Direct Construction of an Open-Sandwich Enzyme Immunoassay for

Jan 7, 2011 - Lifescience Laboratory R&D, Fujifilm Co., 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa, 258-8577, Japan. Anal. Chem. , 2011, 8...
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Direct Construction of an Open-Sandwich Enzyme Immunoassay for One-Step Noncompetitive Detection of Thyroid Hormone T4 Kamrun Nahar Islam,† Masaki Ihara,‡ Jinhua Dong,† Noriyuki Kasagi,§ Toshihiro Mori,§ and Hiroshi Ueda*,†,‡ †

Department of Chemistry and Biotechnology, and ‡Department of Bioengineering, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8656, Japan § Lifescience Laboratory R&D, Fujifilm Co., 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa, 258-8577, Japan

bS Supporting Information ABSTRACT: To establish a sensitive noncompetitive immunoassay for thyroxine (T4), we attempted to isolate anti-T4 antibodies from a phage display library based on a phagemid pDong1 (Dong et al. Anal. Biochem. 2009, 36, 386), which was designed to enable open-sandwich enzyme-linked immunosorbent assay (OS-ELISA) after selection on immobilized antigen. After the Fab-displaying phage library made from the splenocytes of T4-KLH immunized mice was subjected to biopanning on T4BSA, two T4-specific clones were obtained. When they were assayed by indirect competitive ELISA, both clones showed low IC50 (5-13 ng/mL), indicating their high affinity to T4. When they were used for OS-ELISA that detects antigen-dependency of the interaction between variable domains VH and VL, a clone successfully detected 1 ng/mL of T4 with a working range superior to that of competitive IA. OS-ELISA was also performed with maltose binding protein (MBP)-fused VH/VL of this clone, which showed a detection limit less than 0.1 ng/mL T4. Moreover, the assay showed crossreactivity with T3 similar to that of competitive ELISA, and also gave a reasonable total serum T4 concentration (90 ng/mL) from ethanol-extracted sample serum using the recombinant proteins. This is the first direct construction of an OS-ELISA system bypassing hybridoma, which will be applicable to the detection of many other small molecule antigens.

hyroxine (T4; 3,30 ,5,50 -tetraiodothyronine, fw: 776.87) is the most commonly measured thyroid hormone for diagnosis of thyroid function. Stimulation of thyroid gland by the pituitary hormone (TSH) causes the release of T4 in bloodstream. It has its primary influence on protein synthesis and oxygen consumption in virtually all tissues but it is also important for growth, development, and sexual maturation. Greater than 99% of T4 is reversibly bound to three plasma proteins: blood thyroxine binding globulin (TBG) binds 70%, thyroxine-prealbumin (TBPA) binds 20%, and albumin binds 10%. While approximately 0.03% of T4 exists free in the blood, only the free (unbound) portion of T4 is responsible for the biological action. Thyroid function is assessed through the measurement of T4 level. Increased levels of T4 have been found in hyperthyroidism due to Graves’ disease and Plummer’s disease and in acute and subacute thyroiditis.1 Low levels of T4 have been associated with congenital hypothyroidism, myxedema, chronic thyroiditis (Hashimoto’s disease), and some chronic genetic abnormalities. At present, patients suffering from thyroid diseases are increasing, and a convenient and highly sensitive assay kit for thyroid hormones especially T4 is strongly desired. The methods proposed up to now for the determination of T4 are HPLC,2 radioimmunoassay,3,4 fluorescence immunoassay,5 electrochemiluminescence,6,7 and direct amperometry.8,9 Furthermore, various enzyme immunoassays (EIA) for T4 have been reported,10-15 but those techniques are

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based on the competitive IA. Though noncompetitive IA (sandwich ELISA) exhibits high specificity and sensitivity, it requires a long time, having multiple incubation/washing steps. Moreover, small molecules (haptens) are not suitable for the conventional sandwich immunoassay because of the lack of two discrete binding sites. Recently, open-sandwich immunoassay (OS-IA)16-18 has been developed for the noncompetitive detection of haptens. It is based on the phenomenon of increased association of two antibody variable domains (VH and VL) upon binding with antigens. Compared with conventional sandwich IA, OS-IA is superior, as it can measure monovalent antigens such as haptens in noncompetitive format; moreover, it has a wider working range16,19 and shorter measurement time and can be applied for homogeneous immunoassay. However, previous reports of OS-IA including ours16,17,19 and others20 always relied on the use of established hybridoma as a source of antibody genes. While the approach was reliable because of proven affinity and specificity of the used clones, which ensured the resultant sensitivity and selectivity of OS-IA, respectively, the overall procedure and period to elucidate the assay was considerably long. So this time we took a novel Received: October 25, 2010 Accepted: December 15, 2010 Published: January 7, 2011 1008

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Analytical Chemistry approach to directly obtain antibody V region genes from the spleen of immunized mice. With the help of robust phage display methodology, we could successfully isolate anti-T4 antibodies and utilized them to establish OS-IA.

’ MATERIALS AND METHODS All experiments for recombinant DNA techniques and animal experiments were conducted under appropriate biosafety regulations, namely, performed under Biosafety Level 1 practices and under the guidance of animal welfare committee of the University of Tokyo, respectively. Materials. Escherichia coli strain TG-1 (GE Healthcare, Tokyo, Japan) was used for phage production, HB2151 (GE Healthcare) for the production of VH-displaying phage and L chain, XL-10Gold (Stratagene, La Jolla, CA) for recombinant DNA preparation, and BL21 (DE3, pLysS) (Novagen, San Diego, CA) for the production of MBP-VL and MBP-VH. The primers used for amplifying variable region sequences are based on Thomas Grunwald and Greg Winter in “Primer set for generation of highly diversified mouse phage display libraries”, MRC Centre for Protein Engineering, Cambridge, UK (2000) (http://www.mrc-cpe.cam.ac.uk/). A minor modification of the 50 sequence from `GGAACCCTTT’ to `CTTTCTATGC’ was made for VH backward primers. Only the primers for kappa light chains were used for the amplification of VL. Oligonucleotides were obtained from Fasmac Co. (Kanagawa, Japan). The chemicals and other reagents, unless otherwise indicated, were obtained from Sigma (St. Louis, MO), Nacalai Tesque (Kyoto, Japan), or Wako Pure Chemical Industries (Osaka, Japan). Cloning of Antibody V-Genes and Construction of Fab Fragment Library. T4-keyhole limpet hemocyanine (KLH)

and T4-bovine serum albumin (BSA) were prepared as described.21 Two mice were immunized with T4-KLH four times in two week intervals and sacrificed to obtain the spleen 3 days after the last immunization. The cDNA for the antibody variable regions was prepared from total RNA derived from the spleen using Omniscript RT Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s protocol, using oligo dT(18) primers. The following PCR conditions were 94 °C 3 min, 94 °C 1 min, 58 °C 1 min, 72 °C 1 min for 5 cycles and 25 cycles of 94 °C 1 min, 62 °C 1 min, 72 °C 1 min followed by final extension at 72 °C for 1 min. All PCRs were performed with mouse VH/VL-specific primers and Ex-Taq DNA polymerase (Takara-Bio, Otsu, Japan). The PCR products were then purified using Wizard PCR Cleanup System kit (Promega). The purified VL fragments were digested with restriction enzymes SalI and NotI, again purified and ligated using T4 DNA ligase at 16 °C for 16 h with a phagemid pDong1(HyHEL10),22 which had been digested with the same enzymes as well as SbfI that cuts inside HyHEL10 VL, to reduce self-ligation. After confirmation of the inserted VL sequence of several clones out of the obtained ones (∼103), the VH fragments were inserted into the VL-inserted phagemid library using restriction enzymes SfiI and XhoI, while SnaBI that cuts inside HyHEL10 VH was used to reduce self-ligation. Electrocompetent E. coli TG-1 was transformed with the ligation product and plated on 2YTAG agar (16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl, pH 7.2 supplemented with 100 μg/mL ampicillin, 1% glucose, and 1.5% agar) plates overnight at 37 °C. The library size (1.5  105) was estimated from the number of colonies on the plate. Fab Phage Display. Single colonies of E. coli TG1 strain were inoculated to 4 mL of 2YTAG and incubated at 37 °C for

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overnight. The overnight culture was then 100-fold diluted with 2YTAG and incubated at 37 °C until OD600 reached ∼0.5. Then KM13 helper phage was added at moi of ∼20. The cells were incubated at 37 °C without shaking for 30 min and then centrifuged 3000g for 10 min. The cells were resuspended in 2YTAK (containing 100 μg/mL ampicillin and 50 μg/mL kanamycin) and incubated with shaking at 30 °C for overnight. The overnight culture was centrifuged at 6000g for 10 min, PEG/NaCl (20% polyethylene glycol 6000, 2.5 M NaCl) was added to the supernatant, and after 1 h incubation on ice, the supernatant was centrifuged at 6000g for 30 min. The pellet was resuspended in PBS and centrifuged at 10600g for 10 min. The supernatant containing the phage particles was recovered and stored at 4 °C. Biopanning. The phage display library was selected using biotinylated T4-BSA prepared using EZ-Link NHS-LC-biotin (Thermo Scientific), Dynabeads M280 streptavidin (Invitrogen), and Kingfisher apparatus (Thermo Scientific). The beads were washed with 300 μL of PBS three times. Then 1 mL of biotinylated T4-BSA conjugate (10 μg/mL) was added to the beads and incubated for 30 min at 25 °C using a rotating mixer. After being washed four times with 1000 μL of PBS containing 0.1% BSA, the beads were suspended with 100 μL of PBS. Then 900 μL of phage solution (1012 cfu/mL in 0.1% BSA/PBS) was mixed with the beads for 60 min at 25 °C on a rotating wheel. After sedimentation, the supernatant (880 μL) was removed and the remaining solution was used as a suspension. Then the beads were washed six times with PBS containing 0.1% Tween 20 using Kingfisher apparatus, and phages bound to the beads were eluted with 100 μL of 1 mg/mL TPCK-treated trypsin (Sigma) in PBS for 5 min. The eluted phage solution was used to infect TG-1 in log phase, which was plated on several 2YTAG agar plates, and used for phage preparation. Three rounds of biopanning were done. Phage ELISA. A PVC 96-well microplate (Falcon 353912, BD Biosciences) was incubated with 100 μL per well of T4-BSA conjugate (10 μg/mL), BSA (10 μg/mL), or antimyc antibody (1 μg/mL) overnight at 4 °C. Blocked at room temperature for 2 h with 2% skim milk in PBS (MPBS). After being washed three times with PBS containing 0.1% Tween-20 (Sigma) (PBST), the plates were incubated at 25 °C for 1 h with 10 μL of phages from TG1 in 100 μL of 2% MPBS. The plates were washed three times with PBST and incubated at room temperature for 1 h with 100 μL per well of 5000-fold diluted HRP-conjugated mouse anti-M13 monoclonal antibody (GE Healthcare) in 2% MPBS. The microplate was then washed three times with PBST and developed with 100 μL of substrate solution TMB (100 μg/mL 3,30 ,5,50 -tetramethylbenzidine (Sigma) and 0.04 μL/mL H2O2 in 100 mM NaOAc, pH 6.0). The reaction was stopped with 50 μL of 1 M H2SO4 and the absorbance was read at 450 nm with a reference at 655 nm using a microplate reader (Model 680, Bio-Rad, Tokyo, Japan). Competitive ELISA. After a 96-well microplate was coated with or without 100 μL per well of 1 μg/mL T4-BSA in PBS at 4 °C for overnight, the plate was blocked with 2% MPBS for 1 h at 25 °C and washed three times with PBST. The wells were added with total 100 μL/well of phage solution premixed 30 min previously with antigen in a series of concentrations and incubated for 1 h at 25 °C. The wells were washed three times with PBST and incubated with 100 μL/well of 5000-fold diluted HRP/antiM13 monoclonal conjugate in MPBS at 25 °C for 1 h. The solution was washed three times with PBST and developed as above. Open-Sandwich ELISA. To remove hCH1 domain sequence from the vector, pDong1 was digested with SgrAI at 37 °C for 3 h, and the longer band was gel-purified with MinElute (Qiagen) kit. 1009

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Analytical Chemistry The purified fragment was self-ligated using DNA ligation high v2 (Toyobo, Osaka, Japan) and used to transform TG1 cells. Colonies containing hCH1-deleted plasmids were selected by colony PCR using primers M13RV (50 -CAGGAAACAGCTATGAC-30 ) and pHENseq (50 -CTATGCGGCCCCATTCA-30 ), and the culture supernatant containing VH-displaying phage and L chain was prepared as described but without final PEG precipitation. For OS-ELISA, a 96-well microplate was immobilized with 100 μL/well of 1 μg/mL goat antihuman kappa chain antibody (Vector Labs, Burlingame, CA) overnight at 4 °C and blocked with 2% MPBS for 2 h at 25 °C. The microplate was washed three times with PBST, and 50 μL/well of phage solutions that were premixed with varied concentrations of antigen were added. After incubation for 2 h at 25 °C and washing three times with PBST, bound phages were detected as mentioned above. Expression and Purification of MBP-VL Fusion Protein. The VL(D11) gene was amplified by PCR using primers oVLSfiback1 (50 -CGGCCCA-GCCGGCCATGGCCGACTACAAAGAYATTGTDHTVWCHCAGTC-30 ) and VLseqFor (50 -ATTCAGCAGGCACACAACAG-30 ) and pDong1(D11) as a template (Sfi I recognition site is underlined). The purified amplified fragment was digested with SfiI and NotI, gel-purified, and ligated with MBP expression vector pET-MBPp23 digested with the same. E. coli XL10 gold was transformed with the ligation product and incubated on LBAG agar for 16 h at 37 °C. Several colonies were picked up, and the plasmids were extracted for sequence determination. E. coli BL21 (DE3, pLysS) strain was transformed with a recombinant clone carrying the correct insert and plated on an LBAC (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH 7.5, containing 100 μg/mL ampicillin and 33 μg/mL chloramphenicol) agar plate. After incubation for 16 h at 37 °C, a single colony was picked and inoculated to 100 mL of LBAC medium and cultured at 30 °C for 16 h. Afterward, 50 mL of this culture was used to inoculate 800 mL of LBAC medium and cultured further at 30 °C until OD600 reached 0.5-0.8, when the protein expression was induced by the addition of 1 mM isopropyl β-D-thiogalactopyranoside (Wako) to a final concentration of 1 mM. After being cultured at 16 °C for 10 h, the cells were collected by centrifugation at 6000 g at 4 °C for 10 min. The pellet was suspended in 30 mL of PBS and sonicated on ice. The suspension was centrifuged for 10 000g at 4 °C for 30 min. The supernatant was dialyzed against PBS and purified using 2 mL of TALON IMAC (Clontech, Takara-Bio), where samples were eluted in 1.5 mL fractions using elution buffer (20 mM sodium phosphate, 500 mM NaCl, 500 mM imidazole, pH 7.0). The fractions containing target protein were pooled and exchanged for its buffer to PBS using NAP-5 columns (GE Healthcare). The protein was analyzed by SDS-PAGE and was stored in small aliquots at -80 °C. Preparation of HRP-Labeled MBP-VH Fusion Protein. To express MBP-VH, the VH(D11) gene was PCR amplified from phagemid pDong1(D11) using primers M13RV and VH1ForNot2 (50 -CTCATGCGGCCGCGACGGTGACCGTGGTCCCTTGGC-CCC-30 ) and used to make expression vector as above. The expression and purification of MBP-VH protein was performed similarly to MBP-VL. A part (1 mg) of the obtained protein was labeled with horseradish peroxidase (HRP) using HRPperoxidase labeling kit (Dojindo, Kumamoto, Japan) according to the manufacturer and purified once with Ni-NTA spin column (GE Healthcare). Open-Sandwich ELISA Using Recombinant Proteins. The 96-well microplate was immobilized with 100 μL/well of 5 μg/mL

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MBP-VL, 16 h at 4 °C and blocked with 20% Immunoblock (DS-Pharma, Osaka, Japan) in PBS for 2 h at 25 °C. The microplate was then washed once with PBST, and 50 μL/well of various concentrations of antigen in PBS along with 50 μL/well of 2 μg/mL MBP-VH-HRP in PBS supplemented with 10% Immunoblock were added. After incubation for 2 h at room temperature and washing three times with PBST, color development was performed as above. For the serum-derived samples, both the test and control sera were extracted by adding 210 μL of 95% ethanol to test tubes either containing 100 μL of control serum (T3/T4-deficient human, S7144, Sigma) added with different concentrations of free T4, or a test serum without added antigens. After vortex and centrifugation at 15 000g at 4 °C for 10 min, the supernatant was collected and allowed to evaporate in vacuo using a microcentrifuge-concentrator MV-100 (Tomy Seiko, co., Ltd., Tokyo, Japan). After complete evaporation, the pellet was suspended in 100 μL of sterilized water and analyzed as above.

’ RESULTS AND DISCUSSION Cloning of Anti-T4 Antibody Variable Region cDNA. To obtain suitable antibody fragment genes to OS-IA quickly, we took a novel approach to directly obtain antibody V region genes from the spleen of immunized mice. To this end, we utilized a Fab phage display system (pDong system)22 that can select antigen binders as conventional phage display systems do and also perform OS-ELISA soon after the simple vector conversion step (Figure 1). First, to construct a phage library for the selection of T4 binders, the VH and Vκ cDNAs were amplified using specific primers, from the reverse-transcribed RNA extracted from the spleen of mice immunized with T4-KLH. Since we observed amplification of the bands with appropriate length (350-400 bp), the fragments were digested with the restriction enzymes and sequentially ligated into the phagemid pDong1.22 The beauty and power of the phage display system is the coupling of a selectable function (binding to an antigen) to the genetic material that encodes that function. We took the advantage of this methodology to display the Fab fragment using this vector with the help of the KM13 helper phage, which allows specific recovery of antigen-binding phages by protease treatment.24 pDong1 bears two open reading frames (ORFs) for display, namely, VH-hCH1-gIII and VL-hCκ devoid of the C-terminal cysteine residue. When we use an amber suppressor E. coli strain such as TG-1 to produce phage particles, it can express two corresponding proteins that assemble into Fab-pIII (Fd-pIII and L chain), leading to the production of a Fab-phage after infection by a helper phage. With this format, the antigen-binding activity of the displayed Fab was confirmed by phage ELISA. Using this Fab-displaying phage library with an estimated diversity of 1.5  105, three rounds of biopanning were performed using paramagnetic beads immobilized with biotinylated T4-BSA conjugate. Since we could observe significantly higher binding of polyclonal phages to T4-BSA than to BSA in each round, we then screened the phages from the third round by monoclonal phage ELISA (Figure S1, Supporting Information). From the screen of 96 clones, two T4-specific clones (D11 and F11) that show strong binding signals were isolated. Competitive ELISA. To estimate the affinity of the isolated clones, the Fab-displaying phages were prepared on a larger scale, and indirect competitive ELISA with immobilized T4-BSA was performed. As shown in Figure 2A, for both clones, unconjugated T4 completely inhibited the binding of Fab-phages to T4-BSA in a dose-dependent manner. The calculated IC50 values were 1010

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Figure 1. (A) Structure of T4. (B) Principle of OS-ELISA. (C) Construction of Fab-displaying phage library.

Figure 2. Indirect competitive ELISA with Fab-displaying phages. (A) Phages (3  1010 and 1  1010 cfu/mL for D11 and F11, respectively) mixed with free T4 was incubated in the plate immobilized with T4-BSA. (B) Cross-reactivity with T3. Phages displaying F11 Fab were mixed with T3 or T4 and incubated in the plate immobilized with T4-BSA. Bound phages were detected by anti-M13 HRP conjugate. Average of three measurements with 1 SD is shown.

5.3 ( 1.0 and 12.8 ( 1.4 ng/mL (6.9 and 16.6 nM) for D11 and F11, respectively, indicating their high antigen binding affinity. Also, the specificity of the clones against another thyroid hormone 3,30 ,5-triiodothyronine (T3) was investigated. From the curves obtained from similar competitive ELISA, the IC50s for T3 were 303 ( 113 and 701 ( 96 ng/mL for D11 and F11, respectively, showing their specificity for T4 over T3 of 55-57 fold (Figure 2B). OS-Phage ELISA. The advantage of using the pDong1 system is that it affords OS ELISA after a simple procedure of deleting the CH1 domain from its one ORF, which allows us to display

only the VH fragment on the phage. Also, since the ORF for the L chain remains intact, it allows secretion of the protein into the culture supernatant either alone or in association with VH-phage. After the deletion of human CH1 gene from pDong1 by SgrA1 digestion and self-ligation, the resulting phagemids were used for the expression of the VH-displaying phage (VH-phage) and the free light chain (VL-hCκ). This procedure allowed us to rapidly evaluate the interaction between VH and VL and its antigendependency without recloning each fragment. The culture supernatant containing the VH-phage and L chain was added to the 1011

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Figure 3. Phage OS-ELISA with (A) D11 and (B) F11. The open symbols show the values with immobilized antihuman kappa chain antibody. Closed symbols show the value without immobilized antibody, demonstrating nonspecific binding of VH-displaying phages to the plate. Average of three measurements with 1 SD is shown.

Figure 4. OS-ELISA using purified MBP-VL and HRP-MBP-VH of D11. Average of three measurements with 1 SD is shown. (A) A representative dose-response curve. Open and closed circles represent values with and without immobilized antihuman kappa chain antibody, respectively. (B) Cross reactivity with T3. Free T4 and T3 were incubated with HRP-MBP-VH in the plate immobilized with MBP-VL. VL- shows the value without immobilized antibody. (C) Estimation of total T4 in serum using MBP-VL and HRP-MBP-VH.

microplate immobilized with goat antihuman kappa chain antibody, and the bound phages were detected with anti-M13 HRP conjugate. As a result, D11 showed a clear antigen-dependent increase in signal, suggesting its suitability to OS-IA (Figure 3A). According to the dose-response, a significant signal at 1 ng/mL of free T4 was detected. On the other hand, F11 showed a higher signal even in the absence of antigen, but a low signal in the absence of immobilized antilight chain antibody, suggesting stronger VH/VL interaction of this clone than D11 (Figure 3B). V Region Nucleotide Sequences. The results of phage OS ELISA indicated the superiority of D11 in OS-IA compared to F11. To clarify these results, we determined the nucleotide sequences of the two clones. To our surprise, from the deduced amino acid sequences of the VH/VL (Figure S2, Supporting Information), both clones share similar primary structure even within three CDRs for both VH and VL. Namely, according to the IMGT database (http://imgt.cines.fr), the VH gene is composed of IGHV5-12-1/IGHD2-1/IGHJ2 gene segments for the both

clones, while the VL gene is composed of IGKV3-12/IGKJ1 and IGKV4-91/IGKJ5 for D11 and F11, respectively. However, besides primer-derived differences around the VH N-terminus, two residues (37 and 74) in VH and four located in VL (31, 94, 96, and 100) are different between the two, which make them distinctive. Among them, residues H37, L96, and L100 are located at the VH-VL interface and could be attributable to the stronger VH/VL interaction in F11. Especially, the H37 of D11 is a smaller Ala, while that of F11 is Val. This residue is also in the interchain interface and potentially playing an important role in VH/VL interaction. However, a preliminary experiment to exchange VH/VL sequences of D11 and F11 in pDong1 indicated that both VH and VL are important in deciding the VH/VL interaction strength in the absence of antigen (data not shown). OS-ELISA with Recombinant Proteins. To further confirm the T4-dependency in phage OS ELISA of clone D11, we attempted to reproduce the assay with purified fusion proteins. As in our previous studies, we used E. coli maltose binding protein 1012

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Analytical Chemistry (MBP) as a fusion partner25 for the ease of soluble expression of the fusion proteins26 and also their immobilization/conjugation. As a result, expectedly, high yields (6-10 mg/L) of both fusion proteins were recovered from the cytoplasmic soluble fraction of BL21 cells. Hence, the obtained purified proteins were either immobilized directly (MBP-VL) to the microplate, or used as a conjugate with horseradish peroxidase (HRP-MBP-VH) to probe its binding activity to the other variable region fragment (MBP-VL). The result shown in Figure 4A indicates antigen (free T4)-dependent association of purified HRP-MBP-VH conjugate to immobilized MBP-VL similar or superior to that of VH-displaying phage, which allowed detection of less than 0.1 ng/mL T4 in PBS solution. We also performed OS-ELISA using recombinant proteins to evaluate cross reactivity with free T3. The result in Figure 4B suggests that our recombinant protein also shows lower reactivity for T3, which is similar to the result of competitive ELISA (Figure 2B). Estimation of Serum Total T4 Concentration. The total serum T4 concentration is a sensitive index of thyroid function. We tried to apply OS-ELISA to estimate T4 concentration in serum using the recombinant proteins. To make a standard curve, T4deficient serum was spiked with known amounts of T4 before extraction with ethanol. As shown in Figure 4C, the estimated total T4 concentration of an ethanol-extracted pooled serum was 90 ng/mL (9 μg/dL). The value was in good agreement with the normal adult range of 5-12 μg/dL.27

’ CONCLUSIONS To our knowledge, this is the first OS-ELISA system directly constructed from immunized mice. Compared with the conventional method, this system precluded the considerable time and labor required to elucidate hybridoma cells, including cell fusion, culture, cloning, and screening processes. There are also other merits in bypassing hybridoma technology. One is the reduction of artifacts in cDNA cloning, since many hybridoma cells express multiple immunoglobulin genes including aberrant kappa chains from the fusion partner (myeloma), which requires extra effort to select functional transcripts.28 Another is the omission of possible failure to clone/express functional V region genes from an established hybridoma, which is often caused by the inefficient expression or folding of the cloned V gene products in E. coli. While we could obtain a good clone D11 for OS-IA, one of the two candidates F11 was not suitable, despite its similar IC50 in competitive IA and high homology to D11, having only a few residue differences in both VH/VL chains. While detailed analysis is needed to determine the key residue(s) required for OS-IA, the variety of clones to be obtained would be another merit of this library-based method. Considering these merits and the higher frequency of successful OS-IA in small molecule binders, the reported method will be widely applied to noncompetitive and sensitive detection of many other small molecule antigens such as haptens, peptides,29 and their modifications.30 Over the past 40 years, improvements in the sensitivity and specificity of thyroid test methodologies have dramatically impacted the strategies for detecting and treating thyroid disorders. In this study, after the biopanning of Fab-displaying library of immunized mice, we could clone the VH/VL cDNAs of the antiT4 antibody and succeeded in OS-ELISA for T4 including that extracted from serum with sensitivity and working range superior to that of competitive ELISA. We hope that the established

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OS-IA system will contribute to many hospitals and diagnostic sites.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel/Fax: þ81-3-58417362.

’ ACKNOWLEDGMENT K.I.N. was supported by a Japanese Government (MEXT) Scholarship. J.D. was supported by the Postdoctoral Fellowship for Foreign Researchers from JSPS, Japan. This study was supported in part by SENTAN program, JST, Japan, by a Grant-in-Aid for Scientific Research (B20360368) from MEXT, Japan, and by Regional Innovation Cluster Program, City Area Type, from MEXT, Japan. ’ REFERENCES (1) Schmalzing, D.; Koutny, L.; Taylor, T.; Nashabeh, W.; M., F. J. Chromatogr., B 1997, 697, 175–180. (2) Samanidou, V. F.; Gika, H. G.; Papadoyannis, I. N. J. Liq. Chromatogr. Rel. Technol. 2000, 23, 681–692. (3) Nair, N.; Pillai, M. R. A.; Mani, R., S.; Naik, S.; Desai, M.; Upadhye, P.; Colaco, M. P. J. Radioanal. Nucl. Chem. 1988, 122, 129– 135. (4) Tagliaro, F.; Camilot, M.; Valentini, R.; Mengarda, T.; Antoniazzi, F.; Tato, L. J. Chromatogr., B 1998, 716, 77–82. (5) Wu, F.; Xu, Y.; Xu, T.; Wang, Y.; Han, S. Anal. Biochem. 1999, 276, 171–176. (6) Luppa, P. B.; Reutemann, S.; Huber, U.; Hoermann, R.; Poertl, S.; Kraiss, S.; von Bulow, S.; Neumeier, D. Clin. Chem. Lab. Med. 1998, 36, 789–796. (7) Sanchez-Carbayo, M.; Mauri, M.; Alfayate, R.; Miralles, C.; Soria, F. Clin. Biochem. 1999, 32, 395–403. (8) Aboul-Enein, H. Y.; Stefan, R. I.; Litescu, S.; Radu, G. L. J. Immunoassay Immunochem. 2002, 23, 181–190. (9) Stefan, R. I.; Aboul-Enein, H. Y. J. Immuoassay Immunochem. 2002, 23, 429–437. (10) Ullman, E. F.; Blackmore, J.; Leute, R. K.; Emistad, W.; Jaklitsch, A. Clin. Chem. 1975, 21, 1011. (11) Monji, N.; Malkus, H.; Castro, A. Biochem. Biophys. Res. Commun. 1978, 85, 671–677. (12) Schroeder, H. R.; Yeager, F. M.; Boguslaski, R. C.; Vogelhut, P. O. J. Immunol. Methods 1979, 25, 275–282. (13) Finley, P. R.; Williams, R. J.; Lichti, D. A. Clin. Chem. 1980, 26, 1723–1726. (14) Arakawa, H.; Maeda, M.; Tsuji, A. Bunseki Kagaku 1982, 31, E55–E61. (15) Arakawa, H.; Maeda, M.; Tsuji, A. Clin. Chem. 1985, 31, 430– 434. (16) Suzuki, C.; Ueda, H.; Mahoney, W.; Nagamune, T. Anal. Biochem. 2000, 286, 238–246. (17) Ueda, H.; Tsumoto, K.; Kubota, K.; Suzuki, E.; Nagamune, T.; Nishimura, H.; Schueler, P. A.; Winter, G.; Kumagai, I.; Mahoney, W. C. Nat. Biotechnol. 1996, 14, 1714–1718. (18) Ueda, H. J. Biosci. Bioeng. 2002, 94, 614–619. (19) Aburatani, T.; Sakamoto, K.; Masuda, K.; Nishi, K.; Ohkawa, H.; Nagamune, T.; Ueda, H. Anal. Chem. 2003, 75, 4057–4064. 1013

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