Heterogeneous bioluminescence binding assay for an octapeptide using recombinant aequorin

Analytica Chimica Acta 369 (1998) 181±188

Heterogeneous bioluminescence binding assay for an octapeptide using recombinant aequorin Sridhar Ramanathan, Jennifer C. Lewis, Mark S. Kindy1, Sylvia Daunert* Department of Chemistry University of Kentucky, Lexington, KY 40506, USA Received 10 January 1997; received in revised form 27 February 1998; accepted 2 March 1998

Abstract An assay was developed for an octapeptide by using recombinant aequorin as the label. Aequorin (AEQ) is a bioluminescent protein found in the jelly®sh (Aequorea victoria). A plasmid was designed by fusing the gene of AEQ to an oligonucleotide sequence that codes for the octapeptide of interest. The plasmid was transformed into Escherichia coli, and the octapeptide± aequorin fusion protein was expressed and puri®ed. The properties of the octapeptide±aequorin conjugate were studied, and it was observed that the fusion protein retained the bioluminescence characteristics of native aequorin. The octapeptide±AEQ fusion protein was employed in the development of a heterogeneous bioluminescence immunoassay for the octapeptide. The detection limit of the assay was 510ÿ10 M. This study also demonstrates that aequorin can be used as a sensitive label in bioluminescence immunoassays for small biomolecules even when the binding constant between the biomolecule and its corresponding antibody is relatively low. # 1998 Elsevier Science B.V. All rights reserved.

1. Introduction Immunoassays are a powerful tool in the determination of important biomolecules in clinical, environmental, pharmaceutical, and agricultural samples [1±3]. While the selectivity in these assays is mainly determined by the antigen±antibody interaction, the sensitivity is governed by several parameters. One such variable is the ease of detection of the label to be used in the assay. Aequorin (AEQ) is a photoprotein that can be detected at subattomole levels, and therefore, is well-suited in that regard. Aequorin is com*Corresponding author: Tel.: + 1 606 257 7060; fax: +1 606 3231069; e-mail: [email protected] 1 Present address: Department of Biochemistry, University of Kentucky, Lexington, KY 40536-0084. 0003-2670/98/$19.00 # 1998 Elsevier Science B.V. All rights reserved. PII S0003-2670(98)00243-8

posed of the apoprotein, apoaequorin, and an imidazopyrazine chromophore, coelenterazine [4]. Addition of Ca2‡ to aequorin, causes a conformational change in the protein, which triggers the oxidation of coelenterazine to coelenteramide, and results in the release of CO2 and emission of light (max469 nm) [5]. Recently, we have developed a homogeneous bioluminescence assay for biotin using aequorin as the label and avidin as the speci®c binder [6]. The detection limit of this assay for biotin was 110ÿ14 M, which to our knowledge, is the most sensitive homogeneous assay system for biotin reported so far. It is because of this excellent detection limit that we decided to study the feasibility of using aequorin as a bioluminescence label in the assay of small peptides. Another variable that affects the detection limits of immunoassays is the binding constant between the

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antigen and the antibody. In the homogeneous assay for biotin, the obtained detection limit was attributed not only to using aequorin as a label, but also to the high af®nity of avidin for biotin (1015 Mÿ1). It is therefore, another goal of the present study to determine the ability of aequorin to function as a label in systems with a weaker binding constant between the antigen and the antibody. Aequorin has been previously used in the development of assays for large biomolecules [7±17]. In some of these assays, aequorin has been conjugated to proteins (e.g., protein A [15], and an antibody fragment [16]) by a gene fusion approach. However, there is no report on the ability of aequorin to function as a label in immunoassays for small peptidic analytes. Heterogeneous assays for short peptides present a challenge in that fusion proteins prepared between the peptide and the aequorin label have to maintain the recognition of the peptide analyte by an antibody (i.e., the binding of the peptide to the antibody is not sterically hindered by the presence of the aequorin label). In addition, the interaction between the peptide and the antibody should not affect the ability of aequorin to bioluminesce. The assay described herein is based on the attachment of the peptide of interest to aequorin by recombinant DNA techniques involving gene fusion. In that respect, the oligonucleotide sequence that codes for the octapeptide is fused to the gene that codes for apoaequorin (apoAEQ). This fused DNA is then introduced into E. coli, and the octapeptide±apoaequorin fusion protein is expressed and isolated from the bacteria. The isolated protein is charged with coelenterazine to yield the octapeptide±aequorin conjugate. By using the gene fusion approach, the preparation of the peptide±aequorin conjugate is controlled by its corresponding DNA sequence. An advantage of preparing conjugates by using genetic engineering methods is that the same DNA sequence is used every time a new batch of conjugate is prepared, which gives high lot-to-lot reproducibility of the peptide±aequorin conjugate population. This is not usually the case with peptide±protein conjugates prepared by conventional methods involving reagents such as carbodiimide and glutaraldehyde, because of the dif®culty in controlling the nature and/or location of attachment of the peptide on the protein. In the work reported here, the octapeptide, Asp±Tyr±Lys±

Asp±Asp±Asp±Asp±Lys, was selected as a model peptide analyte in the development of a bioluminescence immunoassay based on aequorin. This is the ®rst report that uses a fusion protein conjugate of aequorin in the development of an immunoassay for a peptide. 2. Experimental 2.1. Reagents Tris(hydroxymethyl)amino methane (Tris), ethylenediaminetetraacetic acid (EDTA) sodium salt, dithiothreitol (DTT), bovine serum albumin (BSA), agar, glucose, sodium dodecyl sulfate (SDS), DEAE-cellulose anionic exchanger, and all other reagents were purchased from Sigma (St. Louis, MO). The pFLAGATS vector and the anti-FLAG M2 antibody immobilized on agarose beads (2.8 mg/ml suspension) were obtained from IBI-Kodak (New Haven, CT). The unlabelled octapeptide (Asp±Tyr±Lys±Asp±Asp± Asp±Asp±Lys) and the reverse octapeptide (rev-octapeptide, Lys±Asp±Asp±Asp±Asp±Lys±Tyr±Asp) were synthesized and puri®ed by the University of Kentucky Macromolecular Center. Coelenterazine and the aequorin mammalian expression vector, pMT2, were purchased from Molecular Probes (Eugene, OR). Luria Bertani (LB) broth and isopropylthio-b-galactopyranoside (IPTG) were from Gibco-BRL (Gaithersburg, MD). The bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL). All solutions were prepared using deionized (Milli-Q Water Puri®cation system, Millipore, Bedford, MA) distilled water. All chemicals were reagent grade or better and were used as received. 2.2. Apparatus Bioluminescence measurements were made on an Optocomp I luminometer from GEM Biomedical (Carrboro, NC) using a 100 ml ®xed-volume injector. All luminescence intensities reported are the average of a minimum of three replicates and have been corrected for the contribution of the blank. The purity of the octapeptide±apoaequorin expressed was veri®ed by SDS-PAGE on a PhastSystem electrophoresis setup (Pharmacia Biotech, Uppsala, Sweden).

S. Ramanathan et al. / Analytica Chimica Acta 369 (1998) 181±188

Fig. 1. Schematic representation of the plasmid pSD1 containing the oligonucleotide sequences of the OmpA signaling peptide, the octapeptide, and the gene of aequorin, AQ440, fused in frame.

2.3. Preparation and isolation of the octapeptide± apoaequorin conjugate The gene sequence of aequorin (AQ440) was isolated from the aequorin expression vector, pMT2, as a HindIII±EcoRI fragment [18]. It was then introduced into the multiple cloning site of the pFLAG-ATS vector, to yield the pSD1 vector, which contains the DNA sequence that codes for the peptide±apoAEQ fusion protein (Fig. 1). Bacteria (E. coli strain JM109) were transformed with the pSD1 vector and then cultured to express the fusion protein. Speci®cally, the bacteria were grown in 500 ml of LB broth containing ampicillin (50 mg/ml) and 0.4% (w/w) glucose. IPTG was added up to a ®nal concentration of 500 mM in the broth, and the protein expression was induced for 3 h. Since the fusion protein was expressed in the periplasm, it was isolated by osmotic shock, followed by centrifugation and collection of the supernatant [19]. All molecular biology procedures were performed using standard protocols [20]. 2.4. Purification of the octapeptide±aequorin conjugate The supernatant obtained after the osmotic shock treatment was mixed with the required amounts of

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Tris, EDTA sodium salt, and DTT, and the pH was adjusted using HCl to obtain a solution which is 30 mM Tris±HCl, 10 mM EDTA, 1 mM DTT, pH 7.6. Then, it was loaded onto a DEAE-cellulose anionic exchange column. The column was washed by using 10 mM Tris±HCl, 2.0 mM EDTA, and 2.0 mM DTT, pH 8.0; a salt gradient from 0 M to 0.4 M NaCl in this buffer was used to elute the proteins [21]. The fractions containing the protein were precipitated by saturating the solution with ammonium sulfate [22]. The ammonium sulfate and DTT were removed by three successive dialysis steps against 1 l of 10 mM Tris±HCl, 2.0 mM EDTA, pH 8.0. The purity of the octapeptide±apoAEQ conjugate was veri®ed by SDS-PAGE on 12.5% polyacrylamide PhastGels (Pharmacia Biotech), which were developed by silver staining (Development Method 210, Pharmacia Biotech). The protein concentration was estimated by using the BCA protein assay, with BSA as the standard. A molar excess of the cofactor coelenterazine was added to the octapeptide±apoAEQ fusion conjugate, along with 10 mM DTT and this mixture was stirred in ice for 5 h to yield the octapeptide±AEQ conjugate. It was then dialyzed against 1 l of 10 mM Tris±HCl, 0.15 M NaCl, 2.0 mM EDTA, 2.0 mM DTT, pH 8.0, for 8 h to remove the excess coelenterazine. 2.5. Bioluminescence emission study The ®rst experiments performed involved studying the bioluminescence activity of the fusion protein. A volume of 200 ml of 10 mM Tris±HCl, 0.15 M NaCl, 2.0 mM EDTA, pH 8.0 containing 0.10 mg/ml of BSA (dilution buffer) was added to the octapeptide±AEQ conjugate. A volume of 100 ml of a 100 mM CaCl2, 100 mM Tris±HCl, pH 7.5 (luminescence triggering buffer), was injected into the sample solution in order to trigger the emission of bioluminescence by the octapeptide±AEQ conjugate. The bioluminescence signal was collected at 0.1 s intervals over a 5 s time period. 2.6. Calibration plot for the octapeptide±aequorin In order to perform a calibration plot for the conjugate, a stock solution of the octapeptide±AEQ conjugate was serially diluted with dilution buffer. A

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calibration plot was prepared by injecting 100 ml of luminescence triggering buffer to a test tube containing 300 ml of different concentrations of the octapeptide±AEQ that was placed in the luminometer. A volume of 300 ml of the dilution buffer was used as a blank. The bioluminescence emitted was integrated over a 3 s time period. 2.7. Association study The M2 antibody immobilized on agarose beads was serially diluted from a stock suspension of 2.8 mg of immobilized antibody per milliliter of suspension, and incubated with the octapeptide±AEQ conjugate for 30 min (100 ml of each dilution of beads, 100 ml of buffer and 100 ml of 1.210ÿ11 M of octapeptide± AEQ). After the incubation, the unbound octapeptide± AEQ conjugate was washed away using the dilution buffer. The washing steps involved addition of 2 ml of dilution buffer to each of the test tubes. The beads were pelleted down by centrifuging the test tubes at 2000 rpm for 10 min at room temperature. A volume of 300 ml was retained in the test tubes and the rest of the buffer solution was decanted. This process was repeated three times. A volume of 300 ml of dilution buffer was used as a blank. The bioluminescence emitted was triggered and measured as above. 2.8. Time study The optimum time of incubation for the assay was determined by incubating 100 ml of a 14 mg/ml suspension of the immobilized antibody with 100 ml of 1.210ÿ11 M of octapeptide±AEQ conjugate in 100 ml of dilution buffer for various times. After the addition of the luminescence triggering buffer, the bioluminescence signal was collected as described above. 3. Dose-response curve and selectivity studies A dose-response curve was constructed by incubating 100 ml of a solution of different concentrations of the octapeptide with 100 ml of a 14 mg/ml suspension of the immobilized antibody and 100 ml of 3.410ÿ11 M of octapeptide±AEQ conjugate. The incubations were performed in a sequential manner.

Initially, the octapeptide was incubated with the beads for 30 min. Then, 100 ml of the octapeptide±AEQ was added, and the mixture was incubated for an additional 30 min at room temperature. Unbound octapeptide and octapeptide±AEQ were washed away as described above. The bioluminescence was triggered by injecting the luminescence triggering buffer into the sample solution. The selectivity of the assay was evaluated by constructing a dose-response curve for rev-octapeptide, an octapeptide with the amino acids of the octapeptide in the reverse order. 4. Results and discussion In this study, the octapeptide Asp±Tyr±Lys±Asp± Asp±Asp±Asp±Lys was selected as a model analyte for the following reasons. First, it was to show that fusion proteins between small peptides and aequorin could be prepared that retain the bioluminescence properties of aequorin. The second reason that this octapeptide was chosen was to demonstrate the feasibility of using aequorin as a label in immunoassays that employ antibodies with relatively weak association constants. According to the manufacturer, the association constant between the monoclonal antibody (M2 antibody) and the octapeptide is 10ÿ7 Mÿ1. Another reason for selecting the octapeptide involves the degree of conjugation between the analyte and the label. To prepare a homogeneous population of the octapeptide±AEQ conjugate by conventional methods (e.g., glutaraldehyde- or carbodiimide-based crosslinking chemistry) would be dif®cult with this octapeptide because it contains six carboxylic and three amino groups. Thus, there is limited control on what amino acid(s) on the octapeptide and on aequorin will serve as point of covalent linkage to yield the conjugate. This problem was overcome in the present study by using a gene fusion approach to prepare a site-speci®cally coupled, one-to-one conjugate of the octapeptide with aequorin. Plasmid pSD1 was constructed so that the gene coding for the octapeptide was fused to the gene coding for apoAEQ. The ®nal product was a fusion protein where the C-terminal of the octapeptide was fused to the N-terminal of apoAEQ. The plasmid pSD1 also includes an oligonucleotide sequence encoding for the OmpA signal peptide. This signal

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peptide facilitates the secretion of the octapeptide± apoAEQ into the periplasm of the bacteria [23]. Fewer proteins are present in the bacterial periplasm than in the cytoplasm, and therefore, puri®cation from the periplasm is much easier than from the cytoplasm. In our system, the octapeptide±apoAEQ conjugate was isolated through a single chromatographic step involving anion-exchange chromatography. Then, the protein was concentrated by ammonium sulfate precipitation, and its purity was veri®ed by SDSPAGE using silver staining. A single band corresponding to the molecular weight of the fusion protein (23 kDa) was obtained indicating the purity of the isolated octapeptide±apoAEQ fusion protein. The protein concentration was estimated to be 0.26 mg/ml by a standard BCA assay. The puri®ed octapeptide±apoAEQ conjugate is converted to octapeptide±AEQ by mixing the apoprotein with a molar excess of coelenterazine. A bioluminescence emission study was performed by injecting 100 ml of luminescence triggering buffer into a solution containing 100 ml of 1.210ÿ12 M of the octapeptide±AEQ fusion protein. The conjugate demonstrated ¯ash-type emission characteristics with the emitted bioluminescence signal rising sharply after injection of the triggering solution, followed by an exponential decay of the emission intensity. The half-life of the bioluminescence emission of the conjugate was 0.68 s and approximately 95% of the total light was emitted during a 3 s period. Therefore a 3 s integration time period was chosen for all remaining experiments. This 0.68 s half-life is slightly higher than that obtained for the native aequorin, which has been shown to have a half-life of 0.60 s [24]. This can be attributed to the presence of the octapeptide at the N-terminus of aequorin. From the bioluminescence emission decay of Fig. 2, as well as from bioluminescence emission data obtained with two other batches of octapeptide±AEQ expressed and isolated independently at different times, the half-lives of the conjugates were calculated and found to be 0.680.01 s. The almost identical bioluminescence half-lives of octapeptide±AEQ conjugates prepared at different times demonstrate that preparation of the conjugates by gene fusion renders highly reproducible conjugate populations in terms of their bioluminescence characteristics. This attests to the advantages of using genetic methods in the pre-

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Fig. 2. Bioluminescence emission of a 100 ml solution of 1.210ÿ12 M of octapeptide±AEQ conjugate. The bioluminescence triggered by Ca2‡ is measured as light intensity in counts.

paration of conjugates with high lot-to-lot reproducibility. A calibration plot for the octapeptide±AEQ fusion protein is shown in Fig. 3. The bioluminescence emitted by the fusion protein has a large dynamic range of 6 orders of magnitude, extending from 10ÿ18 to 10ÿ12 mol. In the development of an immunoassay, one of the factors affecting detection limits is the concentration of the conjugate. This has to be kept as low as possible to obtain optimum detection limits, however, it must be high enough so that the signal to be measured is well above the background signal. Because of the ability to detect aequorin at low levels and with high sensitivity, a low concentra-

Fig. 3. Calibration plot of the octapeptide±AEQ fusion protein. Light intensity is measured in counts and integrated over a 3 s time period.

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Fig. 4. Association curve obtained by incubating varying concentrations of the M2 antibody immobilized on agarose beads with 100 ml of a 1.210ÿ11 M solution of the octapeptide±AEQ conjugate. Data are the average  one standard deviation (nˆ3). Some error bars are obstructed by the symbols for the points.

Fig. 5. Effect of the incubation time on the binding of 100 ml of a 1.210ÿ11 M solution of the octapeptide±AEQ conjugate to 100 ml of 14 mg/ml suspension of the M2 antibody immobilized on agarose beads. Data are the average  one standard deviation (nˆ3). All the error bars are less than 10% and are presented as observed.

tion of the octapeptide±AEQ conjugate can be used for the assay. The next step in the development of the assay was to study the interaction between the octapeptide±AEQ conjugate and the anti-octapeptide M2 monoclonal antibody. The M2 antibody had been immobilized on agarose beads using a hydrazide linkage [25]. Association studies were performed (Fig. 4) by incubating a ®xed concentration of the octapeptide±AEQ (100 ml of 1.210ÿ11 M) with different concentrations of the antibody immobilized on agarose beads for 30 min. As the concentration of the antibody increases the light intensity measured on the solid phase increases until it reaches a plateau. The amount of antibodybeads to be used in an assay is selected so that the intensity of the bioluminescence emitted is suf®cient to perform the assay, while the amount of binding sites is kept as low as possible. The effect of incubation time between the conjugate and the immobilized antibody was evaluated. Fig. 5 shows a time study performed with 100 ml of 14 mg/ml of the immobilized M2 antibody suspension and 100 ml of 1.210ÿ11 M of the octapeptide±AEQ. Since there is no substantial increase in binding after 30 min, the incubation time was set at this value. After evaluating the various parameters that in¯uence the assay, a dose-response curve was constructed in a sequential manner. The octapeptide analyte was incubated ®rst with the immobilized antibody, and

then the octapeptide±AEQ conjugate was added. The antibody concentration used was 14 mg/ml, which is a 1:200 dilution of the 2.8 mg/ml of the stock immobilized M2 antibody suspension. The dose-response curve thus obtained (Fig. 6) is sigmoidal, with the bioluminescence signal being lower at high concentrations of the octapeptide, and increasing as the concentration of the octapeptide decreases. At high concentrations of the octapeptide, most of the available binding sites are occupied by the octapeptide leaving a few unoccupied binding sites for the octapeptide±AEQ conjugate. On the other hand, when the concentration of the octapeptide is low, the bioluminescence signal is at a maximum because most of the binding sites are available and taken up by the octapeptide±AEQ conjugate. The region in-between corresponds to the steep portion of the curve, which is the analytically useful region. The detection limit for the octapeptide was determined to be 110ÿ9 M, which corresponds to 1 10ÿ14 mol of the octapeptide in the sample. Another dose-response curve was generated with a synthetic octapeptide, rev-octapeptide. The amino acid sequence of this peptide is in the reverse order of the original octapeptide. The response obtained with the rev-octapeptide was essentially a ¯at line with the signal remaining constant (within experimental error) from 10ÿ12 to 10ÿ4 M of the rev-octapeptide. This is a result of a poor binding between the rev-

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Acknowledgements This work was supported by grants from the National Institutes of Health (GM47915) and the Department of Energy (DE-FG05-95ER62010).

References

Fig. 6. Dose-response curve for the octapeptide generated by sequentially incubating 100 ml of 14 mg/ml suspension of immobilized M2 antibody with varying concentrations of the octapeptide for 30 min, followed by incubation with 100 ml of 3.410ÿ11 M solution of the octapeptide±AEQ conjugate for an additional 30 min. Data are the average  one standard deviation (nˆ4). Some error bars are obstructed by the symbols for the points.

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