US3505019A - Method for determining vitamin b12 and reagent therefor - Google Patents

Method for determining vitamin b12 and reagent therefor Download PDF

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Publication number
US3505019A
US3505019A US675323A US3505019DA US3505019A US 3505019 A US3505019 A US 3505019A US 675323 A US675323 A US 675323A US 3505019D A US3505019D A US 3505019DA US 3505019 A US3505019 A US 3505019A
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United States
Prior art keywords
vitamin
particles
binding
substance capable
labelled
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Expired - Lifetime
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US675323A
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English (en)
Inventor
Rolf E A V Axen
Jerker O Porath
Leif E Wide
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Pfizer Health AB
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Pharmacia AB
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/804—Radioisotope, e.g. radioimmunoassay

Definitions

  • the present invention relates to a method for determining vitamin B 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 waterinsoluble polymers to which a substance capable of binding vitamin B has been bound by covalent bonds, are contacted with the sample fluid and with a certain quantity of vitamin B labelled with a radioactive isotope, whereafter, subsequent to the reaction between vitamin B and the substance capable of binding vitamin B 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 is meant to signify a substance containing proteins or polypeptides or, optionally, carbohydrates possessing a specific ability to bind vitamin B
  • 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
  • the invention is based on the discovery that substances, e.g. intrinsic factor, exist which are capable of specifically binding vitamin B and that this binding takes place irrespective of whether vitamin B is labelled with a radiosotope or not.
  • the binding of labelled and unlabelled vitamin B to the substance capable of binding vitamin B e.g. intrinsic factor takes place in proportion to the concentration of labelled and unlabelled vitamin B respectively.
  • the major advantage presented by the present method is that the substance capable of binding vitamin B is not in solution but is very securely bound to an insoluble carrier and thus the labelled vitamin B which in the determination reacts with and is bound to the substance capable of binding vitamin B can be readily separated from the unbound, labelled vitamin B 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 B 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 B and the bound labelled vitamin B can be effected in one and the same test tube without any extra additions of precipitating agents or the like.
  • the labelling of vitamin B 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 labelled with a cobalt radioisotope is accessible commercially and many hosiptal 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 B
  • 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 B e.g. intrinsic factor, to the polymer by bridges with bonds of covalent character.
  • 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-RZ 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 covalnet linkages, to the substance capable of binding vitamin B e.g. intrinsic factor.
  • Small particles are preferably selected so that a larger contact surface is obtained.
  • the substance capable of binding vitamin B 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 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 (P) and the polymer particle e.g. of the type:
  • the bridge established between the substance capable of binding vitamin B 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 from being washed out.
  • the radioactivity determination can be effected by known methods, e.g. with the assistance of scintillation dctectors.
  • the quantity of particles having the substance capable of binding vitamin B attached thereto is selected, inter alia, with consideration to the degree of sensitivity required for the test.
  • the quantity of labelled vitamin B added for the reaction is selected, for instance, so that about 4060% of the labelled vitamin B is bound to the substance capable of binding vitamin B when no competing unlabelled vitamin B 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 and the substance capable of binding vitamin B 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.
  • the method determines free vitamin B in serum, for instance, vitamin B is bound to a protein.
  • vitamin B is released before the determination, e.g. by heating with hydrochloric acid.
  • Excess of protein capable of binding vitamin B in serum may also be determined by means of the present method, in that particles having a substance capable of binding vitamin B attached thereto and suitable amounts of labelled vitamin B 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
  • This reagent contains particles of water insoluble polymers to which have been bound, by covalent bounds, a substance capable of binding vitamin B in dried, e.g. lyophilized form.
  • such a reagent may be contained in a sealed ampoule.
  • the invention also includes a test pack for the determination of vitamin B 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 in dried, e.g. lyophilized, form and one or more ampoules with vitamin B labelled with a radioisotope in dried, e.g. lyophilized form.
  • EXAMPLE 1 Determination of vitamin B in blood serum
  • --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 mol 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.
  • 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 B (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 B in the unknown samples can later be calculated.
  • one ml. of the supernatant is transferred in counter tubes, whereafter the radiation from free labelled vitamin B 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 B in the unknown test samples can then be estimated graphically in the same way as above.
  • EXAMPLE 2 Determination of vitamin B in an aqueous sample
  • A Preparation of particles having a substance capable of binding vitamin B attached thereto by covalent bnds.-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.
  • a method for determining vitamin B in an aqueous sample which comprises contacting particles of waterinsoluble polymers to which a substance capable of binding vitamin B has been bound, by covalent bonds, with the sample and with a certain amount of vitamin B labelled with a radioisotope, and subsequent to the reaction between vitamin B and the substance capable of binding vitamin B attached to the particles having taken place, separating the particles from the sample liquid and determining the radioactivity of the particle material.
  • a method for determining vitamin B in an aqueous sample which comprises contacting particles of waterinsoluble polymers to which a substance capable of binding vitamin B has been bound, by covalent bonds, with the sample and with a certain amount of vitamin B labelled with a radioistope, and subsequent to the reaction between vitamin B and the substance capable of binding vitamin B attached to the particles having taken place, separating the particles from the sample liquid and determining the radioactivity in the fluid.
  • a reagent for use in the determination of vitamin B containing particles of water insoluble polymers to which have been bound, by covalent bonds, a substance capable of binding vitamin B in dried form.
  • a reagent according to claim 10 wherein the reagent is in lyophilized form.
  • Test pack for the determination of vitamin B 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 in dried form and another ampoule with vitamin B labelled with a radioisotope in dried form.
  • 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 is in lyophilized form.
  • Test pack according to claim 14 wherein the vitamin B labelled with a radioisotope is in lyophilized form.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
US675323A 1966-10-21 1967-10-16 Method for determining vitamin b12 and reagent therefor Expired - Lifetime US3505019A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
SE14397/66A SE326845B (enrdf_load_stackoverflow) 1966-10-21 1966-10-21

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US3505019A true US3505019A (en) 1970-04-07

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US (1) US3505019A (enrdf_load_stackoverflow)
AT (1) AT286243B (enrdf_load_stackoverflow)
BE (1) BE705412A (enrdf_load_stackoverflow)
CH (1) CH476493A (enrdf_load_stackoverflow)
DE (1) DE1673015B1 (enrdf_load_stackoverflow)
DK (1) DK120509B (enrdf_load_stackoverflow)
ES (1) ES346273A1 (enrdf_load_stackoverflow)
FI (1) FI48509C (enrdf_load_stackoverflow)
GB (1) GB1151608A (enrdf_load_stackoverflow)
NL (1) NL159191B (enrdf_load_stackoverflow)
NO (1) NO122457B (enrdf_load_stackoverflow)
SE (1) SE326845B (enrdf_load_stackoverflow)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3853987A (en) * 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US3936440A (en) * 1974-05-22 1976-02-03 Drexel University Method of labeling complex metal chelates with radioactive metal isotopes
US4115065A (en) * 1973-12-11 1978-09-19 The Radiochemical Centre Limited Saturation analysis of folate compound with selenium-75 labeled folate
US4225784A (en) * 1976-06-17 1980-09-30 Smith Kline Instruments, Inc. Covalently bound biological substances to plastic materials and use in radioassay
US5227311A (en) * 1988-10-11 1993-07-13 Abbott Laboratories Intrinsic factor to determine B12
US5451508A (en) * 1989-01-11 1995-09-19 Boehringer Mannheim Gmbh Method and monoclonal antibodies for vitamin B12 determination
US6942977B1 (en) 1991-04-09 2005-09-13 Bio-Rad Laboratories, Inc. Immunoassays for determining vitamin b12, and reagents and kits therefor

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2413940C2 (ru) * 2009-02-02 2011-03-10 Государственное образовательное учреждение высшего профессионального образования Воронежская государственная технологическая академия Способ определения витамина b12 в водных растворах

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3853987A (en) * 1971-09-01 1974-12-10 W Dreyer Immunological reagent and radioimmuno assay
US4115065A (en) * 1973-12-11 1978-09-19 The Radiochemical Centre Limited Saturation analysis of folate compound with selenium-75 labeled folate
US3936440A (en) * 1974-05-22 1976-02-03 Drexel University Method of labeling complex metal chelates with radioactive metal isotopes
US4225784A (en) * 1976-06-17 1980-09-30 Smith Kline Instruments, Inc. Covalently bound biological substances to plastic materials and use in radioassay
US5227311A (en) * 1988-10-11 1993-07-13 Abbott Laboratories Intrinsic factor to determine B12
US5451508A (en) * 1989-01-11 1995-09-19 Boehringer Mannheim Gmbh Method and monoclonal antibodies for vitamin B12 determination
US6942977B1 (en) 1991-04-09 2005-09-13 Bio-Rad Laboratories, Inc. Immunoassays for determining vitamin b12, and reagents and kits therefor

Also Published As

Publication number Publication date
NL6714282A (enrdf_load_stackoverflow) 1968-04-22
AT286243B (de) 1970-11-25
NO122457B (enrdf_load_stackoverflow) 1971-06-28
DE1673015B1 (de) 1972-05-04
ES346273A1 (es) 1969-01-01
DK120509B (da) 1971-06-07
CH476493A (fr) 1969-08-15
FI48509B (enrdf_load_stackoverflow) 1974-07-01
GB1151608A (en) 1969-05-14
SE326845B (enrdf_load_stackoverflow) 1970-08-03
BE705412A (enrdf_load_stackoverflow) 1968-03-01
NL159191B (nl) 1979-01-15
FI48509C (fi) 1974-10-10

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