Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-06-22
2001-07-03
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007940, C435S006120, C436S518000
Reexamination Certificate
active
06255060
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of detecting selected proteins in a sample by a technique referred to herein as immuno aRNA. Amounts of a selected protein detected by this method can then be correlated to the presence of various diseases. Accordingly, this method is useful in diagnosis of diseases.
BACKGROUND OF THE INVENTION
An analysis of specific mRNA levels in a given cell provides insight into the function and differentiative state of that particular cell at any point in its life cycle. However, mRNA levels do not always provide an accurate portrayal of a cell's functional state. It is the translated products of these mRNAs, such as the receptors, ion channels, enzymes, and structural proteins of the cell, that determine its function.
Techniques currently used to detect proteins are based on various types of immunoassays, such as ELISA, immunohistochemistry and radioimmunoassay, which utilize antibodies specific for the protein of interest. These immunoassays, while useful, are limited by the sensitivity of the detection of the antibody. Standard labeling methods include fluorescence, radioisotopes, and enzymes such as peroxidase and phosphatase. In addition, secondary antibodies are oftentimes biotinylated to increase their sensitivity. Still, these techniques are often not capable of detecting small amounts of a particular antigen. Furthermore, these types of techniques are not feasible for detection of a specific protein from a particular cell.
Electrophysiological techniques can detect protein from a specific cell. However, applications are limited to the monitoring of ion channel functioning, as well as the functioning of other receptors or proteins which are coupled to channels. It is difficult to detect the small amounts of other proteins that do not directly couple to ion channels, which techniques such as expression profiling and immunohistochemistry indicate are present and potentially regulated within an individual cell.
Recently, Sano et al., 1992
Science,
258:120-122, described an antigen detection technique termed immuno-polymerase chain reaction (immuno-PCR). This procedure provides an extremely sensitive method to detect proteins. In immuno-PCR, a linker molecule with bi-specific binding affinity for DNA and antibody is used to attach a marker DNA molecule specifically to an antigen-antibody complex, thus resulting in the formation of a specific antigen-antibody-DNA conjugate. The attached marker DNA can be amplified by PCP with the appropriate primers. The presence of specific size PCR products demonstrates that marker DNA molecules are attached specifically to antigen-antibody complexes thereby indicating the presence of antigen. As described by Sano et al. 1992, antigen is immobilized on the surface of microtiter plates and then detected by immuno-PCR. Using this technique, an approximately 10
5
increase in sensitivity over an alkaline phosphatase conjugated ELISA was obtained. Sensitivity advantages of immuno-PCR have subsequently been confirmed in assays for mouse anti-lipoprotein IgG (Ruzicka et al., 1993
Science,
260:698-699); a human proto-oncogene protein (Zhou et al., 1993
Nucleic Acid Res.,
21:6038-6039); and tumor necrosis factor alpha (Sanna et al., 1995
Proc. Natl. Acad. Sci.,
92:272-275).
More recent reports have described advancements in immuno-PCR technology. For example, Joerger et al., 1995
Clin. Chem.,
41(9):1371-1377) demonstrate that double-stranded DNA labels can be directly attached to antibodies thus allowing conjugate reagents to be prepared before the assay. Suzuki et al., 1995
Jpn. J. Cancer Res.,
86:885-889, describe a method called double determinant immuno polymerase chain reaction (double-determinant immuno-PCR) which utilizes two monoclonal antibodies, in which the antigens are sandwiched, and a specific DNA molecule is used as a marker. Instead of the antigen itself, the first monoclonal antibody to bind the circulating antibody is immobilized, the biotinylated second monoclonal antibody is bound to the antigen and free streptavidin is used to attach a biotinylated DNA to the second monoclonal antibody. The biotinylated DNA complexed with antigen-antibody-streptavidin is amplified by PCR, and the products analyzed by Southern blot analysis. While this technique has provided advantages over traditional methods of protein detection, such as an increase in sensitivity, there still exist several notable limitations. For example, the use of the polymerase chain reaction is not quantitative. While PCR can be used to “amplify” a marker sequence to detect rarely occurring proteins, this amplification is not quantitative for >10-fold differences in antigen concentration. Thus, there is no direct correlation between the amount of signal and the amount of protein present. The immuno-PCR method also has inherent limitations that make it difficult to detect the presence of antigen in a particular cell. This is particularly relevant when antigen expressed in a specific cell type, such as a neuron, is being assayed. Further, these detection techniques can only assay protein present in solution or tissue, which is often a mixture of cell types.
Accordingly, there exists a need for an easily adaptable, sensitive detection method that is semi-quantitative and that can provide an accurate protein profile for a specific cell.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method of detecting a selected protein by immuno aRNA. In this method, a first antibody targeted to the selected protein is immobilized to a solid support. The solid support with the immobilized first antibody is then contacted with the selected protein so that the selected protein binds to the immobilized antibody. The solid support is then contacted with a RNA-promoter driven cDNA sequence covalently coupled to a second antibody targeted to the selected protein so that the second antibody binds to selected protein bound to the immobilized first antibody on the solid support. The amount of selected protein in the cell is then determined by amplified RNA techniques which detect the bound promoter-driven cDNA sequence.
Amounts of selected protein can then be correlated to the presence of a disease. For example, in one embodiment, the method of the present invention was used to measure levels of tau protein in single cells such as neurons. Elevated levels of tau have been correlated to Alzheimer's disease. Accordingly, the method of the present invention will be useful in diagnosing diseases such as, but certainly not limited to, Alzheimer's disease.
DETAILED DESCRIPTION OF THE INVENTION
A technique known as amplified RNA (aRNA) synthesis has been developed and utilized in the past few years for a variety of purposes including in vitro RNA synthesis from plasmids containing the appropriate promoter site for use as probes (Melton et al., 1984
Nucl. Acid Res.,
12:7035-7056), for in vitro translation studies (Krieg and Melton, 1984
Nucl. Acid Res.,
12:7057-7070), for producing synthetic oligonucleotides (Milligan et al., 1987
Nucl. Acid Res.,
15:8783-8798), and for detection of low abundance messages (Sarkar and Sinner, 1989
Science,
244:331-333; van Gelder et al., 1990
Proc. Natl. Acad. Sci.,
87:1663-1667; Carpenter et al., 1993
Clin Chem.,
39(9):1934-1938; Eberwine et al., 1992
Proc. Natl Acad. Sci.,
89:30-10-30-14; and Muckler and Eberwine, 1993
Mol. Pharm.,
44:308-315).
The technique of aRNA synthesis has been utilized by those of skill in the art perhaps most effectively for the detection of rare messages. In general, the first step in this method involves synthesizing an oligo(dT) primer that is extended at the 5′ end with an RNA polymerase promoter such as the T7 or SP6 promoter. This oligonucleotide can be used to prime poly (A+) mRNA populations for cDNA synthesis. After the first strand cDNA is synthesized, the second strand cDNA is made, followed by RNA nuclease treatment to degrade the RNA and treatment with T4 DNA polymerase to generate a blunt-end
Eberwine James
Rodgers Lori
Licata & Tyrrell P.C.
Trustees of the University of Pennsylvania
Wortman Donna C.
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