Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Immunoassays for determining vitamin B12, and reagents and kits therefor

a technology of immunoassays and vitamin b12, which is applied in the field of immunoassays for determining vitamin b12 levels in samples, can solve the problems of affecting the apparent specificity of said preparations, unable to detect the presence of a single molecule, and unable to detect a single molecule, etc., to achieve the effect of flexible and specificity

Inactive Publication Date: 2006-04-06
NEWMAN KAREL +2
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] This invention involves the application of anti-intrinsic factor antibodies in rapid, sensitive immunoassays for the detection of vitamin B12. Clearly, specificity of the assay for active forms of cobalamin remains entirely confined to intrinsic factor. Thus, the reagents used in this invention offer an extremely flexible and specific means for determining vitamin B12 levels in samples.
[0016] These preferred allosteric competitive antibodies will bind to intrinsic factor only in the absence of vitamin B12, but bind at a site distinct from the vitamin B12 binding site. Such antibodies can be used in an assay to detect vitamin B12 in a sample. The allosteric competitive antibodies are able to bind to intrinsic factor in the absence of vitamin B12. Upon the addition of a sample, vitamin B12 contained therein will be free to bind to the intrinsic factor at the B12 binding site. The binding of vitamin B12 will lead to the prompt release of previously bound antibody, presumably due to the conformational change in the intrinsic factor that occurs upon the binding of vitamin B12. Alternatively, the allosteric competitive antibody and sample may be added to intrinsic factor without preincubation; the B12 contained in the sample would be more easily able to displace the antibody than in the case of the directly competitive antibodies because prior dissociation of the intrinsic factor from the antibody would not be required.

Problems solved by technology

The lack of intrinsic factor was found to result in the development of pernicious anemia.
R-protein has been identified as a contaminant of intrinsic factor preparations, and as such, can adversely impact the apparent specificity of said preparations.
The kinetics of the interaction of B12 and intrinsic factor is slowed by the necessity of dissociating a particular antibody-intrinsic factor complex before B12 can bind to that intrinsic factor molecule and competitive assays of this type are slow and lack sensitivity.
However, immunoassays in which an antibody is allowed to bind to an analyte and the extent to which it is subsequently released by later conformational changes in the analyte determined have not been described.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Immunoassays for determining vitamin B12, and reagents and kits therefor

Examples

Experimental program
Comparison scheme
Effect test

example 1

Preparation of Antibodies

[0042] Eight week old female BALB / c mice were immunized on a biweekly basis with 10 ug / dose of porcine intrinsic factor in Incomplete Freund's adjuvant by the intraperitoneal route. The mice were sacrificed by cervical dislocation, and the spleens surgically removed. The spleens were transferred to a petri dish containing 5 ml of Dulbecco's modified medium (DME) containing 100 ug / ml gentamycin. The splenocytes were freed by teasing the tissue apart. The splenoctyes were washed once with DME, and combined in a 5:1 ratio with washed SP2 / O cells, from a plasmocytoma cell line that can be obtained commercially.

[0043] The cells were pelleted, excess supernatant removed, and the cells resuspended in 1 ml of buffered 50% polyethylene glycol solution (Boehringer Mannheim). The cells were incubated at room temperature for 120 seconds prior to centrifugation at approximately 400×g for 6 minutes. Fused cells were then centrifuged, gently rinsed with DME, resuspended ...

example 2

Preparation of Enzyme Labelled Antibodies

[0048] Maleimide groups were introduced onto alkaline phosphatase (ALP) by reaction with a 30-fold molar excess of sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexanel-carboxylate in 0.2 M imidazole, pH 9.0; excess reagent was removed by gel filtration on a 1×28 cm column of Sephadex G-50 in PBS. Thiols were introduced onto the antibody (obtained through the procedure described in Example 1) with 40 mM N-acetyl homocysteine thiolactone in 0.1M sodium bicarbonate, 0.15 M NaCl, 1 mM EDTA, pH 8.0; excess reagent was removed on a second gel filtration column in the PBS+1 mM EDTA. Conjugation was achieved by incubating modified enzyme with modified antibody at a 2:1 molar ratio of ALP to antibody for two hours at room temperature. The conjugate was purified by size exclusion chromatography on Sephacryl S-300 (1.5×120 cm) in Tris-buffered saline, pH 8.0.

example 3

Preparation of Enzyme Labelled Intrinsic Factor

[0049] Maleimide groups were introduced onto alkaline phosphatase as above. Purified intrinsic factor (obtained from Scripps Laboratories, Inc.) was reacted with 40 mM N-acetyl homocysteine thiolactone in 0.08 M sodium bicarbonate, 0.12 M NaCl, 8 mM EDTA, pH 8.0; excess reagent was removed on a second gel filtration column in the PBS+1 mM EDTA, as above. Conjugation was achieved by incubating modified intrinsic factor with a two-to-three-fold molar excess of modified enzyme for two hours at room temperature. Under these conditions thiols, such as were introduced into intrinsic factor, are known to react stoichiometrically with maleimides, as on the modified enzyme, to form chemically stable linkages. The conjugate was purified by size exclusion chromatography on Sephacryl S-300 (1.5×120 cm) in Tris-buffered saline, pH 8.0.

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
pHaaaaaaaaaa
pHaaaaaaaaaa
pHaaaaaaaaaa
Login to View More

Abstract

Methods are described for the development of monoclonal antibodies to intrinsic factor and the application of said antibodies in the development of B12 immunoassays. In particular, antibodies are described that are capable of binding to intrinsic factor only in the absence of vitamin B12, and that are released from binding in the presence, and upon the binding, of vitamin B12 to intrinsic factor. Assays may be formatted such that either antibody or intrinsic factor is immobilized or labeled.

Description

FIELD OF THE INVENTION [0001] The present invention relates to immunoassays for determining vitamin B12 levels in a sample, and particularly to immunoassays utilizing anti-intrinsic factor antibodies. BACKGROUND OF THE INVENTION [0002] Assays to determine vitamin B12 levels in humans have long been recognized as a valuable tool in the diagnosis and treatment of such diseases as pernicious anaemia. Vitamin B12 is required for the maintenance of homeostasis. [0003] Vitamin B12 was first purified in 1948. Its complete molecular structure was subsequently elucidated. It has an organometallic corrin nucleus with a stabilized central cobalt ion. The biological activity of such corrin compounds is determined by the composition of the side chain, of which only molecules possessing an “A” ring conjugated nucleotide alpha-ribazole side chain stabilizing the cobalt residue are active. [0004] Intrinsic factor was first noted in 1929. The lack of intrinsic factor was found to result in the devel...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/537G01N33/53G01N33/543C07K16/18C07K16/28G01N33/58
CPCC07K16/18C07K2317/32G01N33/82
Inventor NEWMAN, KARELSCHMIDT, JANEWEGFAHRT, PAUL
Owner NEWMAN KAREL
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com