Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-02-22
2001-08-07
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C530S367000, C530S810000, C530S811000, C530S812000, C530S813000, C530S814000, C530S815000, C530S816000, C435S007100, C435S007920, C435S174000, C435S287100, C435S287800, C435S287900, C435S969000, C436S174000, C436S176000, C436S178000, C436S518000, C436S524000, C436S528000, C436S532000, C436S533000, C422S051000, C422S068100, C427S207100, C427S343000, C428S403000
Reexamination Certificate
active
06270983
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to surfaces coated with streptavidin/avidin. Fields of application of the invention are medical diagnostics and pharmaceutical industry.
To determine immunologically effective components according to the principle of the solid phase immunoassay where a reactant is bonded on a solid phase today usually reaction vessels in the form of tubes or wells of microtitration plates are used as solid phase on the inner surfaces of which or outer surfaces of pellets the reactant is bonded. It is the aim to coat these surfaces with organic chemical substances in a way as to reach highly specific bonds in the subsequent assay and to keep the number of non-specific bonds as small as possible.
In the last few decades the immunoassay in its multifarious forms has become established for the determination of a specific bondable immunological substance. It is possible to determine smallest substance quantities in the presence of a billionfold excess of foreign matter. The basis is the sensitive and specific competitive reaction of a non-marked substance P with a fixed quantity of a marked substance P* around specific bonding sites of a binding agent Q in a limited quantity. The concentration of the latter may be determined from the distribution pattern of the marked ligand as a function of the concentration of non-marked ligands. Various methods for separating converted from non-converted reactants have been developed.
In the last few years the solid phase method where one reactant is bonded on a solid phase gained major importance. Reactants bonded chemically or physically on the solid phase may be antibodies, antigens, receptors, cells, DNA, RNA etc. These reactants may be adsorbed on the solid phase either directly or through a precoating. Today it is usually modified with a specifically bondable substance, biotin, which allows to bond it on the coated supporting material (Patent DE 36 40 412 A1 and: Soukup, G. A. et al.: Bioconjugate Chemistry 6, 135/1995).
Today usually plastic surfaces of polystyrene, polypropylene, polyethylene, polyamide, polymethacrylate, polycarbonate, polyacrylate or copolymeres of polystyrene are used as supporting material in the form of inner surfaces of tubes or wells of microtitration plates or outer surfaces of pellets.
Various modifications of these surfaces are known. Thereby, the specifically bonding substance shall be bonded in a way as to make the bondage of the reactant modified by biotin stable, unaffected and sufficient for all manipulations required during the determination procedure. Here, the modified, specifically bonding reactant shall not lose its bondability. Today most of the coatings of solid phases are based on adsorptive bonds.
To ensure stability and an optimum coating adhering agents bringing about an adsorptive bonding were recommended. Here, there shall be made sure that the bondability of the specifically bonding reactant will not be affected (Patent DE 38 06 431).
The determination procedures so far known show the following drawbacks:
the adsorptive loading with reactants frequently results in a too low loading,
a too low absorbancy of the coated surfaces for bonding the reactants subsequently biotinylated,
high non-specific bonding occurring frequently,
instability towards aggressive substances such as detergents, strong bases or acids.
SUMMARY OF THE INVENTION
Proceeding on this the invention was aimed at developing a coated solid phase which provides high surface bonding yields and a sufficient stability, also in the presence of detergents, which is universally applicable, thus facilitating the required sensitivity of the determination procedure to be conducted.
The aim of the invention is reached according to patent claims
1
and
17
, the sub-claims are preferential variants.
The supporting material is in a first stage adsorptively loaded with a hydrophobic adhering agent which, on its turn, is modified by a specific bonding agent for a further, specifically bonding substance. The adhering agent may be a polypeptide, protein, carbohydrate or glycoprotein. It may be used non-cross-linked, cross-linked or derivatized. Proteins with a molecular weight between 10 000 and 900 000 are used preferably for hydrophobing and/or cross-linkage. RSA, lipase or immune globulins such as immune globulin gamma, are especially preferred. The adhering agent has to be more hydrophobic than the supporting material.
DETAILED DESCRIPTION OF THE INVENTION
According to the invention the adhering agent is modified by a bonding agent, the biotin. This is used to produce a stable bond with a further specifically bondable substance, the streptavidin and/or avidin, which results in a stably coated solid phase of a new quality according to the invention. Streptavidin and/or avidin have a few bonding sites for biotin, being thus able to subsequently bond with reactants of a determination procedure modified by biotin. Thereby, on the one hand, the strong biotin-streptativin and/or avidin bond and on the other hand, the fact that streptavidin or avidin have four bonding sites for biotin are taken advantage of. To our surprise, the coated surfaces have the ability that the remaining bonding vacancies for biotin react with a greater affinity with the reactants modified by biotin to be bonded subsequently in spite of the fact that bonding sites were taken by streptavidin and/or avidin due to being bonded on the biotinylated adhering agent. This leads to an extremely stable and highly sensitive solid phase allowing to make the determination procedures developed on this solid phase by far more sensitive. This becomes apparent notably when detecting biotinylated oligonucleotides and DNA fragments where smallest quantities may be detected. The coated solid phase applied according to the invention is able to react sensitively to smallest differences.
The adhering protein may be used non-cross-linked as an individual molecule, cross-linked or derivatized. The methods of preparation are known to the specialist, equally the method of biotinylation. Harmonizing of the degree of cross-linkage with the degree of biotinylation, the choice of the respective adhering agent, the quantity of the adhering agent and of streptavidin and/or avidin with each other and with the biotinylated adhering substance is essential to the implementation of the invention. Immune globulin gamma is an especially preferred form as adhering agent. It may be applied non-cross-linked as well as preferably cross-linked with the known spacers.
As a general rule, there applies the higher the degree of cross-linkage and biotinylation the less adhering protein has to be used. It is important that not too many biotin bonding sites on streptavidin and/or avidin will be occupied, otherwise the effect will be reversed. The streptavidin/avidin quantity has to be harmonized with the quantity of the biotinylated adhering protein.
TABLE
Cross-linkage agents used
short form
chemical designation
SPDP
N-succcinimidyl-3-(2-pyridylditho)-propionate
DSS
disuccinimidyl suberate
DMS
dimethyl suberimidate
MHS
malimidohexanoyl-n-hydroxysuccinimide ester
MABI
methyl-4-azidobenzoimidate * HCl
SAMBA
S′-acetyl-mercapto succinoanhydride
MBS
m-maleinimidobenzoyl-n-hydroxysuccinimide ester
SATP
n-succinimidyl-S-acetyl thiopropionate
SATA
n-succinimidyl-s-acetylthioacetate
SADP
n-succinimidyl-(4-azidophenyl)-1,3′-dithiopropionate
After cross-linkage preferably immune globulin gamma is biotinylated according to the usual methods.
TABLE
Biotinylation reagents used
NHS-biotin
biotin hydrazide
NHS-LC-biotin
biotin LC-hydrazide
sulpho-NHS-LC-biotin
biotinamido pentylamine
NHS-SS-biotin
According to the invention the degree of biotinylation is in the range between 10 and 35. The quantities of cross-linked, biotinylated adhering protein used are between 5 and 15 &mgr;g/ml of coating solution depending on the degree of biotinylation.
Hereinafter the biotin-streptavidin and/or avidin bonding is carried out on the solid phase. The quantities are between 2.5 and 20 &mgr;g/ml dependin
Immer Ulrike
Strohner Pavel
BioTeZ Berlin-Buch GmbH
Le Long V.
Norris McLaughlin & Marcus P.A.
Padmanabhan Kartic
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