Ž .Journal of Immunological Methods 224 1999 171–184

Open sandwich ELISA with V -rV -alkaline phosphatase fusionH L

proteins

Chikako Suzuki a, Hiroshi Ueda a,b,), Kouhei Tsumoto c, Walt C. Mahoney d,Izumi Kumagai c, Teruyuki Nagamune a

a Department of Chemistry and Biotechnology, Graduate School of Engineering, The UniÕersity of Tokyo, Hongo, Bunkyo-ku,Tokyo 113-8656, Japan

b Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UKc Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku UniÕersity, Aoba, Aramaki, Aoba-ku, Sendai,

980-8579, Japand Roche DiagnosticsrBMC, 4300 Hacienda DriÕe, Pleasanton, CA 94588-2722, USA

Received 26 June 1998; received in revised form 25 January 1999; accepted 1 February 1999

Abstract

The Sandwich ELISA is a widely used technique to measure antigen concentration. Recently, a novel ELISA based onŽ . Žthe interchain interaction of separated V and V chains from a single antibody variable region Fv was proposed OpenH L

.Sandwich ELISA . Since it employs a single antibody recognizing one epitope, the assay requires, in essence, only one cycleof incubation and washing steps. To demonstrate this directly, we have constructed a recombinant gene fusion encoding the

Ž . Ž .V chain of an anti-hen egg lysozyme HEL antibody HyHEL-10 and Escherichia coli alkaline phosphatase V -PhoA .H HŽ .The same type of gene fusion using V chain instead of V chain V -PhoA was also constructed and the proteins wereL H L

obtained with an E. coli expressionrsecretion system. Open Sandwich ELISAs were performed using microtiter plates withimmobilized V or V fragment, and V -PhoA or V -PhoA, respectively, as the detection reagent which was simultane-L H H L

ously added to each well with samples. As a result, HEL concentrations in the samples were determined after one round ofincubation and washing steps, with a signal generated in a direct relationship to the concentration of HEL added to thereaction mixture. The minimum detectable HEL concentration was ;10 ngrml, which was almost equal to the valuepreviously obtained with plate-immobilized V and V fragment displayed on M13 phage. When the active-site mutantL H

Ž .V -PhoA D101S was employed instead of V -PhoA and reacted at an optimum pH of 10, a significant enhancement inH H

signal was attained. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Immunoassay; Alkaline phosphatase; Chimeric protein; Fv stability

AbbreÕiations: BSA, bovine serum albmin; ELISA, enzyme-linked immunosorbent assay; Fv, antibody variable region; ScFv,single-chain Fv; V , antibody H chain variable region; V , antibody L chain variable region; HEL, hen egg lysozyme; PBS,H L

phosphate-buffered saline; PCR, polymerase chain reaction; IPTG, isopropyl-b-D-thiogalactoside; p-NPP, p-nitrophenyl phosphate;SDS-PAGE, polyacrylamide gel electrophoresis in sodium dodecyl sulfate; PhoA, Escherichia coli alkaline phosphatase

) Corresponding author. Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK. Tel.: q44-1223-402116;Fax: q44-1223-402140; E-mail: hueda@bio.t.u-tokyo.ac.jp

0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 99 00020-4

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184172

1. Introduction

Assays using antibodies or their derivatives arecommonly used in both the diagnostic and researchfields. The specificity of antibodies has permitted thedetection and analysis of various trace substances.By using specific antibodies, various antigens from

Ž .biological substances e.g., DNAs, proteins and arti-Ž .ficial chemicals e.g., small chemicals, drugs can be

measured. However, the current heterogeneous im-munoassays usually require tedious and time-con-suming procedures due to multiple incubation andseparation processes. For example, the SandwichELISA is one of the most commonly used im-munoassays. The assay has several merits such as

high sensitivity and specificity, low background, andlinearity of the standard curve. On the other hand,there is one basic limitation in the assay in that itrequires at least two antibodies to capture and detectthe antigen, which leads to several consecutive incu-

Ž .bationrwashing steps Fig. 1B . This also means thatthe antigen must possess at least two epitopes inorder to be detected. Antigens possessing only asingle epitope, such as haptens, are not detectedusing this assay.

To obviate these limitations, our group recentlyproposed a novel ELISA based on the interchaininteraction of separated V and V chains from aH L

Ž .single antibody variable region Ueda et al., 1996 . Itis widely accepted that many antibody Fvs have

Ž .Fig. 1. Schematic diagrams of Open Sandwich ELISA and Sandwich ELISA. A Open Sandwich ELISA with immobilized V andL

V -phage. V was immobilized onto the microtiter plate. Then, antigen and V -phage were added at the same time. Antigen-dependentH L HŽ .binding of V -phage was detected with enzyme-conjugated anti-phage antibody. B Sandwich ELISA. First, antibody was immobilizedH

Ž .onto the microtiter plate. Antigen polyvalent and enzyme-conjugated second antibody were sequentially added and incubated separately.Ž .Antigen-dependent binding of the second antibody was detected by enzymatic activity. C Open Sandwich ELISA with immobilized VL

and V -PhoA. Antigen and V -PhoA were simultaneously added to the V -immobilized microtiter plate. Antigen-dependent binding ofH H L

V -PhoA was detected directly by alkaline phosphatase activity.H

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184 173

limited stability in the absence of antigen. The inter-action between isolated V and V is rather weak,H L

and in the absence of the constant domains, they tendŽto dissociate at low concentrations Glockshuber et

al., 1990 and references therein; Tsumoto et al.,.1994a . On the other hand, some unstable Fv frag-

ments are known to be markedly stabilized by theŽaddition of the antigen Glockshuber et al., 1990;

.Ueda et al., 1993 . Though the precise mechanism isstill unknown, the extent of stabilization is more thanexpected from simple bridging between the two

Ž .chains by the antigen Ueda et al., 1996 . This isprobably because some conformational change in theV –V interface region is induced upon antigenH L

binding to stabilize the Fv–antigen complex. Thus, ifwe can detect the V –V interaction in some way, itH L

should be possible to measure antigen concentration.In fact, when the interaction between V and phage-L

displayed V fragments from the anti-HEL antibodyH

HyHEL-10 was measured with an ELISA detectingimmobilized phage particles, a good correlation be-tween HEL concentration and absorbance was ob-

Žserved indirect Open Sandwich ELISA, Ueda et al.,.1996 . Fig. 1A shows a schematic diagram of the

assay. The method has merit in that it is applicable tothe in vitro evolution of antibody, and was indeedapplied to the selection of mutant HyHEL-10 Fv that

Ž .can bind to human lysozyme Tsumoto et al., 1997 .However, from the viewpoint of the assay, it re-quired additional steps to detect phage particles andhence the time required was no shorter than that ofconventional sandwich assays.

Here, we have examined the possibility of a gen-uine Open Sandwich ELISA using fusion proteinscomprising V or V fragments and a reporter en-H L

Žzyme, Escherichia coli alkaline phosphatase PhoA,.EC 3.1.3.1 . By adopting such fusion proteins, the

last incubation and washing steps necessary for de-Ž .tecting phage particles, are omitted Fig. 1C . It has

been reported that, when highly conserved residuesin the family of alkaline phosphatases were mutated,many of the resulting enzymes exhibited an im-

Žproved k Mandecki et al., 1991; Matlin et al.,cat.1992; Janeway et al., 1993 . By introducing one such

mutation into the V -PhoA alkaline phosphatase do-H

main and searching for the optimal reaction condi-tions, we examined the possibility of increased per-formance.

2. Materials and methods

2.1. Materials

Oligonucleotides were synthesized using a 380AŽautomated DNA synthesizer Applied Biosystems,

. Ž .USA . Pyrococcus furiosus Pfu DNA polymeraseŽ .was obtained from Stratagene CA, USA . Restric-

tion and other modification enzymes were from ei-Ž .ther Takara Kyoto, Japan or New England Biolabs

Ž .MA, USA . PCR were performed in a DNA Ther-Ž .mal Cycler PV-2000 Perkin-Elmer, Takara . The

Žw Xstrains of E. coli used were XL1-Blue F <Tn10q q q ( )proA B lacI D lacZ M15 r recA1 endA1 gyrA96

Ž r . Ž y q. xNal thi hsdR17 r m supE44 relA1 lac , Strata-k k.gene for DNA preparation and, for protein expres-

Ž . Žw X [ ][ ]sion, BL21 lDE3 pLysS F ompT gal dcm lon( y y)x .hsdS r m , Novagen, WI, USA , which harborsB B B

the T7 RNA polymerase gene under the control ofthe lacUV5 promoter, with pLysS plasmid encoding

Ž .T7 lysozyme Studier, 1991 . Plasmid pET20b, whichcontains a T7 promoter and the coding sequencesspecifying 5X-terminal pelB signal peptide and 3X-terminal 6=histidine affinity tag, was obtained from

Ž .Novagen Madison, WI, USA . DNA sequences weredetermined using a DNA sequencer SQ-5500Ž .Hitachi, Tokyo, Japan . Cell densities and alkalinephosphatase activity were measured using a spectro-

Ž .photometer U-2000 Hitachi . Absorbances of mi-crotiter plate wells were measured using a Sjeia

ŽER-8000 microplate reader Sanko Junyaku, Tokyo,. Ž .Japan or Microplate Reader Model 550 Bio-Rad

with a wavelength of 630 nm as control. Microtiterplates were shaken using Microplate Mixer MPX96Ž .Iwaki Glass, Chiba, Japan when indicated. Themolecular weights of the proteins were calculated

Žusing Genetyx-Mac ver. 9.0 Software Development,.Tokyo, Japan . Other chemicals and reagents were

Ž .obtained from Sigma St. Louis, MO, USA , WakoŽ . Ž .Osaka, Japan or Kanto Chemicals Tokyo, Japan .

2.2. Preparation of the expression plasmids

The HyHEL-10 Fv-encoding plasmid pKTN2 wasŽ .constructed as described Tsumoto et al., 1994b . To

express V -PhoA and V -PhoA fusion proteins, plas-H L

mids pV -PhoA and pV -PhoA were constructed. AH L

1450 bp fragment encoding E. coli alkaline phos-

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184174

Ž .phatase PhoA, EC 3.1.3.1 was amplified fromXL1-Blue chromosomal DNA and inserted into

ŽpET20b at the NotI site as described designated.pPhoA, Suzuki et al., 1997 . Genes encoding V andH

V fragments were amplified separately from pKTN2L

by PCR, and a sequence corresponding to threeamino acids spacer and HindIII site were introducedinto the 3X-terminal end of each fragment. The oligo-

Ž Xnucleotides used as primers were T7 primer 5 -TTAX. Ž XATA CGA CTC ACT AT-3 and VHhindfor 5 -

AAA TTC GAA CCT GAG TGG GCG GCT CTGX.CCA CTG CTC-3 for V , and T7 primer andH

Ž XVLhindfor 5 -AAA TTC GAA CCT GAG TGGX.GAA CTA GAG GTC GAA CCA-3 for V . TheL

fragments obtained were digested with EcoRV andHindIII, and inserted between pET20b-derivedEcoRV and HindIII sites of pPhoA, resulting inplasmids pV -PhoA and pV -PhoA, respectively. AllH L

of the PCR were carried out with 35 cycles ofŽ . Ždenaturation 1 min at 948C , annealing 2 min at

. Ž .558C , and extension 2 min at 728C , with 10 ng oftemplate in a 100 ml reaction containing 50 pmoleach of primer and 2.5 U Pfu DNA polymerase.

2.3. Expression and purification of the fusion pro-teins

Ž .BL21 lDE3 pLysS cells were transformed withpV -PhoA or pV -PhoA, and cultured at 378C for 16H L

h on LB medium containing 1.5% agar and antibi-Žotics 50 mgrml ampicillin and 34 mgrml chlor-

. Ž .amphenicol . BL21 lDE3 harboring pLysS wasŽ .used instead of BL21 lDE3 because of its repro-

ducible protein expression. Single colonies werepicked and cultured at 288C in 1 l LB mediumcontaining antibiotics as above. When the cell den-sity reached an OD of 0.3–0.4, IPTG was added600

to a final concentration of 0.1 mM to induce theexpression of V -PhoA or V -PhoA protein. TheH L

cells were then cultured for 16 h, and in order toconfirm the target protein expression, 20 ml of theculture was used for SDS-PAGE analysis. The re-maining culture broth was centrifuged and the cellpaste was collected. Cells were thoroughly resus-

Žpended in sonification buffer 50 mM NaH PO , 102 4.mM Tris–HCl, 100 mM NaCl, pH 8.0 and lysed by

sonification with a Branson Sonifier with 60 Woutput, 50% duty cycle per second for 20 min on ice.

After centrifugation at 15,000=g for 20 min at 48C,the supernatant was applied to 1 ml Talone metal

Ž .affinity column Clontech, Palo Alto, CA, USA .The column was washed with 20 ml of the sonifica-tion buffer, and bound proteins were eluted in 1 ml

Žfractions with elution buffer 50 mM NaH PO , 202 4.mM MES, 100 mM NaCl, pH 5.0 . V -PhoA wasH

purified further using FPLC with a MonoQ HR5r5Žanion exchange column Pharmacia, Uppsala, Swe-

.den . The fractions containing V -PhoA were con-HŽ .centrated with Cetricon-50 Amicon, MA, USA ,

and dialyzed against 50 mM Tris–HCl, 150 mMNaCl, pH8.0. The protein purity was judged bySDS-PAGE.

2.4. Preparation of V and V fragmentsH L

V and V fragments of HyHEL-10 were ex-H LŽ .pressed and purified as described Ueda et al., 1996 .

The fragments obtained were stored in 20 mM sodiumphosphate buffer pH 7.4 containing 150 mM NaCl,at y808C until use.

2.5. Open Sandwich ELISA

The Open Sandwich ELISA was performed essen-Ž .tially as described Ueda et al., 1996 . Briefly, VL

and V fragments were biotinylated using NHS-Bio-HŽ .tin Pierce, IL, USA according to the supplier’s

manual. A total of 100 ml of 10 mgrml streptavidinin PBS were added to each well of a microtiter plateŽ .Falcon 3912, Becton Dickinson, Oxnard, CA, USAand incubated for 16 h at 48C. After removal of thesolution, the wells were blocked with 2% skimmedmilk powderrPBS. The plate was incubated for 2 hat 258C, and after discarding the buffer, 100 ml of

Ž .biotinylated V or V fragment 10 mgrml in PBSL H

were added to each well. The plate was incubated for1 to 2 h at 258C, and the wells washed three times

Ž .with 0.1% Tween-20rPBS PBS-T . Then, 10 mlvolumes of 10 times concentrations of HEL and 90

Ž .ml of 4 mgrml V V -PhoA in PBS were added.H L

The plate was incubated for 1 to 2 h at 258C andwashed three times with PBS-T. Then 100 ml of

Žalkaline phosphatase substrate solution 1 mM p-NPP, 1 M Tris–HCl, 10 mM MgCl , 50mM ZnCl ,2 2

.pH 8.0 were added to each well and color developedfor up to 1 h before the absorbance at 405 or 410 nm

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184 175

was measured. When binding to a HEL-coated plateŽwas measured, 100 ml freshly prepared HEL 10

.mgrml in PBS were coated onto the wells of aFalcon 3912 plate instead of streptavidin.

( )2.6. Preparation of V -PhoA D101S mutantH

Site-directed mutagenesis was performed onAsp101 of the alkaline phosphatase-active site by the

Ž .method of Kunkel 1985 . Using uridine-incorpo-¨rated ssDNA of pV -PhoA as a template and syn-H

thetic oligonucleotides of 5X-TGA TGC AGC CGAGCT CGT GAC GTA GTC CG-3X as a primer tointroduce the mutation, Asp101 was mutated to ser-

Ž .ine, resulting in pV -PhoA D101S . DNA sequenc-H

ing of the enzyme-coding region verified the pres-Ž .ence of the desired mutation. V -PhoA D101S wasH

expressed and purified by the same method as thatfor V -PhoA.H

2.7. Preparation of wild type and mutant alkalinephosphatases

The fragment encoding alkaline phosphatase mu-Ž .tant PhoA D101S was obtained by digesting pV -H

Ž .PhoA D101S with NotI, and was inserted intoŽ .pET20b at the NotI site, resulting in pPhoA D101S .

Ž .PhoA and PhoA D101S were expressed and puri-fied with a Talone metal affinity column as de-

Ž .scribed Suzuki et al., 1997 . The protein purity wasjudged by SDS-PAGE.

2.8. Determination of enzymatic actiÕity

The phosphatase activities of V -PhoA and V -H HŽ .PhoA D101S were measured spectrophotometrically

utilizing p-NPP as the substrate, and the kineticparameters were determined. The reaction was moni-tored by following the increase in absorbance at 410

Ž 4 y1 y1.nm Es1.62=10 M cm . All measurementswere performed at 258C in 1 M Tris–HCl buffercontaining 10 mM MgCl and 50 mM ZnCl . The2 2

kinetic parameters were determined by monitoringŽ .initial reaction velocities Õ at several substrate

Žw x . y3 y5concentrations S from 1=10 to 1=10 M0

with 0.25–0.5 mgrml protein. In order to evaluatethe pH profile of the enzyme, the substrate pH wasvaried from 7.5 to 10. The values V , K and kmax m cat

w xwere calculated by least-squares-fitting of S rÕ0w x 2against S plot of six to eight points with r )0.98.0

The kinetic parameters of wild type PhoA andŽ .PhoA D101S were also determined as controls.

3. Results

3.1. Preparation of the fusion proteins

In order to construct the fusion protein expressionvectors pV -PhoA and pV -PhoA, DNA sequencesH L

encoding either the H or L chain V region of Hy-HEL-10 were fused to the alkaline phosphatase gene

X Ž .at its 5 terminus Fig. 2A . Appropriate DNA frag-ments were obtained by the PCR. V and V genesH L

were obtained from plasmid pKTN2, encoding theFv region of HyHEL-10, while the alkaline phos-phatase gene was obtained from E. coli chromoso-mal DNA. A sequence corresponding to nine amino

Žacids NH - Gly Glu Ser Lys Leu Ala Ala Ala Gly2.-COOH was placed between the two DNAs to

create enough space between the two domains toendow the fusion protein with full antigen bindingand enzymatic activities. According to the Chou–

ŽFasman protein secondary structure prediction Chou.and Fasman, 1978 , most of these amino acids have

a tendency to form a helix, so it is possible that thesenine amino acids form a stable helical spacer. Fig.2B shows the nucleotide and amino acid sequencesof this region in pV -PhoA.H

Ž .The E. coli strain BL21 lDE3 pLysS was trans-formed with each expression plasmid. Protein ex-pression was induced by adding IPTG and SDS-PAGE analysis of all the bacterial proteins wasperformed to confirm the expression of the targetproteins. Fig. 3A shows the Coomassie stained gel ofthe whole cellular proteins under IPTG induced andnon-induced conditions. In the induced conditions, apredominant band of approx. 60 kDa was visible inthe lane containing V -PhoA, while a band of ap-L

proximately 70 kDa was visible in the lane contain-ing V -PhoA. Since the calculated molecular weightsH

of V -PhoA and V -PhoA are 61.7 and 60.6 kDa,H L

respectively, the 60 kDa band should be V -PhoA.L

However, the heavier band which is supposed to beV -PhoA is approximately 10 kDa larger than itsH

calculated molecular weight. This difference may be

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184176

Ž .Fig. 2. Structure of the fusion proteins. A Schematic representation of the plasmid DNA region encoding fusion proteins. The sequence forŽ .V was encoded in pV -PhoA or in pV -PhoA D101S , and that for V was in pV -PhoA. Genes encoding each fusion protein are underH H H L L

the control of the T7 promoter. The plasmids have a coding sequence specifying the 5X-pelB signal peptide and 3X-6 =His affinity tag. InŽ . Ž .pV -PhoA D101S , a point mutation which enhances PhoA activity was introduced at the site as indicated. B Nucleotide and amino acidH

sequences of the joint region in pV -PhoA.H

attributed to the large mobility difference betweenŽ .V and V Ueda et al., 1993 . Although a part ofH L

the expressed fusion protein was found in the insolu-ble fraction as inclusion bodies, more than 50% ofthe total expressed protein was in the soluble fraction

Ž .for V -PhoA and V -PhoA data not shown . TheH L

expressed protein was also partly leaked into theŽ .media data not shown .

The induced cells were collected from overnightculture and cell lysates were prepared. Since theprotein should be expressed as a dimer containingtwo C-terminal 6=His affinity tags, the lysate wassubjected to direct purification with metal chelationchromatography. V -PhoA protein could be purifiedL

to homogeneity with this step, whereas V -PhoAHŽprotein resolved into two major bands approx. 70

.and 50 kDa . As judged from the result of theSDS-PAGE shown in Fig. 3A, the 70 kDa bandappeared to correspond to the target fusion protein.To eliminate the smaller band, the metal affinity-purified V -PhoA protein was subjected to anionH

exchange chromatography for further purification.Fig. 3B shows Coomassie stained bands of fractionsfrom the purification process of V -PhoA proteinH

Ž .and Fig. 3C lanes 2 and 3 shows each fusion

protein after purification. The yields of the purifiedV -PhoA and V -PhoA from soluble fractions of 1H L

litre culture were approximately 5 and 7 mg, respec-tively.

3.2. Open Sandwich ELISA

First, the antigen binding activity of the purifiedV -PhoA protein was estimated by ELISA usingH

HEL-coated microtiter plates. In the absence of VL

fragment, 20 mgrml of V -PhoA protein showedH

negligible binding to a HEL-coated plate as assessedŽby bound PhoA activity after washing A of 0.02405

.after 1 h reaction at pH 8.0 . However, when thesame concentration of V -PhoA was coincubatedH

Ž .with V fragment 1.0 mgrml , significant PhoAL

activity was observed, probably due to stable com-Žplex formation of V -PhoA, V and HEL A ;0.8H L 405

.under the same conditions . The result agreed withŽthe previous studies using V -displayed phage UedaH

.et al., 1996 , suggesting that functional fusion pro-teins were obtained. Using the purified proteins andmicrotiter plates, an Open Sandwich ELISA wasattempted with either V -PhoA protein and isolatedH

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184 177

Ž .Fig. 3. Expression and purification of the fusion proteins. A Analysis of the expressed proteins by SDS-PAGE. Cells from 20 ml ofŽ . Ž .Ž . Žovernight culture were subjected to 10% PAGE. The gel was stained with CBB-R250. Lanes: 1 and 2 BL21 lDE3 pLysS, pV -PhoA ; 3H

. Ž .Ž . Ž . Ž .Ž Ž ..and 4 BL21 lDE3 pLysS, pV -PhoA ; 5 BL21 lDE3 pLysS, pV -PhoA D101S . 1, 3, 5 are induced with IPTG, while 2 and 4 areL HŽ . Ž .non-induced. The positions of the molecular weight markers Pharmacia are indicated. B Purification of V -PhoA. V -PhoA was purifiedH H

Žto homogeneity with metal affinity and anion exchange chromatography. The conditions of the SDS-PAGE were the same as above. Lanes. Ž . Ž . Ž . Ž . Ž .1 Total soluble protein; 2 affinity-purified protein eluted fractions ; 3 eluate from MonoQ column. C Proteins 2 mg each after

Ž . Ž . Ž . Ž . Ž .purification. Lanes: 1 Molecular weight markers; 2 V -PhoA; 3 V -PhoA; 4 V -PhoA D101S .H L H

V fragments, or V -PhoA protein and isolated VL L H

fragments. Fig. 4A shows the result for the formercase. There appeared to be little nonspecific bindingof V -PhoA to streptavidin-coated wells, since veryH

low signal was obtained in the absence of immobi-lized V fragment. In the presence of immobilizedL

V fragment, HEL-concentration dependent bindingL

of V -PhoA was clearly observed. The minimumH

detectable concentration was 10 ngrml, which was

equivalent or even superior to the value obtainedŽ .with V -displayed phage Ueda et al., 1996 . Fig. 4BH

shows the HEL-concentration dependency in theconverse situation, namely immobilized V and V -H L

PhoA. Again, little nonspecific binding of the V -L

PhoA to streptavidin-coated wells was observed. Inaddition, HEL-concentration dependency was repro-ducibly observed in the presence of immobilized VH

fragment. The minimum detectable concentration was

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184178

Ž .Fig. 4. Open Sandwich ELISA. A HEL dose response assayedŽ .with V -PhoA. V was immobilized and V -PhoA 4 mgrmlH L H

and various concentrations of HEL were added. The amount ofV -PhoA bound to the plate was assayed by alkaline phosphataseH

activity. Some 1 mM p-NPP in 1 M Tris–HCl pH 8.0 was usedŽ .for color development, and 410 nm absorbance was measured v

Ž .with immobilized V , after 1.0 h reaction; ^ with immobilizedLŽ . Ž .V , after 0.5 h reaction; ` without V , after 1.0 h reaction. BL L

HEL dose response assayed with V -PhoA. V was immobilizedL HŽ .and V -PhoA 4 mgrml and various concentrations of HEL wereL

Ž .added. The amount of bound V -PhoA was assayed as above vLŽ .with immobilized V , after 1.0 h reaction; ^ with immobilizedH

Ž .V , after 0.5 h reaction; ` without V , after 1.0 h reaction.H H

Assays were carried out in triplicate and the values shown aremeans. Error bars represent standard deviations.

50 ngrml. Although the maximum absorbance at410 nm after a 1 h reaction was almost the same inboth experiments with V -PhoA and V -PhoAH L

Ž .0.27–0.28 at a HEL concentration of 1 mgrml thedetection limit was somewhat different.

3.3. Mutagenesis of alkaline phosphatase

The drawbacks to this assay so far were that thedetection signals were relatively low and it took aconsiderable period of time to obtain sufficientchanges in absorbance. In order to address therepoints, we attempted to enhance the alkaline phos-phatase activity by mutating the residue close to theactive site which determines the release rate of phos-

Žphate. Following previous work Mandecki et al.,.1991; Chen et al., 1992 , Asp101 of the alkaline

phosphatase domain in V -PhoA was changed to SerH

by site-directed mutagenesis. Using the vector pV -HŽ . Ž .phoA D101S , the V -PhoA D101S mutant proteinH

was successfully obtained, with a yield similar toŽthat of V -PhoA of 5 mg per 1 l Fig. 3A, lane 5,H

.Fig. 3C, lane 4 .Ž .Employing V -PhoA D101S and immobilizedH

V , another Open Sandwich ELISA was performed.L

As expected, the signal was enhanced to almost

Fig. 5. Open Sandwich ELISA with mutant V -PhoA. HEL doseHŽ .response assayed with V -PhoA and V -PhoA D101S . V wasH H L

Ž . Ž .immobilized and V -PhoA or V -PhoA D101S 4 mgrml andH H

various concentrations of HEL were added. The assay was per-formed in triplicate with the same conditions as described in Fig.

Ž .4. The color development was measured after 1.0 h reaction. v

Ž . Ž .with V -PhoA D101S and immobilized V . ^ With V -PhoAH L HŽ . Ž .and immobilized V . ` With V -PhoA D101S only.L H

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184 179

double compared to the assay with wild type V -H

PhoA, and the maximum absorbance at 410 nm after1 h reaction was 0.54 at a HEL concentration of 1

Ž .mgrml Fig. 5 . The minimum detectable concentra-tion was around 10 ngrml, which was almost thesame value as that with wild type V -PhoA.H

3.4. The steady-state kinetics of the proteins

In order to evaluate the effects of fusion andamino acid substitution on alkaline phosphatase ac-tivities, the steady-state kinetic parameters of V -H

Ž .PhoA, V -PhoA D101S , wild type PhoA andHŽ .PhoA D101S were obtained. Table 1 lists the ki-

netic constants of each protein at 1 M Tris–HCl, pH8.0 with the substrate p-NPP. Under these condi-tions, the k and the K values of wild type PhoAcat mŽ . y1control were 91 s and 37 mM, respectively. The

Ž .mutant PhoA D101S displayed approximately 2.6-fold enhancement of the k value compared withcat

that for wild type PhoA, whereas some increase inK was also observed. The k value for V -PhoAm cat H

was decreased approximately 2.1-fold compared withthat of wild type PhoA, and the K was increased tom

125 mM, resulting in a 7.2-fold decrease in thecatalytic efficiency as expressed by the k rKcat m

ratio. The same tendency was also observed in theŽ .case of V -PhoA D101S . A certain increase in theH

Table 1Kinetic parameters of the wild type and the mutant alkalinephosphatase at pH 8.0

bk K k rKcat m cat my1 y6 y1 y1Ž . Ž . Ž .s mM 10 M s

VH-PhoA 43.0 124.9 0.35Ž .VH-PhoA D101S 70.3 173.5 0.41

Ž .Wild type PhoA 90.9 36.9 30 2.46aŽ .reported

Ž . Ž .PhoA D101S 235.6 51.6 56 4.57Ž .reported

Kinetic parameters were calculated based on Michaelis–Mentensteady-state kinetics.Assays were carried out at 258C in 2 ml substrate solutioncontaining various concentrations of p-NPP, 10 mM MgCl , 502

mM ZnCl , 1 M Tris–HCl, pH 8.0.2a Ž .Values reported by Mandecki et al. 1991 .b The k values are calculated from the V assuming a dimercat max

molecular weight of 100,700 for wild type PhoA andŽ . Ž .PhoA D101S , and 123,400 for VH-PhoA and VH-PhoA D101S .

Fig. 6. pH profile of the alkaline phosphatase activity. pH vs.Ž .alkaline phosphatase activity k profiles for each protein arecat

shown. The k values were calculated from the V as de-cat maxŽ . Ž . Ž . Ž .scribed in Table 1. B V -PhoA, v V -PhoA D101S , IH H

Ž . Ž .Wild type PhoA, ` PhoA D101S .

k and K was observed compared with those ofcat m

V -PhoA, but the k value was reduced by approx-H catŽ .imately 3.3-fold compared to that of PhoA D101S

with a corresponding decrease of 11.2-fold in thek rK ratio.cat m

3.5. pH profiles of the proteins

The pH profiles of the alkaline phosphatase activ-Ž .ity for V -PhoA, V -PhoA D101S , wild type PhoAH H

Ž .and PhoA D101S are shown in Fig. 6. The specificactivities of the proteins increased as the pH in-

Ž . Ž .creased. PhoA D101S and V -PhoA D101S andH

wild type PhoA showed significant increases in kcat

between pH 8.0 and 9.0, while V -PhoA showedH

little difference from pH 8.0 to 10.0. At pH 10.0,Ž .V -PhoA D101S was 3.2-fold more active thanH

Ž .V -PhoA, while PhoA D101S showed 2.3-fold moreH

activity than wild type PhoA.

3.6. Open Sandwich ELISA at optimal pH

Following the above result, the Open SandwichELISA was performed again at pH 10 to test whetherincreased readings were obtained. In addition, each

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184180

Fig. 7. Open Sandwich ELISA at optimal pH. The assay wasŽperformed as in Fig. 5 except for the shorter incubation times See

.Section 3 . The amount of bound protein was assayed by PhoAactivity at pH 10.0. The color development was measured after 0.5

Ž . Ž . Ž .h reaction. v With V -PhoA D101S and immobilized V . ^H LŽ . Ž .With V -PhoA and immobilized V . ` With V -PhoA D101SH L H

only.

incubation period with biotinylated V and withL

fusion proteinrsample was shortened to 1 h at 258C,with continuous shaking on a microplate mixer. Fig.7 shows the comparison between V -PhoA and itsH

mutant with 30 min reaction time. As expected, theŽ .signal obtained with V -PhoA D101S and 1.0H

Ž .mgrml HEL A of 0.58 was almost twice the405Ž .value obtained with V -PhoA A of 0.27 . De-H 405

spite a considerable time reduction, the minimumdetectable HEL concentration was still less than 25ngrml, and the absorbance changes were similar tothose achieved under the previous assay conditionsŽ .Fig. 5 .

4. Discussion

The Open Sandwich ELISA is a newly developedimmunoassay method based on the interaction be-

Ž .tween V and V fragments Ueda et al., 1996 . Fig.H L

1 shows the comparison of the Open SandwichŽ .ELISA Fig. 1A,C and conventional sandwich

Ž .ELISA Fig. 1B . The most remarkable feature of the

Open Sandwich ELISA is that the assay essentiallyrequires only one specific antibody. Only one cycleof incubation and washing procedures is necessary,and this novel assay may be applied to the quantita-tion of antigens possessing only a single epitope,where the antigen can increase the Fv stabilityŽ .Glockshuber et al., 1990 . In this study, we haveexamined the possibility of performing a direct OpenSandwich ELISA using alkaline phosphatase-con-jugated V or V fragments. By using these fusionH L

Žproteins instead of phage-displayed V Winter etH.al., 1994; Ueda et al., 1996 , genuine one cycle

measurement has been achieved.The major merit of employing such fusion pro-

teins is that we can save time on the incubation andwashing steps compared with the phage system,which considerably reduces the time required for thewhole assay. Furthermore, there appear to be somemore advantages in adopting such fusion proteins.Antibodies chemically coupled to enzyme labels arewidely used as immunoassay reagents today. How-ever, chemical coupling reactions sometimes yieldundesirable cross-linked molecules which need to beremoved before measurements are performed. Genet-ically engineered fusion proteins such as our fusionproteins are intrinsically homogeneous and directlyproduced, and are thus attractive alternatives tochemical conjugates. Another advantage is that, sincethe fusion proteins were strongly expressed in E.coli, and easily purified with good yields, the pro-duction costs of the protein are much lower than thatof antibody from in vitro hybridoma culture or fromthe abdominal cavity of mice. It should be mentionedalso that a considerable proportion of fusion proteinwas expressed in soluble form, so that a tediousrefolding procedure was not necessary.

We have chosen E. coli alkaline phosphatase as areporter molecule. Alkaline phosphatase is a nonspe-cific phosphomonoesterase with a high catalytic

Ž . Žturnover number k Gettins and Coleman,cat.1983a,b; Gettins et al., 1985 . It is one of the most

commonly used enzyme labels, and the constructionof fusion proteins of alkaline phosphatase and anti-body-derivatives has been extensively attemptedŽWels et al., 1992; Ducancel et al., 1993; Weiss andOrfanoudakis, 1994; Gandecha et al., 1994; Carrier

.et al., 1995; Suzuki et al., 1997; Furuta et al., 1998 .We have previously constructed a bacterial expres-

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184 181

sion vector for producing a fusion protein of hapten-specific single-chain Fv and alkaline phosphataseŽ .Suzuki et al., 1997 . In that case, the fusion proteinwas efficiently expressed in the soluble fraction withboth antigen binding and enzymatic activities re-tained. Taking advantage of this, we used the sameexpression system to produce V -PhoA and V -H L

PhoA.In Fig. 3B, V -PhoA was resolved into two majorH

Ž .bands approx. 70 and 50 kDa after metal affinitypurification. Since the calculated molecular weights

Žof V -PhoA and PhoA including 6=His affinityH.tag are 61.7 and 50 kDa, respectively, it is probable

that the 70 kDa band is V -PhoA and the 50 kDaH

band is a degradation product, presumably the PhoAdomain. Because the intrinsic alkaline phosphatase

Ž .activity of BL21 lDE3 pLysS lysate was negligibleŽ .data not shown , it is unlikely that V -PhoA forms aH

heterodimer with a host-derived PhoA protein. SinceV -PhoA was purified to homogeneity using metalL

affinity chromatography, V -PhoA was thought to beH

more susceptible than V -PhoA to protease attackL

during production and purification. In fact, SDS-PAGE of a batch of purified 70 kDa V -PhoAH

protein stored for 4 months at 48C showed almostequal quantities of 50 and 70 kDa moieties, with the

ŽELISA signal decreased almost to 50% data not.shown . It might be possible to prevent such degra-

dation by the addition of protease inhibitors andstorage at lower temperature. Nevertheless, it shouldbe noted that V -PhoA is still much more stable thanH

the V monodomain. V -PhoA could still be usedH H

for an Open Sandwich ELISA after several weeks ofstorage at 48C, while isolated V begins to denatureH

and aggregate after only one day under the sameŽ .conditions data not shown . It is possible that the

V domain is stabilized when fused to a largerH

moiety by being protected from nonspecific collisionand subsequent aggregation.

Fig. 4A and B show that both V -PhoA andH

V -PhoA could be used for the Open SandwichL

ELISA, with a sensitivity similar to that with V -HŽ .Phage Ueda et al., 1996 . In addition, the sensitivity

of these assays was almost equal or even superior tothat of the previous indirect sandwich ELISA. Al-though signal obtained with 1 mgrml HEL wasalmost the same, the minimum detectable concentra-

Ž .tion of HEL differed from 10 ngrml V rV -PhoAL H

Ž .to 50 ngrml V rV -PhoA . Though the preciseH L

mechanism is not clear, the difference in minimumdetection level is possibly explained by the tendency

Žof V to form dimers Maeda et al., 1978; Stevens etL.al., 1980 . There are two V domains in V -PhoAL L

and it is possible that these can form an intramolec-ular homodimer. Since intramolecular V homod-L

imerization reactions compete with V –V het-H L

erodimerization, V -PhoA may show less binding toL

V than monomeric V . This effect would be moreH L

prominent at low HEL concentrations because HELpromotes V –V associations and thus inhibits VH L L

homodimerization. When sensitivity is required andconsidering the relative stabilities of the proteins, VL

and V -PhoA would be the preferred combination inH

the Open Sandwich ELISA, although care should betaken to prevent inactivation of the V -PhoA protein.H

For other Fv, the alternative pairing may be used ifimmobilized V and V -PhoA proteins are thoughtH L

to be more stable.In this study, biotinylation of V or V wasL H

adopted to efficiently immobilize them onto thestreptavidin plate. We have considered biotinylatingthe fragments in vivo by genetically conjugatingthem with a biotinylation peptide which mimics bi-

Ž .otin carboxyl carrier protein BCCP , to further facil-Ž .itate this process Schatz, 1993 . However, prelimi-

nary results for the expression of V -biotinylationL

peptide fusion protein showed inefficient in vivobiotinylation presumably due to rapid secretion into

Ž .the periplasm not shown . The recently reportedcytoplasmic expression system for the production offusion proteins with biotinylation peptide-tagged

Žthioredoxin might solve this problem Smith et al.,.1998 .

Alkaline phosphatase is a metalloenzyme thatcontains two Zn2q ions and one Mg2q ion in each

Ž .monomer active site region Sowadski et al., 1985 .Several point mutations in the vicinity of the activesite have been shown to increase the enzymatic

Žactivity of alkaline phosphatase Mandecki et al.,.1991; Matlin et al., 1992; Janeway et al., 1993 .

Ž .Among these, Mandecki et al. 1991 showed thatalteration of the amino acid residue Asp-101 to SerŽ .D101S resulted in a dramatic increase in k com-cat

pared with wild type alkaline phosphatase. Accord-ing to the report, the steady-state kinetic parametersof the D101S mutant displayed 35-fold enhancement

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184182

of specific activity over wild type at pH 10.0. Thisenhancement of catalytic activity is due to a loweraffinity towards phosphate which leads to a quickercatalytic turnover by allowing faster release of the

Ž .phosphatase product Chen et al., 1992 . Followingthese reports, we have changed Asp101 of the V -H

PhoA alkaline phosphatase domain into Ser by site-directed mutagenesis, with the aim of enhancing thealkaline phosphatase activity.

We have performed an Open Sandwich ELISAusing immobilized V fragment and V -PhoAL HŽ .D101S . A dramatic enhancement of the detection

Ž .signal was expected by adopting V -PhoA D101SH

instead of V -PhoA. However, only a modest two-H

fold increase in signal was obtained. As we supposedthat this low enhancement of alkaline phosphataseactivity might have been caused by tethering anothermolecule at its N terminus, we genetically prepared

Ž . Ž .unfused PhoA wild type PhoA and PhoA D101Senzymes, calculated their kinetic parameters, andcompared the values with those of V -PhoA andH

Ž .V -PhoA D101S . Convincing decreases in enzy-H

matic activities were observed on tethering V frag-HŽ .ments to both PhoA and PhoA D101S . The kcat

values of the fusion proteins were less than 50% ofthose for unfused enzymes. Moreover, there weresignificant increases in the K of the fusion proteinm

which correlate with lower catalytic efficiencies.In a previous study, we constructed a fusion

Ž .protein of anti-hapten single-chain antibody V –VL HŽ .and alkaline phosphatase, named ScFv NP -PhoA

Ž .Suzuki et al., 1997 . Although the expression vec-Ž .tors of ScFv NP -PhoA and V -PhoA are identical toH

each other except for the antibody coding region,virtually no decrease in alkaline phosphatase activitywas observed in the former protein. We believe thatthe decrease in PhoA activity observed with the VH

fusion proteins might be related to the purificationŽ .process. In contrast to ScFv NP -PhoA that was

affinity purified with NP-Sepharose, V -PhoA itselfH

had low antigen binding affinity, and we followed aHis-tag mediated purification approach. It is possiblethat a considerable amount of misfolded inactiveV -PhoA was contained in the soluble fraction, andH

affected the specific enzymatic activity of the puri-fied protein. However, it is also possible that thehybrid protein with correctly folded V domains hadH

also correctly folded PhoA domains with full enzy-

Ž .matic activity as in the case of ScFv NP -PhoA, anddid not affect the sensitivity of the Open Sandwichassay. The low background signal observed impliesnegligible coexistence of misfolded V domains andH

functional PhoA domains.Ž . Ž .The V k of PhoA D101S showed 2.6-foldmax cat

increase relative to PhoA when measured in 1 MTris–HCl, pH 8.0, which is only one-sixth of the

Ž .enhancement reported by Mandecki et al. 1991Ž . Ž .Table 1 . V -PhoA D101S also showed only aH

1.6-fold increase in V compared with V -PhoA.max H

Because we have confirmed the mutation by DNAsequencing of the entire coding region, one possibleexplanation of this lower enhancement is the effectof additional vector-derived N-terminal residues orthe C-terminal His-tag sequence. According to the

Ž . Ž .report of Mandecki et al. 1991 , PhoA D101Sshowed 35-fold increase of specific activity overwild type at pH 10.0. We also obtained the pHprofiles of each protein as shown in Fig. 6. Thespecific activities increased as the pH increased, butno such dramatic difference was observed at pH 10.0in our study. The reason why this discrepancy inkinetics occurred is still to be established.

Association affinities between V and V seg-L H

ments are known to differ among antibodies. Severalstudies of the interactions between V and V in theL H

absence of antigen have been reported. Many anti-bodies, originating from hybridomas, showed rela-tively weak association of V and V segments inL H

Žthe absence of antigen Klein et al., 1979; Ueda et.al., 1993 , whereas some showed stronger associa-

Žtions Horne et al., 1982; Polymenis and Stollar,.1995 . On the other hand, it is also known that most

V –V pairs from ScFv fragments selected fromH LŽphage libraries show low affinity G. Winter, per-

.sonal communication . Since the principle of theOpen Sandwich ELISA is based on the weak V –VL H

association in the absence of antigen and strongV –V association in the presence of antigen, it isL H

essential to use appropriate antibodies. In this study,the Open Sandwich ELISA was successfully estab-lished using fusion proteins of alkaline phosphataseand HyHEL-10 V rV . In such a situation, thisH L

simple assay can be a better alternative to the con-ventional sandwich ELISA. However, in order tomake this assay more generally useful, applicationsto other antibodies should be shown in future.

( )C. Suzuki et al.rJournal of Immunological Methods 224 1999 171–184 183

Acknowledgements

We thank Kazuishi Kubota and Yu Wang for theirhelp with the preparation of V rV fragments. ThisH L

work was partly supported by Grant-in-aids for Sci-Ž .entific Research B09450301 and for Encourage-

Ž .ment of Young Scientists A08750916 from theMinistry of Education, Science, Sports and Culture,and funded by Biodesign Research Promotion Groupof the Institute of Physical and Chemical ResearchŽ .RIKEN , Japan.

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