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Sensitive immunoassays utilizing antibody conjugates with replicable DNA templates
| Details |
Inventors: Martinelli, Richard A.; Carroll, III, Eddie;
Assignee: Bayer Corporation (East Walpole, MA)
Primary Examiner: Minnifield; Nita
Assistant Examiner: Masood; Khalid
Attorney, Agent or Firm: Lauder; Leona L., Morgenstern; Arthur S.
A novel signal amplification system for immunoassays that minimizes non-specific signals is disclosed. Immunoassay methods, reagents and test kits are described for obtaining immunoassays of enhanced sensitivity. The reagents include antibody-variant DNA conjugates, wherein the variant DNA is a substrate for an RNA-dependent RNA polymerase, such as, QB replicase. Immunoassay methods to detect, or to detect and quantitate, analyte in test samples comprise transcribing the variant DNA of said antibody-DNA conjugates that are bound to analyte, to RNA, and replicating the RNA transcripts, wherein the presence or quantity of variant RNA replication products can be correlated with the presence or quantity of analyte in the test samples. Further, methods are provided to detect, or to detect and quantitate, simultaneously two or more analytes in a test sample. |
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DETAILED DESCRIPTION Aspects of this invention revolve around antibody-variant DNA conjugates that are immunoassay reagents.
The conjugates are components of an immunoassay signal amplification system.
For enhanced sensitivity of immunoassays, the signal for an analyte molecule, especially when the analyte is in trace concentrations, needs to be amplified, so that the signal is visualizable and thus detectable.
The instant invention provides such a signal amplification system.
The system can produce an amplified signal representative of the number of analyte molecules in a sample that is assayed by methods of this invention.
The variant DNA of the conjugates when bound to an immunocomplex according to this invention is transcribed to RNA, and then the RNA transcripts are replicated, by contact with an appropriate RNA-dependent RNA polymerase in the presence of ribonucleotides, resulting in RNA replication products.
Thus, for each bound variant DNA template molecule transcribed, many RNA variant molecules can be produced.
Those RNA replication products are rendered visualizable by an appropriate means, such as by labeling with a radionuclide, fluorescer, chemiluminescer, or enzyme or by staining with an intercalating dye, among other methods (as discussed infra).
Examples 1-3, below demonstrate some representative methods of preparing the antibody-variant DNA conjugates of this invention.
Example 4 demonstrates the ability of variant DNA conjugated to an antibody to serve as a template for an RNA-dependent RNA polymerase to synthesize RNA.
In Example 4, nanovariant DNA conjugated to antibodies via the representative cross-linking reagent SPDP, retains its capability to serve as a template for QB replicase catalyzed synthesis of nanovariant RNA.
Thus, evidence is provided to show that representative antibody-variant DNA conjugates of this invention, may be used for immmunoassays of enhanced sensitivity.
The overall antibody concentration is chosen so that the amount of conjugated antibody non-specifically bound and capable of amplification by a RNA-dependent RNA polymerase, such as, QB replicase, is approximately 1000 to 10,000 molecules
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Semiconductor manufacture 
