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(Clinical Chemistry. 1999;45:1954-1959.)
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Articles

Two-Site Expression Immunoassay Using a Firefly Luciferase-coding DNA Label

Norman H.L. Chiu and Theodore K. Christopoulosa

Department of Chemistry and Biochemistry, University of Windsor, 401 Sunset Ave., Windsor, Ontario, N9B 3P4 Canada.
a Author for correspondence. Fax 519-973-7098; e-mail tkc{at}uwindsor.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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-µL sample) with a signal-to-background ratio of 2.3, and the linear range extended to 3 µg/L. The results obtained from the proposed assay agreed well to those determined by IMx immunoassay (y = 0.98x + 0.74 µg/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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Firefly luciferase is a 62-kDa monomeric protein found in the light-emitting organ, known as lantern, of Photinus pyralis (1). Firefly luciferase, in the presence of ATP and Mg2+, catalyzes the oxidation of luciferin by molecular oxygen, yielding the excited state of oxyluciferin (2)(3)(4). When the excited molecule relaxes back to its ground state, it emits light at 562 nm (5)(6) with a quantum yield of 0.88, the highest value reported for a bioluminescent reaction. Because of its high sensitivity, the reaction has been used extensively for the determination of ATP (7). The cDNA of luciferase has also been widely used as a reporter gene in a plethora of different biological studies (8).

Luciferase is inactivated by the chemical reactions involved in direct labeling of antigens or antibodies (9). Consequently, its use as a label in immunoassay is limited. The luciferase-catalyzed reaction, however, has been exploited as an indicator reaction in immunoassay. In one assay configuration (10), acetate kinase is used as a label, and the generated ATP is monitored by the bioluminescent reaction of luciferase. The bioluminescence observed is a measure of the kinase concentration. A competitive immunoassay has also been reported (11) that uses ATP-labeled antigens (as tracers) prepared by reacting an amino group-containing antigen with adenosine-5'-trimetaphosphate. The immunoreaction is followed by the release of ATP in a mild acidic solution and its measurement by the luciferase reaction. Another immunoassay configuration uses antibodies labeled with alkaline phosphatase or ß-galactosidase. Luciferin phosphate or luciferin galactoside are used as substrates, respectively (12). The enzymatically released luciferin can be quantified by the luciferase reaction. The recent progress in DNA technology has allowed the preparation of recombinant fusion proteins in which luciferase is "genetically" conjugated to protein A and used as a detection reagent in immunoassay (13). In another attempt to avoid inactivation, luciferase was biotinylated in vivo and then complexed to streptavidin (SA)1 (14).

Recently, we developed a new analytical system named expression immunoassay (ExIA) (15). In ExIA, an expressible DNA fragment that encodes for an enzyme (firefly luciferase) is used as a label instead of the enzyme itself. The immunoreaction is performed in microtiter wells, and the DNA label bound to the immunocomplex is subjected to a cell-free (in vitro) coupled transcription/translation reaction that produces several active enzyme molecules in solution. We have also extended this principle (16) by using a DNA label that encodes for a relatively small polypeptide with no inherent enzymatic activity ({alpha}-peptide of ß-galactosidase). Each peptide, however, is able to trigger an enzymatic reaction by interacting with a larger, inactive protein molecule (M15 protein isolated from lacZ{Delta}M15 bacterial strains) in a "complementation reaction" to form a fully active enzyme molecule in solution.

To date, the above systems have been tested in a simple assay in which various amounts of a monoclonal antibody were immobilized in microtiter wells and detected by using a goat anti-mouse antibody (15)(16). In the present study, we report the development of the first two-site, "sandwich-type" ExIA. The principle of the proposed assay is illustrated in Fig. 1 . The proposed assay uses an expressible firefly luciferase-coding DNA fragment (FLucDNA) as a reporter molecule. The biotin/SA interaction (17) is used as a linkage between the antibody and FLucDNA. In vitro expression of the FLucDNA that is bound to the immunocomplex produces several luciferase molecules in solution. The activity of synthesized luciferase is measured by its bioluminescent reaction with luciferin. The proposed system was evaluated by analyzing serum prostate-specific antigen (PSA) as a model assay. PSA is a 33-kDa glycoprotein expressed predominantly by the epithelial cells of the prostate gland (18). Recent studies have also shown that PSA is present at low concentrations in breast tumors (19)(20) and other tissues (21). Over the past decade, the determination of serum PSA has been widely used for monitoring patients diagnosed with prostate cancer (22).



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  		Figure 1. Principle of the two-site ExIA.

After the completion of the immunoreaction, the luciferase-coding DNA fragment is expressed into active luciferase molecules by a one-step in vitro transcription/translation reaction. The activity of synthesized luciferase is measured by its bioluminescent reaction with luciferin. PSA was chosen as a model analyte. B, biotin; SA, streptavidin; T7, T7 RNA polymerase promoter. *, excited state.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
solutions
The wash solution contained 50 mmol/L Tris, pH 7.4, 0.15 mol/L NaCl, and 1 mL/L Tween 20. The blocking solution consisted of 10 g/L blocking reagent (cat. no. 1096176; Boehringer Mannheim), 0.1 mol/L maleate, and 0.15 mol/L NaCl, pH 7.5. The assay buffer contained 50 mmol/L Tris, pH 7.8, 60 g/L bovine serum albumin, 0.5 mol/L KCl, 0.5 g/L sodium azide, and 5 g/L Triton X-100. The Tris-EDTA buffer was 10 mmol/L Tris, pH 7.6, and 1 mmol/L EDTA. The substrate solution contained 20 mmol/L tricine, 1.1 mmol/L magnesium carbonate pentahydrate, 2.7 mmol/L magnesium sulfate, 0.1 mmol/L EDTA, 33 mmol/L dithiothreitol, 270 µmol/L CoA, 530 µmol/L ATP, and 470 µmol/L D-luciferin (Promega), pH 7.8.

A rabbit reticulocyte-based transcription/translation mixture (rabbit reticulocyte TNT system; Promega) was prepared according to the manufacturer's instructions. The complete mixture consisted of the reticulocyte extract (containing rNTPs, ribosomes, tRNAs, and other translation factors), T7 RNA polymerase, and amino acids in the appropriate buffer.

The two anti-PSA antibodies used throughout this study were as described in Ref. (23) and were obtained from Medix Biotech (MBP0405, a monoclonal anti-PSA antibody; PBP0101, a polyclonal anti-PSA antibody).

Solutions of various PSA concentrations were prepared by diluting PSA (cat. no. P0724; Scripps Laboratories) in 50 mmol/L Tris, pH 7.8, 60 g/L bovine serum albumin, 1 g/L NaN3 and storing the dilutions at 4 °C.

biotinylation of anti-psa detection antibody
The polyclonal anti-PSA (0.25 mg) was dialyzed overnight against 3.5 L of 0.1 mol/L sodium bicarbonate at 4 °C. The purified antibody was diluted twofold with 0.5 mol/L carbonate buffer, pH 9.1. The resulting protein concentration was 1 g/L. The N-hydroxysuccinimide ester of biotin (0.5 mg; NHS-LC-biotin; Pierce) was dissolved in 25 µL of dimethyl sulfoxide and added to the antibody solution. The mixture was incubated for 2 h at room temperature with mixing at regular time intervals. The biotinylated antibody was stored at 4 °C and used without further purification.

preparation of SA-FLucDNA COMPLEX
The SA-FLucDNA complex was prepared as described in Ref. (15). In brief, a plasmid containing the firefly luciferase gene downstream from a T7 RNA polymerase promoter was digested with Alw44I into three fragments possessing 3' recessive ends. One of these fragments contained the T7 promoter, the luciferase gene, and a poly(dA/dT) tail. The fragments were subsequently labeled with biotin, using the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I to fill in the recessive ends with dGTP, dCTP, dTTP, and biotin-14-dATP. This process biotinylated both termini of each fragment. A 0.49-kbp fragment just upstream from the T7 promoter was then removed by digestion with PvuII, thus leaving a 2.1-kbp fragment (FLucDNA) labeled with biotin at only one terminus. The FLucDNA was separated from the other fragments by agarose gel electrophoresis and purified by elution from the gel. A SA-FLucDNA complex was prepared by mixing biotinylated FLucDNA with an excess of SA. The concentration of SA was confirmed by measuring its absorbance at 282 nm (molar absorptivity, 2.4 x 105 mol · L-1 · cm-1). The SA-FLucDNA was purified by size exclusion HPLC and concentrated by ultrafiltration. The concentration of the SA-FLucDNA was determined by scanning densitometry.

coating of microtiter wells
Transparent U-bottom polystyrene microtiter wells (Maxisorp; Nunc, obtained from Life Technologies) were coated by incubation overnight at room temperature with 25 µL per well of 5 mg/L monoclonal anti-PSA antibody diluted in 50 mmol/L Tris pH 7.8, 0.5 g/L sodium azide. Before use, the wells were washed twice with wash solution and blocked for 1 h with 200 µL of blocking solution per well.

two-site immunoassay for psa
A 12.5-µL aliquot of the assay buffer was pipetted into each well, followed by the addition of 12.5 µL of PSA sample. The antigen was allowed to react with the immobilized antibody for 3 h at room temperature with continuous mechanical shaking. At the end of this period, the wells were washed six times with wash solution followed by the addition of 25 µL per well of 0.23 mg/L biotinylated anti-PSA detection antibody diluted in blocking solution. After a 1-h incubation, the excess detection antibody was removed by washing the wells six times as above. A 25-µL aliquot of 0.86 µg/L SA-FLucDNA complex (diluted in 10 g/L blocking reagent, 0.1 mol/L maleate, pH 7.5, 0.15 mol/L NaCl, and 2 mmol/L EDTA) was then added into each well and allowed to bind to the immunocomplex for 20 min. The unbound SA-FLucDNA complex was removed by washing the wells five times with wash solution and once with Tris-EDTA buffer. The luciferase-coding DNA that was bound to the solid phase was expressed in vitro by an one-step coupled transcription/translation reaction. A 25-µL aliquot of the transcription/translation mixture was added into each well and incubated at 30 °C for 90 min in an incubator shaker (Model G24; New Brunswick Scientific). The activity of synthesized firefly luciferase was then measured by mixing 10 µL of the transcription/translation reaction mixture with 50 µL of substrate solution at room temperature in a microcentrifuge tube. The tube was placed immediately in a glass scintillation vial, and the bioluminescence was measured for 1 min in the liquid scintillation counter (Model LS-6500; Beckman Instruments). The coincidence photon detection of the counter was disabled to facilitate the counting of single photon events (single photon monitoring mode).

analysis of clinical specimens
Serum samples were diluted 30-fold in blocking solution before the analysis. Samples with high turbidity were centrifuged at 12 000g for 30 min at room temperature before the analysis. The PSA concentration of the serum samples was determined by using a calibration curve constructed from PSA calibrators whose concentrations were confirmed by the Abbott IMx PSA assay (Abbott Laboratories).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We first compared the yields of coupled transcription/translation systems (both from Promega) based on either a wheat germ extract [as used previously (15)] or a rabbit reticulocyte lysate by measuring the activity of firefly luciferase obtained from the expression of the luciferase-coding DNA fragment (0.83 amol). The latter method produced ~30% more luciferase than the wheat germ extract (not shown). This could be explained by the relative insufficiency of translational initiation factors in the wheat germ extract (24), which produces a lower rate of protein synthesis. In addition, the lower yield might be attributable to premature termination of translation, which yields inactive protein molecules (25).

To optimize the concentration of biotinylated anti-PSA antibody, we prepared dilutions of the antibody in the range of 0.2–3.7 mg/L and analyzed a sample with 0.5 µg/L PSA. In Fig. 2 , the luminescence and the signal-to-background (S/B) ratio are plotted against the biotinylated antibody concentration. The background is defined as the luminescence obtained when no antigen is present in the well and is a measure of the nonspecific binding of the biotinylated anti-PSA antibody and the SA-FLucDNA complex. The luminescence increased with the antibody concentration, but the maximum S/B ratio was observed at 0.23 mg/L, followed by a continuous decrease as the antibody concentration increased (because of increased nonspecific binding).



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  		Figure 2. Optimization of the concentration of biotinylated anti-PSA detection antibody.

The luminescence (——–) and the S/B ratio (- - - - - - -) obtained for 0.5 µg/L PSA are plotted against the concentration of biotinylated anti-PSA antibody. cpm, counts/min. 10^-7, 10-7.

In the range of 0.2–3.4 mg/L, the luminescence increased with the SA-FLucDNA concentration (Fig. 3 ), but the S/B ratio reached a plateau at 0.43 mg/L. At higher concentrations, the background concomitantly increased and the S/B ratio remained practically constant.



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  		Figure 3. Optimization of the concentration of SA-FLucDNA complex.

A 0.5 µg/L PSA sample was analyzed as described in Materials and Methods by varying the concentration of SA-FLucDNA complex. The concentrations refer to the DNA label. The solid and dashed lines represent the luminescence and the S/B, respectively. cpm, counts/min. 10^-7, 10-7.

The linear range of the assay was 30–3000 ng/L (Fig. 4 ). The S/B ratio for 30 ng/L PSA was 2.3 with a sample volume of 12.5 µL.



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  		Figure 4. Calibration graph for the two-site ExIA of PSA.

The assays were carried out as described in Materials and Methods, and the luminescence (corrected for the background) was plotted against the PSA concentration. cpm, counts/min.

To further evaluate the linearity of the method, we prepared dilutions (using female serum as a diluent) of two serum samples with added PSA. Each sample was diluted another sixfold in the blocking solution before analysis. The relationship between the concentration (y, in ng/L) and the dilution factor (x) was linear. The linear regression equations were: log(y) = 3.739 - 0.964 log(x); r = -0.999; and log(y) = 3.537 - 1.116 log(x); r = -1 (n = 5).

In a method-comparison study of 44 serum samples with PSA concentrations ranging from 1.1 to 53 µg/L with the Abbott IMx method (Fig. 5 ), the linear regression equation was: (ExIA) = 0.98(IMx) + 0.74 µg/L; r = 0.971; Sy|x = 0.562.



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  		Figure 5. Correlation of PSA concentrations obtained by ExIA and the IMx method (n = 44).

The within- and between-day imprecision (CVs) for three different serum samples was 8–10% (Table 1 ).


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  		Table 1. Reproducibility study for the two-site ExIA for PSA.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Enzymes are the most widely used reporter molecules in immunoassays because they introduce signal amplification through the turnover of substrates to detectable products. To date, efforts toward improving the sensitivity of enzyme immunoassays have been focused on the synthesis of better substrates. Thus, substantial improvements have come from the replacement of chromogenic substrates with fluorogenic or chemiluminogenic ones (26). Enzyme amplification cascades have also been proposed as a means to achieve higher sensitivity in measuring enzyme activity. One such system uses alkaline phosphatase (ALP) as a reporter that dephosphorylates NADP+ to produce NAD+, which is used as a cofactor by alcohol dehydrogenase. Alcohol dehydrogenase converts ethanol to acetaldehyde with a concomitant reduction of NAD+ to NADH. Subsequently, diaphorase catalyzes the reduction of resazurin to the fluorescent compound resorufin. This reaction regenerates NAD+ (27). Another system also uses ALP as a label and flavin-adenine dinucleotide phosphate as substrate. The released cofactor FAD binds to apo D-amino acid oxidase, and the resulting active holoenzyme catalyzes the oxidation of D-proline to produce H2O2, which is determined by the horseradish peroxidase-catalyzed conversion of 3,5-dichloro-2-hydroxybenzenesulfonic acid and 4-aminoantipyrine to a colored product (28).

ExIA is complementary to the above efforts because instead of providing an alternative method for measuring enzyme activity, it entails an increase in the number of enzyme molecules, thus introducing additional amplification. Consequently, ExIA might be used in combination with the above systems to further enhance the sensitivity. For example, the ALP-coding DNA might be used as a reporter to provide several ALP molecules that then could be detected by a chemiluminogenic substrate or a cascade amplification system. Furthermore, by using an enzyme-coding DNA fragment as a label, the problem of enzyme inactivation after conjugation to antibodies is eliminated, and the enzyme remains free in solution. Moreover, in direct comparison with fluorescent and chemiluminescent immunoassays and hybridization assays (15)(16)(29), substantial improvements on the sensitivity were achieved by using the enzyme-coding DNA labels. Disadvantages of the ExIA that might be considered are the use of a transcription/translation cocktail, which requires storage at -70 °C, and the need for a 90-min incubation step for expression. The transcription/translation cocktail is added as one reagent in a single step and therefore does not complicate the protocol.

In conclusion, we have developed a two-site ExIA and demonstrated its clinical utility. Because of its high sensitivity, we anticipate that the developed assay will be useful for determining other clinical analytes that exist at low concentrations in biological fluids.


   Acknowledgments
 
This work was supported by grants from the National Science and Engineering Research Council (NSERC) of Canada (to T.K.C.). N.H.L.C. acknowledges a summer research award from the University of Windsor. We wish to thank Dr. D.S. Keys at Medical Laboratories of Windsor for providing the serum samples and performing the PSA analysis with the IMx system. We also thank Eleftheria Laios and Bakhos A. Tannous for assistance in performing the reproducibility and correlation studies.


   Footnotes
 
1 Nonstandard abbreviations: SA, streptavidin; ExIA, expression immunoassay; FLucDNA, firefly luciferase-coding DNA; PSA, prostate-specific antigen; S/B, signal-to-noise; and ALP, alkaline phosphatase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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