Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1994-05-20
2001-06-05
Le, Long V. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C422S051000, C422S051000, C422S068100, C435S007100, C435S007920, C435S174000, C435S805000, C435S815000, C435S961000, C435S962000, C435S967000, C436S174000, C436S175000, C436S177000, C436S528000, C436S531000, C436S547000, C436S821000, C436S825000
Reexamination Certificate
active
06242265
ABSTRACT:
The invention relates to the use of doubly or triply charged cations in methods for immunochemical detection and for the determination of an analyte in biological material.
Known immunochemical assay systems make use of added proteins, polysaccharides and/or surfactants which are not involved in the immunochemical reaction but which are suitable for having a beneficial effect on the result of a reaction of this type.
For solid-phase ELISAs, which represent a selection of assay systems of this type, the incubation media which are required (buffer solutions, incubation milieus) and which contain the analyte and are brought into contact with the solid phase must have a composition such that the non-specific binding of concomitant substances of the sample and/or of the conjugate to the solid phase is prevented. This is why the known additives such as proteins, for example albumin, IgG, casein, hydrolyzed gelatin and the derivatives thereof and mixtures of proteins or else human or animal sera, as well as surfactants, are used in the incubation media.
EP 0,152,847 describes enzyme-antigen and enzyme-antibody conjugates which contain calcium salts and polyoxyethylene together, where the stability of a conjugate in aqueous solution is increased by adding both substances but not by each of the substances alone. DE 3,638,767 describes an incubation medium for solid-phase immunochemical assays, for example an ELISA, which contains lactoferrin, fetal calf serum, polyoxyethylene 20 sorbitan monolaurate (Tween 20®) and buffer salts.
DE 3,807,478 mentions amine oxides as an advantageous addition for immunochemical agents, in particular for incubation media contained therein.
It has now been found, surprisingly, that doubly or triply charged cations are suitable for addition to agents with which an analyte in a sample is detected or determined by an immunochemical reaction, in particular in combination with the abovementioned additives, in order to have a beneficial effect on the immunochemical reaction, which increases the specificity of the detection and determination of the analyte.
Hence the invention relates to the use of doubly or triply charged cations, preferably of water-soluble magnesium and/or calcium salts, in a method for the immunochemical detection and for the immunochemical determination of an analyte contained in a biological material, wherein the appropriate salts are present in dissolved form in a concentration of 5 to 500 mmol/l when the analyte is brought into contact with an unlabeled reactant.
The water-soluble magnesium or calcium salts preferred from the group thereof are those whose anions do not interfere with the immunochemical reaction. Preferred anions are acetate, chloride and citrate, particularly preferably chloride.
Methods within the meaning of the invention are those in which precipitates are generated as dispersion or in a gel or agglutinates of particles, or whose absence is effected, or those in which the immunochemical reaction takes place on a solid phase.
The preferred methods are those called solid-phase immunochemical assays.
In this connection, a solid phase is a carrier which is insoluble in water and to which one or more reactants is bound. The analyte can either bind to a reactant or, if several reactants are present bound to the carrier, detach at least one of these by its own binding and release it into the aqueous phase.
Examples of carriers are latex particles, granular, swellable or non-swellable material, beads, inside surfaces of tubes, microassay plates as particular embodiment of an arrangement of tubes as well as porous materials to be termed an absorbent matrix.
In immunochemical methods, antigen and antibody are used both as analytes and as reactants as well as other bioaffinity binding partners for the reactants or else the analytes, for example lectins, complement, protein A or G as well as derivatized biotin and avidin.
The multiply charged cations can be contained in dry form in a device for receiving a sample, for example in a sample-receiving vessel or in a receiving zone, for example an absorbent matrix, for the sample on a socalled called “dry chemical” assay system; or else in an aqueous solution which also contains buffer salts and a detergent and, where appropriate, stabilizing additives such as proteins or polysaccharides as substances which likewise stabilize the analyte, the multiply charged cations being contained in a concentration of 5 to 500 mmol/l, preferably of 30 to 100 mmol/l, particularly preferably of 50 to 60 mmol/l.
Examples of the biological materials which contain the analyte, which are also called the sample, are tissue from biopsies or autopsies, blood cells, serum or plasma, secretions, CSF, blood from inflamed and non-inflamed tissue, the products of necrosis and metabolic excretions.
Preferred immunochemical methods are those in which one of the reactants is present in solid phase, in which case the sample is brought into contact with the solid phase in the presence of multiply charged cations, where appropriate together with other immunochemical reactants apart from those on the solid phase and reagents for detecting the analyte, whereupon the solid phase is separated from the liquid phase and either the analyte bound to the solid phase is determined or the unbound analyte is determined.
In the examples which follow, the advantages of the use of multiply charged cations in methods for the detection and for the determination of antibodies directed against the agents causing (1) infectious bovine rhinotracheitis (IBR) or infectious pustular vulvovaginitis (IPV) and (2) bovine leukosis are explained.
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Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
Le Long V.
Padmanabhan Kartic
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