Research Article

Received: 6 May 2008, Revised: 23 June 2008, Accepted: 18 August 2008, Published online 17 March 2009 in Wiley Interscience

(www.interscience.wiley.com) DOI 10.1002/bio.1086

Luminescence 2009; 24: 131–133 Copyright © 2009 John Wiley & Sons, Ltd.

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John Wiley & Sons, Ltd.A bioluminescent enzyme immunoassay for prostaglandin E2 using Cypridina luciferaseChun Wu,a* Saki Irie,b Shozo Yamamotob and Yoshihiro Ohmiyaa,c

ABSTRACT: Prostaglandin E2 is one of the major cyclooxygenase metabolites of arachidonic acid. We developed a competi-tive immunosorbent assay for prostaglandin E2 utilizing a bioluminescent enzyme Cypridina luciferase. The prostaglandin E2amount could be quantified over the concentration ranging from 7.8 to 500 pg/mL. The amount of unlabeled prostaglandinE2 required to displace 50% of the maximal binding of Cypridina luciferase-labeled prostaglandin E2 (B/B0) was approximately35 pg/mL. The results show a great potential of Cypridina luciferase as a new labeling enzyme for enzyme-linked immunosor-bent assay. Copyright © 2009 John Wiley & Sons, Ltd.

Keywords: luciferase; luciferin; immunoassay; prostaglandins; bioluminescence

Introduction

Prostaglandin E2 (PGE2) is one of the major cyclooxygenasemetabolites of arachidonic acid. PGE2 is biosynthesized by PGEsynthases from prostaglandin H2 and is immediately releasedfrom cells. PGE2 exerts its function in vivo via 4-type receptorswith seven transmembrane domains (1). The function of PGE2 isinvolved in fever generation, contraction and relaxation of smoothmuscle, control of vascular permeability and modulation ofinflammation (1). Enzyme-linked immunosorbent assay (ELISA)is a quick and convenient analytical method to measure the levelof PGE2 from biological samples. The levels of PGE2 in normalplasma are from 3 to 12 pg/mL (2). In order to develop a highlysensitive and quick ELISA system for PGE2, we studied a competi-tive immunoassay using the flash-type bioluminescent enzymeCypridina luciferase, which is involved in the bioluminescence ofsea-firefly Cypridina noctiluca (3). This luciferase is very stable,because it is a protein rich in disulfide bonds. We have recentlyapplied the luciferase to a non-destructive dual reporter assaysystem using its gene, and developed a sandwich immunoassayfor IFNα by using the biotinylated luciferase (4, 5). These experi-mental results have shown that luciferase is a very sensitive andversatile bioluminescent reporter. In this study we attempted toapply luciferase to the competitive immunassay of PGE2.

Materials and methods

Materials and apparatus

The recombinant Cypridina luciferase, Cypridina luciferin andmonoclonal anti-PGE2 antibody were prepared according toprevious reports (4, 6, 7). N-Hydroxysuccinimde (NHS), 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC) andsynthetic PGE2 were purchased from Sigma-Aldrich (St Louis, MO,USA). The size-exclusion column (PD-10 column) was purchasedfrom GE Healthcare (Fairfield, CT, USA). PGE2 standard and 96-wellstrip plates pre-coated with secondary antibody (goat anti-mouseIgG) were purchased from Cayman Chemical (Ann Arbor, MI,USA). All chemicals were analytical reagent-grade or better and

were used as received. Distilled water was prepared using withMilli-Q water purification system. Absorption spectra werecollected using a UV–vis spectrophotometer from Hitachi (Tokyo,Japan). Bioluminescence measurement for the assay was performedon AB 2300 plate reader from ATTO (Tokyo, Japan).

Conjugation of PGE2 and luciferase

The N-hydroxysuccinimidyl ester of PGE2 was prepared byreacting with NHS (12 equiv.) and EDC (12 equiv.) for 7 h at roomtemperature. The NHS ester was then conjugated to the luciferasein a molar ratio of 18:1 in 0.1 M potassium phosphate buffer (pH7.2) containing 0.15 M NaCl. After incubation on ice for 13 h, thereaction mixture was loaded onto a size-exclusion column, andPGE2-luciferase was then eluted with 0.1 M potassium phosphatebuffer (pH 7.2) containing 0.15 M NaCl. All the fractions weretested for bioluminescent activity by mixing 50μl of 1 μM Cypridinaluciferin solution (0.1 M Tris–HCl, pH 7.4–0.3 M sodium ascorbate–0.02 M Na2SO3). The fractions showing bioluminescent activitieswere combined and were then stored at −30°C until use.

Km value of PGE2-luciferase

PGE2-luciferase was diluted with 0.1 M Tris buffer (pH 7.4) con-taining 0.05% bovine serum albumin. Serial dilution of Cypridinaluciferin ranging from 0.32 to 5 μM was carried out with 0.1 M

* Correspondence to: Chun Wu, Research Institute for Cell Engineering,National Institute of Advanced Industrial Science and Technology, Osaka,563-8577, Japan. E-mail: chun-wu@aist.go.jp

a Research Institute for Cell Engineering, National Institute of AdvancedIndustrial Science and Technology, Osaka, 563-8577, Japan

b Department of Food and Nutrition, Faculty of Home Economics, KyotoWomen’s University, Kyoto, 605-8501, Japan

c Department of Photobiology, Graduate School of Medicine, HokkaidoUniversity, Hokkaido University, Sapporo, 060-8638, Japan

C. Wu et al.

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Tris–HCl buffer (pH 7.4) containing 0.3 M sodium ascorbate and0.02 M Na2SO3. The bioluminescent measurements were performedby mixing 50 μl of 7 pM PGE2-luciferase with 50 μl of differentconcentrations of the serially diluted luciferin solutions.

Kd value of luciferase-labeled PGE2 to monoclonal anti-PGE2 antibody

Serially diluted PGE2-luciferase ranging from 0.54 to 70 nM (50 μL)and 1 nM monoclonal anti-PGE2 antibody (50 μL) were placed ina 96-well strip plate pre-coated with a secondary antibody (goatanti-mouse IgG). The plates were incubated in the dark at 4°C for18 h. Then the plates were washed with a washing buffer (20 mM

Tris–HCl pH 7.8–0.1% Tween20–0.9% NaCl) four times to removeany unbound primary antibody and PGE2-luciferase. Finally, thebioluminescent measurements were performed with a 96-wellplate reader by injection of 100 μL of 1 μM Cypridina luciferinsolution (0.1 M Tris–HCl, pH 7.4–0.3 M sodium ascorbate–0.02 M

Na2SO3) to each well.

Enzyme immunoassay

Serially diluted solutions of PGE2 standard ranging from 7.9 to1000 pg/mL were prepared with 0.1 M Tris–HCl buffer, pH 7.4,containing 0.05% bovine serum albumin. The same Tris–HClbuffer was used for the preparation of blank, non-specific bindingand maximum binding wells. A volume of 50 μL of these dilutedstandard solutions was first placed in a 96-well strip plate, followedby the addition of 50 μL of 35 nM PGE2-luciferase. Finally, 50 μL of1 nM monoclonal anti-PGE2 antibody was added. After incubationof the 96-well strip plate at 4°C in the dark for 18 h, all wells werewashed and dried. The bioluminescent measurements wereperformed with a 96-well plate reader after injection of 100 μLof 1 μM Cypridina luciferin solution (0.1 M Tris–HCl pH 7.4–0.3 M

sodium ascorbate–0.02 M Na2SO3) to each well.

Results and discussionCypridina luciferase has 30 lysine residues. We used some ofthese amino groups of lysine residues to covalently couple theluciferase to amine-reactive succinimidyl ester derivative ofPGE2. An 18-fold molar excess of PGE2 was used in this couplingreaction. The conjugate of PGE2 and luciferase was purified bysize-exclusion chromatography. The final concentration of themodified luciferase was estimated to be 700 nM from theabsorbance at 280 nm. The specific activity of the modifiedluciferase was 14% of the original enzyme (data not shown). Asshown in Fig. 1, the Km value of the modified luciferase forCypridina luciferin was approximately 1.6 μM according to theLineweaver–Burk plot.

To determine the Kd value of PGE2-luciferase to anti-PGE2

monoclonal antibody, we incubated various amounts of PGE2-luciferase with a fixed concentration of anti-PGE2 monoclonalantibody in pre-coated goat anti-mouse IgG wells. As shown inFig. 2, the bioluminescent activities increased depending on theamount of PGE2-luciferase. A small background of PGE2-luciferasewas observed in the absence of the antibody. According toScatchard plot analysis on the basis of the data shown in Fig. 2,the Kd value was estimated to be 50 nM. Considering the ratio of thebioluminescent signal to background, we used the concentrationof PGE2-luciferase at 35 nM and the concentration of antibody at1 nM in the subsequent experiments.

To evaluate the performance of PGE2-luciferase in immunosor-bent assay of PGE2, we prepared serially diluted solutions of PGE2

standards ranging from 7.8 to 1000 pg/mL. A typical calibrationcurve is shown in Fig. 3. The results indicated that PGE2 could bequantified at the concentration ranging from 7.8 to 500 pg/mL.The amount of unlabeled PGE2 required to displace 50% of themaximal binding of luciferase-labeled PGE2 (B/B0) was approximately35 pg/mL. The coefficients of variation (CV) ranges were 1–10%.

Various types of detection methods of PGE2 including radio-immunoassay, enzyme immunoassay and gas chromatographywith mass spectrometry (GC/MS) have been reported in theliterature. Radioimmunoassay and GC/MS are highly sensitivemethods, but they have disadvantages, including the use ofharmful radioactivity and expensive instrumentation. So far, aslisted in Table 1, three enzyme immunoassay systems for PGE2

using acetylcholinthioesterase, β-galactosidase and horseradishperoxidase as a label have been described. The detection inthese immunoassays has been performed with absorbance,fluorescence or chemiluminescence, respectively. In this study,we demonstrated a bioluminescent immunoassay using PGE2-luciferase, because our recent studies have already shown thatCypridina luciferase is a highly stable and sensitive bioluminescent

Figure 1. Lineweaver-Burk plot analysis of the modified Cypridina luciferase.The bioluminescent measurement was performed by mixing 50 μL of 7 pM PGE2-luciferase with 50 μL of the serially diluted luciferin solutions. The Linewearver–Burk plots were constructed on the basis of the bioluminescent kinetics. RLU,relative luminescent unit. 59x42mm (600 x 600 DPI)

Figure 2. Measurement of the bioluminescent activities of bound PGE2-luciferase. Various amounts of PGE2-luciferase were incubated with or without afixed concentration of anti-PGE2 monoclonal antibody in pre-coated goat anti-mouse IgG wells. The bioluminescent measurement of the bound PGE2-luciferasewas performed with the injection of the luciferin. The dots represent the incuba-tion with antibody and the triangles represent the incubation in the absence ofantibody. 59x36mm (600 x 600 DPI)

A bioluminescent enzyme immunoassay for prostaglandin E2

Luminescence 2009; 24: 131–133 Copyright © 2009 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/bio

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reporter (4, 5). Since PGE2 is easily dehydrated in solution abovepH 8, the coupling reaction was performed in phosphate bufferat pH 7.2. Although the bioluminescent activity of the luciferaseconjugated to PGE2 was lower than that of native enzyme, thebioluminescent signal generated by PGE2-luciferase could beused to quantify free PGE2 in a buffer at a concentration rangingfrom 7.8 to 500 pg/mL. The lower limit was lower than that ofother enzymes. The high sensitivity of Cypridina luciferase inimmunoassay is due to a high quantum yield in the biolumines-cence rather than the turnover rate of the enzyme, because theturnover rate in the bioluminescent enzyme of sea-firefly (1600/min) is much smaller than that of acetylcholinthioesterase of theelectric eel (3 840 000/min) for the hydrolysis of acetylthiocholine(10). The flash light emission kinetics of the luciferase allowsthe immediate measurement of PGE2, whereas non-luminousenzymes usually require incubation for 60–90 min. Furthermore,Cypridina luciferase is highly stable and resistant to detergent,which is a big advantage for the application of the enzyme toimmunoassays (4, 5).

In conclusion, we have developed an immunoassay system forPGE2 using the bioluminescent enzyme Cypridina luciferase. Our

results indicated that the developed ELISA using the luciferasewas highly sensitive and quick for the measurement of PGE2,suggesting a great potential of Cypridina luciferase as a newlabeling enzyme in various immunoassays.

Acknowledgements

We thank Kosei Kawasaki and Satoru Ohgiya for providingCypridina luciferase and for critical reading of this paper. Thisstudy was supported in part by a Grant-in-Aid for the Support ofYoung Researchers (grant no. 19710196) from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan.

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Tabel 1. Various enzyme immunoassay systems for PGE2 185x75mm (600 x 600 DPI)

Enzyme Detection Low limit of quantification Incubation References

Acetylcholin esterase Absorbance 15 pg/mL Required (60–90 min) 8β-Galactosidase Fluorescence 4.2 pg/mL Required (60–90 min) 7Horseradish peroxidase Chemiluminescence 100 pg/mL Not required 9Cypridina luciferase Bioluminescence 7.8 pg/mL Not required This paper

Figure 3. A typical calibration curve of competitive immunoassay of PGE2.A mixture of serially diluted solutions of PGE2 standard ranging from 7.9 to 1000pg/mL, PGE2-luciferase and anti-PGE2 monoclonal antibody was incubated inpre-coated goat anti-mouse IgG wells. The bioluminescent measurement wasperformed with the injection of the luciferin. 59x41mm (600 x 600 DPI)