Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1997-11-04
2003-01-07
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C422S051000, C422S051000, C422S067000, C435S007100, C435S007200, C435S007210, C435S007310, C435S007320, C435S007400, C435S007900, C435S007920, C435S007940, C435S287200, C435S287900, C435S961000, C435S971000, C435S973000, C435S974000, C435S975000, C436S518000, C436S528000, C436S530000, C436S532000, C436S017000, C436S808000, C436S825000
Reexamination Certificate
active
06503702
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to rapid immunoassays for the detection of a variety of test substances.
BACKGROUND OF THE INVENTION
Immunoassays, where one or more antibodies are used to detect a test substance in a test sample, are widely known. The evolution of immunoassay methods has led to increasing sensitivity and ease of use. Despite this evolution, there remains a desire to achieve the detection and measurement of antigenic substances more rapidly and at less cost in terms of time and resources, without sacrificing sensitivity or reliability of results.
In pursuit of this desire, several forms of immunoassay have been developed. Some immunoassays have been developed which rely upon multiple antibodies, one of which may be bound to a solid material. This binding has been accomplished using nitrocellulose, polystyrene beads, plastic cloth, polycarbonate filters and the like. For example, U.S. Pat. No. 4,803,154 describes a “sandwich”-type immunoassay method in which a hydrophobic sheet is treated to create defined and delimited hydrophilic regions which can be used to bind antibodies through reactive aldehyde groups. The test sample containing the test substance is placed in contact with the treated regions having the bound antibody, which captures the test substance through an immunological reaction. Next, another antibody, to which is conjugated a means of detection, is added to the treated region and binds to the test substance immobilized on the sheet in the first step. Executing the means of detection allows one to measure the presence of the test substance in the test sample. This method is described as a “rapid” method, but requires from 60 minutes to 48 hours to complete, not including pre-assay sample preparation.
Convenient formats such as “dipsticks” have also been developed. In one example, a “dipstick” format having a polycarbonate detection membrane fused to a polyvinyl chloride sheet is used to provide an immunoassay method usable under field conditions. C. L. Penny et al. Journal of Immunological Methods 123: 185-192 (1989). In this method, the test substance is bound to a detection membrane by immersing the dipstick into the test sample, which must be a fluid. In a second step, the dipstick is immersed into a solution containing an antibody, to which is conjugated a means of detection, which is then immobilized by immunological reaction with the bound antigenic substance. In the final step, the means of detection is executed, which entails immersing the dipstick into a solution containing the appropriate detection reagents. The assay is described as requiring more than one hour to complete, not including pre-assay sample preparation.
In another example of the “sandwich”-type immunoassay, a first antibody is bound to the surface of a multiwell plate. S. Kodama et al. Journal of Immunological Methods 127: 103-108 (1990). A previously prepared test sample is then mixed with an appropriate buffer containing a second antibody, to which is conjugated a means of detection, and the combined solution is placed in a well of the plate. In the final step, the means of detection is executed, which is accomplished by adding the appropriate detection reagent to the well. The presence of the antibody-test substance sandwich is then determined. The assay as described requires 50 minutes for completion. Sample preparation time is not included.
Other immunoassays are also known which purport to be rapid and economic, e.g., U.S. Pat. No. 4,962,023, U.S. Pat. No. 5,169,757, but all have the limitation of requiring at least one hour to complete the assay, without including the time to prepare the test sample.
Immunoassays that require less time are also known, such as the commonly available latex agglutination tests for pregnancy based on detection of human chorionic gonadotropin (hCG). One example of such a test is the B-Clone® hCG Assay manufactured by Monoclonal Antibodies and distributed by Baxter Scientific Products (McGaw, Ill.). Such tests are simple and require from a few minutes or less up to 30 minutes for detection of hCG. These tests are designed to use urine as the test sample, which does not require any preparation prior to detection of the test substance. In cases where the test sample containing the test substance is more complex, such as whole blood, feces, and plant or animal tissues, test sample preparation can be a separate and time consuming step, which can increase the time and labor needed to obtain reliable and accurate results. In these circumstances, an immunoassay that can be completed in a similar period of time, and that combines the steps of extraction with those of detection of the test substance would provide an advantage in both time and labor.
Some immunoassays have also been developed that require only a single incubation step. Such immunoassays are known as “simultaneous” immunoassays. One example of such an assay is described in U.S. Pat. No. 4,376,110. In such an assay, an antibody bound to a solid phase support is incubated with the test sample simultaneously with another antibody having a means of detection conjugated to it. Another type of immunoassay described in U.S. Pat. No. 4,376,110 is a “reverse” immunoassay, which involves the stepwise addition to the liquid test sample of first the antibody having a means of detection conjugated to it followed by the addition of an antibody bound to a solid phase support. Such immunoassays provide for ease of handling but suffer due to the potential presence of interfering substances in an unprepared test sample. Furthermore, a liquid test sample is required as the antibody-antigen reaction occurs in the liquid phase in such a immunoassay. Such “simultaneous” or “reverse” immunoassays also require an additional separation and wash steps to remove the captured antibody-test substance complex from the liquid test sample. Further examples of “simultaneous” and “reverse” immunoassays can be found in U.S. Pat. No. 5,011,771, where the need to prepare a test sample prior to immunoassay is ignored and the need to separate the captured antibody-test substance complex from the liquid test sample requires an additional sedimentation and centrifugation step. Hence, the necessity of additional steps which can require laboratory equipment can limit the usefulness of such methods for conducting immunoassays under non-laboratory conditions.
Another immunoassay has been described in U.S. Pat. No. 5,169,789 for bacteria and viruses which can be conducted in a short period of time and includes simultaneous lysing of the test sample and antibody reaction. This immunoassay uses a nitrocellulose membrane having a submicrometer pore size where the flow of the liquid containing the test substance from the sampling means to the capture membrane is limited to diffusion facilitated by an optional underlying absorbent layer. Such an immunoassay cannot be physically arranged to allow simultaneous visual determination of detection results for multiple test substances in a single assay because of the limited flow rates and opaque nature of the membrance material. Other immunoassays based on nitrocellulose with submicrometer-sized pores, such as those diclosed in U.S. Pat. No. 4,366,241 and U.S. Pat. No. 5,006,464, have similar shortcomings.
The present invention provides an immunoassay which is sensitive, reliable and economic and requires as little as 60 seconds to complete, including time to prepare the test sample and execute the means of detection of the test substance or substances.
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Bruce Vrana
Chin Christopher L.
Grun James L.
Syngenta Investment Corporation
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