USRE29480E - Method for determining vitamin B12 and reagent therefor - Google Patents
Method for determining vitamin B12 and reagent therefor Download PDFInfo
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- USRE29480E USRE29480E US05/598,142 US59814275A USRE29480E US RE29480 E USRE29480 E US RE29480E US 59814275 A US59814275 A US 59814275A US RE29480 E USRE29480 E US RE29480E
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- Prior art keywords
- vitamin
- iaddend
- iadd
- substance capable
- binding
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- FDJOLVPMNUYSCM-WZHZPDAFSA-L cobalt(3+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical compound [Co+3].N#[C-].N([C@@H]([C@]1(C)[N-]\C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C(\C)/C1=N/C([C@H]([C@@]1(CC(N)=O)C)CCC(N)=O)=C\C1=N\C([C@H](C1(C)C)CCC(N)=O)=C/1C)[C@@H]2CC(N)=O)=C\1[C@]2(C)CCC(=O)NC[C@@H](C)OP([O-])(=O)O[C@H]1[C@@H](O)[C@@H](N2C3=CC(C)=C(C)C=C3N=C2)O[C@@H]1CO FDJOLVPMNUYSCM-WZHZPDAFSA-L 0.000 title claims abstract description 93
- 239000011715 vitamin B12 Substances 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims abstract description 31
- 239000003153 chemical reaction reagent Substances 0.000 title claims description 17
- 229930003779 Vitamin B12 Natural products 0.000 title 1
- 235000019163 vitamin B12 Nutrition 0.000 title 1
- 239000000126 substance Substances 0.000 claims abstract description 53
- 239000002245 particle Substances 0.000 claims abstract description 45
- 229920003176 water-insoluble polymer Polymers 0.000 claims abstract description 12
- 229920000642 polymer Polymers 0.000 claims description 20
- 229940029329 intrinsic factor Drugs 0.000 claims description 13
- -1 amino, hydroxyl Chemical group 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 229920002307 Dextran Polymers 0.000 claims description 8
- 230000001588 bifunctional effect Effects 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 claims description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 4
- 235000014633 carbohydrates Nutrition 0.000 claims description 4
- 229910017052 cobalt Inorganic materials 0.000 claims description 4
- 239000010941 cobalt Substances 0.000 claims description 4
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 4
- JMANVNJQNLATNU-UHFFFAOYSA-N glycolonitrile Natural products N#CC#N JMANVNJQNLATNU-UHFFFAOYSA-N 0.000 claims description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 3
- 125000004432 carbon atom Chemical group C* 0.000 claims description 3
- 238000004132 cross linking Methods 0.000 claims description 3
- 229910052736 halogen Inorganic materials 0.000 claims description 3
- 150000002367 halogens Chemical class 0.000 claims description 3
- 210000002381 plasma Anatomy 0.000 claims description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 3
- 150000005846 sugar alcohols Chemical class 0.000 claims description 3
- 239000003708 ampul Substances 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 4
- 239000004593 Epoxy Substances 0.000 claims 2
- 239000007788 liquid Substances 0.000 claims 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 14
- 239000000243 solution Substances 0.000 description 14
- 239000000047 product Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 210000002966 serum Anatomy 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 238000001914 filtration Methods 0.000 description 7
- 239000000203 mixture Substances 0.000 description 7
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 7
- 235000017557 sodium bicarbonate Nutrition 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 229920005654 Sephadex Polymers 0.000 description 6
- 239000012507 Sephadex™ Substances 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 238000007792 addition Methods 0.000 description 3
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000005855 radiation Effects 0.000 description 2
- MNWBNISUBARLIT-UHFFFAOYSA-N sodium cyanide Chemical compound [Na+].N#[C-] MNWBNISUBARLIT-UHFFFAOYSA-N 0.000 description 2
- JVBXVOWTABLYPX-UHFFFAOYSA-L sodium dithionite Chemical compound [Na+].[Na+].[O-]S(=O)S([O-])=O JVBXVOWTABLYPX-UHFFFAOYSA-L 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OLKYVRJEDDPVFH-UHFFFAOYSA-N 3-[3-hydroxy-3-(4-nitrophenoxy)propoxy]-1-(4-nitrophenoxy)propan-1-ol Chemical group C=1C=C([N+]([O-])=O)C=CC=1OC(O)CCOCCC(O)OC1=CC=C([N+]([O-])=O)C=C1 OLKYVRJEDDPVFH-UHFFFAOYSA-N 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 102000001621 Mucoproteins Human genes 0.000 description 1
- 108010093825 Mucoproteins Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 125000003700 epoxy group Chemical group 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- ZWZVWGITAAIFPS-UHFFFAOYSA-N thiophosgene Chemical compound ClC(Cl)=S ZWZVWGITAAIFPS-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
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
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Definitions
- the present invention relates to a method for determining vitamin B 12 in aqueous samples e.g. from body fluids such as blood serum, and a reagent to be used for the method.
- the invention characterized in that particles of water-insoluble polymers to which a substance capable of binding vitamin B 12 has been bound by covalent bonds, are contacted with the sample fluid and with a certain quantity of vitamin B 12 labelled with a radioactive isotope, whereafter, subsequent to the reaction between vitamin B 12 and the substance capable of binding vitamin B 12 attached to the particles having taken place, the particles are separated from the sample fluid and the radioactivity of the particle material and/or in the fluid is determined.
- the method can be utilized for qualitative and quantitative determination.
- the term "substance capable of binding vitamin B 12" is meant to signify a substance containing proteins or polypeptides or, optionally, carbohydrates possessing a specific ability to bind vitamin B 12 .
- a well known example of such a substance is intrinsic factor, a muco protein from the ventriculus mucous membrane.
- Another example is a protein fraction from blood plasma which is capable of binding vitamin B 12 .
- the invention is based on the discovery that substances, e.g. intrinsic factor, exist which are capable of specifically binding vitamin B 12 and that this binding takes place irrespective of whether vitamin B 12 is labelled with a radiosotope or not.
- substances e.g. intrinsic factor
- vitamin B 12 with a radioisotope can be effected in a conventional manner, a suitable isotope for the purpose being selected.
- a radioisotope of cobalt is particularly suitable for the purpose.
- Vitamin B 12 labelled with a cobalt radiosiotope is accessible commercially and many hospital laboratories are now equipped to measure this isotope.
- polymer particles comprising a three-dimensional network, held together by covalent bonds.
- Such particles even if they are capable of swelling in water, are completely insoluble therein and can therefore not release any of the polymer material or the substance bound thereto by covalent bonds, e.g. during washing procedures.
- examples of such polymer particles are grains of polymers obtained by cross linking substances containing a plurality of hydroxyl groups, such as carbohydrates and sugar alcohols, e.g. dextran, starch, dextrins and other polysaccharides and polyvinyl alcohol with a bifunctional substance, e.g.
- bifunctional substances of the type X-R-Z wherein, for instance, X and Z are each halogen or an epoxy group and R the residue of the bifunctional substance, e.g. an aliphatic radical containing from 3 to 10 inclusive carbon atoms.
- Sephadex for this purpose grains of the commercially accessible product Sephadex can be used, for instance; this product containing dextran cross linked with glycerine etherbridges, obtained by treating dextran with epichlorohydrin.
- Sephhadex and products obtained in a similar manner are capable of swelling in water but are insoluble gel grains. They contain hydroxyl groups and can thereby be easily substituted by other groups e.g. such as those containing amino groups or carboxyl groups, and are well suited for producing bridges, by covalent linkages, to the substance capable of binding vitamin B 12 , e.g. intrinsic factor.
- the substance capable of binding vitamin B 12 is bound to these carrier particles, by covalent bonds, under conditions which are so mild that its reactivity is not reduced to any appreciable extent. Because of the covalent bonds the substance capable of binding vitamin B 12 cannot be loosened and washed out from the particles.
- reactive groups contained in this polymer such as amino groups, hydroxyl groups and carboxyl groups, a bridge having covalent bonds being established between the substance capable of binding vitamin B 12 (P) and the polymer particle e.g. of the type:
- the bridge established between the substance capable of binding vitamin B 12 , e.g. intrinsic factor, and the polymer need not be determined as to its structure and permits a great variation in its selection because the purpose thereof is only to prevent the substance capable of binding vitamin B 12 from being washed out.
- the quantity of particles having the substance capable of binding vitamin B 12 attached thereto is selected, inter alia, with consideration to the degree of sensitivity required for the test.
- the quantity of labelled vitamin B 12 added for the reaction is selected, for instance, so that about 40-60% of the labelled vitamin B 12 is bound to the substance capable of binding vitamin B 12 , when no competing unlabelled vitamin B 12 is present.
- the incubation may be effected at different temperatures but is preferably carried out at temperatures between +4 and 25° C. It is not necessary to continue the reaction between vitamin B 12 and the substance capable of binding vitamin B 12 to completion.
- the incubation is interrupted after, for instance, two hours, but may also be interrupted later, e.g. after 24 hours. It is important that the reaction time and temperature are selected equal for sample solutions and standard solutions.
- Excess of protein capable of binding vitamin B 12 in serum may also be determined by means of the present method, in that particles having a substance capable of binding vitamin B 12 attached thereto and suitable amounts of labelled vitamin B 12 are added to untreated serum whereafter, subsequent to the particles having been separated, the radio-activity is measured.
- the invention also includes a reagent for use in the determination of vitamin B 12 .
- This reagent contains particles of water insoluble polymers to which have been bound, by covalent bounds, a substance capable of binding vitamin B 12 , in dried, e.g. lyophilized form.
Abstract
Method for determining vitamin B12 in aqueous samples by contacting said sample with radioactivity labelled vitamin B12 and with particles of water insoluble polymers to which substances capable of binding vitamin B12 have been bound, thereafter separating said particles and determining the radioactivity.
Description
The present invention relates to a method for determining vitamin B12 in aqueous samples e.g. from body fluids such as blood serum, and a reagent to be used for the method.
The invention characterized in that particles of water-insoluble polymers to which a substance capable of binding vitamin B12 has been bound by covalent bonds, are contacted with the sample fluid and with a certain quantity of vitamin B12 labelled with a radioactive isotope, whereafter, subsequent to the reaction between vitamin B12 and the substance capable of binding vitamin B12 attached to the particles having taken place, the particles are separated from the sample fluid and the radioactivity of the particle material and/or in the fluid is determined.
The method can be utilized for qualitative and quantitative determination.
In the present instance the term "substance capable of binding vitamin B12 " is meant to signify a substance containing proteins or polypeptides or, optionally, carbohydrates possessing a specific ability to bind vitamin B12. A well known example of such a substance is intrinsic factor, a muco protein from the ventriculus mucous membrane. Another example is a protein fraction from blood plasma which is capable of binding vitamin B12.
The invention is based on the discovery that substances, e.g. intrinsic factor, exist which are capable of specifically binding vitamin B12 and that this binding takes place irrespective of whether vitamin B12 is labelled with a radiosotope or not. The binding of labelled and unlabelled vitamin B12 to the substance capable of binding vitamin B12, e.g. intrinsic factor, takes place in proportion to the concentration of labelled and unlabelled vitamin B12 , respectively.
The major advantage presented by the present method is that the substance capable of binding vitamin B12 is not in solution but is very securely bound to an insoluble carrier and thus the labelled vitamin B12, which in the determination reacts with and is bound to the substance capable of binding vitamin B12 , can be readily separated from the unbound, labelled vitamin B12 by, for instance, a simple centrifugation or filtration. The test is easily performable as known quantities of particles with the substance capable of binding vitamin B12 attached thereto can be dispensed before hand in, for instance, test tubes, and may be stored without losing their binding property. The total procedure, including the separation of the free labelled vitamin B12 and the bound labelled vitamin B12 can be effected in one and the same test tube without any extra additions of precipitating agents or the like.
The labelling of vitamin B12 with a radioisotope can be effected in a conventional manner, a suitable isotope for the purpose being selected. A radioisotope of cobalt is particularly suitable for the purpose. Vitamin B12 labelled with a cobalt radiosiotope is accessible commercially and many hospital laboratories are now equipped to measure this isotope.
Particles of water-insoluble polymers are used as the carrier of the substance capable of binding vitamin B12. The polymer is selected so that it contains or can be provided with suitable reactive groups, e.g. amino groups, hydroxyl groups and carboxyl groups, to make possible binding of the substance capable of binding vitamin B12 , e.g. intrinsic factor, to the polymer by bridges with bonds of covalent character.
It is particularly suitable to select polymer particles comprising a three-dimensional network, held together by covalent bonds. Such particles, even if they are capable of swelling in water, are completely insoluble therein and can therefore not release any of the polymer material or the substance bound thereto by covalent bonds, e.g. during washing procedures. Examples of such polymer particles are grains of polymers obtained by cross linking substances containing a plurality of hydroxyl groups, such as carbohydrates and sugar alcohols, e.g. dextran, starch, dextrins and other polysaccharides and polyvinyl alcohol with a bifunctional substance, e.g. bifunctional substances of the type X-R-Z, wherein, for instance, X and Z are each halogen or an epoxy group and R the residue of the bifunctional substance, e.g. an aliphatic radical containing from 3 to 10 inclusive carbon atoms.
For this purpose grains of the commercially accessible product Sephadex can be used, for instance; this product containing dextran cross linked with glycerine etherbridges, obtained by treating dextran with epichlorohydrin. Sephhadex and products obtained in a similar manner are capable of swelling in water but are insoluble gel grains. They contain hydroxyl groups and can thereby be easily substituted by other groups e.g. such as those containing amino groups or carboxyl groups, and are well suited for producing bridges, by covalent linkages, to the substance capable of binding vitamin B12 , e.g. intrinsic factor.
Other examples are reactive derivatives obtained by treating a copolymer of dextran with epichlorohydrin (Sephadex) with cyanogen halides such as cyanogen bromide. Such reactive derivatives will readily react with intrinsic factor.
Small particles are preferably selected so that a larger contact surface is obtained.
The substance capable of binding vitamin B12 is bound to these carrier particles, by covalent bonds, under conditions which are so mild that its reactivity is not reduced to any appreciable extent. Because of the covalent bonds the substance capable of binding vitamin B12 cannot be loosened and washed out from the particles. Used for chemically binding the substance capable of binding vitamin B12 to the polymer particle are reactive groups contained in this polymer such as amino groups, hydroxyl groups and carboxyl groups, a bridge having covalent bonds being established between the substance capable of binding vitamin B12 (P) and the polymer particle e.g. of the type:
P-NH.CS.NH-Polymer particle
P-NH.CO.NH-Polymer particle
P-N=N-Polymer particle
The bridge established between the substance capable of binding vitamin B12, e.g. intrinsic factor, and the polymer need not be determined as to its structure and permits a great variation in its selection because the purpose thereof is only to prevent the substance capable of binding vitamin B12 from being washed out.
During the analysis operation a solution of vitamin B12 of known concentration is suitably used as a standard.
The radioactivity determination can be effected by known methods, e.g. with the assistance of scintillation detectors.
The quantity of particles having the substance capable of binding vitamin B12 attached thereto is selected, inter alia, with consideration to the degree of sensitivity required for the test.
The quantity of labelled vitamin B12 added for the reaction is selected, for instance, so that about 40-60% of the labelled vitamin B12 is bound to the substance capable of binding vitamin B12, when no competing unlabelled vitamin B12 is present. The incubation may be effected at different temperatures but is preferably carried out at temperatures between +4 and 25° C. It is not necessary to continue the reaction between vitamin B12 and the substance capable of binding vitamin B12 to completion. The incubation is interrupted after, for instance, two hours, but may also be interrupted later, e.g. after 24 hours. It is important that the reaction time and temperature are selected equal for sample solutions and standard solutions.
In that the method is simple, rapid, practical and gives accurate analysis results it is well suited for quantitative determinations, even for routine work, and permits the determination of very small amounts of sample substance.
The method determines free vitamin B12. In serum, for instance, vitamin B12 is bound to a protein. Thus, in the determination vitamin B12 is released before the determination, e.g. by heating with hydrochloric acid.
Excess of protein capable of binding vitamin B12 in serum, for instance, may also be determined by means of the present method, in that particles having a substance capable of binding vitamin B12 attached thereto and suitable amounts of labelled vitamin B12 are added to untreated serum whereafter, subsequent to the particles having been separated, the radio-activity is measured.
The invention also includes a reagent for use in the determination of vitamin B12. This reagent contains particles of water insoluble polymers to which have been bound, by covalent bounds, a substance capable of binding vitamin B12, in dried, e.g. lyophilized form.
According to an embodiment of the invention such a reagent may be contained in a sealed ampoule.
The invention also includes a test pack for the determination of vitamin B12 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 B12 in dried, e.g. lyophilized, form and one or more ampoules wth vitamin B12 labelled wth a radioisotope in dried, e.g. lyophilized form.
The invention will be further illustrated in the following by means of detailed examples concerning the determination of vitamin B12 in blood serum.
Determination of vitamin B12 in blood serum
(A) Preparation of particles having a substance capable of binding vitamin B12 attached by covalent bonds.--Finely grained particles of the product Sephadex (G25, superfine) were used as a starting material, the product being dextran cross linked with glycerine ether-bridges and substituted with p-nitrophenoxy-hydroxy-propyl-ether groups to a substitution degree of 200 μmol of nitro groups per gam of dry substance, 10 g. of the substituted Sephadex product was introduced together with 50 ml. water into a two-necked flask, whereafter the temperature of the mixture was maintained at 35° C. The mixture was agitated and at the same time 25 ml. of a 5 N aqueous solution of sodium hydroxide and 6 g. of sodium dithionite were added, for reducing the nitro groups into amino groups. After approximately 30 mins. further 5 g. of sodium dithionite were added. The reduction process was interrupted after about one hour whereafter neutralization took place with dilute hydrochloric acid, the solid substance being removed by filtration and washed with distilled water on a suction filter.
10 g. of the above obtained Sephadex product substituted wth p-amino-phenoxy-hydroxy-propyl groups were introduced into a reaction flask together with 100 ml. of a 10 percent solution of thiophosgene in carbon tetrachloride. The flask was sealed with a plug and the mixture agitated for about two hours. The obtained mixture was cooled in an ice bath, whereafter the flask was opened and the contents removed by filtration. The residue of filtration was washed with a 0.1 M aqueous solution of sodium hydrogen carbonate, distilled water and acetone. The residue was then dried in a drying oven at 60-80° C.
2 grams of the Sephadex product obtained according to the above substituted with a p-isothio-cyanato-phenoxy-hydroxyl-propyl groups were swollen in 6 ml. of a 0.1 M aqueous solution of sodium hydrogen carbonate. The agitator was connected, whereafter 4 ml. of the same sodium hydrogen carbonate solution containing 95 mg. of intrinsic factor were added in a dropwise manner. The mixture was agitated for 24 hours at 20° C., and then filtered. The residue of filtration was washed with a 0.5 M aqueous solution of sodium hydrogen carbonate to remove unreacted substances. The product can be dried carefully e.g. by lyophilization.
(B) Determination.--The analyses are suitably effected in glass or plastic tubes of 50 × 10 mm. in dimension. A 0.05 M tris-buffer with pH 7.4 containing 0.9% of NaCl, 0.1% of bovine serum albumin and 0.01% of sodium azide was used as a diluent. Prior to the analysis the vitamin B12 in the serum was separated from serum protein by heating with hydrochloric acid in the following manner: 0.5 ml. of serum +0.5 ml. of a 0.9% percent solution of NaCl added with 2 micro grams of NaCN per ml.+1 ml. of 0.1 N HCl were placed in a boiling water bath for 20 minutes, whereafter the solution was cooled with running cold water.
The determination is then carried out according to the following, for instance:
(1) 0.25 ml. of serum solution treated according to the above is introduced into each of two tubes.
(2) 0.25 ml. of standard solutions of different concentrations of vitamin B12, e.g. 1000, 400, 100, 40, 10 and 0 pg./ml. diluted in the aforesaid buffer with addition of two micro grams of NaCl per ml. are each introduced into two tubes.
(3) 0.1 ml. of a solution containing 1 nanogram of vitamin B12 labelled with 58 Co per ml. diluted in buffer with an addition of two μg. NaCN per ml. is added to all tubes.
(4) 1 ml. of a homogenized suspension of the polymer particles (1 mg. per ml.) to which intrinsic factor has been bound by covalent bonds is added to each of all tubes.
(5) Incubation for 3 hours at room temperature or 4° C., the tubes being slowly rotated.
(6) The particles are centrifuged down at 3000 r.p.m. for 5 minutes.
(7) The particles are washed twice with a 0.9 percent solution of NaCl. Subsequent to the last removal by suction of the supernatant the tubes are placed in counter tubes for estimating the radiation from the bound labelled vitamin B12.
(8) The number of "counts" for a certain time from the standard tubes is set on a "counts-dose" diagram on lin-log scale, from which the amount of vitamin B12 in the unknown samples can later be calculated.
Alternatively, subsequent to centrifuging in item 6 above one ml. of the supernatant is transferred in counter tubes, whereafter the radiation from free labelled vitamin B12 can be estimated. "Counts" from the standard tubes can be entered in the same way into a count-dose diagram in lin-log scale and the amount of vitamin B12 in the unknown test samples can then be estimated graphically in the same way as above.
Determination of vitamin B12 in an aqueous sample
(A) Preparation of particles having a substance capable of binding vitamin B12 attached thereto by covalent bonds.--10 grams of a copolymer obtained by reacting dextran with epichlorohydrin (Sephadix G25, superfine) were swollen, with agitation for 3 min., in 200 ml. of a solution of cyanogen bromide, containing 10 grams of the latter per 100 ml. of water. There were then added an aqueous 5 M solution of sodium hydroxide with agitation to a pH-value of 10.7. This value was maintained constant for 8 minutes. The temperature was maintained at 20° C. in the whole procedure. The mixture was then transferred to a glass filter and washed carefully with water to neutral reaction. The particles separated off were shrunk by washing with acetone. The particles were dried carefully and could be stored at, e.g., -20° C.
2 grams of the obtained particles activated with cyanogen bromide were swollen in 6 ml. of an aqueous 0.1 M solution of sodium hydrogen carbonate. The stirrer was switched on after which 4 ml. of the same solution of sodium hydrogen carbonate, containing 100 mg. of intrinsic factor, were added in a drop-wise manner. The mixture was agitated for 24 hours after which filtration took place. The residue of filtration was washed with an aqueous 0.5 M solution of sodium hydrogen carbonate to remove unreacted substances. The product could be dried carefully, e.g., by lyophilization.
(B) Determination.--This procedure was carried out in a manner similar to Example 1(B).
Claims (15)
1. A method for determining vitamin B12 in an aqueous sample, which comprises contacting particles of water-insoluble polymers to which a substance capable of binding vitamin B12 has been bound, by covalent bonds, with the sample and with a certain amount of vitamin B12 labelled with a radioisotope, and subsequent to the reaction between vitamin B12 and the substance capable of binding vitamin B12 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 B12 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 B12 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 B12 in an aqueous sample, which comprises contacting particles of water-insoluble polymers to which a substance capable of binding vitamin B12 has been bound, by covalent bonds, with the sample and with a certain amount of vitamin B12 labelled with a radioisotope, and subsequent to the reaction between vitamin B12 and the substance capable of binding vitamin B12 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 B12 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 B12 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 B12, containing particles of water insoluble polymers to which have been bound, by covalent bonds, a substance capable of binding vitamin B12, 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 B12 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 B12 in dried form and another ampoule with vitamin B12 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 B12 is in lyophilized form.
15. Test pack according to claim 14, wherein the vitamin B12 labelled with a radioisotope is in lyophilized form. .Iadd. 16. A method according to claim 1, wherein the substance capable of binding vitamin B12 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 B12 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 B12 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 B12 is a protein fraction from blood plasma. .Iaddend..Iadd. 25. A reagent according to claim 10, wherein the substance capable of binding vitamin B12 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 B12 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US05/598,142 USRE29480E (en) | 1966-10-21 | 1975-07-23 | Method for determining vitamin B12 and reagent therefor |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE14397/66A SE326845B (en) | 1966-10-21 | 1966-10-21 | |
| SW14397/66 | 1966-10-21 | ||
| US05/598,142 USRE29480E (en) | 1966-10-21 | 1975-07-23 | Method for determining vitamin B12 and reagent therefor |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US04675323 Reissue | 1967-10-16 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| USRE29480E true USRE29480E (en) | 1977-11-22 |
Family
ID=26655998
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US05/598,142 Expired - Lifetime USRE29480E (en) | 1966-10-21 | 1975-07-23 | Method for determining vitamin B12 and reagent therefor |
Country Status (1)
| Country | Link |
|---|---|
| US (1) | USRE29480E (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4115065A (en) | 1973-12-11 | 1978-09-19 | The Radiochemical Centre Limited | Saturation analysis of folate compound with selenium-75 labeled folate |
| CN117347530A (en) * | 2023-12-04 | 2024-01-05 | 天津玉健生物工程有限公司 | Detection method of plasticizer in vitamin B2 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3555143A (en) * | 1966-06-02 | 1971-01-12 | Pharmacia Ab | Method for the determination of proteins and polypeptides |
| US3646346A (en) * | 1968-12-26 | 1972-02-29 | Pharmacia Ab | Antibody-coated tube system for radioimmunoassay |
-
1975
- 1975-07-23 US US05/598,142 patent/USRE29480E/en not_active Expired - Lifetime
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3555143A (en) * | 1966-06-02 | 1971-01-12 | Pharmacia Ab | Method for the determination of proteins and polypeptides |
| US3646346A (en) * | 1968-12-26 | 1972-02-29 | Pharmacia Ab | Antibody-coated tube system for radioimmunoassay |
Non-Patent Citations (8)
| Title |
|---|
| Axen et al., Chemical Abstracts, v. 62:11911e (1965). * |
| Axen et al., Nature, v. 210, pp. 367-369 (1966). * |
| Axen et al., Nature, v. 214, pp. 1302-1304 (1967). * |
| Bacher, F. A. et al., Analytical Chemistry, vol. 26, pp. 1146-1149 (1954). * |
| Barakat et al., Lancet, v. ii, pp. 25-26 (1961). * |
| Bunge, M. B. et al., Chemical Abstracts, vol. 52, p. 5570 (1958). * |
| Ceska et al., Clin. Chim. Acta, v. 32, pp. 339-354 (1971). * |
| Shaw, W. H. C. et al., Analyst, vol. 85, pp. 389-409 (1960). * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4115065A (en) | 1973-12-11 | 1978-09-19 | The Radiochemical Centre Limited | Saturation analysis of folate compound with selenium-75 labeled folate |
| CN117347530A (en) * | 2023-12-04 | 2024-01-05 | 天津玉健生物工程有限公司 | Detection method of plasticizer in vitamin B2 |
| CN117347530B (en) * | 2023-12-04 | 2024-02-13 | 天津玉健生物工程有限公司 | Detection method of plasticizer in vitamin B2 |
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