WO1989012826A1 - Vitamin b12 assay - Google Patents

Vitamin b12 assay Download PDF

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Publication number
WO1989012826A1
WO1989012826A1 PCT/US1989/002193 US8902193W WO8912826A1 WO 1989012826 A1 WO1989012826 A1 WO 1989012826A1 US 8902193 W US8902193 W US 8902193W WO 8912826 A1 WO8912826 A1 WO 8912826A1
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Prior art keywords
vitamin
intrinsic factor
bound
immobilized
binding partner
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PCT/US1989/002193
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English (en)
French (fr)
Inventor
Chan S. Oh
Linda J. Stone
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Beckman Instruments, Inc.
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Priority to JP1990600022U priority Critical patent/JPH03500003U/ja
Publication of WO1989012826A1 publication Critical patent/WO1989012826A1/en

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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

Definitions

  • This invention relates to a vitamin B. 2 assay usable for determining vitamin B 12 concentrations in body fluids such as serum.
  • Cyanocobalamin (vitamin B 12 ) is an essential vitamin in the human diet. It participates as a coenzyme in several reactions of intermediary metabolism, including the conversion of acetyl-coenzyme A to methylmalonyl-coenzyme A by the enzyme methylmalonyl-coenzyme A mutase in lipid metabolism, and the reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase, essential for the synthesis of DNA.
  • Vitamin B 12 has traditionally been assayed by microbiological assays using the growth response of organisms requiring the vitamin, such as the bacterium Lactobacillus lactis and the protozoan Euglena gracilis. Although such microbiological assays are still widely accepted in research and clinical laboratories, they are difficult to perform and time consuming. In recent years, attempts have been made to devise alternative vitamin B 12 assays based on a competitive binding reaction between the vitamin B 12 contributed by a test sample and a known quantity of labeled vitamin B 12 as an assay reagent. The test sample vitamin B 12 and the labeled vitamin B 12 compete for a limited amount of binding partner for the vitamin B 12 .
  • Typical binding partners include binding proteins such as IF and protein R as well as antibodies raised against vitamin B 12 .
  • Competitive binding assays provide a quicker and equal reliable alternative to the traditional microbiological assays.
  • the particular binding protein used in a competitive binding assay for vitamin B 12 can be important.
  • some binding proteins whose use has previously been suggested are believed to bind cobalamin analogues as well as vitamin B 12 itself.
  • transcobalamin II and R proteins bind to cobalamin analogues present in human serum as well as to vitamin B 12 . This lack of specificity leads to erroneous results in vitamin B 12 assays.
  • IF is the preferred binding protein for use in competitive binding assays because it is highly selective for 5'-deoxyadenosylcobalamin, the physiologically active form of vitamin B 12 in human serum.
  • assays based on competitive binding employ a labeled ligand or ligand analogue carrying a chemical moiety or group that can be readily identified or quantified.
  • labels typically include radioactive isotopes, fluorescent groups, and enzymes.
  • Radioactively labeled vitamin B 12 derivatives have been the most commonly used ligand analogues in competitive binding assays for vitamin B 12 , and several different isotopes have been successfully used. The earliest isotope to be used successfully in suc h assays was 57 Co.
  • Natural vitamin B 12 contains a number of amide substituents on its porphyrin rings. Mild hydrolysis cleaves some of these amide sub ⁇ tituents to form a mixture of monocarboxylic acids containing mostly the (e)-isomer. The mixture of acids is then reacted with a p-(aminoalkyl) phenol to introduce a phenol group by reaction with one of the free carboxylic acid groups. The mixed substituent vitamin B 12 derivatives are then iodinated on the phenol group substituent. These 125 I-labeled mixed derivatives are useful in the radioimmunoassay of vitamin B 12 , using antibodies raised against the mixture.
  • U.S. Patent No. 4,465,775 to Houts discloses that labeled derivatives formed from the (d)-monocarboxylic acid isomer of partially hydrolyzed vitamin B 12 have a much greater affinity for IF than do the (b)- and (e)-isomer derivatives. Accordingly, these derivatives are preferred for use as labeled analogues in vitamin B 12 competitive binding assays.
  • radioactive compounds in these immunoassays presents many disadvantages. Extensive safety precautions must be taken in their storage, use, and disposal, such as use of lead shielding and special waste treatment procedures. Expensive equipment is needed for radioactive counting- The isotope 125 I decays with a half- life of approximately 60 days. This radioactive decay not only reduces the amount of radioactivity available for detection, but also may initiate chemical reactions that damage the remaining reagents, reducing sensitivity further. Thus storage of 125 I-labeled reagents is limited to several months. These difficulties are magnified if the other commonly used iodine isotope, 1 31 I, is used to label. This isotope has a half-life of less than 9 days.
  • Enzyme-labeled vitamin B 12 derivatives are also prepared by labeling the monocarboxylic acid derivatives of vitamin B 12 .
  • Leonidas et al., Biotechni ⁇ ues 4 , 42-55 (1986) suggest that the use of ligand-selective binding proteins such as IF can be superior to the use of antibody to bind vitamin B 12 in such a competitive binding assay.
  • IF is first immobilized on the surface of a solid support material such as agarose beads.
  • the immobilized IF is then contacted with the test sample containing vitamin B 12 and allowed to incubate for a designated period of time.
  • a given amount of the enzyme-labeled vitamin B 12 is subsequently a d ded to the reaction mixture, followed by a further incubation.
  • the solid phase is washed to remove unbound material and the amount of bound enzyme determined by adding the appropriate substrates for the enzymatic reaction.
  • the amount of bound enzyme can be determined colorimetrically, typically by spectrophotometry.
  • a change in absorbance results from the action of the enzyme on the substrate, and the extent of absorbance change is inversely proportional to the concentration of unlabeled vitamin B 12 present in the test sample.
  • test sample vitamin B 12 and enzyme-labeled vitamin B 12 "compete" for the available binding sites on the immobilized IF. Because of the sequential addition, this assay is more properly described as “pseudo-competitive.”
  • This enzyme-labeled sequential binding method of Leonidas et al. has not been found to produce consistently good data in a clinical environment.
  • This dose-response curve achieved with enzyme-labeled vitamin B 12 was not as steep as that achieved in sequential binding assays for other analytes using comparable natural binding proteins for those analytes.
  • Leonidas et al. theorize that the lower dose-response curve may be due in part to the impaired ability of immobilized IF to assume the proper three-dimensional conformation for folding around vitamin B 12 once the IF has been bound to a solid support.
  • a competitive binding assay method for vitamin B 12 in a test sample satisfies these needs.
  • the essential feature of this assay method is the use of a binding partner for vitamin B 12 , generally intrinsic factor, free in solution, and of an immobilized vitamin B 12 derivative. Even though the same competition reaction occurs as in previous assays, the reversal of the roles of these reagents causes an unexpected improvement of the efficiency of the assay, especially within the clinically significant concentration ranges.
  • biotinylated intrinsic factor in which substantially all of the molecules of intrinsic factor are covalently linked to at least one biotin molecule, and in which the biotin molecules are coupled to the amino groups of the intrinsic factor.
  • the biotin can be replaced by another molecule, such as a hapten, capable of being specifically recognized by a macromolecule carrying a "reporter group.”
  • reporter group includes enzymes, enzyme cofactors, enzyme inhibitors, enzyme modulators, fluorescent labels, chemiluninescent labels, and e l ectrolytically detectable labels.
  • one version of the assay method comprises the steps of:
  • the immobilized vitamin B 12 is cross-linked to a solid support.
  • the solid support can be activated cross-linked polyacrylamide, activated cellulose, or diazotized amine-derivatized polystyrene.
  • the linkage between the vitamin B 12 and the solid support can be through a protein linker.
  • the protein linker can be bovine serum albumin or IgG. The linkage can be formed by the steps of:
  • the vitamin B 12 -monocarboxylic acid is the (e)-isomer.
  • the vitamin B 12 -monocarboxylic acid can also be succinylated on its primary alcohol group to improve its affinity for the binding partner.
  • the reporter group can be an enzyme, an enzyme cofactor, an enzyme inhibitor, an enzyme modulator, or a fluorescent, chemiluminescent, or electrolytically detectable label.
  • the enzyme can be horseradish peroxidase, alkaline phosphatase, or ⁇ -galactosidase.
  • the step of determining the quantity of the biotinylated intrinsic factor bound to the immobilized vitamin B 12 can comprise:
  • This assay is an assay of the bound enzymatic activity when the reporter group is an enzyme.
  • the avidin can be succinylated to reduce its non-specific binding.
  • anti-hapten antibody When the intrinsic factor is conjugated to a hapten instead of to biotin, anti-hapten antibody then replaces avidin in the above method.
  • ⁇ nconjugated intrinsic factor can alternatively be used, and the avidin or anti-hapten antibody is then replaced by anti-intrinsic factor antibody bound to a receptor group.
  • the binding partner for vitamin B 12 capable of subsequently binding a reporter group can be an antibody.
  • the antibody can be biotinylated, and avidin covalently bound to a reporter group can be used.
  • a second antibody specific for the first antibody and covalently bound to a reporter group can be used.
  • the binding partner for vitamin B 12 can itself be covalently linked to a reporter group.
  • the assay method can comprise the steps of:
  • the binding partner can be intrinsic factor or anti-vitamin B 12 antibody.
  • one important version of the test comprises the steps of:
  • biotinylated intrinsic factor such that substantially all of the molecules of intrinsic factor are covalently linked to at least one biotin molecule, the biotin molecules being coupled to the amino groups of the intrinsic factor
  • a version of the assay specifically useful for serum comprises the steps of:
  • compositions of matter comprising modified intrinsic factors suitable for the assays of the present invention.
  • a composition of matter comprises modified intrinsic factor covalently coupled to a ligand through the amino groups of the intrinsic factor such that substantially all the molecules of intrinsic factor are covalently linked to at least one ligand.
  • the composition of matter for example, can comprise biotinylated intrinsic factor or intrinsic factor covalently coupled to a hapten.
  • the ligand molecules can contain a carboxyl group and can then be coupled to the molecules of intrinsic factor through amide linkages.
  • the amide linkages can be produced by the reaction of the carboxyl group of the ligand with N-hydroxysuccinimide to form a N-hydroxysuccinimidyl ester and the subsequent reaction of the N-hydroxysuccinimidyl ester with the amino groups of intrinsic factor.
  • Still another important aspect of the invention is a process for the biotinylation of intrinsic factor, as well as a composition of matter comprising biotinylated intrinsic factor produced by the process.
  • the process comprises the steps of:
  • an improvement can comprise using as the binding partner any of the modified intrinsic factor preparations described above.
  • the single figure is a standard curve for the assay of the present invention using immobilized vitamin B 12 and biotinylated intrinsic factor.
  • a novel competitive binding assay for vitamin B 12 is provided in accordance with the present invention.
  • the term "competitive binding assay” is intended to include both true competitive binding assays in which the competing species are present simultaneously, and pseudocompetitive binding assays in which the competing species are present sequentially.
  • the critical element of the assay of the present invention is the use of biotinylated IF prepared in such a manner that substantially all the IF molecules are biotinylated without any significant degradation in the ability of the biotinylated IF to bind vitamin B 12 .
  • This biotinylated IF also serves as the way of introducing an enzyme or other reporter group, such as enzyme cofactors, enzyme modulators, enzyme inhibitors, fluorophores, c h emi luminophores, or electrolytically detectable species, into the assay.
  • the reporter group is used for subsequent determination of the quantity of IF bound to the solid support.
  • the enzyme or other reporter group is introduced as avidin labeled with the enzyme or other reporter group and binds tightly and specifically to the biotin moiety of the biotirfylated IF.
  • biotinylated IF also allows the vitamin B 12 to be coupled to a solid support instead of being labeled.
  • This role reversal of the competitive B 12 reagent and the IF reagent has unexpectedly been found to improve the efficiency of the assay, especially within the clinically significant concentration ranges. This unexpected improvement occurs even though the same competitive reaction takes place, the insolubilized vitamin B 12 competing with the test sample vitamin B 12 for the same binding partner, typically biotinylated IF.
  • an excess of the immobilized vitamin B 12 is typically added to the test sample containing vitamin B 12 along with a limited amount of the biotinylated IF.
  • the insolubilized vitamin B 12 reagent competes with the test sample vitamin B 12 for the limited number of available IF binding sites.
  • Enzyme-labeled avidin is then added following incubation of the biotinylated IF with the immobilized vitamin B 12 and the test sample vitamin B 12 for a predetermined period of time.
  • the solid phase is then separated from the solution and the enzyme activity associated with the solid phase measured to determine the quantity of labeled IF reagent bound to the solid phase through the immobilized vitamin B 12 .
  • the quantity of bound enzyme activity is inversely proportional to the vitamin B 12 content of the sample.
  • biotinylated IF Two essential requirements exist for biotinylated IF that is to serve as binding partner for vitamin B 12 in this assay.
  • the first requirement is the preservation of the binding capacity of the IF for vitamin B 12 after biotinylation.
  • the second requirement is the biotinylation of substantially all of the IF. If one of these requirements is not met, the resulting preparation of biotinylated IF is unsuitable for use as a binding partner for vitamin B 12 in a competitive binding assay.
  • Many naturally occurring binding proteins have amino functional groups distributed throughout the binding surface. Some of those amino groups can generally be found at or near the critical binding site. Biotinylating agents are usually in the form of species reactive with amino groups.
  • This biotinylated IF was prepared by reacting IF with the N-hydroxysuccinimidyl ester of biotinylamidocaproic acid. This ester activates the carboxyl group of the above biotin derivative, and results in the coupling of the biotin moiety to either the ⁇ -amino group of lysine or the aminoterminus of the IF protein.
  • the amino groups of IF are believed not to be located in or adjacent to the active site, and therefore can be derivatized extensively without altering either the three-dimensional structure or the activity of IF.
  • the ratio of biotin moieties to IF molecules in the starting reaction mixture can range from 6.3 to 1375; even at the lowest ratio of biotin moieties to IF molecules, all of the IF molecules were biotinylated. Further details on the coupling reaction are given below in Example 1.
  • any ligand or chemical species that either has a free carboxyl group or can be converted to a carboxylic acid derivative can then be converted to an NHS ester and reacted with IF exhaustively without denaturing the IF.
  • These species include haptens such as 2,4-dinitrophenol; drugs; reporter groups such as enzymes, enzyme cofactors, enzyme inhibitors. enzyme modulators, fluorescent labels, chemiluminescent labels, and electrolytically active labels detectable by a change of pH or electrical potential in solution.
  • this method is not limited to either ligands containing carboxyl groups or coupling through NHS esters.
  • the amino groups of IF provide suitable sites for derivatization by many techniques.
  • thiolcontaining ligands can be coupled to amino groups by use of such cross-linkers as m-maleimidobenzoyl-N-hydroxysuccinimide ester and 4-(N-maleimidomethyl)-cyclohexane-1-carboxylic acid N-hydroxysuccinimide ester.
  • Carboxyl groups can be coupled to the amino groups of IF through carbodiimides or through conversion of the carboxyl group of the ligand to an acid anhydride.
  • the vitamin B 12 is coupled to a solid support.
  • solid supports and coupling reactions are usable.
  • solid supports cross-linked acrylamide, agarose, cellulose, and nylon were successfully used.
  • the vitamin B 12 derivative has weaker binding characteristics towards IF than unmodified vitamin B 12 . The binding becomes even weaker when the vitamin B 12 derivative is bound to a bulky protein molecule.
  • IF which itself is a relatively bulky protein molecule, has difficulty in binding protein-bound vitamin B 12 moieties.
  • One way to improve the stable binding of IF to the vitamin B 12 derivative is to use a plurality of vitamin B 12 molecules immobilized on a solid phase.
  • vitamin B 12 molecules There are several methods for immobilizing vitamin B 12 molecules. Solid surface functional groups can be suitably chemically derivatized and reacted with vitamin B 12 -monocarboxylic acid. Alternatively, vitamin B 12 -monocarboxylic acid can be attached to a suitable proteinaceous carrier molecule and this vitamin B 12 -protein conjugate can be attached to a solid phase by many known methods.
  • the vitamin B 12 is partially hydrolyzed to a monocarboxylic acid, preferably the (e)-isomer of monocarboxylic acid, which is the most tightly binding of the monocarboxylic acid isomers.
  • the monocarboxylic acid is then covalently coupled to a protein, either bovine serum albumin (BSA) or immunoglobulin G (IgG), typically horse IgG or bovine IgG.
  • BSA bovine serum albumin
  • IgG immunoglobulin G
  • This coupling occurs by activation of the carboxyl group of the vitamin B 12 -monocarboxylic acid with carbonyldiimidazole (CDI), followed by reaction of the activated vitamin B 12 derivative with N-hydroxysuccinimide (NHS) to give the N-hydroxysuccinimidyl ester of vitamin B 12 - monocarboxylic acid.
  • CDI carbonyldiimidazole
  • NHS N-hydroxysuccinimide
  • the activated ester then reacts with the amino groups of the protein to couple the vitamin B 12 derivative to the protein by the formation of an amide linkage.
  • the vitamin B 12 is also succinylated by the reaction of the primary alcohol group of the vitamin B 12 with succinic anhydride.
  • the succinylation of the vitamin B 12 improves the binding of vitamin B 12 to IF when vitamin B 12 is subsequently coupled to a solid support through protein.
  • Vitamin B 12 can be coupled to a number of solid supports, depending upon the requirements of the assay. a . Pel 102
  • Pel 102 (t ⁇ is a proprietary cross-linked polyacrylamide available from Amicon, preactivated with NHS and ready to couple species containing amino groups. If a vitamin B 12 monocarboxylate-BSA conjugate is added to Pel 102, the amino groups on the BSA will become linked to the solid support.
  • CDI activates the hydroxyl groups of the cellulose for reaction with amino groups of the protein in a vitamin B 12 monocarboxylate-protein conjugate.
  • the protein can be either BSA or IgG.
  • CDI- activated cellulose can be derivatized with hexanediamine.
  • the free end of the hexanediamine spacer, bearing an amino group, can then be reacted with succinic anhydride.
  • Additional hexanediamine spacers can then be incorporated by coupling the free carboxyl group of the succinyl moiety to additional hexanediamine.
  • the optimum solid support in terms of minimizing leakage of immobilized vitamin B 12 and suitability for use with automated analyzer equipment has proven to be polystyrene beads of 1/4 inch (6.35 mm).
  • the derivatization of polystyrene beads to produce immobilized vitamin B 12 proceeds by a series of reactions:
  • a vitamin B 12 -IgG conjugate to the diazoniumpolystyrene beads by reaction in 0.1 M sodium borate buffer, pH 9.2.
  • a monocarboxylate derivative of vitamin B 12 can be activated with CDI and coupled to ethylenediamine, giving a free amino group.
  • the amino derivative can then be coupled with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate.
  • the product can then be coupled to any protein containing thiol groups.
  • Avidin has an exceptionally high affinity for biotin, with the binding constant being approximately 10 15 M - 1 .
  • the coupling of an enzyme to avidin is an excellent method for introducing an enzymatic label into an immunological reaction in which one of the reagents is labeled with biotin.
  • the enzyme used can be any easily assayable enzyme producing a detectable product whose activity is not affected by the conjugation with avidin or by the binding to biotinlabeled IF.
  • the detectable product is visually detectable.
  • the enzyme is horseradish peroxidase (HRPO), but ⁇ -galactosidase, glucose oxidase, and alkaline phosphatase are all suitable enzymes.
  • HRPO When HRPO is used as the enzyme, several coupling methods can be used to couple the HRPO to the avidin. Two efficient methods are periodate-mediated conjugation and conjugation by the reaction of maleimide with thiols.
  • periodate-mediated conjugation procedure used is modified, by slight changes of reaction time and reagent volume, from the procedures described by P. Tijssen and E. Kurstak, "Highly Efficient and Simple Methods for the Preparation of Peroxidase and Active Peroxidase-Antibody Conjugates for Enzyme Immunoassays, " Anal. Biochem. 136, 451-457 (1984), and P.K. Nakane and A. Kawaoi, "Peroxidase Labeled Antibody: A New Method of Conjugation," J. Histochem. Cytochem. 22, 1084-1091 (1974).
  • the conjugation reaction with maleimide is a modification of the method described by M. Imagawa et al., "Characteristics and Evaluation of Antibody-Horseradisr Peroxidase Conjugates Prepared by Using a Maleimide Compound, Glutaraldehyde and Periodate," J. Applied Biochem. 4, 41-57 (1982).
  • the reaction conditions and purification procedure are slightly modified from those given.
  • the maleimide group is introduced into HRPO by treatment with a 1:200 molar excess of sulfo-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate for 1 to 2 hours in sodium phosphate buffer, pH 7.0.
  • the avidin is treated with dithio-bio-succinimidyl propionate followed by dithiothreitol to introduce thiol groups.
  • the two proteins are mixed in equimolar amounts for from 20 minutes to 120 minutes.
  • the conjugate is then isolated using the same carboxymethyl-Sephadex procedure used for the periodate conjugate.
  • Avidin-HRPO conjugate is typically introduced into the assay following a 30 minute incubation of the sample with biotinylated IF and a further 30 minute incubation after addition of the immobilized vitamin B 12 .
  • the incubation of avidin-HRPO is optimally performed in "avidin buffer,” containing, per liter of solution, 29.8 g KCl, 1.2 g Tris base, 10.0 g BSA, and 0.5 g Thimerosal (a preservative) .
  • avidin buffer containing, per liter of solution, 29.8 g KCl, 1.2 g Tris base, 10.0 g BSA, and 0.5 g Thimerosal (a preservative) .
  • Avidin is an extremely basic protein, with an isoelectric point of approximately pH 11. Its high basicity leads to a high level of non-specific binding of the avidin conjugate. To reduce this non-specific binding, incubation with the conjugate must be performed with avidin buffer as stated above. However, avidin buffer is incompatible with performance of the assay in certain automated analyzer systems. To eliminate this, the avidin can be succinylated by reaction with succinic anhydride. This succinylated avidin, after conjugation with HRPO, can be used in the assay without avidin buffer, using normal saline in the wash buffer.
  • Such an assay step can comprise a 15 minute incubation of the serum sample with NaOH and dithiothreitol (DTT) for 15 minutes at room temperature, followed by neutralization of the NaOH with Tris buffer (Example 4).
  • DTT dithiothreitol
  • KCN is very important in the assay mixture.
  • the KCN is believed to stabilize the vitamin B 12 in the form of cyanocobalamin during the performance of the assay.
  • reporter group on avidin it is not necessary for the reporter group on avidin to be an enzyme.
  • Other reporter groups such as enzyme cofactors, modulators, or inhibitors; chemiluminescent or fluorescent labels; or electrolytically detectable labels, can be used instead.
  • the reporter group need not be on the avidin or other molecule binding specifically to the IF, but can be located on the IF itself.
  • the binding partner for vitamin B 12 can be covalently conjugated with the enzyme or other reporter group. In this embodiment, there is no need to use the avidin-biotin system.
  • antibody that is specific for the binding partner of vitamin B 12 , such as anti-intrinsic factor antibody.
  • the enzyme or other reporter group can be directly attached to the antibody.
  • a first and second antibody can be used, with the first antibody specific for the binding partner, and the second antibody specific for the first antibody.
  • the enzyme or other reporter group would be attached to the second antibody.
  • the binding partner were IF
  • the first antibody could be rabbit anti-IF antibody and the second antibody could be goat anti-rabbit IgG covalently conjugated to HRPO.
  • binding partner for vitamin B 12 is typically intrinsic factor
  • other binding partners are also usable in the assay of the present invention, such as Protein R and anti-vitamin B 12 antibodies.
  • the IF is labeled with a hapten such as 2,4-dinitrophenol rather than biotin
  • highly specific tight-binding antibodies for the hapten can replace avidin in the assay.
  • the antibodies can be labeled with any of the reporter groups previously mentioned.
  • the IF itself can be labeled with any of these reporter groups.
  • the tubes containing the reaction mixtures were stirred at room temperature for 2 to 3 hours and then stirred overnight at 4°C. The reaction mixtures remained clear. To the first and second aliquot were then added 0.1 ml of 0.1 M ethylenediamine in phosphate buffered saline (PBS). To the third aliquot was added 0.2 ml of 0.1 M ethylenediamine solution. After the addition of the ethylenediamine, the solutions were stirred for at least 2 hours at room temperature. Finally, to each tube was added 0.1 ml of bovine serum albumin (BSA) at 0.1 g/ml.
  • BSA bovine serum albumin
  • each tube was then transferred to dialysis tubing and dialyzed against 3 changes of 50 mM Tris-HCl, pH 7.4, and then against two further changes of phosphate buffered saline, pH 7.4, diluted to 10 mM.
  • the ratio of biotin moieties to IF molecules was 6.3:1 in the first reaction mixture, 63:1 in the second, and 1375:1 in the third.
  • Analysis of the extent of biotinylation was also carried out using 57 Co-labeled vitamin B 12 , BSA-treated charcoal, and avidin immobilized on cellulose.
  • the beads were suspended in 750 ml of concentrated HCl. To the beads was added 150 g of tin powder, and the mixture was shaken on a rocker with occasional hand shaking for 1 hour. At that point, 200 g more tin and 1000 ml concentrated HCl were added, and the mixture was shaken overnight at room temperature, after which 500 ml of concentrated HCl was again added. The beads were washed with copious amounts of water. The presence of amino groups on the beads was confirmed by a test with 2,4,6-trinitrobenzenesulfonic acid (TNBS).
  • TNBS 2,4,6-trinitrobenzenesulfonic acid
  • the vitamin B 12 -IgG conjugate was prepared separately before the diazotization of the beads.
  • 87.5 mg of the (e) -isomer of the monocarboxylic acid derivative of vitamin Bit was heated to 70° -80° C in 2 ml of dimethylformamide. Not all of the monocarboxylic acid derivative of vitamin B 12 dissolved. 87.5 mg of carbonyldiimidazole was then added, and the temperature maintained at 70° - 80° C for 10 minutes. A clear red solution formed. The solution was then cooled to room temperature, and 87.5 mg of N-hydroxysuccinimide (NHS) added, with stirring for 4 hours at room temperature.
  • NHS N-hydroxysuccinimide
  • IgG was then dissolved into 0.1 M phosphate buffered saline (PBS) at 10 mg/ml.
  • PBS phosphate buffered saline
  • the ratio of activated B 12 to IgG present in the reaction mixture ranged from 1:1 (Tube 1) to 1:500 (Tube 5).
  • the reactions between the activated vitamin B 12 and the IgG were then allowed to proceed overnight at 4°C.
  • the products were dialyzed against 10 mM PBS, then passed through a Sephadex (TM) G-25 column, and then dialyzed again except for the two products with the greatest ratio of vitamin B 12 to IgG (Tubes 1 and 2).
  • the IgG conjugate was diluted with 15 ml of 0.1 M sodium borate, pH 9.2.
  • For the diazotization and coupling to an aliquot of beads was added 10 ml of 4% NaNO 2 and 10 ml 2M HCl; the tubes containing the beads were shaken and allowed to stand for 5 minutes at 4°C. The solution was then filtered from the beads, and the beads washed with 0.01 M HCl. The vitamin B 12 -IgG conjugate was then added, and enough PBS added to cover the beads. The conjugate and beads were allowed to react for 3 days with shaking.
  • Example 3 nitration (Example 3) and prewashed three times with 0.1 M Tris, pH 8 was added, and the reactions were incubated for an additional 30 minutes at 37° C on a rotator at 190 rpm.
  • the beads were washed in avidin buffer, and 300 ml a 1/2000 dilution of avidin-HRPO (obtained commercially from Boehringer-Mannheim) was added. After an additional incubation for 30 minutes at 37° C on the rotator, the beads were then washed three times with avidin buffer, and 300 ⁇ l of p_-phenylenediamine at 2 mg/ml in citric acid buffer was added as a substrate for th HRPO.
  • avidin-HRPO obtained commercially from Boehringer-Mannheim
  • the reactions were incubated for 15 minutes at room temperature. The reactions were stopped with 1 ml of 0.9 M H 2 SO 4 , and the absorbance at 490 nm (A 490 ) was read for each sample. A substrate blank and a non-specific binding control without biotinylated IF were also run. The value for the substrate blank was subtracted from the mean value of the A « , 0 from the assays for each concentration of vitamin B 12 to obtain a corrected mean absorbance proportional to the quantity of biotinylated IF bound to the immobilized vitamin B 12 (B). The B value for each concentration of vitamin B 12 was then divided by the B value at zero concentration of vitamin B 12
  • B/B 0 the fraction of biotinylated IF binding compared to the fraction binding at zero concentration.
  • Fig. 1 shows the results of a plot of B/B 0 versus vitamin B 12 concentration. The results on the standards are shown in Table 2 and Fig. 1.
  • Table 3 shows the results obtained on a number of sera with this assay compared with the results on the same sera obtained using two commercially available radioimmunoassay kits from Amersham and Corning. The correlation between the assay of the present invention, designated EIA in Table 3, and either the
  • Amersham and Corning assays is at least as good or better than that obtained between the Amersham and Corning assays themselves.
  • a SmithKline Bioscience radioimmunoassay was also performed on the same samples, and the correlation between the assay of the present invention and the SmithKline Bioscience assay was better than that between the Corning assay and the SmithKline Bioscience assay.
  • the square of the correlation coefficient. R 2 was 0.561 between the assay of the present invention and the Corning assay, and was
  • SmithKline Bioscience assay It was approximately zero between the assay of the present invention and the Amersham assay, and between the Amersham and Corning assays.
  • EIA The column labeled "EIA” refers to assays performed by the enzy immuno.assay method of the present invention
  • the method of assaying vitamin B 12 concentration of the present invention achieves the goals that have been sought, and possesses a number of significant advantages over the assays previously used.
  • the assay is accurate and precise, especially in the clinically significant concentration ranges. It gives a better dose-response curve than the assay of Leonidas et al. It can use the theoretically most desirable binding partner for vitamin B 12 , intrinsic factor.
  • the assay avoids the problems of either underbiotinylation of the intrinsic factor or inactivation of the vitamin B 12 -binding activity of the intrinsic factor, problems that have previously hampered the use of the biotin-avidin system in assays for vitamin B 12 using intrinsic factor.
  • the assay is readily performed on serum and accommodates simple methods for inactivating the intrinsic vitamin B 12 -binding proteins of serum. It does not require the use of radioactivity, giving it the advantages of greater stability of reagents and greater shelf life.
  • the avoidance of radioactivity means that the assay can be performed by workers with less training, at less risk to themselves, co-workers, or the environment.

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PCT/US1989/002193 1988-06-15 1989-05-19 Vitamin b12 assay WO1989012826A1 (en)

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0460576A1 (en) * 1990-06-06 1991-12-11 Abbott Laboratories Assay for cobalamins
FR2678387A1 (fr) * 1991-06-27 1992-12-31 Bio Rad Laboratories Procede de dosage immuno-enzymatique de la vitamine b12 et de preparation d'un echantillon pour ce dosage.
GB2269898A (en) * 1991-06-27 1994-02-23 Bio Rad Laboratories B12 Enzyme immunoassay
FR2695406A1 (fr) * 1992-09-04 1994-03-11 Becton Dickinson Co Procédé de préparation de conjugués facteur intrinsèque-peroxydase de raifort, conjugués ainsi obtenus et kits les contenant.
FR2695405A1 (fr) * 1992-09-04 1994-03-11 Becton Dickinson Co Procédé de préparation de conjugués facteur intrinsèque-peroxydase de raifort.
EP0599325A1 (de) * 1992-11-26 1994-06-01 Roche Diagnostics GmbH Verbesserte B12-Konjugate
US5451508A (en) * 1989-01-11 1995-09-19 Boehringer Mannheim Gmbh Method and monoclonal antibodies for vitamin B12 determination
EP1377220A4 (en) * 2001-03-16 2008-06-18 Univ Utah Res Found FLUORESCENT COBALAMINE AND ITS USE
CN102798726A (zh) * 2012-07-26 2012-11-28 博奥赛斯（天津）生物科技有限公司 维生素b12化学发光免疫定量检测试剂盒及其制备方法
EP3377879A4 (en) * 2015-11-18 2019-04-24 Cornell University COMPETITIVE LATERAL FLOW ASSAY

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1540895A (fr) * 1966-10-21 1968-09-27 Pharmacia Ab Procédé et réactif de dosage de la vitamine b
FR2412074A1 (fr) * 1977-12-14 1979-07-13 Technicon Instr Procede pour doser la vitamine
GB2084320A (en) * 1980-09-22 1982-04-07 Amersham Int Ltd Assay of vitamin B12
US4333918A (en) * 1979-03-23 1982-06-08 Technicon Instruments Corporation Radioassay for vitamin B12
EP0069450A1 (en) * 1981-06-22 1983-01-12 TECHNICON INSTRUMENTS CORPORATION (a New York corporation) Labelled vitamin B12 derivatives, their preparation and use

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR1540895A (fr) * 1966-10-21 1968-09-27 Pharmacia Ab Procédé et réactif de dosage de la vitamine b
FR2412074A1 (fr) * 1977-12-14 1979-07-13 Technicon Instr Procede pour doser la vitamine
US4333918A (en) * 1979-03-23 1982-06-08 Technicon Instruments Corporation Radioassay for vitamin B12
GB2084320A (en) * 1980-09-22 1982-04-07 Amersham Int Ltd Assay of vitamin B12
EP0069450A1 (en) * 1981-06-22 1983-01-12 TECHNICON INSTRUMENTS CORPORATION (a New York corporation) Labelled vitamin B12 derivatives, their preparation and use

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5451508A (en) * 1989-01-11 1995-09-19 Boehringer Mannheim Gmbh Method and monoclonal antibodies for vitamin B12 determination
EP0460576A1 (en) * 1990-06-06 1991-12-11 Abbott Laboratories Assay for cobalamins
GB2269898B (en) * 1991-06-27 1995-07-12 Bio Rad Laboratories B12 Enzyme immunoassay
FR2678387A1 (fr) * 1991-06-27 1992-12-31 Bio Rad Laboratories Procede de dosage immuno-enzymatique de la vitamine b12 et de preparation d'un echantillon pour ce dosage.
GB2269898A (en) * 1991-06-27 1994-02-23 Bio Rad Laboratories B12 Enzyme immunoassay
GB2257249B (en) * 1991-06-27 1995-07-19 Bio Rad Laboratories B12 enzyme immunoassy and sample pretreatment
FR2695406A1 (fr) * 1992-09-04 1994-03-11 Becton Dickinson Co Procédé de préparation de conjugués facteur intrinsèque-peroxydase de raifort, conjugués ainsi obtenus et kits les contenant.
US5350674A (en) * 1992-09-04 1994-09-27 Becton, Dickinson And Company Intrinsic factor - horse peroxidase conjugates and a method for increasing the stability thereof
FR2695405A1 (fr) * 1992-09-04 1994-03-11 Becton Dickinson Co Procédé de préparation de conjugués facteur intrinsèque-peroxydase de raifort.
EP0599325A1 (de) * 1992-11-26 1994-06-01 Roche Diagnostics GmbH Verbesserte B12-Konjugate
EP1377220A4 (en) * 2001-03-16 2008-06-18 Univ Utah Res Found FLUORESCENT COBALAMINE AND ITS USE
CN102798726A (zh) * 2012-07-26 2012-11-28 博奥赛斯（天津）生物科技有限公司 维生素b12化学发光免疫定量检测试剂盒及其制备方法
CN102798726B (zh) * 2012-07-26 2015-09-23 博奥赛斯（天津）生物科技有限公司 维生素b12化学发光免疫定量检测试剂盒及其制备方法
EP3377879A4 (en) * 2015-11-18 2019-04-24 Cornell University COMPETITIVE LATERAL FLOW ASSAY

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EP0413001A1 (en) 1991-02-20

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