to claim 10, wherein said polymer is obtained by cross-linking a material selected from the group consisting of carbohydrates, sugar alcohols, and polyvinyl alcohol, with a bifunctional compound of the formula X--R--Z, wherein X and Z are each independently halogen or epoxy, and R is an aliphatic radical containing from 3 to 10 carbon atoms. .Iaddend. .Iadd. 31. A reagent according to claim 30, wherein said material is dextran and said bifunctional compound is epichlorohydrin. .Iaddend.