Chemistry: analytical and immunological testing – Involving diffusion or migration of antigen or antibody
Reexamination Certificate
1999-05-20
2002-03-19
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving diffusion or migration of antigen or antibody
C436S518000, C436S806000, C435S007100, C435S007930, C435S007940, C435S970000, C204S194000, C204S400000, C204S290010
Reexamination Certificate
active
06358752
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method for detecting or quantifying an analyte, and a test device used in the method. More particularly, the invention relates to a biosensor test device and method employing marker-loaded liposomes and electrochemical detection for signal amplification and quantitation.
BACKGROUND OF THE INVENTION
There exist a variety of techniques useful for detecting and/or measuring the concentration of an analyte in a test sample. Such techniques include immunoassays, as described in U.S. Pat. Nos. 5,789,154; 5,756,362; and 5,753,519, each of which is hereby incorporated by reference.
Immunoassays employing electrochemical detection are described in U.S. Pat. No. 4,822,566 to Newman, and Niwa, O.; Xu, Y.; Halsall, H. B.; and Heineman, W. R. Anal Chem. 1993, 65, 1559-1563 (“Niwa”). Newman describes a multilayer immunoassay device which relies on the movement of biological species into or out of a biological binding layer in the course of biospecific binding reactions. This movement changes the dielectric constant of the fluid medium containing the analyte, resulting in capacitance changes detected by a sensor. A capacitor comprised of an array of interdigitated copper and gold fingers (2 mil wide, 0.5 mil high, separated by 3 mil spaces) formed by photolithographic etching techniques is disclosed. Niwa describes an electrochemical enzyme immunoassay which employs an interdigitated array microelectrode cell to detect 4-aminophenol (PAP), produced during enzyme immunoassay of mouse IgG. The gold interdigitated array consisted of 50 pair of 3 or 5 &mgr;m wide microbands, spaced 2 &mgr;m apart. Silver-plated and unplated gold square electrodes were used as reference and auxiliary electrodes, respectively. The assay was conducted in microwells.
The devices and techniques in Newman and Niwa, however, are relatively complex. For example, the enzyme immunoassay described in Niwa is carried out through multiple steps to completion on an immunowell device, and the reaction solution is then transferred to a separate electrochemical detection device.
Nucleic acid detection methods are potentially useful for detecting and measuring the presence of organisms, such as pathogens in food and water supplies. Southern, northern, dot blotting, reverse dot blotting, and electrophoresis are the traditional methods for isolating and visualizing specific sequences of nucleic acids. Each has advantages and disadvantages. For example, gel electrophoresis, often performed using ethidium bromide staining, is a relatively simple method for gaining fragment length information for DNA duplexes. This technique provides no information on nucleotide sequence of the fragments, however, and ethidium bromide is considered very toxic, although safer stains have been developed recently.
If, in addition to length information, there is a desire to determine the presence of specific nucleotide sequences, either Southern blotting, for DNA, or northern blotting, for RNA, may be chosen. These procedures first separate the nucleic acids on a gel and subsequently transfer them to a membrane filter where they are affixed either by baking or UV irradiation. The membrane is typically treated with a pre-hybridization solution, to reduce non-specific binding, before transfer to a solution of reporter probe. Hybridization then takes place between the probe and any sequences to which it is complementary. The initial hybridization is typically carried out under conditions of relatively low stringency, or selectivity, followed by washes of increasing stringency to eliminate non-specifically bound probe and improve the signal-to-noise ratio.
Originally, probes were often labeled with
32
P which was detected by exposure of the membrane to photographic film. Today, however, many researchers are making use of non-isotopic reporter probes. These blotting procedures require more time and effort than simple gel electrophoresis, particularly when low levels of nucleic acid are present.
Dot, or slot, blotting are essentially equivalent methods which provide sequence homology information only. No separation of nucleic acid sequences is performed prior to hybridization thus saving considerable time. Typically, the entire DNA, or RNA, composition of the sample being evaluated is attached to a nylon, or nitrocellulose, membrane to form a small “dot” of the nucleic acid mixture. The membrane is then probed in a fashion similar to that described for Southern and northern blotting. The technique is simpler than Southern and northern blotting but can give rise to non-specific binding of the probe thus reducing sensitivity. Probes can be labeled with 32P, biotin, various haptens, or enzymes such as horseradish peroxidase and alkaline phosphatase to produce a colored spot on the membrane in the presence of appropriate substrate.
In the reverse dot blot technique, an oligonucleotide capture probe is immobilized on a membrane while the target is kept in solution. In this scheme the target must also bear the reporter entity, usually by indirect registration. An advantage of this strategy is that multiple capture probes can be immobilized on the same membrane so that several target sequences can be determined simultaneously.
There are a wide variety of DNA and RNA detection schemes in the literature, many of which are available as commercial kits. Nucleic acid detection schemes have seen the same trends in assay design as immunoassays, with efforts directed towards simpler, more rapid, and automatable detection schemes.
It is useful to categorize assays based on the fashion in which the signal is produced and detected. Vener et al. (1991) Anal. Biochem. 198, 308-11, classified hybridization probe use into two categories: direct registration and indirect registration. Direct registration, not to be confused with direct hybridization, is defined as the use of a reporter probe which itself is capable of producing a detectable signal. This may be by labeling with a radioisotope, fluorescent tag, enzyme, or sol particle. Most of the initial work done with nucleic acid hybridization made use of direct registration with
32
P labelled probes.
An example of this type of assay is that reported by Pollard-Knight et al., (1990) Anal. Biochem. 185, 84-9. These researchers used probes labelled directly with horseradish peroxidase in an enhanced chemiluminescence detection scheme. Single-copy sequences of human genomic DNA were immobilized on nitrocellulose membranes, by Southern blotting, and targeted with enzyme-labelled probes of lengths between 50 and 3571 bases. One enzyme existed for every 50-100 bases of the probe so that better sensitivity was obtained with longer probe lengths. The use of a special blue sensitive film, along with a commercial enzyme substrate, allowed the detection of one amol of several different target sequences.
In indirect registration the probe itself does not bear the signal producing, or reporter entity, rather it bears a ligand such as biotin, fluorescein, digoxigenin, or, in some cases, a non-complimentary nucleotide sequence, which is then specifically recognized by a separate biomolecule or receptor. The latter then either generates or bears the signal producing molecules. This type of assay is very commonly used as a non-isotopic replacement for
32
P labeling and is available as commercial kits produced by Amersham International (Arlington Heights, Ill.) and Boehringer Mannheim (Indianapolis, Ind.).
The water-borne pathogen
Cryptosporidium parvum
illustrates the need for efficient and inexpensive nucleic acid detection methods.
Cryptosporidium parvum
is found in water supplies and food. Its life cycle includes oocysts that can be difficult to control by drinking water treatment processes such as chemical disinfection and filtration. The ingestion of oocysts can cause serious illness. Table 1 shows a number of documented water-borne outbreaks of Cryptosporidiosis in recent history.
TABLE 1
NUMBER
NUMBER
LOCATION OF OUTBREAK (DATE)
EXPOSED
INFECTED
Braun Station, TX (1984)
5
Bäumner Antje J.
Durst Richard Allen
Montagna Richard A.
Rule Geoffrey S.
Siebert Sui Ti A.
Cornell Research Foundation Inc.
Nguyen Bao-Thuy L.
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