Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-05-31
2004-04-20
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007720, C435S007900, C435S028000, C435S176000, C435S188000, C435S287200, C435S288300, C435S288700, C435S808000, C435S810000, C436S172000, C436S173000, C436S524000, C436S525000, C436S527000, C436S539000, C436S808000, C422S082070, C422S082080, C422S082110, C310S311000, C310S312000, C310S323160, C310S361000, C310S369000, C310S370000, C310S371000
Reexamination Certificate
active
06723524
ABSTRACT:
FIELD OF THE INVENTION
The subject invention is in the field of immunoassays for determining, an analyte, especially an analyte adsorbed at a surface, or analyte as it occurs in a liquid sample in solution or at a particular location therein, such as at a specific surface.
More in particular, the invention relates to enzymelinked immunosorbance assays (ELISA's) for determining the concentration of an analyte in a liquid sample, including its concentration in the bulk of the liquid sample or its concentration at a particular surface. Specifically, the present invention relates to new immunoassay methods and kits specifically adapted for carrying out these new immunoassay methods.
BACKGROUND OF THE INVENTION
Immunoassays, such as ELISA's, are widely used for the determination, either qualitative or, mostly, quantitative, of a nearly unlimited variety of organic substances, either of natural origin or synthetic chemical compounds, such as peptides, proteins, enzymes, hormones, vitamins, drugs, carbohydrates, etc., for various purposes, such as in particular for diagnostic purposes, but also for forensic applications, food quality control, and generally for any analytic purpose. All such substances to be assayed will be referred to herein generally as analytes.
Many different variants of ELISA methods exist. The description given hereunder aims at illustrating a typical ELISA technique. It does not pretend to be complete and should not be construed in any way as restricting the scope of the present invention. For example, in the following description, ELISA methods are described as comprising separate steps of incubating a sample with a first binding partner of the analyte and incubating the reaction product formed with a second binding partner of the analyte (herein binding partners of the analyte will sometimes be referred to as binding partners for the analyte). However, some existing ELISA embodiments do not comprise such separate incubation steps and allow the analyte to react simultaneously, or shortly one after the other, in one and the same incubation step, with both its first and second binding partners. Competitive ELISA's are another example of ELISA variants not discussed in detail herein. The subject invention is in principle applicable to any and all ELISA variants, and to similar immunoassay methods which, strictly speaking, are not ELISA methods, e.g. because they do not involve the use of an enzyme.
In a typical ELISA, to detect the presence, or measure the concentration, of an analyte of interest, especially in a liquid sample, which may be a body fluid such as blood, plasma, serum, urine, saliva, sputum, etc., the sample is contacted with a first binding partner for the analyte, and the sample and the binding partner for the analyte are incubated for a sufficient time to allow analyte contained in the sample to bind to the binding partner.
A typical example of such a binding partner is an analyte-specific antibody, e.g. a monoclonal antibody with specificity for the analyte in question. However, other kinds of substances and structures may also qualify as a binding partner. For example, the natural receptor of the analyte in question could be useful as a binding partner, as well as other substances and structures to which the analyte can bind. The binding partner would normally be a specific binding partner, but this is not a requirement. It is even possible to work without a first binding partner and to immobilize (i.e. bind to a solid phase) the analyte either directly (e.g. by adsorption) or through a non-specifically binding linker substance. All of such variants are explicitly intended to be included in the scope of this invention. The words “analyte adsorbed at a surface” as used herein refer to any method resulting in attachment or binding of analyte to a surface.
Usually, said first binding partner is used in an immobilized form, i.e. attached to a solid phase, such as polystyrene beads or the inner surface of the reaction container (e.g. a reaction tube or a well of a microtiter plate). The binding partner may be physically adsorbed onto the solid phase or, usually, be attached by covalent binding. According to some ELISA embodiments, the binding partner is attached by using a suitable coupling agent, and in others by using appropriate linker substances, such as biotin and (strept)avidin. In some ELISA embodiments, the immobilization of the binding partner is carried out after the incubation of the sample and binding partner, thereby allowing the reaction between analyte and binding partner to proceed in the liquid phase. To allow its subsequent immobilization, said binding partner may be applied in a biotinylated form. Immobilization can then be effected by using a solid phase carrying (strept)avidin.
As a result of this first reaction, any analyte present in the sample will have become bound to its binding partner and thereby to the solid phase. Usually after the liquid has been removed and the solid phase has been washed, steps are taken to make the result detectable. The solid phase having attached thereto the binding partner and analyte, if any, is contacted with a second binding partner for the analyte. Again, a specific binding partner is the rule, but not strictly required. Usually, this second binding partner carries a label/marker allowing its detection. In some ELISA embodiments, however, the second binding partner is used in unlabelled form and is labelled after its binding by using a labelled binding partner for the second binding partner. As an example thereof, the second binding partner may be a mouse antibody (either polyclonal or monoclonal) against the analyte in question, and after its binding to the analyte which had been attached via its first binding partner to the solid phase, a labelled goat anti-mouse IgG is used to attach a label to the immobilized complex.
In ELISA'S, the label consists of an enzyme capable of a detectable conversion of a substrate, e.g. a peroxidase such as horseradish peroxidase, capable of converting, in the presence of hydrogen peroxide, a substrate, such as 3,3′5,5′-tetramethylbenzidine, into a coloured product.
Normally, after the enzyme-labelled reactant has been attached to the immobilized complex, the solid phase with complex bound thereto is washed before the actual detection phase is entered.
In the detection phase, substrate solution is added to the solid phase with attached complex and the conversion, if any, of the substrate is detected. To allow quantitative measurement of the analyte, the solid phase is incubated with the substrate solution for a fixed time, which should be sufficiently long to allow a substantial enzymatic conversion of the substrate into a coloured substance. After termination of the substrate-converting reaction the intensity of the colouration, which is proportional to the immobilized amount of enzyme, is measured by optical means, such as a photometer to measure the absorbance at a chosen wavelength, such as 450 nm.
A disadvantage of the existing ELISA techniques is that the adsorption and detection phases, to secure assay sensitivity, are very time consuming for low analyte concentrations. The rate of adsorption of analyte to the surface is proportional to the concentration of analyte in the solution, and thus will also become very low, even in well-stirred systems. To allow a reliable measurement of analytes present in a liquid at a concentration in the order of nanograms or even picograms per ml, the adsorption phase may require a reaction time of one to several hours.
Another disadvantage of the existing ELISA techniques is that they do not allow to measure extremely low surface concentrations of analytes, such as occur at the surface of biological (model) membranes.
An object of this invention is to provide a modified immunoassay, e.g. ELISA, technique allowing to reduce the incubation time in the adsorption and/or detection phase, or to increase the sensitivity of the assay, or both.
A further object of this invention is to provide a
Aarden Lucien Adrianus
Hack Cornelis Erik
Hermens Willem Theodoor
Robers Markus
Banner & Witcoff , Ltd.
Le Long V.
Nederlandse Organisatie voor toegepastnatuurwetenschappelijk Ond
Padmanabhan Kartic
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