Nucleic Acids Research, 2002, Vol. 30, No. 24 e140
© 2002 Oxford University Press
T7 RNA polymerase as a self-replicating label for antigen quantification
Department of Chemistry and Biochemistry, University of Windsor, Ontario N9B 3P4, Canada, 1 Department of Chemistry, University of Patras, Patras GR-26500, Greece and 2 Institute of Chemical Engineering and High Temperature Processes, PO Box 1414, Patras GR-26500, Greece
*To whom correspondence should be addressed at Department of Chemistry, University of Patras, Patras GR-26500, Greece. Tel: +30 2 610 997130; Fax: +30 2 610 997118; Email: tkc{at}chemistry.upatras.gr
Received August 21, 2002; Revised and Accepted October 30, 2002
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ABSTRACT |
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Enzymes are used widely as labels in binding assays for protein analytes, because they provide signal amplification. Efforts at improving the assay sensitivity have been focused mainly on the synthesis of novel substrates, e.g. fluorogenic and chemiluminogenic ones. We report the investigation of T7 RNA polymerase (T7RP) as a label with unique characteristics for antigen quantification. In an in vitro, coupled (one-step) transcription/translation reaction, T7RP catalyzes the expression of an enzyme-coding DNA template to produce free enzyme (luciferase) in solution. We demonstrate that the generated luciferase is linearly related to the input T7RP in a range covering over four orders of magnitude. It is also shown that T7RP exhibits a significant level of self-replication (100-fold) in vitro by acting on a DNA template comprising the T7RP cDNA downstream of a T7 promoter. By combining the self-replication reaction with the expression of luciferase DNA, as low as 1400 T7RP molecules are detectable. Furthermore, the T7RP is biotinylated, complexed with streptavidin and used for antigen quantification in a microtiter well-based assay with high sensitivity and reproducibility.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Whole-genome sequencing projects have led to the identification of thousands of new genes. The challenge ahead is to unravel gene function and regulation on a genome-wide scale. Most studies of gene function are based on the comparison of expression profiles between control and perturbed states, which allows for the identification of genes whose expression is induced or suppressed. DNA microarrays provide valuable information on gene expression at the mRNA level (1,2). Gene function, however, is manifested through the activity of the encoded protein. mRNA abundances do not always correlate with protein concentrations due to significant post-translational regulation (3). Consequently, the direct quantitative analysis of proteins provides more accurate information about biological systems. Moreover, the comparison of protein expression profiles in patients and normal samples (differential profiling) reveals potential biomarkers for diagnosis, prognosis and monitoring of disease progression, as well as new therapeutic targets. The challenge, however, lies in the fact that proteins present at low concentrations are usually the ones that mediate the cellular response to various stimuli and are involved in the early stages of pathological processes. A recent study has shown that half of the yeast proteome was undetectable using two-dimensional electrophoresis followed by mass spectrometry (4). Thus, high sensitivity, along with specificity, are essential requirements for any new technique in the field of proteomics, because they permit quantification of minute amounts of antigen and/or the use of smaller numbers of cells. Furthermore, these qualities must be combined with the ability for automation and high-throughput protein analysis, in order to exploit the information provided by large-scale sequencing projects.
Target amplification techniques analogous to PCR that offer exquisite sensitivity to nucleic acid analysis are not available for protein analytes. The most sensitive protein assays are based on the interaction of the analyte with a specific binder (antibody, receptor or peptide) that is linked to a signal-generating molecule (label). The assay sensitivity is determined, mainly, by the detectability of the label and the affinity of the binder. DNA fragments have been used as labels that provide signal amplification through replication [PCR (5) or rolling circle DNA replication (6)] or expression (7). However, the most widely used labels are enzymes (alkaline phosphatase, horseradish peroxidase, etc.) because they provide signal amplification through the turnover of many substrate molecules to detectable product. For almost 30 years, research efforts have been focused on the synthesis of novel substrates to allow more sensitive detection of enzyme labels. Thus, chromogenic substrates were gradually replaced by fluorogenic (8) and, more recently, chemiluminogenic ones (9). In contrast, this work introduces an enzyme label, T7 RNA polymerase (T7RP), which (i) has the unique ability to self-replicate in vitro and (ii) catalyzes the in vitro synthesis of a second enzyme (firefly luciferase). The resulting signal amplification is due to the generation of many enzyme molecules in solution. The assay allows for antigen quantification with high sensitivity, wide dynamic range and very good reproducibility. Because it is performed in microtiter wells, it is amenable to automation and high-throughput analysis.
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MATERIALS AND METHODS |
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In vitro coupled transcription/translation
The reaction mixture contained rabbit reticulocyte lysate (TNT) from Promega Corp., Madison, WI, supplemented with amino acids, but lacked T7RP. The appropriate DNA templates were added to the mixture.
Determination of firefly luciferase
A 2 µl aliquot of the transcription/translation reaction mixture was added to 50 µl of luciferase substrate buffer (20 mmol/l Tricine, pH 7.8, 1.1 mmol/l magnesium carbonate pentahydrate, 2.7 mmol/l MgSO4, 0.1 mmol/l EDTA, 33 mmol/l dithiothreitol, 270 µmol/l coenzyme A, 530 µmol/l ATP and 470 µmol/l luciferin) (10). The luminescence was monitored for 1 min using a liquid scintillation counter (model LS-6500; Beckman Instruments, Fullerton, CA) in the single-photon monitoring mode.
Biotinylation of T7 RNA polymerase
An aliquot of 1 mg (1.8 µmol) of sulfo-N-hydroxysuccinimide ester of biotin (NHS-LC-biotin; Pierce, Rockford, IL) was dissolved in 3 ml of dimethyl sulfoxide and then diluted to 15 µmol/l in T7RP buffer (20 mmol/l magnesium phosphate, pH 7.7, 0.1 mol/l NaCl, 1 mmol/l dithiothreitol and 1 mmol/l EDTA). A sample of 2 µl of the NHS-LC-biotin solution was mixed with 1 µl (6 pmol) of T7RP (Stratagene, La Jolla, CA) and incubated for 1 h at 4°C. The volume was increased to 50 µl with T7RP buffer containing 0.2 g/l bovine serum albumin (BSA). The biotinylated T7RP (BT7RP) was purified from free biotin by size exclusion chromatography using NAP columns (Amersham Pharmacia Biotech, Piscataway, NJ). The enzyme was eluted with 1 ml of sodium phosphate buffer pH 6.8. An aliquot of 100 µl of 10x concentrated T7RP buffer containing 1.4 g/l BSA was added to the purified BT7RP solution and the mixture was concentrated by ultrafiltration using microcon-30 filters (mol. wt cut-off = 30 000; Amicon, Beverly, MA)
Preparation of streptavidin-biotinylated T7 RNA polymerase complex (SA–BT7RP)
Purified BT7RP (3 pmol) was mixed with 4.8 pmol streptavidin (Sigma, St Louis, MO), diluted in T7RP buffer (final volume 150 µl). The complexation reaction was allowed to proceed for 10 min at room temperature and the SA–BT7RP complex was used without purification.
Biotinylation of monoclonal anti-prostate-specific antigen (PSA) antibody
The monoclonal anti-PSA antibody solution (catalog no. 8311; Diagnostic Systems Laboratories, Webster, TX) was dialyzed overnight against 3.5 l of 0.1 mol/l sodium bicarbonate at 4°C. A sample of 0.2 mg of the antibody was diluted with 0.5 mol/l carbonate buffer, pH 9.1, to a final concentration of 0.5 g/l. For biotinylation, 1 mg NHS-LC-biotin was dissolved in 50 µl of dimethyl sulfoxide and a 12.5 µl (0.25 mg) aliquot was added to the antibody solution. The mixture was incubated for 2 h at room temperature. The biotinylated antibody was stored at 4°C and used without purification.
T7 RNA polymerase as a label for antigen quantification
U-bottom, polystyrene microtiter wells (Nunc Maxisorp; Life Technologies, Burlington, Ontario, Canada) were coated overnight at room temperature with 25 µl of 5 mg/l capture anti-PSA antibody (catalog no. 8301; Diagnostic Systems Laboratories) diluted in 50 mmol/l Tris, pH 7.8, and 0.5 g/l NaN3. Before use, the wells were washed six times with wash solution (50 mmol/l Tris, pH 7.4, 150 mmol/l NaCl and 1 ml/l Tween-20). A 10 µl aliquot of PSA standard (Scripps Laboratories, CA) diluted in 50 mmol/l Tris, pH 7.8, and 60 g/l BSA, along with 15 µl of 0.5 mg/l biotinylated anti-PSA antibody, diluted in assay buffer (50 mmol/l Tris, pH 7.8, 60 g/l BSA, 0.5 mmol/l KCl, 0.5 g/l NaN3 and 0.5 g/l Triton X-100), were added to each well. The immunoreaction was allowed to proceed for 1 h with continuous shaking. At the end of the incubation, any unbound biotinylated anti-PSA antibody was removed by washing the wells six times as above. Afterwards, 25 µl of 2.4 nmol/l SA–BT7RP complex (diluted in T7RP buffer containing 1% fat-free dry skim milk) was added to each well and incubated for 10 min. The wells were then washed six times followed by twice with 50 mmol/l potassium acetate. Subsequently, 25 µl of transcription/translation mixture containing 52.5 fmol luciferase cDNA (Luc-DNA) (4.3 kb plasmid containing the T7RP promoter upstream from the firefly luciferase gene) was added to each well. The coupled in vitro transcription/translation reaction was allowed to proceed for 90 min at 30°C and the activity of synthesized firefly luciferase was measured as described above.
Antigen quantification using a self-replicating T7 RNA polymerase label
The formation of the immunocomplex on microtiter wells and the binding of the SA–T7RP complex were carried out as described above. Subsequently, 23.5 µl of transcription/translation mixture containing 37.5 fmol T7RP-DNA [plasmid pT7G1 (11), a kind gift from J. A. Wolff, Departments of Pediatrics and Medical Genetics, Waisman Center, Madison, WI] and 150 ng of salmon testes DNA (Sigma) was added to each well and incubated for 60 min (self-replication phase). Afterwards, 1.5 µl of Luc-DNA (26 fmol) was added to the wells and incubated for another 60 min (detection phase). The activity of synthesized firefly luciferase was measured as above.
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RESULTS AND DISCUSSION |
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T7RP was chosen for this study because it is one of the simplest DNA-dependent enzymes, capable of transcribing a complete gene without the need of additional proteins. Moreover, it is a single polypeptide chain (mol. wt 98 000), specific for its promoter. T7RP has been cloned and overexpressed in Escherichia coli (12).
The principle of antigen quantification using T7RP as a label is illustrated in Figure 1. Two approaches for measuring T7RP, based on in vitro transcription/translation (with and without self-replication), are also shown diagrammatically in Figure 1.
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Quantification of T7 RNA polymerase by coupled in vitro transcription and translation
The goal of these experiments was to establish a relationship between the input T7RP and the synthesized protein in an in vitro transcription/translation system. The expression of firefly luciferase was chosen because this enzyme can be detected with high sensitivity by using its characteristic bioluminogenic reaction (13).
Various amounts of T7RP were added to a coupled (one-step) transcription/translation reaction (rabbit reticulocyte mixture, final volume 12.5 µl) that contained the firefly Luc-DNA under the control of the T7 promoter. The reaction was allowed to proceed for 90 min at 30°C and then the activity of synthesized luciferase was measured by adding 2 µl of the expression mixture to 50 µl of luciferin substrate solution. It was observed (Fig. 2) that the luminescence was linearly related to the number of T7RP molecules in a range extending over four orders of magnitude (5.2 x 104–8 x 108 molecules of T7RP). The signal-to-background (S/B) ratio for the 5.2 x 104 molecules was 2.8.
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The coupled transcription/translation process consists of a series of complex reactions that require the concerted action of numerous factors, such as RNA polymerase, ribosomal subunits, translation initiation, elongation and termination factors, aminoacyl-tRNA synthetases, etc. Nevertheless, our data demonstrate that the final outcome is a simple linear relationship between input T7RP and the in vitro synthesized protein over a wide range of T7RP concentrations. This forms the basis for the development of a T7RP-based signal amplification system exploiting T7RP as a label.
Enhancing the detectability of T7 RNA polymerase through self-replication
A self-replication system for T7RP was designed as follows. Two consecutive 90 min in vitro expression reactions were carried out (12.5 µl each). In the first reaction the T7RP catalyzed the transcription of its cognate gene positioned downstream of the T7 promoter (T7RP-DNA) (11,14) and the generated RNA was translated simultaneously into active T7RP molecules. The newly synthesized T7RP also acted on the T7RP-DNA template to produce more of the enzyme (self-replication). The T7RP was then measured by transferring 2 µl into another expression reaction, containing 35 fmol Luc-DNA, and monitoring the synthesized luciferase. The extent of self-replication is a function of the T7RP-DNA level, as indicated by the increase in the luminescence as the T7RP-DNA concentration increases (Fig. 3A). At the optimum level of T7RP-DNA, the self-replication process caused a 110-fold increase in the signal compared to a reaction that contained no T7RP-DNA.
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Similar experiments with decreasing amounts of T7RP (aimed at estimating the detectability of the polymerase) revealed a low level of ‘illegitimate’ transcription of T7RP-DNA in the absence of T7RP. This was attributed to a eukaryotic RNA polymerase activity that is present in the rabbit reticulocyte extract and initiates a low level of transcription of T7RP-DNA, generating a few T7RP molecules which, in turn, are amplified by entering the self-replication cycle. This activity was not detectable in the absence of T7RP-DNA. Because illegitimate transcription compromises the detectability of T7RP, we carried out experiments to minimize it by adding various amounts of salmon DNA to the rabbit reticulocyte extract. Addition of 100 ng salmon DNA suppressed illegitimate transcription by 98%, whereas it caused only a 15% decrease in the T7RP-catalyzed transcription of T7RP-DNA (Fig. 3B).
In order to estimate the detectability of T7RP in a self-replication system, various amounts of the enzyme were added to the first expression reaction containing 25 fmol T7RP-DNA followed by a separate expression reaction containing 35 fmol Luc-DNA. The linearity extends from 1.4 x 103–107 molecules (Fig. 2). The S/B ratio at 1400 T7RP molecules was 2.5.
We investigated the possibility of combining self-replication with the detection of T7RP in a single reaction mixture containing both T7RP-DNA and Luc-DNA templates. However, it was observed that self-replication was suppressed dramatically due to competition between the two templates for binding to a limited number of T7RP molecules. Therefore, a delayed addition protocol was designed in which the T7RP was first allowed to act on its cognate gene (self-replication) followed by the addition of Luc-DNA, in the same reaction mixture.
The T7RP-DNA/Luc-DNA ratio, as well as the incubation times required before and after the addition of Luc-DNA, were optimized to ensure efficient self-replication and detection with the delayed addition protocol. The T7RP-DNA/Luc-DNA molar ratio was studied in the range 0.05–3 at three levels of Luc-DNA (Fig. 3C). The luminescence increases as the ratio becomes greater, due to increased self-replication, and reaches a plateau when the molar ratio of the two templates becomes 1–1.5. For the same molar ratio, the signal increases by increasing the concentration of Luc-DNA.
Various combinations of reaction times before and after Luc-DNA addition were studied (Fig. 3D). The maximum signal was achieved with 60 min–60 min, respectively, giving a 50-fold enhancement over the assay that contains no T7RP-DNA. Other combinations, such as 30–60, 30–90 and 60–30, compromised either the yield of the self-replication reaction or the detection reaction, thus giving a lower signal. In particular, the 0–90 combination (both templates added simultaneously at the beginning of expression) gave the lowest yield of self-replication (Fig. 3D).
The detectability of T7RP using the optimized single expression reaction protocol (delayed addition protocol) was 8.5 x 103 molecules, with a S/B ratio of 4.3. The luminescence was a linear function of the amount of T7RP, up to 107 molecules (Fig. 2).
Biotinylation of T7 RNA polymerase, complexation with streptavidin and application to antigen quantification
The effect of biotinylation on the activity of T7RP was studied by reacting with increasing concentrations of the sulfo-N-hydroxysuccinimide ester of biotin (NHS-LC-biotin) at pH 7.7 and 9.0. All reactions were incubated for 1 h at 4°C and the activity of T7RP was measured by in vitro coupled transcription/translation. Inactivation of T7RP becomes significant at biotin:T7RP molar ratios greater than 5 (Fig. 4A), due to modification of free amino groups that are necessary for full activity. The inactivation was more extensive at pH 9.0 than pH 7.7 because the biotinylation reaction is more efficient when the NH2 groups are deprotonated.
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A microtiter well-based ‘two-site’ immunoassay was developed for PSA, as a model (Fig. 1). The antigen was bound both by an immobilized capture antibody and a biotinylated detection antibody. The two anti-PSA antibodies are directed to different epitopes. BT7RP was complexed to streptavidin and added to the immunocomplex. The solid phase-bound T7RP was measured by in vitro expression.
The complexation of streptavidin with BT7RP was studied at SA:BT7RP molar ratios ranging from 0.5 to 12 (Fig. 4B) and the complexes were applied directly to the assay of 20 fmol antigen. The luminescence reached a maximum at a SA:BT7RP molar ratio of 1.5. The signal dropped sharply at lower or higher ratios. When BT7RP is in excess, all four biotin-binding sites of streptavidin are occupied and the complexes cannot bind to the biotinylated antibody on the solid phase. On the other hand, when streptavidin is in excess, free streptavidin competes with SA–BT7RP complex for binding to the well (15).
The time-course of the in vitro transcription/translation process with immobilized T7RP was studied up to 180 min (Fig. 4C). The luminescence increased with time but the S/B ratio reached a plateau at 120 min. The background was defined as the luminescence obtained when no antigen was present in the well. The concentration of SA–BT7RP added to the well also affected both the signal and the background of the assay and therefore its detectability (Fig. 4D). The signal increased with the SA–BT7RP concentration and a plateau was reached at 7 nmol/l. The S/B ratio, however, was highest at 3 nmol/l and then dropped because of the increasing non-specific binding of the complex to the solid phase.
The sensitivity and dynamic range of the optimized immunoassay were assessed by analyzing serial dilutions of the antigen, in the appropriate buffer. The S/B ratio was plotted as a function of the amount of PSA in the assay mixture (Fig. 5). In the absence of T7RP-DNA (no self-replication), 195 amol antigen were detected with a S/B ratio of 2.3. When the self-replication system was used (single expression reaction with a delayed addition of Luc-DNA), as low as 12 amol PSA could be detected with a S/B ratio of 1.9. The dynamic range of the assay extended up to 50 000 amol (Fig. 5). For comparison of detectabilities, we also performed a classical ELISA assay in which the immunocomplex was detected by using a streptavidin–alkaline phosphatase conjugate (New England BioLabs) and the enzymic activity was determined by adding p-nitrophenylphosphate as a substrate (Sigma) and measuring the absorbance at 405 nm on a microplate photometer (model EL-307C; BioTek Instruments, Winooski, VT). As shown in Figure 5, a S/B ratio of 2.1 was obtained for 7300 amol antigen. Consequently, the proposed assay offers 
600-fold higher detectability and a much wider analytical range.
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To assess the reproducibility of the proposed immunoassay (including all the steps, i.e. coating of the wells, immunocomplex formation, binding of SA–BT7RP, in vitro coupled transcription/translation and luciferase measurement), we analyzed samples containing 0.1, 1 and 10 fmol PSA. The percent coefficients of variation were 7.8, 7.5 and 8.1, respectively (n = 7).
It should be noted that the assay is carried out in three steps, namely immunocomplex formation, reaction with the SA–T7RP complex and finally self-replication of T7RP and luciferase synthesis. The wells are washed at the end of each step to remove all unbound components of the samples as well as the excess of reagents. Consequently, the transcription/translation reaction mixture does not come into contact with the sample components (removed during first washing).
It has been shown previously that T7RP transcripts that are neither capped nor polyadenylated can be efficiently translated in eukaryotic systems (16,17). Optimizing the structure of T7RP-DNA and Luc-DNA templates may further enhance the sensitivity of the system. For instance, suitable enhancer and transcription termination sequences may be incorporated to increase the yield of both self-replication of T7RP and luciferase synthesis. Insertion of both T7RP-DNA and Luc-DNA templates, under the control of the T7 promoter, into a single vector may also be investigated for higher yields. Besides firefly luciferase DNA, cDNAs for other highly detectable proteins may be employed, e.g. green fluorescent protein (18), alkaline phosphatase, aequorin (19), etc. A eukaryotic (rabbit reticulocyte) coupled transcription/translation system was used throughout this work. However, prokaryotic (E.coli) systems may also be tested by using DNA templates containing a T7 promoter and a Shine–Dalgarno sequence for ribosome binding.
We have used streptavidin as a linker between the biotinylated detection antibody and BT7RP. An alternative means of biotinylation would be to express a recombinant T7RP/strep-tag (II) fusion protein. Strep-tag (II) is an eight amino acid polypeptide, generated by combinatorial engineering, which exhibits binding properties for streptavidin or for a mutant form of streptavidin called strep-tactin (20).
The assay is amenable to automation because it is performed in microtiter wells. Although we used the 96-well format, the technology is transferable to microtiter plates with larger numbers of wells as well as to microfabricated wells for high throughput parallel analysis of multiple proteins.
While this manuscript was in progress, another technique for protein analysis was reported (21) that exploited the T7RP reaction for signal amplification (IDAT, immunodetection amplified by T7RP). However, the two concepts are fundamentally different. In IDAT, a double-stranded DNA fragment (the substrate for T7RP) was used as the antibody label. Following immunocomplex formation, the label was transcribed for 4 h by excess T7RP to generate multiple RNA copies. Since RNA by itself is not a suitable reporter molecule, a 32P-labeled ribonucleotide was incorporated during transcription in order to confer high detectability. The labeled transcripts were then separated by electrophoresis on a denaturing (urea) polyacrylamide gel and measured by autoradiography. Conversely, the present work uses the enzyme T7RP as a label that by acting on both T7 cDNA and Luc cDNA templates produces the corresponding mRNAs that upon translation generate T7RP (self-replication of T7RP) and firefly luciferase (indicator enzyme).
Although at present the proposed assay does not achieve the reported detectability of IDAT, it offers a number of significant advantages. (i) The assay does not use radioactive isotopes. During the last two decades there have been intense research efforts in the fields of immunoassays and DNA/RNA hybridization assays towards replacing radioactive labels with non-radioactive alternatives, in order to avoid the hazards associated with the use and disposal of radioactivity. As a consequence, most clinical laboratories now use exclusively non-radioactive assays and the use of non-radioactive detection systems in research laboratories is expanding rapidly. (ii) In contrast to IDAT, which requires tedious denaturing gel electrophoresis and autoradiography, the present assay is performed entirely in microtiter wells, thereby allowing for automation and high-throughput analysis. (iii) A quantitative relationship is established between the luminescence signal and the amount of antigen with a dynamic range covering almost four orders of magnitude. (iv) Compared to IDAT, the proposed assay is much shorter. Indeed, after immunocomplex formation, IDAT requires a 4 h transcription step followed by time-consuming electrophoresis and autoradiography. In contrast, the present technique requires 2 h for quantification of the immunocomplexes. (v) Because of the self-replication reaction, amplification in the proposed system is exponential, whereas IDAT involves linear amplification of the label.
In recent years, research efforts have focused increasingly on the identification of binders, with the requisite affinity and specificity, for a large number of protein analytes. Various approaches include recombinant antibodies selected by phage (22) or ribosome display (23), RNA or DNA aptamers (24) and small organic compounds selected through combinatorial library methods (25,26). The extent and/or the position of biotinylation of proteins, nucleic acids and small molecules can be controlled to achieve minimal interference with the interaction between the binder and the protein analyte. Consequently, SA–BT7RP is a universal detection reagent that can bind with high affinity (Kd = 10–14 M) to any biotinylated binder–analyte complex.
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ACKNOWLEDGEMENTS |
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We would like to thank J. A. Wollf for providing the plasmid pT7G1. This work was supported by grants from the National Science and Engineering Research Council of Canada (NSERC).
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