Chemistry: electrical and wave energy – Apparatus – Electrolytic
Reexamination Certificate
2001-04-19
2003-05-13
Warden, Sr., Robert J. (Department: 1743)
Chemistry: electrical and wave energy
Apparatus
Electrolytic
C204S403140, C435S286100, C435S817000
Reexamination Certificate
active
06562209
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to quantitative in vitro detection and assaying of bioagents and nucleic acids, and, more particularly, to an automated apparatus for assaying of bioagents and nucleic acids on an industrialized scale.
BACKGROUND
It is known that the composition and relative quantities of reactants and products occurring in any chemical reaction, whether inorganic, organic or biochemical, must be determined while the reaction is in progress or at an equilibrium end point. Known procedures to perform such monitoring uses biologic or non-biologic molecules, here referred to as “recognition” molecules. Recognition molecules are capable of binding to either reactant or product molecules in a structure-restricted manner. That is, the recognition molecule binds to a specific three-dimensional structure of a molecule or to a two-dimensional surface that is electrically charged and/or hydrophobic in a specific surface pattern. Historically such molecules have been identified by exploring animal immune systems or through trial-and-error procedures. The foregoing analytic technique has in general been referred to as immunochemical, in reference to the selective recognition and binding capacity of immunoglobulins, even though substances other than antibodies may serve as recognition molecules.
Although applied in basic organic and biochemistry, the foregoing techniques have achieved the greatest development in clinical medicine. The most frequently used method is the enzyme linked immunosorbent assays (“ELISA”), which is applicable to a wide variety of fields, such as biotechnology, environmental protection and public health. In the immuno-chemistry field, enzyme immunoassays (“EIA”) and, more particularly, ELISA are well known and important for detecting traces of foreign substances, such as antigens or antibodies in body fluids and tissues.
ELISA is a quantitative in vitro test for an antibody or antigen in which the test material is adsorbed on a surface and exposed to a complex of an enzyme linked to an antibody specific for the substance being tested for with a positive result indicated by a treatment yielding a color in proportion to the amount of antigen or antibody in the test material. The ELISA procedure is described more specifically, for one, in a book entitled Methods in Molecular Biology Vol 42, John R. Crowther, Humana Press, 1995. It should be recognized that the “antibody specific for the substance being tested for” in the foregoing definition constitutes a recognition molecule, earlier defined. It may also be recognized that ELISA-like approaches using other recognition molecules can also be used, such as aptamers, DNA, RNA & molecular imprint polymers.
More recently, the foregoing definition has been expanded beyond the colometric approach, wherein color is used as an indicia, to include yielding a rate of change of voltage or current conductivity in proportion to the amount of antigen or antibody in the test material, a voltametric or amperiometric approach to detection. Patent Cooperation Treaty application PCT/US98/16714, filed Aug. 12, 1998 (International Publication No. WO 99/07870), entitled “Electrochemical Reporter System for Detecting Analytical Immunoassay and Molecular Biology Procedures” (hereafter the “'16714 PCT application), claiming priority of U.S. patent applications No. 09/105,538 and 09/105,539”), describes both a colormetric and an electrochemical reporter system for detecting and quantifying enzymes and other bioagents in analytical and clinical applications.
The electrochemical reporter system of the 16714 PCT application employs a sensor for detecting the voltametric and/or amperiometric signals that are produced in proportion to the concentration of organic (or inorganic) reporter molecules, by redox recycling at the sensor. Although many different sensors are possible of application in the foregoing ELISA (and ELISA-like) methods, the preferred sensor in the foregoing method of the 16714 PCT application is stated to be of the kind described in a prior International Patent Application, published as PCT Publication No. WO 94/29708, published Dec. 22, 1994 (hereafter “IPN '708”) in which a closely spaced (eg. nanometer scale spacing) interdigitated array (“IDA”) of thin film noble metal microelectrodes are used. One set of the electrically conductive fingers in the interdigitated array serves as the cathode and the opposed set of electrically conductive fingers as the anode. The foregoing IDA is stated to be many times more sensitive than prior sensors due to redox recycling occurring at the surface of the electrodes.
With both an improved electrochemical ELISA protocol and an improved sensor the preferred ELISA system of the '16714 PCT application provides a more effective assaying system, one possessing greater sensitivity than prior assaying systems. The present invention also relates to detection and quantitative assaying by the electrochemical ELISA procedure. As brought out in the following description, the preferred embodiment of the present invention incorporates and improves upon the apparatus and method of the '16714 PCT application. Accordingly, the present application incorporates the content of each of the '16714 PCT application and the IPN '708 application here within as background to the present invention.
The electrochemical ELISA procedure and apparatus of the prior PCT application appears well suited to practice in a microbiology laboratory by highly skilled personnel who are alert to the details of the process. Other facilities or environments in which such an analysis is desirable, however, may not enjoy either the availability of highly skilled technicians or an adequately equipped laboratory. As example, one would like a simple and easily implemented test for the presence of the e-salmonella bacterium in meat products that could be conducted at meatpacking plants. In a military setting the field medics may need to quickly determine the presence of a deadly bioagent to which troops may have been or could become exposed. In such environments, the availability of a foolproof, user-friendly test apparatus that is able to analyze a sample and report a meaningful result with minimal human intervention is certainly desirable.
The system and apparatus of the present invention satisfies the foregoing need. As an advantage, the invention extends the benefit of the analytic procedures of the foregoing PCT applications to new fields and environments beneficial to the public.
Accordingly, a principal object of the present invention is to automate the ELISA procedure (and ELISA-like procedures), reducing the technical skills required to conduct the assay.
Another object of the invention is to provide apparatus for performing an ELISA of bioagents that is user friendly and capable of being performed by relatively unskilled personnel.
A further object of the invention is to make ELISA testing more portable, more easily accomplished, and of wider availability.
And a still further object of the invention is to computerize ELISA testing and make such testing available on an industrial scale.
SUMMARY OF THE INVENTION
In accordance with the foregoing objects and advantages, an electronic controller, such as a programmed microcontroller controls a series of pumps to automatically sequence the pumping of the individual fluids required by the ELISA procedure into a cell (or cells) necessary to produce a reporter, control the positioning of the carrier of the reporter adjacent the reporter sensor, analyze the data obtained from the reporter sensor and display the concentration of the bioagent determined from the analysis of the foregoing data. Once started, the apparatus, governed by the program, conducts the test automatically without the necessity for human intervention. In accordance with a specific aspect, the foregoing components may be housed in a single package for easy portability; may be battery powered or line powered.
An ancillary invention disclosed, that is incorporated within
MacPhee, Jr. Robert D.
McHugh Richard K.
Sullivan Brian M.
Zsolnay Denes L.
Goldman Ronald M.
Northrop Grumman Corporation
Olsen Kaj K.
Warden, Sr. Robert J.
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