Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-02-18
2002-04-23
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C422S051000, C422S051000, C422S067000, C435S007200, C435S007920, C435S007950, C435S287700, C435S962000, C435S969000, C435S970000, C436S518000, C436S524000, C436S530000, C436S538000, C436S541000, C436S823000, C436S825000
Reexamination Certificate
active
06376195
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to the field of binding assay devices and methods, and more specifically to a binding assay device and method for detecting and quantifying analyte, particularly small molecular weight molecules.
BACKGROUND OF THE INVENTION
The field of immunochemistry, and the development of immunoassay technology, has been evolving since the late 19th century. However, the majority of these methods have been developed for use by the medical community. Immunoassays are based on the highly specific binding between and antibody and the antigen recognized by the antibody. Antibodies are binding proteins that are produced by the immune system of vertebrates in response to substances that are perceived to be foreign.
Various approaches have been described for carrying out immunoassays. The early ELISA's were what is commonly called a “competitive” assay in which the enzyme labeled antigen or antibody competed with the antigen or antibody to be determined for a reaction site on a bead, pad or surface to which one member of an immunologically coupling pair was attached. Subsequently, the “sandwich” assay was developed. In the sandwich assay, the antibody or antigen to be determined was “sandwiched” by an immunochemical reaction between a solid surface treated with an immunological species reactive with the species to be determined and the same or a different reactive immunological species which has been coupled to a signal generating label.
Immunoassay methods combine the specific binding characteristics of an antibody molecule with a read-out system that is used to detect and quantify compounds. Immunochemical assays are reliable when used in the screening of soil for contamination and have been used commercially for the rapid analysis of a variety of compounds, and have been developed to detect a number of different compounds of environmental concern.
In the immunology field the term “hapten” refers to compounds that are unable to directly stimulate antibody production when injected into an animal, but are capable of binding to antibodies if they are produced by an alternate means. Many small molecules do not stimulate the immune system to produce antibodies. Antibodies can be raised, however, that specifically bind to such small molecule haptens. For example, many environmental contaminants, although potentially toxic to humans and animals, do not elicit a strong antibody response.
A “binding assay” is an assay for at least one analyte which may be detected by the formation of a complex between the analyte and an analyte receptor capable of specific interaction with that analyte. The analyte may be haptens, hormones, peptides, proteins, deoxyribonucleic acid (DNA), ribonucleic acids (RNA), metabolites of the aforementioned materials and other substances of either natural or synthetic origin which may be of diagnostic interest and have a specific ligand receptor therefor. Binding assays are generally useful for the in vitro determination of the presence and concentration of analyte in body fluids, food products, animal fluids, and environmental samples. For example., the determination of specific hormones, peptides, proteins, therapeutic drugs, and toxic drugs in human blood or urine has significantly improved the medical diagnosis of the human condition.
Current binding assay technology benefits from the diversity of detection systems developed that use enzyme-catalyzed chromogenic reactions, radionuclides, chemiluminescence, bioluminescence, fluorescence, fluorescence polarization, a variety of potentiometric and optical biosensor techniques and visual labels such as latex beads, gold particles and carbon black.
There is a continuing need for simple, rapid assays for the qualitative, semi-quantitative, and quantitative determination of such analytes in a sample. Furthermore, in many situations, such assays need to be simple enough to be performed and interpreted by non-technical users.
Binding assays rely on the binding of analyte by analyte receptors to determine the concentrations of analyte in a sample. Analyte-receptor assays can be described as either competitive or non-competitive. Non-competitive assays generally utilize analyte receptors in substantial excess over the concentration of analyte to be determined in the assay. Sandwich assays, in which the analyte is detected by binding to two analyte receptors, one analyte receptor labeled to permit detection and a second analyte receptor, frequently bound to a solid phase, to facilitate separation from unbound reagents, such as unbound labeled first analyte receptor, are examples of non-competitive assays.
Competitive assays generally involve a sample suspected of containing analyte, an analyte-analogue conjugate, and the competition of these species for a limited number of binding sites provided by the analyte receptor. Competitive assays can be further described as being either homogeneous or heterogeneous. In homogeneous assays all of the reactants participating in the competition are mixed together and the quantity of analyte is determined by its effect on the extent of binding between analyte receptor and analyte-conjugate or analyte analogue-conjugate. The signal observed is modulated by the extent of this binding and can be related to the amount of analyte in the sample. U.S. Pat. No. 3,817,837 describes such a homogeneous, competitive assay in which the analyte analogue conjugate is a analyte analogue-enzyme conjugate and the analyte receptor, in this case an antibody, is capable of binding to either the analyte or the analyte analogue. The binding of the antibody to the analyte analogue-enzyme conjugate decreases the activity of the enzyme relative to the activity observed when the enzyme is in the unbound state. Due to competition between unbound analyte and analyte analogue-enzyme conjugate for analyte-receptor binding sites, as the analyte concentration increases the amount of unbound analyte analogue-enzyme conjugate increases and thereby increases the observed signal. The product of the enzyme reaction may then be measured kinetically using a spectrophotometer.
Heterogeneous, competitive assays require a separation of analyte analogue conjugate bound to analyte receptor from the free analyte analogue conjugate and measurements of either the bound or the free fractions. Separation of the bound from the free may be accomplished by removal of the analyte receptor and anything bound to it from the free analyte analogue conjugate by immobilization of the analyte receptor on a solid phase or precipitation. The amount of the analyte analogue conjugate in the bound or the free fraction can then be determined and related to the concentration of the analyte in the sample. Normally the bound fraction is in a convenient form, for example, on a solid phase, so that it can be washed, if necessary, to remove remaining unbound analyte analogue conjugate and the measurement of the bound analyte analogue conjugate or related products is facilitated. The free fraction is normally in a liquid form that is generally inconvenient for measurements. If multiple analytes are being determined in a single assay, the determination of the free fraction of analyte analogue conjugate for each analyte is made impossible if all are mixed in a single liquid unless the responses of the individual analyte analogue conjugates can be distinguished in some manner. However, detecting the free fraction of analyte analogue conjugate in assays that are visually interpreted is a distinct advantage because the density of the color developed in such assays is generally proportional to the analyte concentration over much of the range of analyte concentration.
One method that can be used to detect the free analyte analogue conjugate in a heterogeneous, competitive analyte-receptor assay process is to provide a second, immobilized receptor specific for the analyte on a solid phase so that the analyte analogue conjugate not bound to the first analyte receptor can be bound to the second analyte receptor immo
Kilpatrick & Stockton LLP
Nguyen Bao-Thuy L.
Strategic Diagnostics Inc.
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