Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-04-16
2001-06-12
Schwartzman, Robert A. (Department: 1635)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C536S023100, C536S024300, C536S024330, C536S025300, C536S025400, C536S025500
Reexamination Certificate
active
06245513
ABSTRACT:
BACKGROUND OF THE INVENTION
Biochemical assays such as immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)) and methods for detecting nucleic acid sequences in a test sample are well known. In such assays, high sensitivity is important to ensuring the ability of the assay to detect low levels of the analyte of interest. Radioactive and colorimetric methods have often been employed in such assays. However, achieving high levels of sensitivity has not always been possible, and methods of increasing the sensitivity of such tests are desirable.
One reported method (European Patent Publication EP 128 332) for detecting analytes, including nucleic acid sequences and antigens (or antibodies), is the use of a “bridging moiety,” which provides a bridge between an analyte and a “signalling moiety” which provides a detectable signal. The bridging moiety includes an analyte-specific region and a signalling moiety-specific region. This method has the advantage that, by varying the bridging moiety according to the target analyte, the same signalling moiety can be employed to detect a variety of analytes. However, the preparation of the bridging moieties can be rather lengthy and inefficient. Also, large bridging moieties (such a long polynucleotide sequences) may be less sensitive at detecting target analyte due to the presence of large segments which do not bind to the target. Moreover, this method requires that the analyte-specific region and a signalling moiety-specific region of the bridging moiety be different.
Another publication (U.S. Pat. No. 5,627,030 to Pandian et al.) describes the use of a primary probe for detecting a target nucleic acid sequence, and an “amplification probe”, which is a nucleic acid sequence which includes two regions: a first region complementary to the primary probe, and a second region which contains repeated sequences for binding to a plurality of labeled probes. The amplification probe permits several labeled probes to bind to each primary probe, amplifying the signal from the binding of the primary probe to the target molecule in the sample. However, construction of the amplification probe can be cumbersome.
SUMMARY OF THE INVENTION
The present invention relates to methods and compositions for amplifying or amplifying signals in biochemical assays. In particular, the invention provides methods and compositions useful for improving sensitivity in biochemical assays involving the binding of specific binding pairs.
In one aspect, the invention provides amplifying entities (also referred to herein as polymeric amplifying moieties (PAMs)) which are capable of binding to both an analyte (e.g., an antigen or nucleic acid) or analyte-detecting moiety (e.g., an antibody, a nucleic acid probe, and the like) and to a plurality of signalling moieties which include a detectable label. By binding to multiple signalling moieties, the PAM amplifies the signal generated in the presence of the analyte, thereby detecting the presence or absence of the analyte in a sample.
In another aspect, the invention provides a method for amplifying signals in assay systems. The method includes the steps of contacting the sample with a reagent having a first portion which specifically binds to the analyte and a second portion comprising a polynucleotide sequence, such that a complex of the analyte and the reagent is formed; contacting the complex of the analyte and the reagent with an amplifying entity having a first polynucleotide sequence and a second polynucleotide sequence, wherein the first polynucleotide sequence is complementary to the polynucleotide sequence of the second portion of the reagent, such that a complex of the analyte, the reagent, and the amplifying entity is formed; contacting the complex of the analyte, the reagent, and the amplifying entity with a plurality of signalling moieties, each of the signalling moieties comprising a detectable label and a polynucleotide sequence complementary to the second polynucleotide sequence of the amplifying entity, to form a detectable complex of the analyte, the reagent, the amplifying polynucleotide and the signalling moieties; and detecting the label as indicative of the presence or absence of analyte in the sample.
The analyte can be a nucleic acid sequence; the reagent first portion can be a nucleic acid sequence which is substantially complementary to the analyte; the analyte can be an antibody or antigen; the reagent first portion can be an antibody or antigen which specifically binds with the analyte; the amplifying entity first polynucleotide sequence and second polynucleotide sequence comprise the same or substantially the same sequence. In certain embodiments, the amplifying entity is a homopolynucleotide; the homopolynucleotide can comprise poly(dA); the poly(dA) can have a length of at least about 3000 bases; the reagent second portion can comprise poly(dT); each of the signalling moieties comprises poly(dT); each of the signalling moieties can comprise a detectable label selected from the group consisting of antigens, antibodies, enzymes, radioisotopes, and fluorescent moieties. In certain embodiments, prior to the step of contacting the complex of the analyte, the reagent and the amplifying entity with the plurality of signalling moieties, the method comprises the further step of washing the complex of the analyte, the reagent and the amplifying entity to remove unbound polynucleotide. In preferred embodiments, the analyte is immobilized with an immobilized capture reagent.
In another embodiment, the invention provides a method for detecting the presence or absence of an analyte in a sample, the method including the steps of contacting the sample with a reagent having a first portion which specifically binds to the analyte and a second portion comprising a homopolynucleotide sequence, such that a complex of the analyte and the reagent is formed; contacting the complex of the analyte and the reagent with a homopolynucleotide strand complementary to the homopolynucleotide sequence of the reagent, such that a complex of the analyte, the reagent, and the homopolynucleotide is formed; and contacting the complex of the analyte, the reagent, and the homopolynucleotide with a plurality of signalling moieties, each of the signalling moieties comprising a detectable label and a homopolynucleotide sequence complementary to homopolynucleotide strand, to form a detectable complex of the analyte, the reagent, the homopolynucleotide strand and the signalling moieties; and detecting the label as indicative of the presence or absence of analyte in the sample.
In certain embodiments, the homopolynucleotide strand is poly(da) and the reagent second portion and the signalling moieties comprise poly(dT) or poly(dU).
In still another aspect, the invention provides a method for detecting the presence or absence of an analyte in a sample, the method comprising the steps of contacting the sample with a first reagent having a first portion which specifically binds to the analyte and a second portion comprising a polynucleotide sequence, such that a complex of the analyte and the first reagent is formed; contacting the complex of the analyte and the first reagent with an amplifying entity having a first polynucleotide sequence and a second polynucleotide sequence, wherein the first polynucleotide sequence is complementary to the polynucleotide sequence of the second portion of the first reagent, such that a complex of the analyte, the first reagent, and the amplifying entity is formed; contacting the complex of the analyte, the first reagent, and the amplifying entity with a second reagent, the second reagent having a first portion which includes a polynucleotide sequence complementary to the second polynucleotide sequence of the amplifying entity, and a second portion, to form an extendable complex of the analyte, the first reagent, the amplifying entity and the second reagent; contacting the extendable complex with an extension reagent, the extension reagent comprising a first portion capable of specifically binding to the secon
Benight Albert S.
Faldasz Brian D.
Lane Michael J.
DeConti, Jr. Esq. Giulio A.
Lahive & Cockfield LLP
Schmidt M
Schwartzman Robert A.
Tm Technologies, Inc.
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