development of highly sensitive bioluminescent enzyme immunoassay with ultra-wide measurable range...

Analytica Chimica Acta 429 (2001) 19–26

Development of highly sensitive bioluminescent enzymeimmunoassay with ultra-wide measurable range forthyroid-stimulating hormone using firefly luciferase

Yoshiaki Setoa,c,∗, Hiroshi Ohkumaa, Susumu Takayasub,Toshihiko Ibab, Atsuko Umedab, Katsushi Abea

a Biochemical Research Laboratory, Eiken Chemical Co. Ltd., 1381-3 Shimoishigami,Ohtawara-shi, Tochigi 324-0036, Japan

b Biochemical Research Laboratory, Eiken Chemical Co. Ltd., 143 Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japanc Department of Chemistry, Faculty of Engineering, Gunma University, Kiryu, Gunma 376-8515, Japan

Received 4 July 2000; received in revised form 9 October 2000; accepted 19 October 2000

Abstract

A bioluminescent enzyme immunoassay (BLEIA) of thyroid-stimulating hormone (TSH) using biotinylated firefly lu-ciferase as a labelling enzyme for an antibody was developed. This immunoassay, of which method was one step sandwichmethod, had high sensitivity and very wide measurable range. The detection limit for TSH was found to be 0.0025mU ml−1

and the measurable range for TSH was 0.0025–200mU ml−1. The conjugate for this BLEIA was prepared by binding theantibody to the end of cross-linking reagent and streptavidin (SA)-biotinylated luciferase complex to the opposite end ofthe cross-linking reagent, respectively. The stability of the conjugate was excellent. It showed 70% conjugate activity at thestorage of a week at 37◦C and 100% at the storage of 6 months at 4◦C. © 2001 Elsevier Science B.V. All rights reserved.

Keywords:Bioluminescent enzyme immunoassay; Firefly luciferase; Thyroid-stimulating hormone; Ultra wide measurable range

1. Introduction

The bioluminescent enzyme, firefly luciferase,catalyses the oxidation ofd-luciferin in the presenceof adenosinetriphosphate (ATP), Mg2+ and molec-ular oxygen. The exited state oxyluciferin that wasproduced in the oxidation process decays with pro-duction of light followed by a release of steady stateoxyluciferin from the enzyme. This luminescencequantum yield is 0.88 and higher than any other bio-or chemiluminescence [1]. For this high quantum

∗ Corresponding author.E-mail address:yoshiaki [email protected] (Y. Seto).

yield, the firefly luciferase bioluminescent systemhas been applied to genetic reporter in the molecularbiology and ATP detection in the microbiology [2,3].However, there were little reports for BLEIA usingwild type firefly luciferase [4,5] because of the poorenzymatic stability and the weakness for chemicalmodification of wild type firefly luciferase. So, itwas necessary to improve these disadvantages for theapplication of wild type firefly luciferase to BLEIA.

Recently, Kajiyama and Nakano isolated ther-mostable mutant firefly luciferases from Japanesefireflies, Luciola latelalis and Luciola cruciata, byprotein engineering [6,7]. Maeda et al. developed bio-luminescent immunoassay using thermostable mutant

0003-2670/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved.PII: S0003-2670(00)01254-X

20 Y. Seto et al. / Analytica Chimica Acta 429 (2001) 19–26

luciferase [8–10]. In those immunoassay, acetatekinase (AK) was used as a labelling enzyme andthe concentration of ATP that was produced by AKwas quantified by using the luminescence reagentcontaining the luciferase. Although this method washigh sensitive with improved luciferase stability, it’sincreased background signal might be observed bycontamination of ATP.

Tatsumi et al. have made the thermostable lu-ciferases (bL203 and bL248) by genetically introduc-tion of d-biotin into its molecule [11]. The BLEIAusing the biotinylated luciferase as a labelling en-zyme was not affected by contamination of ATP.However, when this BLEIA was compared with aconventional immunoassay, it required some steps toform avidin–biotin complex. Ohkuma et al. made aconjugate using the biotinylated luciferase (bL248),streptavidin (SA) and biotinylated antibody complex[12]. In the immunoassay using this conjugate, theassay steps could be decreased, but there was a seri-ous problem that was the dissociation of the enzymefrom the conjugate.

In this report, we made a stable antibody conju-gate using the biotinylated luciferase and constructedBLEIA for thyroid stimulating hormone that requiredhigh sensitive and very wide measurable range.

2. Materials and methods

2.1. Materials

ATP and SA were purchased from Sigma (St. Louis,MO). N-(11-Maleimidoundecanoyloxy)sulfosucci-mide, sodium salt (Sulfo-KMUS),N-(2-acetamido)-iminodiacetic acid (ADA),N-cyclohexyl-3-aminopro-panesulfonic acid (CAPS), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) were obtai-ned from Dojin (Kumamoto, Japan). Block Ace waspurchased from Dainippon Pharmaceutical (Osaka,Japan). Anti-TSHb subunit monoclonal antibodieswere obtained from Medix Biochemica (Kauniainen,Finland) and Scantibodies Laboratory (Santee, CA).d-Luciferin was purchased from Biosynth (Staad,Switzerland). Magnetic particle (Dynabeads M280Tosyl activated) was obtained from Dynal (Oslo,Norway).

2.2. Preparation of bL248-SA-antibody conjugate

Anti-TSH antibody was diluted to 1 mg ml−1

(6.6mM) with 50 mM phosphate buffer (PB) con-taining 5 mM EDTA (pH 6.3). To 1 ml of this an-tibody solution, 100 ml of 0.2 M 2-mercaptoethanolin 50 mM PB containing 5 mM EDTA (pH 6.3) wasadded and incubated at 37◦C for 90 min. The reactionmixture was purified on a PD-10 (Pharmacia) columnusing 50 mM PB containing 5 mM EDTA (pH 6.3)as an elution buffer. The fractions containing the re-duced antibody were collected and concentrated bya Centricon 30 (Millipore) centrifuge concentrator at5000 rpm for 30 min. This solution was stored at 4◦C.

To 240ml of 1.79 mg ml−1 (29.8 mM) SA in phos-phate buffer saline(PBS), 14.6ml of 14.6 mM Sulfo-KMUS in dimethyl formamide (DMF) was addedand incubated at 30◦C for 60 min. The reaction mix-ture was purified on a PD-10 column using 50 mMPB containing 5 mM EDTA (pH 6.3) as an elutionbuffer. The fractions, containing the activated SA,was collected and concentrated by a Centricon 30centrifuge concentrator at 5000 rpm for 30 min. To300ml of 2.14 mg ml−1 (14.1mM) of the reducedantibody solution, 172ml of 1.0 mg ml−1 (16.7mM),the activated SA solution was added and incubatedat 30◦C for 60 min. To the reaction mixture, 450mlof 0.23 mg ml−1 (3.83mM) bL248 in the storagebuffer (0.1 M PB, 2 mM 2-mercaptoethanol, 1 mMEDTA, 10% glycerol) was added and incubated at25◦C for 60 min. The solution was purified on Su-perdex 200HR 10/30 (Pharmacia) using 0.1 M PB,0.15 M NaCl (pH 7.2) as elution buffer. Fractionsshowing high immunological reactivity and enzymeactivity were collected. This conjugate solution wasdiluted to 0.33 nM (enzyme concentration) with con-jugate buffer (0.1 M PB containing 0.4 M NaCl, 5 mMadnosine-5′-monophosphate, 2.5 mM EDTA, 0.2%BSA, 0.02% casein and 0.05% NaN3) and stored at4◦C.

2.3. Immobilisation of anti-TSH antibody tomagnetic particles

The antibody was immobilised on magnetic parti-cles (Dynabeads M280 tosylactivated). 1 ml of mag-netic particle suspension (10 mg ml−1) was washed

Y. Seto et al. / Analytica Chimica Acta 429 (2001) 19–26 21

three times with 2 ml of purified water. The magneticparticles were re-suspended with 500ml of 0.1 MCAPS buffer (pH 8.5). To the suspension, 500ml of0.4 mg ml−1 antibody in 0.1 M CAPS buffer (pH 8.5)was added and the suspension was stirred at 25◦C for18 h. The suspension was washed with 2 ml of 0.1 MPB (pH 7.2), 2 ml of 0.05% Tween 20 in PBS and2 ml of 0.1 M PB (pH 7.2). To the magnetic particles,2 ml of blocking buffer (50 mM PB containing 20%Block Ace, pH 7.2) was added and the suspensionwas stirred at 25◦C for 3 h. After blocking buffer wasremoved from the suspension, 4 ml of stock buffer(50 mM HEPES containing 2% Block Ace, 0.1%bovine serum albumin, 0.5% chloroacetamide and0.01% methylisothiazoline, pH 7.4) was added andstored at 4◦C.

2.4. Bioluminescent enzyme immunoassay forthyroid stimulating hormone (TSH)

The immunoassay method was based on one stepsandwich immunoassay. After 80ml of sample wastransferred to a polystyrene test tube(12 mm×75 mm),100ml of the 0.33 nM conjugate solution and 20ml of2 mg ml−1 antibody immobilised magnetic particlessuspension was added to the test tube. The mixturewas stirred for 2 s and then left it standing at 25◦C for15 min and washed 0.05% Tween 20 in PBS (750ml ×3). To the magnetic particles, 100ml of suspensionbuffer (50 mM Tris–HCl, pH 8.5) was added andstirred for 2 s. The test tube containing the suspensionwas placed to a luminometer (Lumat LB 9507, EG&GBerthold) and 100ml of a substrate solution (940mMluciferin, 2 mM ATP, 40 mM ADA, 16 mM MgSO4,0.26 mM EDTA, 0.2 mM potassium pyrophosphate,pH 6.5) was added. Luminescence intensity wasmeasured an integrated time of 5 s (delay time 0.5 s).

3. Results and discussion

3.1. Preparation of the conjugate

As shown in Fig. 1, a streptavidin was introducedto anti-TSH antibody with hetero bifunctional cross-linking reagent containing both maleimide group andsuccimide ester. Their functional groups were linked

with a long alkylchain undecanoyl group. Such a longalkylchain was used to reduce the dissociation ofthe biotinylated luciferase from bL248-SA-antibodyconjugate by the steric interference. The biotiny-lated firefly luciferase, bL248, was introduced to theSA-antibody conjugate. In this conjugation proce-dure, purification on gelfiltration chromatography wascarried out for only bL248-SA-antibody conjugatebut not for the SA-antibody conjugate.

3.2. Stability of the conjugate

In this immunoassay, biotinylated enzyme formedcomplex with avidin covalently binding to the cross-linking reagent of which end was covalently binding tothe antibody. The covalent bond was necessary to in-crease the conjugate stability. Avidin–biotin complexhas a high binding constant and is useful for conjuga-tion method, however, it is possible to dissociate theenzyme from the conjugate when it was stored for longtime as this binding is not covalent. When dissocia-tion of the enzyme was occurred to some extent, thedetection sensitivity of the assay should be decreased.Thus the stability of the conjugate is very important.We found that high concentration of NaCl in the con-jugate buffer was effective to prevent dissociation ofthe enzyme from the conjugate. In the fraction profileof the gelfiltration chromatography, the enzyme wasdissociated only 4.7% at 37◦C for a week (Fig. 2). Thisdegree of dissociation is practically admitted. And theloss of enzyme activity due to chemical/biochemicaldegradation was 20% at the same condition. There-fore, the remaining conjugate activity containing thedissociation of enzyme and the loss of enzyme activityand the immunological activity was 70% when storedat 37◦C for a week. This stability is same as that ofantibody conjugates with a horse radish peroxidase oran alkaline phosphatase.

3.3. Detection limit for TSH

Detection limit was determined by replicate anal-ysis (n = 7) of 0, 0.00125, 0.0025, 0.005, 0.01 and0.02mU ml−1 of TSH standards and was based on+3SD of zero standard (Fig. 2). The lowest detectableconcentration of TSH was 0.0025mU ml−1. The de-tection limit of this BLEIA was greater 20-fold than


Fig. 1. Preparation of bL248-SA-antibody conjugate.

that of immunoradiometric assay (IRMA, Ab TSH II‘Eiken’, Eiken Chemical, Japan).

3.4. Standard curve for TSH

As shown in Fig. 3, standard curve for TSH hadgood linearity from 0.02 to 20mU ml−1. The mea-surable range of TSH was 0.0025–200mU ml−1. Theintra-assay coefficient of variations (CVs) for eachpoints on standard curve were 2.9–8.7% (n = 7). The

inter-assay CVs for each points on standard curvewere 2.4–6.8% (n = 5). The intra- and inter-assayCVs for five replicates of 1.65–151mU ml−1 of TSHin serum were 1.3–2.6 and 1.5–3.8%, respectively(Table 1) (Fig. 4).

3.5. Linearity of sample dilution and recovery test

The linearity of dilution was carried out by usinghuman serum containing high concentration TSH


Fig. 2. Gel filtration chromatography of conjugates. Eluent: 0.1 M PB, 0.15 M NaCl, pH 7.2.

Fig. 3. Detection limit for TSH.


Table 1Intra-assay

Sample Average (mU ml−1) S.D. CV (%)

(a) Intra-assayA 0.753 0.005 0.7B 1.486 0.019 1.3C 8.921 0.155 1.7D 31.50 0.813 2.6E 148.9 6.281 4.2

(b) Inter-assay of the BLEIAF 1.503 0.024 1.6G 1.676 0.025 1.5H 6.981 0.263 3.8I 27.20 0.660 2.4J 153.9 5.506 3.6

diluted with a zero standard solution. Excellent lin-earity was observed on all samples as shown inTable 2.

The recovery test was carried out by using themixture of the high concentration TSH standards andhuman sera in a 9:1 volume ratio. The recovery was90–105% in all samples as shown Table 3.

Fig. 4. Standard curve for TSH. Value in parenthesis represents the intra-assay imprecision (CV%), (n = 5).

Table 2Dilution liniarity of TSH in human seraa

Sample Correlation equation Correlationcoefficient (r)

H y = 140.33x + 0.0634 0.9992M y = 15.985x − 0.1811 0.9988L y = 2.5844x − 0.0003 0.9989

a The initial concentrations of sample H, M and L were 139.1,15.99 and 2.61mU ml−1, respectively.

3.6. Method comparison

The TSH concentrations measured by the presentBLEIA method were compared with those measuredby the conventional methods, immunoradiometricassay (IRMA, Ab TSH II ‘Eiken’, Eiken chemical,Japan) and fluorescent enzyme immunoassay (FEIA,AIA-21, E-test TOSOH II TSH 3G, Tosoh, Japan).There was good correlation between the presentBLEIA and other conventional methods (Fig. 5).


Table 3Recovery tests of present BLEIA for TSH in human sera

Added TSH (mU ml−1) Concentration (mU ml−1) Recovered (mU ml−1) Recovered (%)

L0.00 0.565.00 5.35 4.79 95.8

10.00 10.01 9.45 94.520.00 19.60 19.04 95.2

M0.00 8.625.00 13.10 4.48 89.6

10.00 18.11 9.49 94.920.00 27.82 19.20 96.0

H0.00 28.685.00 33.54 4.86 97.2

10.00 37.47 8.80 88.020.00 48.15 19.47 97.4

Fig. 5. Correration of TSH in human sera determined by conventional methods (IRMA, FEIA) and present BLEIA.

4. Conclusions

The conjugate used in this study consisted of bothcovalent bond and avidin–biotin complex. The cova-lent bond was necessary to increase for stability ofconjugate and the avidin–biotin complex was nec-essary for the conjugation of the luciferase to theantibody without the loss of enzyme activity. The

conjugate was very stable, and the remaining im-munological activity was 70% when stored at 37◦Cfor a week. The stability was of same order as otherenzyme conjugates. The detection limit of the assayfor TSH was 0.0025mU ml−1 and 20-fold greaterthan IRMA for TSH. Moreover, the measurable rangewas very wide, 0.0025–200mU ml−1. The correlationbetween BLEIA and other conventional methods was


extremely good. This BLEIA system showed the ex-cellent assay performance with the high sensitivityand ultra wide measurable range.

Acknowledgements

We thank Dr. Hiroki Tatsumi (Kikkoman Corp.) forproviding the biotinylated thermostable recombinantfirefly luciferase (bL248) used in this study.

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development of highly sensitive bioluminescent enzyme immunoassay with ultra-wide measurable range...

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