Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
2000-01-04
2003-01-21
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C422S051000, C422S051000, C422S051000, C422S051000, C422S067000, C435S007100, C435S287100, C435S287200, C435S287900, C435S287700, C435S805000, C435S810000, C435S970000, C436S518000, C436S169000, C436S805000, C436S810000
Reexamination Certificate
active
06509196
ABSTRACT:
BACKGROUND OF THE INVENTION
Quantitative analysis of cells and analytes in fluid samples, particularly bodily fluid samples, often provides critical diagnostic and treatment information for physicians and patients. Quantitative immunoassays utilize the specificity of the antigen (Ag)—antibody (Ab) reaction to detect and quantitate the amount of an Ag or Ab in a sample. In solid phase immunoassays, one reagent (e.g., the Ag or Ab) is attached to a solid surface, facilitating separation of bound reagents or analytes from free reagents or analytes. The solid phase is exposed to a sample containing the analyte, which binds to its Ag or Ab; the extent of this binding is quantitated to provide a measure of the analyte concentration in the sample. Transduction of the binding event into a measurable signal, however, is affected by a number of interferences, such as non-specific binding of components of the assay to the solid phase, which are not associated with the presence or amount of the analyte. These interferences limit the specificity and applicability of quantitative immunoassays.
SUMMARY OF THE INVENTION
The invention relates to methods of measuring the amount of an analyte of interest in a fluid sample, using a solid phase assay such as a quantitative immunochromatographic assay or an enzyme-linked immunosorbent assay, in which an internal control is used to compensate for non-specific binding of assay components.
For a quantitative immunochromatographic assay, the methods use a membrane strip made of a suitable material, such as cellulose nitrate or glass fiber, which has sufficient porosity and the ability to be wet by the fluid containing the analyte, and which allows movement of particles by capillary action. The membrane strip has an application point, a contact region, and a detection zone; the contact region is between the application point and the detection zone. Imbedded in the contact region is a population of test particles, such as liposomes or organic polymer latex particles, and a population of internal control particles of the same type. The test particles are coated with a binding agent (e.g., an antibody) to the analyte of interest, and the internal control particles are coated with a binding agent (e.g., an antibody) to a control analyte. The particles can be labeled, using a colorimetric, fluorescent, luminescent, or other appropriate label, to facilitate detection; test particles and internal control particles are labeled using distinguishable labels, preferably of the same type (e.g., two distinguishable fluorescent labels). A detection reagent (e.g., antibody to the analyte of interest) is immobilized in the detection zone.
In the methods, the application point of the membrane strip is contacted with the fluid sample to be assayed for the analyte of interest. The membrane strip is then maintained under conditions which are sufficient to allow capillary action of fluid to transport the analyte of interest, if analyte is present in the sample, through the membrane strip to the contact region. The apparatus is further maintained so that when analyte of interest reaches the contact region, the analyte binds to the analyte binding agent coated on the test particles imbedded in the contact region. Test particles, including those which are bound with analyte, as well as internal control particles, are mobilized by fluid and move by capillary action through the strip to the detection zone. The detection reagent interacts with analyte-bound test particles; interaction of the detection reagent and the analyte-bound test particles results in arrest of analyte-bound test particles in the detection zone. The amount of test particles that are arrested in the detection zone is then detected, as is the amount of internal control particles in the detection zone. The amount of non-specific binding of test particles that are arrested in the detection zone is approximated by the amount of non-specific binding of internal control particles in the detection zone. Thus, the amount of analyte of interest in the fluid sample can be more accurately determined by eliminating the non-specific binding component: for example, the amount of analyte of interest in the fluid sample can be determined as the difference between the amount of test particles that are arrested in the detection zone and the amount of internal control particles that are arrested in the detection zone. The amount of analyte can determined from a standard curve for the analyte of interest.
If desired, the membrane strip can also comprise a control reaction zone, having control detection reagent coated on the membrane. The internal control particles are coated with a binding agent (e.g., an antibody) to the control detection reagent. Capillary action of the fluid mobilizes the internal control particles not only to the detection zone, but also to the control reaction zone, where they bind to the control detection reagent. The amount of analyte of interest in the fluid sample is determined by taking into consideration the amount of test particles that are non-specifically arrested in the detection zone, represented by the amount of internal control particles that are arrested in the detection zone. For example, the amount of analyte of interest in the fluid sample can be determined as a ratio between 1) the difference between the amount of test particles that are arrested in the detection zone and the amount of internal control particles that are arrested in the detection zone, and 2) the amount of internal control particles in the control reaction zone.
In an alternative immunochromatographic assay, the fluid sample to be assayed for the analyte of interest is applied directly to the detection zone of the apparatus. In this embodiment, the detection reagent is antibody to the analyte of interest. The membrane strip is maintained under appropriate conditions so that analyte in the fluid sample interacts with the detection reagent, and is immobilized in the detection zone. Water or an appropriate buffer is then added to the application point of the membrane, to mobilize the test particles and the internal control particles, which are moved by capillary action into the detection zone. The membrane strip is further maintained under conditions which allow interaction of the test particles with analyte that is immobilized in the detection zone. Interaction of the test particles with immobilized analyte arrests movement of the test particles. The amount of analyte in the fluid sample is determined by taking into consideration the amount of test particles that are non-specifically arrested in the detection zone; for example, the amount of analyte can be related to the difference between the amount of test particles that are arrested in the detection zone, and the amount of internal control particles that are arrested in the detection zone, and can be determined from a standard curve, as described above.
For an enzyme-linked immunosorbent assay, a solid phase reactant (e.g., a microtiter plate) having a first member of an immunobinding pair, such as an analyte-binding agent (e.g., antibody) to the analyte of interest adsorbed thereon, is contacted with a sample to be assayed for the amount of a second member of the immunobinding pair, such as an analyte of interest, under conditions allowing the members of the immunobinding pair to interact (e.g., to allow analyte of interest to bind to the analyte-binding agent, such as antibody, adsorbed on the solid phase reactant). Alternatively, a solid phase reactant having a test sample to be assayed for the amount of a first member of an immunobinding pair adsorbed thereon, is contacted with a solution containing the second member of the immunobiding pair, under conditions allowing the members of the immunobinding pair to interact. The solid phase reactant is then contacted with a solution containing an initial detection antibody and an internal control antibody, under conditions allowing the initial detection antibody to bind to the second member of the immunobinding pair that is
Brooks Donald Elliott
Harris Paul C.
Olal Andrew D.
Richards Brian G.
Xie Zongcen Charles
Chin Christopher L.
Hamilton Brook Smith & Reynolds P.C.
Response Biomedical Corp.
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