Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-06-29
2001-10-16
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C436S177000, C436S809000, C435S007100, C435S007940, C435S287700, C435S287800, C435S288200, C435S288300, C435S288400, C422S051000, C422S051000, C422S051000, C422S091000, C422S105000, C422S105000
Reexamination Certificate
active
06303389
ABSTRACT:
FIELD OF THE INVENTION
The invention relates generally to improved devices for performing binding assays and, more particularly, to rapid flow-through binding assay apparatus and methods.
BACKGROUND OF THE INVENTION
Binding assays are routinely used to screen for and diagnose a host of diseases and conditions, including Lyme disease, herpes, acquired immunodeficiency syndrome (AIDS), streptococcal infections, lupus and pregnancy. Such assays are relatively simple in theory, utilizing the binding affinity between two or more binding members to detect and/or quantify the presence of one of the members, referred to herein as the analyte. Binding members comprise a wide range of substances, including antigens, antibodies, haptens, complimentary nucleic acid sequences, ligands, small molecules and receptors. Antigen-antibody binding member pairs used in immunoassays currently enjoy the most widespread use.
A common format of a binding assay involves immobilizing a binding member specific for the analyte on a paper-like sheet or membrane. The membrane is then contacted with the test sample and appropriate reagents under conditions allowing binding to occur between the immobilized binding member and any analyte in the sample, with means for detecting binding events also provided. Often a labeled second binding member which binds to the first binding member-analyte complex is added to provide a detectable signal on the membrane.
The sandwich immunoassay is an example of one commonly used binding assay for antibody detection. In a generic sandwich immunoassay, the antigen is immobilized on a solid substrate. Antibody containing solution, e.g., diluted serum, is incubated with the immobilized antigen. Antibodies specific to the antigen bind to it, and unbound antibodies are then removed by buffer washes. A detection agent which may typically be a secondary antibody conjugated to an enzyme, is then incubated with the primary antibody-antigen complex. Finally, an enzyme substrate is added which is converted into a visual, detectable product whenever the enzyme is. Such multi-step sandwich immunoassays can be developed in many different ways depending on assay requirements.
A Western Blot is another example of a commonly used immunoassay. In current practice, the Western Blot method comprises a sequence of incubation and wash steps performed on a membrane bearing electrophoretically resolved antigen bands. Typically, the membrane is cut into narrow strips, each bearing the identical pattern of antigen bands. Strips are then processed in reagent solutions individually in narrow trays, each typically holding 0.5-2.0 ml. In the first step, the strip is incubated with a blocking solution containing a non-specific protein, e.g., non-fat dry milk, bovine serum albumin, newborn calf serum or gelatin. After washing off excess blocking solution with a wash buffer, typically a physiological saline buffer containing a low percentage of detergent, the strip is then incubated with antibody solution. Antibody solution may be diluted human or animal serum, cerebrospinal fluid, dried blood spot eluate, monoclonal antibody, to name a few. Unbound antibody is then washed off with buffer, and the strip is incubated in the detection reagent. In a typical application, the detection reagent could be goat-anti-human IgG-alkaline phosphatase conjugate. Unbound detection reagent is washed off with buffer, and finally the substrate (for alkaline phosphatase, a common substrate is 5-bromo-4-chloro-3-indolyl phosphate plus nitroblue tetrazolium) for the detection enzyme is added. The conversion of the substrate to a visually detectable product is allowed to proceed until optimal visualization of bands, and then substrate is washed away. The strip is typically dried, providing a permanent record of the assay result. Bands on the strip indicating antibody reactivity can be compared with control strips to determine the specificity of the immunoreaction. In currently used algorithms for HIV and Lyme testing, a positive test result is defined as the appearance of certain combinations of specific bands. For example, an HIV Western Blot test requires the presence of two bands to be considered positive, while a Lyme Western Blot test requires five out of ten bands to be positive for IgG, or two out of three bands to be positive for IgM.
As described above, the Western Blot method involves incubating the membrane strips sequentially in reagent solutions usually contained in a tray. In typical protocols, incubations with antibody solutions and detection reagents may take 30 minutes to several hours each. Wash steps may take 5-10 minutes each. The total time for processing a blot is therefore, not less than one hour, and is often several hours.
It would thus be desirable to provide a method and apparatus for the rapid processing of a binding assay, e.g., an immunoassay, including a Western Blot.
SUMMARY OF THE INVENTION
The invention includes, inter alia, systems and methods for providing assay cassettes that can be employed during rapid flow-through binding assays. The assay cassettes can be disposable units suitable for one-time use and readily assembled to include a filter membrane carried between an upper plate and a lower plate. A pattern of channels can extend through the top plate to allow a fluid sample to be applied through the top plate and onto the filter. The bottom plate can include a plurality of channels that are aligned with the channel of the top plate and which will allow a negative pressure to be applied to the underside of the filter membrane to draw the sample through the filter. In one embodiment, the cassette includes a frangible section that allows the cassette to be divided into at least a first and second component.
More specifically, in one embodiment, the cassettes comprise a multi-channeled top plate that can be received into a multi-channeled bottom plate to define an interior chamber between the top plate and the bottom plate. A membrane having antibodies bound thereon can be received within the interior chamber and sandwiched between the top plate and bottom plate. To this end, the bottom plate and top plate can include an engagement mechanism, such as a notch and catch assembly that allows the top plate and bottom plate to be joined together. The bottom plate and top plate can be joined together in such a manner that a membrane placed into the interior chamber of the cassette is sandwiched between the bottom surface of the top plate and the top surface of the bottom plate. The assembled cassette with the membrane therein can be placed in an assay machine, such as a machine of the type described in U.S. Ser. No. 09/045,630, filed Mar. 19, 1998, entitled SYSTEMS AND METHODS FOR RAPID BLOT SCREENING. As disclosed therein the cassette can be compressed by the plates of a container that can compress together the top plate and bottom plate of the assay cassette to hold together the top plate and bottom plate with sufficient force that a series of substantially tight seals are formed around the portions of a filter membrane, sandwiched between the two plates, that are enclosed by the walls of the channels found in the top plate. Effectively, this causes the filter membrane to act as a series of strips of membrane wherein each strip is isolated from the other strips formed on the membrane by the grooves of the upper plate. In this way, the assay cassette can be employed for carrying out a plurality of reactions with reduced or eliminated cross-contamination.
In one embodiment the invention assay cassettes described herein, comprise a top plate having a frangible portion extending transversely across the top plate and a bottom plate having a peripheral side wall and being adapted to couple with said top plate to define an interior chamber capable of receiving a filter membrane. In one embodiment, the frangible portion is formed by a score extending across the width of the top plate. However, in other embodiments the frangible portion can include a cavity or a frangible seal or any other mechanism suitab
Bowers William
Levin Andrew E.
Chin Christopher L.
Foley Hoag & Eliot LLP
Immunetics
Nguyen Bao-Thuy
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