Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-19
2001-02-27
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C422S051000, C422S051000, C422S105000, C422S105000, C435S287100, C435S287200, C435S287700, C435S288500, C436S518000, C436S809000
Reexamination Certificate
active
06194160
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to medical instrumentation for in vitro diagnostics, and more particularly to medical instrumentation for more efficiently conducting accurate assay for the in vitro diagnosis of infectious diseases.
BACKGROUND OF THE INVENTION
The successful medical treatment of a disease depends, in part, on an accurate diagnosis. Traditionally, diagnosis is done by examining a patient's symptoms and comparing the identified symptoms against the list of known symptoms that are associated with each known disease. Given the range of known diseases this can be a difficult task, particularly for young children in distress who are less capable of describing their symptoms and less capable of confirming for the treating physician contact with significant environmental factors, such as exposure to certain plants, animals or insects.
To improve the accuracy of this process, scientists have developed new testing techniques that identify from a sample of the patient's blood, the existence of pathogens indicative of the disease. More specifically, patients infected with microbial pathogens develop an immune response characterized by the appearance in the patient's serum of antibodies specific to microbial antigens. Such antibodies can be detected in various ways, and their detection can serve as an indication of infection and the existence or likely development of a particular disease. Consequently, the medical community has adopted these tests as part of the process of diagnosing serious infectious diseases, such as HIV, hepatitis and Lyme disease. The standard test performed is the Enzyme-Linked Immunosorbent Assay (ELISA). The ELISA test provides an efficient way to test for the presence of antibodies of interest. However, ELISA can provide false or questionable test results up to 20% of the time, depending on the specific test. To overcome this drawback, doctors retest specimens with a more accurate membrane-based assay, such as the Western blot test.
The Western blot test involves preliminary electrophoretic separation of antigen components by molecular weight on polyacrylamide gels, followed by transfer of the separated antigen bands to a membrane. In the Western Blot method, antibody reactions to specific antigen bands can be detected and distinguished from antibody reactions with non-specific or cross-reactive bands, thereby significantly increasing the specificity of the immunoassays. The Western blot method has become the method of choice in confirming the presence of antibodies specific for a range of infections, including HIV, HTLV-1/2, Lyme Disease, and Cysticercosis.
However, the Western blot assay is cumbersome and labor intensive. In current practice, the Western blot method comprises a sequence of incubation and wash steps performed on a membrane bearing 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. In the first step, the strip is incubated with a blocking solution containing a non-specific protein. After washing off excess blocking solution with a wash buffer, the strip is incubated with antibody solution. Unbound antibody is then washed off with buffer and the strip is incubated in the detection reagent. Unbound detection reagent is washed off with buffer and finally the substrate 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.
The Western blot method as described is a multiple step manual procedure, that includes several rate-limiting steps, such as incubating the strips sequentially in the reagent solutions contained in a tray. In typical protocols, incubation 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 not less than one hour, and often several hours. Consequently, the Western blot method is a time-consuming and labor intensive test and therefore disfavored by today's cost conscious health care providers as well as by doctors that are in need of immediate verification of a diagnosis.
However, the Western blot, as well as other membrane-based tests can provide accurate test results for a number of infectious diseases including HIV, Lyme, Babesiosis and Ehrlichiosis. Accurate tests results are needed to treat properly these dangerous diseases, which if untreated can result in serious illness. Moreover, the need to use these more accurate tests is increasing, as Lyme disease is the most rapidly spreading vector-borne disease in the United States, and the HIV positive population is expected to grow from an estimated 20 million people to 30 million people within the next two years.
Accordingly, there is a need for technology that rapidly and efficiently provides accurate test results for infectious diseases.
SUMMARY OF THE INVENTION
In one aspect, the invention provides systems for conducting membrane flow-through assays. The systems can include a filtration procedure which provides for more effective interaction of solutes and membrane-bound ligands. To that end, the systems described herein include a plate that is adapted to receive a cassette which contains a membrane which can contain binding members for binding an analyte of interest in a sample. The systems also include an agitator that agitates fluid applied to the membrane to achieve an even distribution of fluid over the membrane. An even distribution is understood to achieve even and maximal diffusion of fluid into the membrane. The systems further include a pump that is in fluid communication with the cartridge assembly and is capable of generating a pressure differential for drawing the liquid reagent through the membrane. Accordingly, both solvents and solutes can thereby contact molecules bound to the fibers within the thickness of the membrane.
More specifically, the invention is understood to include an apparatus for conducting a membrane flow-through assay. The apparatus can include a plate for carrying a cartridge, or cassette, of the type having a membrane with a binding member for binding an analyte of interest, a base being coupled to the plate for supporting the plate and for allowing the plate to move about at least one axis, an agitator coupled to the plate and capable of moving the plate in a selected motion about the axis, and a pump capable of generating a pressure differential and preferably coupled in fluid communication with the plate for allowing the pressure differential to be applied to the cartridge for drawing a fluid through a membrane of the cartridge, whereby action of the agitator and the pump can allow fluid to distribute evenly across the surface area of the membrane and be drawn through substantially the entire thickness of the membrane.
In one embodiment, the apparatus is adapted such that the plate can removably and replaceably receive the cartridge. This is understood to facilitate the use of disposable cartridge assemblies. The apparatus can be designed for automatic operation and to that end can include a timer, as well as a timer control unit for controlling the duration for which the agitator operates, or for which the vacuum operates. Additionally, the apparatus can include a controlling unit for automatically controlling the operation of the agitator or the vacuum pump. In one embodiment, the control unit includes a programmable memory for allowing a user to select a program of operation for aspirating and agitating a sample material appli
Chin Christopher L.
Foley Hoag & Eliot LLP
Immunetics, Inc.
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