Enzyme-amplified aequorin-based bioluminometric hybridization assays.

The sensitivity of aequorin-based bioluminometric hybridization assays was enhanced by introducing, enzymically, multiple aequorin labels per DNA hybrid. The target DNA was hybridized in microtiter wells with an immobilized capture probe and a digoxigenin-labeled detection probe. The hybrids were reacted with an anti-digoxigenin antibody conjugated to horseradish peroxidase. Peroxidase catalyzed the oxidation of digoxigenin-tyramine by hydrogen peroxide, resulting in the attachment of multiple digoxigenin moieties to the solid phase. Aequorin-labeled anti-digoxigenin antibody was then allowed to bind to the immobilized digoxigenins. The bound aequorin was determined by its characteristic Ca2+-triggered bioluminescence. As low as 20 fmol/L (1 amol/ well) target DNA was detected with a signal-to-background ratio of 2.7. A hybridization assay that used only aequorin-labeled anti-digoxigenin antibody without the peroxidase amplification step gave a signal-to-background ratio of 2 for 160 fmol/L target DNA. The signal enhancement of the amplified assay was in the range of 14-38 times. The analytical range of the amplified assay extended up to 2600 fmol/L. The CVs were in the range of 5.5-7.3%.

Expression immunoassay.

Expression immunoassay is a sensitive analytical method that takes advantage of coupled in vitro transcription and translation as a signal amplification technique. Essentially, the immunoassay is performed using a detection antibody that is labeled with an expressible fragment of DNA. The product of expression is a protein that can be used to generate a signal. Here we describe two distinct expression immunoassays; both are based on expression of DNA labels to produce active enzyme molecules which are subsequently detected through their enzymatic activities. The luciferase expression immunoassay uses a 2.1-kb DNA template as a reporter molecule. The DNA is attached to a biotinylated detection antibody via biotin-streptavidin linkage. After the detection antibody is immunoreacted with immobilized antigen and excess antibody is removed, the DNA label is expressed in vitro. A linear relationship exists between the bioluminescent signal, from luciferase activity, and the immobilized antigen. This expression immunoassay allows the detection of 5 x 10(4) antigen molecules. The second expression immunoassay makes use of the well-characterized alpha-complementation of beta-galactosidase by employing an alpha-peptide encoding DNA as a reporter molecule. By monitoring the resulting beta-galactosidase activity with a fluorogenic substrate it was possible to detect as little as 3 fmol of immobilized antigen. Both expression immunoassays are amenable to automation and demonstrate the potential sensitivity that can be achieved using in vitro expression as a signal amplification method.

Expression hybridization assays combining cDNAs from firefly and Renilla luciferases as labels for simultaneous determination of two target sequences.

Two cDNAs encoding firefly luciferase (FLuc) and Renilla luciferase (RLuc) were used as labels for the development of a microtiter well-based expression hybridization assay that allows simultaneous determination of two target DNA sequences. The target DNAs were denatured and hybridized with specific capture and detection probes. One detection probe was biotinylated while the other was tailed with poly(dT). The hybrids were reacted with a streptavidin-FLuc DNA complex and a poly(dA)-tailed RLuc DNA, respectively. Subsequently, the cDNA labels were expressed in vitro simultaneously and independently in the same transcription/translation reaction mixture. The activities of generated firefly and Renilla luciferases were co-determined in the same sample based on the differential requirements of their characteristic bioluminescent reactions for magnesium ions.

Two-site expression immunoassay using a firefly luciferase-coding DNA label.

BACKGROUND: We report the first two-site, "sandwich type" expression immunoassay using as a label an expressible DNA fragment encoding firefly luciferase. METHODS: The DNA label consisted of a T7 RNA polymerase promoter, a firefly luciferase-coding sequence, and a poly(dA/dT) tail. The 3' end of the DNA label was biotinylated and complexed with streptavidin. A sandwich immunoassay for prostate-specific antigen (PSA) was developed in which the antigen was first bound to an immobilized monoclonal antibody and then reacted with a biotinylated polyclonal antibody. The streptavidin-luciferase-coding DNA complex was then bound to the immunocomplex. The DNA label was subsequently expressed in vitro by coupled transcription and translation. The generated luciferase was measured by its characteristic bioluminescent reaction. RESULTS: The bioluminescence was linearly related to the concentration of PSA in the sample. As low as 30 ng/L PSA was measured (12.5-microL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 microg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 microg/L; r = 0.971; n = 44). CONCLUSIONS: The use of the newly developed DNA label in a two-site immunoassay was demonstrated for the first time. The assay was applied successfully to the measurement of serum PSA.

Signal amplification system for DNA hybridization assays based on in vitro expression of a DNA label encoding apoaequorin.

The development of hybridization assays based on an apoaequorin-encoding DNA label is reported. The constructed label contains the T7 RNA polymerase promoter, the apoaequorin coding sequence and a downstream (dA/dT)(30). In the captured target configuration, biotinylated target DNA (233 bp) was captured on streptavidin-coated microtiter wells and hybridized to a poly(dT)-tailed detection probe. In the sandwich-type assay, the target DNA was hybridized simultaneously with an immobilized capture probe (through biotin/streptavidin) and a poly(dT)-tailed detection probe. In both configurations, the hybrids were reacted with poly(dA)-tailed apoaequorin DNA. The DNA label was subjected to in vitro transcription/translation to produce apoaequorin, which was converted to active aequorin in the reaction mixture. Generated aequorin was determined by its characteristic Ca(2+)-triggered bioluminescence. Each DNA label was estimated to produce 156 aequorin molecules. As low as 0.25 and 0.5 amol of target DNA were detected with the sandwich-type and captured target hybridization assays, respectively, with a linear range spanning four orders of magnitude. In comparison, captured target hybridization assays using photoprotein aequorin or firefly luciferase-encoding DNA labels were able to detect 25 and 20.5 amol of target DNA, respectively. The dramatic improvement in sensitivity observed with the proposed systems is attributed to amplification introduced by in vitro expression of apoaequorin DNA into multiple active aequorin molecules.

Quantitative polymerase chain reaction based on a dual-analyte chemiluminescence hybridization assay for target DNA and internal standard.

We have developed a dual-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR). The method allows simultaneous determination of both amplified target DNA and internal standard (IS) in the same reaction vessel. The target DNA from the sample (233 bp) was coamplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DNA, differing only by a 24-bp sequence, centrally located. Biotinylated PCR products from target DNA and IS were captured on a single microtiter well coated with streptavidin. The amplified target DNA was hybridized with a digoxigenin-labeled specific probe, and the hybrids were determined by using antidigoxigenin antibody labeled with aequorin. The amplified DNA IS was hybridized, in the same well, with a fluorescein-labeled probe, and the hybrids were determined by using an antifluorescein antibody conjugated to alkaline phosphatase. Aequorin was measured by adding a Ca(2+)-containing light-triggering solution. Alkaline phosphatase was measured by using a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear range extended from 430 to 315,000 target DNA molecules. Average CVs ranged from 7 to 17%. The proposed system is expected to facilitate the automation and routine use of quantitative PCR.

Expression immunoassay based on antibodies labeled with a deoxyribonucleic acid fragment encoding the alpha-peptide of beta-galactosidase.

An immunoassay is reported which uses, as a label, an expressible DNA fragment encoding the alpha-peptide of beta-galactosidase. This inactive peptide consists of 97 amino acid residues containing an amino-terminal portion of the enzyme. Antigen (an anti-thyrotropin immunoglobulin) immobilized in microtiter wells is allowed to react with specific antibodies which are then linked to the DNA label via biotin-streptavidin interaction. After completion of the immunoreaction, the solid phase bound DNA is subjected to a cell-free, one-step transcription/translation reaction to produce the alpha-peptide. The alpha-peptide is allowed to react (complementation reaction) with the remaining part of the beta-galactosidase (M15 protein, also inactive) to give fully active enzyme molecules. 4-Methylumbelliferyl galactoside is used as a substrate. The fluorescence is linearly related to the amount of antigen in the well. As little as 3 fmol of antigen can be detected. The RSDs (within-run) obtained for 8 and 20 fmol of antigen were 10.7 and 9.3%, respectively (n = 4). The present work illustrates the utility of expressing a non-detectable peptide capable of triggering a signal generating system.

Sandwich-type deoxyribonucleic acid hybridization assays based on enzyme amplified time-resolved fluorometry.

We report microtiter well-based sandwich-type DNA hybridization assays using enzyme amplified time-resolved fluorometry of Tb3+ chelates. The target DNA was hybridized with two adjacent and non-overlapping oligonucleotide probes, one oligonucleotide serving as the capture probe and the other as the detection probe. Two ligand-specific binding protein pairs were used alternately for capture of the hybrids to the solid phase and detection; the biotin-streptavidin and the digoxigenin-anti-digoxigenin interaction. In both cases, alkaline phosphatase was used as a reporter molecule and diflunisal phosphate as a substrate. The catalytic hydrolysis of the substrate produces diflunisal which forms ternary fluorescent complex with Tb(3+)-EDTA. Furthermore, we studied the effect of the probe labeling method and the position of the label on the sensitivity of the assays. The data suggest that capture of the hybrids through biotin-streptavidin and detection via digoxigenin-anti-digoxigenin offer 2-3 times higher sensitivity than the reverse configuration. The highest sensitivity was achieved with enzymatic labeling of capture and detection probes at the 3' end. A signal-to-background ratio of 4 was achieved for 0.2 fmol of target DNA. The RSD were better than 4%.

Novel hybridization assay configurations based on in vitro expression of DNA reporter molecules.

OBJECTIVES: To develop and study novel microtiter well based DNA hybridization assays in which the DNA serves as a reporter molecule. METHODS: Two hybridization assay configurations are proposed. In configuration A the target DNA is end-labeled with biotin and captured to streptavidin-coated wells. The one strand is removed by NaOH treatment and the other is hybridized with a dATP-tailed oligonucleotide probe. Configuration B involves simultaneous hybridization of heat-denatured target DNA with a biotinylated capture probe (immobilized on streptavidin-coated wells) and a dATP-tailed detection probe. In both configurations the hybrids are reacted with dTTP-tailed luciferase-coding DNA fragment followed by in vitro expression of the DNA on the solid phase. This is accomplished either by a coupled transcription/translation or by sequential transcription and translation reactions optimized separately. RESULTS: The signal-to-background ratios for configuration A at 0.93 fmoles target DNA were 2.6 and 16.7 with the coupled and the separated transcription/translation protocols, respectively. As low as 0.1 fmoles target DNA can be detected with the separate transcription/translation protocol with a signal-to-background ratio of 2.7. The signal-to-background ratios obtained for 0.1 fmoles target DNA with configuration B using the coupled and the separate expression protocols were 2.2 and 4.6, respectively. The average CV was 10%. CONCLUSION: The expression yield is significantly improved with the separate transcription/translation protocol. Both assay configurations offer high sensitivity and are easily automatable.

Quantification of prostate-specific antigen mRNA by coamplification with a recombinant RNA internal standard and microtiter well-based hybridization.

We report a quantitative analytical methodology for prostate-specific antigen (PSA) mRNA, which is based on the coamplification of the target with a recombinant RNA internal standard (IS) using reverse transcriptase-polymerase chain reaction. PSA mRNA and the RNA IS contain the same primer recognition sites and generate amplification products that have identical sizes but differ in a 24-bp sequence located in the center of the molecule. Amplified sequences are labeled with biotin using a biotinylated upstream primer. The products are captured on streptavidin-coated microtiter wells and hybridized to specific probes labeled with the hapten digoxigenin. The hybrids are determined using alkaline phosphatase-labeled anti-digoxigenin antibody and time-resolved fluorometry. The ratio of the fluorescence values obtained for the PSA mRNA and the RNA IS is a linear function of the amount of PSA mRNA present in the sample. Samples containing total RNA from PSA-expressing cells (LNCaP cells) in addition to 1 microg of RNA from healthy cells give fluorescence ratios related linearly to the number of cells in the range of 4 to 3000 cells.

Two-round enzymatic amplification combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity in DNA hybridization assays.

Microtiter well-based DNA hybridization assays are developed in which two rounds of enzymatic amplification are combined with time-resolved fluorometry of Tb3+ chelates for enhanced sensitivity. The target DNA is immobilized on the wells (through digoxigenin/anti-digoxigenin interaction) and then hybridized with a biotinylated oligonucleotide probe. The hybrids are reacted with a streptavidin-horseradish peroxidase conjugate. Peroxidase catalyzes the oxidation of biotinylated tyramine by hydrogen peroxide, resulting in the attachment of multiple biotin moieties to the solid phase. Alkaline phosphatase-labeled streptavidin is then allowed to bind to the immobilized biotins. The activity of alkaline phosphatase is measured by using the phosphate ester of 5'-fluorosalicylate as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb3+, which is measured by time-resolved fluorometry. We observed a 30-fold improvement of the signal and a 10-times enhancement of the signal-to-background ratio compared to the assay that uses a single round of enzymatic amplification (only alkaline phosphatase). The CV was in the range of 11.2-14.4%.

Quantitative reverse transcriptase-polymerase chain reaction for prostate-specific antigen mRNA.

OBJECTIVE: To develop a quantitative reverse transcriptase-polymerase chain reaction assay for monitoring the prostate-specific antigen (PSA) mRNA. METHODS: PSA mRNA is amplified, in parallel, with the mRNA of beta-actin, a housekeeping gene. The ratio of the amplification products obtained reflects the relative amount of PSA mRNA with respect to actin mRNA. During PCR, digoxigenin-dUTP is incorporated in the amplified sequences. The PCR products are analyzed separately by time-resolved immunofluorometric hybridization assays, using specific probes immobilized in microtiter wells. The hybrids are reacted with alkaline phosphatase-labeled anti-digoxigenin antibody. The phosphate ester of fluorosalicylate is used as a substrate. The fluorosalicylate produced forms a fluorescent complex with Tb(3+)-EDTA which is measured by time-resolved fluorometry. RESULTS: The hybridization assays for both PSA and actin amplification products show linearity in the range of 1.4-110 pmol/L. The exponential phase of PCR amplification extends up to 200,000 and 100,000 PSA and actin cDNA molecules, respectively. We prepared mixtures containing various numbers of LNCaP cells in one million cells that do not express PSA and used them as samples in the proposed assay. The ratio of the fluorescence values obtained after analysis of PSA and actin amplification products is linearly related to the number of LNCaP cells in the range of 20 to 3000 cells. Reproducibility studies demonstrate %CVs for the fluorescence ratios of 14.7, 11.8, and 12.2 when samples containing 150, 300 and 1600 LNCaP cells were analyzed (n = 4). CONCLUSIONS: A quantitative analytical methodology is provided for monitoring PSA mRNA. The assay is expected to be beneficial in the study of prostate cancer spread.

Quantitative RT-PCR combined with time-resolved fluorometry for determination of BCR-ABL mRNA.

A microtiter well-based quantitative reverse transcriptase-PCR assay for determination of BCR-ABL mRNA, which relies on coamplification of the target with an RNA internal standard (IS), was developed. The hapten digoxigenin (Dig) is incorporated during PCR. Target RNA and IS contain identical primer recognition sites and generate same-sized amplification products distinguishable by hybridization with probes specific to the molecules' central part. The hybrids are determined with an anti-Dig-alkaline phosphatase conjugate with fluorosalicylphosphate as substrate. Fluorescent complexes of fluorosalicylate-Tb(III)-EDTA are measured by time-resolved fluorometry. The ratio of fluorescence values for target and IS is linearly related to initial target RNA in the range of 1000 to 200000 molecules. Samples containing K562 total RNA amidst 1 microgram of RNA from normal cells give fluorescence ratios that are linearly related to 30-10000 K562 cells. CVs for 30, 200, and 900 K562 cells are approximately 11%.

Bioluminescence hybridization assays using recombinant aequorin. Application to the detection of prostate-specific antigen mRNA.

We developed microtiter well-based bioluminescence hybridization assays using the photoprotein aequorin as a reporter molecule. The target DNA was hybridized simultaneously with a capture probe and a detection probe. The capture probe was immobilized on the wells through digoxigenin/anti-digoxigenin interaction. The detection probe was biotinylated. The hybrids were determined by using aequorin covalently attached to streptavidin or complexes of biotinylated aequorin with streptavidin. The luminescence was then measured in the presence of excess Ca2+. The optimized protocols showed linearity in the range from 5 amol to 10 fmol of target DNA. In combination with reverse transcriptase polymerase chain reaction, the proposed assay was applied to the detection of the mRNA for prostate-specific antigen (PSA). PSA mRNA from a single cell, in the presence of one million cells that do not express PSA, was detected with a signal-to-background ratio of 2.5. Typical CVs obtained were 6%.

Hybridization assays using an expressible DNA fragment encoding firefly luciferase as a label.

We report the use of a new label, an expressible enzyme-coding DNA fragment, for nucleic acid hybridization assays. The DNA label contains a firefly luciferase coding sequence downstream from a T7 RNA polymerase promoter. The target DNA (200 bp) is denatured and hybridized simultaneously with two oligonucleotide probes. One of the probes is immobilized in microtiter wells, via the digoxigenin/anti-digoxigenin interaction, and the other probe is biotinylated. After completion of the hybridization, the hybrids are reacted with a streptavidin-luciferase DNA complex. Subsequently, the solid-phase bound DNA is expressed by coupled transcription/ translation. The synthesized luciferase catalyzes the luminescent reaction of luciferin with O2 and ATP. The luminescence is linearly related to the amount of target DNA in the range of 5-5000 amol. The CVs obtained for 20 and 100 amol of target are 6.5% and 10.8%, respectively (n = 4).

Fluorometric and time-resolved immunofluorometric assays for protein-tyrosine phosphatase activity.

OBJECTIVE: To develop sensitive nonisotopic assays for protein-tyrosine phosphatase (PTP) activity. METHODS: The fluorometric assay is based on the fact that phosphotyrosine but not tyrosine forms highly fluorescent complexes with Tb3+. Thus, PTP activity can be followed by measuring the decrease of fluorescence due to hydrolysis of phosphotyrosine. The time-resolved immunofluorometric assay employs tyrosine-phosphorylated substrates, immobilized on microtitre wells. After incubation with PTP, the remaining phosphotyrosine residues are reacted with an antiphosphotyrosine antibody. The immunocomplexes formed are detected with an alkaline phosphatase (ALP)-labeled second antibody. The phosphate ester of 5' fluorosalicylate (FSAP) is used as substrate. The fluorosalicylate produced forms highly fluorescent complexes with Tb3+ - EDTA in alkaline solution. The fluorescence is measured with a time-resolved fluorometer. RESULTS: The truncated form of the T-cell protein tyrosine phosphatase (TCdeltaC11 PTP) was determined in the range 1100-36,500 U/L by the fluorometric assay and 36-7100 U/L by the time-resolved immunofluorometric assay. CONCLUSIONS: The two nonisotopic assays should prove beneficial for the determination and study of various PTP.

Quantitative polymerase chain reaction using a recombinant DNA internal standard and time-resolved fluorometry.

Using a 308 bp DNA fragment (target DNA) as a template, we have synthesized an internal standard (IS) that is of the same size and uses the same primers as the target but differs by a 26 bp centrally located sequence. We then designed quantitative polymerase chain reaction (PCR) assays in which the target DNA is coamplified with a constant amount of IS (20,000 molecules). The presence of IS compensates for the reaction-to reaction variability of the amplification efficiency. The PCR products are assayed by two distinct hybridization protocols. The first approach (QPCR-1) requires that specific probes be immobilized onto microtiter wells, followed by hybridization with digoxigenin-labeled PCR product. In the second protocol (QPCR-2), PCR product is captured onto the wells and hybridized with digoxigenin-tailed specific probes. In both assays, the hybrids are detected using an antidigoxigenin-alkaline phosphatase conjugate and 5'-fluorosalicylphosphate as substrate. The hydrolysis product forms a highly fluorescent complex with Tb(3+)-EDTA, as measured by time-resolved fluorometry. The ratio of the fluorescence values obtained for the amplified target DNA and IS is linearly related to the number of target DNA molecules present in the sample prior to amplification. The linear ranges are 1000-200,000 molecules for QPCR-1 and 2000-200,000 molecules for QPCR-2. The CVs ranged from 3.4 to 9.7%.

Expression immunoassay. Antigen quantitation using antibodies labeled with enzyme-coding DNA fragments.

A novel immunoassay is reported which uses an enzyme-coding DNA fragment as label (expression immunoassay). The DNA label is determined with high sensitivity by measuring the enzymatic activity produced after expression. A DNA fragment encoding the firefly luciferase is biotinylated and complexed with streptavidin. Biotinylated, specific antibodies are used for quantitation of antigen immobilized on microtiter wells. After completion of the immunoreaction, streptavidin-DNA is bound to the immunocomplex. Subsequent expression of the solid phase-bound DNA, by an one-step (coupled) cell-free transcription/translation, produces luciferase. The enzyme catalyzes the luminescent reaction of luciferin with O2 and ATP. As few as 3000 molecules of DNA label can be detected. Also, 50,000 antigen molecules can be detected, and the luminescence is a linear function of the number of antigen molecules in a range extending over 3 orders of magnitude. The high sensitivity achieved is a result of the combined amplification due to transcription/translation and the substrate turnover.

Detection of prostate-specific antigen mRNA by reverse transcription polymerase chain reaction and time-resolved fluorometry.

We have developed a time-resolved fluorometric hybridization assay for detecting prostate-specific antigen (PSA) mRNA amplified by reverse transcription polymerase chain reaction. During PCR, digoxigenin-11-dUTP is incorporated into the amplified product. An oligonucleotide internal to the primers is used as a specific probe, being biotinylated and captured on streptavidin-coated microtiter wells. Denatured PCR product hybridizes with the probe, and the hybrids are detected with an alkaline phosphatase-labeled antidigoxigenin antibody. We used the phosphate ester of fluorosalicylic acid as the substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which we can measure by time-resolved fluorometry. A signal-to-background ratio of 10 was obtained when 160 PSA cDNA molecules were present in the preamplification sample. Also, mRNA corresponding to one LNCaP cell in the presence of 10(6) PSA-negative cells can be detected (signal-to-background ratio of 3.1). Samples containing 100, 1000, and 50,000 LNCaP cells gave CVs of 12.4%, 4.9%, and 6.8%, respectively (n = 10).