AU662235B2 - Intrinsic factor - horseradish peroxidase conjugates - Google Patents
Intrinsic factor - horseradish peroxidase conjugates Download PDFInfo
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- AU662235B2 AU662235B2 AU44764/93A AU4476493A AU662235B2 AU 662235 B2 AU662235 B2 AU 662235B2 AU 44764/93 A AU44764/93 A AU 44764/93A AU 4476493 A AU4476493 A AU 4476493A AU 662235 B2 AU662235 B2 AU 662235B2
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- horseradish peroxidase
- oxidized
- conjugates
- hrp
- intrinsic
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- 108010001336 Horseradish Peroxidase Proteins 0.000 title claims description 49
- 229940029329 intrinsic factor Drugs 0.000 title claims description 29
- 238000003556 assay Methods 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 11
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 241000872931 Myoporum sandwicense Species 0.000 claims description 8
- 229930003779 Vitamin B12 Natural products 0.000 claims description 6
- 239000011715 vitamin B12 Substances 0.000 claims description 6
- 235000019163 vitamin B12 Nutrition 0.000 claims description 6
- 150000001720 carbohydrates Chemical class 0.000 claims description 5
- 235000014633 carbohydrates Nutrition 0.000 claims description 5
- 238000012875 competitive assay Methods 0.000 claims description 4
- 150000004678 hydrides Chemical class 0.000 claims description 3
- 230000000155 isotopic effect Effects 0.000 claims description 3
- 239000000700 radioactive tracer Substances 0.000 claims description 3
- 230000002860 competitive effect Effects 0.000 claims description 2
- 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 claims 2
- 240000003291 Armoracia rusticana Species 0.000 claims 1
- 102000003992 Peroxidases Human genes 0.000 claims 1
- 230000021615 conjugation Effects 0.000 claims 1
- 108040007629 peroxidase activity proteins Proteins 0.000 claims 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000007864 aqueous solution Substances 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(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+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- KQDJTBPASNJQFQ-UHFFFAOYSA-N 2-iodophenol Chemical compound OC1=CC=CC=C1I KQDJTBPASNJQFQ-UHFFFAOYSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 102000002464 Galactosidases Human genes 0.000 description 1
- 108010093031 Galactosidases Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 241000123069 Ocyurus chrysurus Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000013043 chemical agent Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000002967 competitive immunoassay Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000012280 lithium aluminium hydride Substances 0.000 description 1
- -1 lithium aluminum hydride Chemical compound 0.000 description 1
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Description
S1or
AUSTRALIA
Patents Act 1990 BECTON, DICKINSON AND COMPANY
ORIGINAL
COMPLETE SPECIFICATION STANDARD PATENT i,
I
A
Invention Title: "Intrinsic factor horseradish peroxidase conjugates" The following statement is a full description of this invention including the best method of performing it known to us:i I i BACKGROUND OF INVENTION Assays for the serum level of Vitamin B12 have proven to be extremely challenging to develop. This is due primarily to the high sensitivity required, on the level of picograms, as well as the fact that normal serum contains endogenous B12 binders. These binders must be treated to release the B12 prior to the assay; such treatment is quite harsh and generally requires a separate step to accomplish it. The treatment involves the use of either heating to elevated temperatures (100 0 commonly termed "boil" assays, or the use of strong chemical agents, "no-boil" assays. One example of a no boil assay is presented in U.S. Patent No. 4,300,907 to Mansbach et al.
Because of these requirements, until quite recently virtually all commercially available assays for B12 have been radioassays which utilize a radioactive isotopic labeled binding protein for detection. A number of other formats have been discussed in the literature including 2Q. the use of chemiluminescent (Clin. Chem., Vol. 35, No. 6, p. 1194, Abst. #609 (1989)), fluorescent (Clin. Chem., Vol. 37, No. 6, p. 978 Abst. #326 (1991)), and color-labelled B12 (available from Ciba Corning) detectors. These assays utilize microbeads coated with B12 binding protein for detection, and, as such, may not be compatible with many automated detection methods.
Additionally, enzyme linked assays utilizing alkaline phosphatase Clin. Chem., Vol. 38, No. 6, p. 1089, -1A- Abst. 40662 (1992), Clin. Chem., Vol. 38, No. 6, p. 1095 Abst. #0691 (1992), Clin. Chem., Vol. 37, No. 6, p. 978, Abst. #320 (1991) and B galactosidase (Clin. Chem., Vol.
33, No. 6, p. 963, Abst. #403, (1987)) have been reported; however, both of these enzymes are quite large. Since the time required for competitive immunoassays is heavily dependent on the diffusion rate, and since the diffusion rate is approximately inversely proportional to the cube root of the molecular weight, the utility of these formats in such assays limited.
SUMMARY OF INVE1TION This invention presents a non-isotopic competitive assay for Vitamin B12 (B12). Briefly, the assay utilizes intrinsic factor (IF) labelled with horse radish peroxidase (HRP). The HRP can be conjugated to the IF by oxidizing surface carbohydrates on the HRP.
This method, it has been found, permits formation of the IF/HRP conjugate without deleteriously affecting the B12 binding sites on the IF. Thus the conjugates can be used in assays for B12. Further, because the IF is labelled with HRP, the assays can be run on automated equipment adapted to utilize the signal generated by the HRP/substrate reaction. Additionally, because the method permits relatively large amounts of HRP to be conjugalt..
to IF a high HRP/IF ratio), the signal generated will also be high, increasing the assay sensitivity.
DETAILED DESCRIPTION OF THE INVENTION The assays of this invention make use of the well known binding affinity of intrinsic factor (IF) for Vitamin B12 (B12). Briefly, in a preferred competitive assay format, the (liquid) sample (which may contain B12) -2i
~II~
is mixed with IF conjugated to horse radish peroxidase (HRP), and permitted to react. An aliquot of this mixture is then placed in contact with a solid phase containing bound B12. Subsequently, the liquid phase is separated from the solid phase, leaving behind any IF/HRP bound to the solid phase B12; any IF/HRP bound to sample B12 remains in the liquid. The amount of IF/HRP conjugate in the sample can then be measured by addition of HRP substrate and measurement of the reaction product. This is directly related to the quantity of B12 in the sample.
Alternatively, the activity of the bound IF/HRP conjugate would be determined to ascertain B12 in the sample indirectly.
The substrate utilized is any amenable to HRP action; preferred substrates include: i tetramethylbenzidine, O-phenylene diamine, luminol/iodophenol, and tyramime Coupling of the HRP to the IF is a critical i' requirement of this assay. Since many coupling methods require treatment with chemicals which can deleteriously affect the IF and/or HRP, the treatment must be .sufficiently mild to permit both of these components to remain unaffected, yet sufficiently strong to permit formation of a conjugate which will not dissociate under storage and/or assay conditions.
One such method utilizes the periodate reaction described by Wilson and Nakane in Immunofluorescence and Related Staining Techniques, Krapp et al North Holland Biomedical Press, Amsterdam, pp. 215-224 (1978), incorporated herein by reference. Briefly, the -3r urn
I
carbohydrate surface of the HRP is oxidized to an aldehyde by addition of NaIO 4 in a dark reaction chamber at room temperature. The HRP is in aqueous solution, at a concentration of 1-50 gm/I, preferably 5-25 gm/l, more preferably 10-15 gm/l; the NaIO 4 is also in aqueous solution at a concentration of 10-50 mM, preferably 15-40 mM, more preferably 20-30 mM. The reactants are mixed at a ratio of 10-20, preferably 12-16 moles NaIO 4 /mole HRP.
Subsequently, the reaction is terminated by the addition of an excess of a glycol, preferably ethylene glycol, and the oxidized HRP is dialyzed against mildly acidic (pH 3-6, preferably 4-5) buffer. About 0.1-3 mg, preferably 0.6-2.4 mg of the purified oxidized HRP is then reacted with about 5-20 ug, preferably 10-15 ug IF, in aqueous solution at a basic pH (8-10, preferably The resultant conjugates are then reduced by an .i aqueous solution of a metallic or non-metallic hydride, preferably sodium borhydride or lithium aluminum hydride.
The final product is then exchanged into a neutral salt solution and glycerol is added to facilitate storage.
The above conjugates can be utilized in the competitive assay for Vitamin B12 as described above. The above procedures are particularly suited for making HRP-IF conjugates as they minimize the use of expensive IF, by utilizing an excess of HRP to assure all IF is reacted, in addition to leaving the HRP and IF relatively intact and functionally unaffected.
Further, the above assay procedure, using HRP-IF conjugates, is particularly suited for use in automated assay instruments, such as the AFFINITY@ analyzer manufactured and marketed by Becton, Dickinson and Company, due to the fact that HRP activity is measured.
-4r I Ip ill c n I II _I_ Since many assays can be formatted to use HRP as the tracer or detector, the versatility of such an assay and, thus, such an instrument is enhanced.
EXAMPLES
The following examples demonstrate certain preferred embodiments of the instant invention, but are not intended to be illustrative of all embodiments.
Example 1 Preparation of HRP-IF Conjugate by Periodate Reaction A 12mg quantity of HRP was dissolved in 1.0 ml water and shielded from light. A total of 164pl aqueous 25 mM NaIO 4 was added and the system was allowed to react for 25 minutes at 25 0 C (ambient temperature water bath).
Immediately thereafter, the reaction was terminated by the addition of 2QOpl of 1% ethylene glycol in water and the system was left undisturbed for 20 minutes at 25 0 C. The resultant solution was buffer exchanged into 1mM aqueous sodium acetate buffer (pH 4.4) using a 20 Centricon® 30 ultrafiltra- tion device to 99.5% carbonate buffer.
The HRP concentration was then determined by absorbance at 403 nm and found to be 9.2 mg/ml. A total of 600pg HRP (66pl) was then admixed with 85pl of 0.5M aqueous sodium carbonate buffer (pH 9.0) and i of IF, and incubated at 25°C for 4 hours, protected from light.
SImmediately thereafter, the tube was placed 4 n an ice bath and 50pl of an aqueous solution of NaBH 4 (4mg/ml) was added to stop the reaction. The solution was incubated at 4 0 C for 40min. and subsequently buffer exchanged into 10mM potassium phosphate in normal saline (pH 7.6) using a Centricon® 30 to 99.5% phosphate buffer. The conjugate was stored in a 50/50 mix of phosphate buffered saline and glycerol.
The conjugate was used in a competitive B12 assay in the AFFINITY® unit wherein bound B12 (in a coated tube) competes with free B12 for IF-HRP. All IF-HRP reacting with sample (free) B12 is removed from the tube, and the B12 concentration is determined by monitoring HRP activity remaining in the coated tube.
It is apparent that many modifications and variations of this invention as hereinabove set forth may be made without departing from the spirit and scope hereof. The S specific embodiments descried are given by way of example Sonly and the invention is limited only by the terms of the appended claims.
I-6- -6-
A
Claims (5)
1. A method for preparing intrinsic factor horseradish peroxidase conjugates comprising: reacting horseradish peroxidase with NaIO 4 to oxidize surface carbohydrates to form an oxidized horseradish peroxidase; (ii) terminating said reaction and concentrating said oxidized horseradish peroxidase; (iii) reacting said concentrated oxidized horseradish peroxidase with intrinsic factor at a basic pH to form an oxidized conjugate; and (iv) subsequently reducing and concentrating said oxidized conjugates, and wherein the ratio of NaIO 4 /horseradish peroxidase (mole/mole) is 10/1 to 20/1. S 15 2. The method of claim 1 wherein the ratio of concentrated oxidized horseradish peroxidase to intrinsic Sfactor is 100-3000/5-20 (wt/wt).
3. The method of claim 1 wherein the oxidized conjugates are reduced by the addition of a metallic or non-metallic hydride.
4. An intrinsic factor/horseradish peroxidase conjugate produced by the method comprising: reacting horseradish peroxidase with NaIO to oxidize surface carbohydrates to form an oxidized horseradish peroxidase; (ii) terminating said reaction and concentrating said oxidized horseradish peroxidase; (iii) reacting said concentrated oxidised Shorseradish peroxidase with intrinsic factor at a basic pH S 30 to form an oxidised conjugate; and (iv) subsequently reducing and concentrating said oxidised conjugates, and wherein the ratio of NaIO 4 /horseradish peroxidase (mole/mole) is 10/1 to 20/1. The conjugates of claim 4 wherein the ratio of concentrated oxidised horseradish peroxidase to intrinsic c 8 factor is 100-3000/5-20 (wt/wt).
6. The conjugates of claim 4 wherein the oxidised conjugates are reduced by the addition of a metallic or non-metallic hydride.
7. In a kit for the competitive assay of Vitamin B12 utilising a horseradish peroxidase-labelled intrinsic factor tracer, the improvement comprising using, as said tracer, the intrinsic/horseradish peroxidase conjugate of claim 4. DATED this 19th day of June 1995 BECTON DICKINSON AND COMPANY Patent Attorneys for the Applicant: F.B. RICE CO. 4 0 1 "4 i 0 I.. I ABSTRACT SThis invention presents a non-isotopic assay for Vitamin B12 (B12), utilizing intri (IF) labelled with horse radish peroxidase conjugation is accomplished by oxidizir carbohydrates on the HRP, and reacting this wit] competitive nsic factor (HRP). The ig surface h IF. Iirtt
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US94059992A | 1992-09-04 | 1992-09-04 | |
| US940599 | 1992-09-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU4476493A AU4476493A (en) | 1994-03-10 |
| AU662235B2 true AU662235B2 (en) | 1995-08-24 |
Family
ID=25475128
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU44764/93A Ceased AU662235B2 (en) | 1992-09-04 | 1993-08-19 | Intrinsic factor - horseradish peroxidase conjugates |
Country Status (6)
| Country | Link |
|---|---|
| JP (1) | JPH06205673A (en) |
| AU (1) | AU662235B2 (en) |
| CA (1) | CA2104413C (en) |
| DE (1) | DE4329530A1 (en) |
| FR (1) | FR2695406B1 (en) |
| GB (1) | GB2270315A (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| AU660837B2 (en) * | 1992-09-04 | 1995-07-06 | Becton Dickinson & Company | Indirect chromatographic antigen sandwich test for detection of specific antibody and device therefor |
| US7790363B2 (en) | 2005-02-07 | 2010-09-07 | Abbott Laboratories Inc. | Diagnostic test for vitamin B12 |
| US8288124B2 (en) | 2008-11-20 | 2012-10-16 | Abbott Laboratories | Cloning, expression and purification of recombinant porcine intrinsic factor for use in diagnostic assay |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU650836B2 (en) * | 1991-09-23 | 1994-06-30 | Daramic, Inc. | Battery separators with T-shaped ribs |
| AU659754B2 (en) * | 1992-09-04 | 1995-05-25 | Becton Dickinson & Company | Chromatographic antigen sandwich test for detection of specific antibody and device therefor |
| AU660837B2 (en) * | 1992-09-04 | 1995-07-06 | Becton Dickinson & Company | Indirect chromatographic antigen sandwich test for detection of specific antibody and device therefor |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4256833A (en) * | 1979-01-08 | 1981-03-17 | Majid Ali | Enzyme immunoassay for allergic disorders |
| FR2502786B1 (en) * | 1981-03-24 | 1985-06-21 | Stallergenes Laboratoire | METHOD FOR FIXING ANTIGENS AND ANTIBODIES TO POLYSACCHARIDE SUPPORT, AND USE OF THE PRODUCT OBTAINED THEREFOR FOR IMMUNOASSAYS |
| JPS6178385A (en) * | 1984-09-25 | 1986-04-21 | Toyobo Co Ltd | Stable peroxidase composition |
| DE3525911A1 (en) * | 1985-07-19 | 1987-01-29 | Boehringer Mannheim Gmbh | CONJUGATE FOR ENZYME IMMUNE DETERMINATION |
| DE3541186A1 (en) * | 1985-11-21 | 1987-05-27 | Boehringer Mannheim Gmbh | WATER-SOLUBLE, STABILIZED PEROXIDASE DERIVATIVES, METHOD FOR THE PRODUCTION THEREOF AND USE FOR DETERMINING HYDROGEN PEROXIDE |
| EP0413001A1 (en) * | 1988-06-15 | 1991-02-20 | Beckman Instruments, Inc. | Vitamin B12 assay |
-
1993
- 1993-08-19 CA CA 2104413 patent/CA2104413C/en not_active Expired - Fee Related
- 1993-08-19 AU AU44764/93A patent/AU662235B2/en not_active Ceased
- 1993-08-20 GB GB9317356A patent/GB2270315A/en not_active Withdrawn
- 1993-09-02 DE DE19934329530 patent/DE4329530A1/en not_active Withdrawn
- 1993-09-03 FR FR9310526A patent/FR2695406B1/en not_active Expired - Fee Related
- 1993-09-06 JP JP22126793A patent/JPH06205673A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU650836B2 (en) * | 1991-09-23 | 1994-06-30 | Daramic, Inc. | Battery separators with T-shaped ribs |
| AU659754B2 (en) * | 1992-09-04 | 1995-05-25 | Becton Dickinson & Company | Chromatographic antigen sandwich test for detection of specific antibody and device therefor |
| AU660837B2 (en) * | 1992-09-04 | 1995-07-06 | Becton Dickinson & Company | Indirect chromatographic antigen sandwich test for detection of specific antibody and device therefor |
Also Published As
| Publication number | Publication date |
|---|---|
| AU4476493A (en) | 1994-03-10 |
| CA2104413C (en) | 1996-05-21 |
| GB9317356D0 (en) | 1993-10-06 |
| FR2695406A1 (en) | 1994-03-11 |
| DE4329530A1 (en) | 1994-03-10 |
| CA2104413A1 (en) | 1994-03-05 |
| JPH06205673A (en) | 1994-07-26 |
| FR2695406B1 (en) | 1995-09-29 |
| GB2270315A (en) | 1994-03-09 |
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