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DOI: 10.1055/s-2005-873188
© Georg Thieme Verlag KG Stuttgart · New York
Construction and Expression of a Single Chain Fv Fragment against Pharmacologically Active Paeoniflorin in Escherichia coli, and its Potential Use in an Enzyme-Linked Immunosorbent Assay
Hiroyuki Tanaka
Department of Medicinal Resources Regulation
Graduate School of Pharmaceutical Sciences
Kyushu University
3-1-1 Maidashi
Higashi-ku
Fukuoka 812-8582
Japan
Phone: +81-92-642-6668
Fax: +81-92-642-6668
Email: htanaka@phar.kyushu-u.ac.jp
Publication History
Received: March 9, 2005
Accepted: June 22, 2005
Publication Date:
05 January 2006 (online)
Abstract
A recombinant single chain variable-fragment (scFv) antibody against paeoniflorin (PF) was produced using the hybridoma cell line C31B9. Variable regions of heavy (VH) and light (VL) chain antibody genes were directly cloned from cDNA resources of hybridoma C31B9 and assembled using splicing by overlap extension (SOE)-PCR using a (Gly4Ser)3 linker DNA. The constructed scFv genes were cloned into pET28a vectors for the generation of recombinant proteins in Escherichia coli. Most of the recombinant proteins were expressed in inclusion bodies. The yield of refolded and purified scFv was 1.89 mg per 100 mL of cell culture. The recombinant scFv displayed cross-reactivity as its mother monoclonal antibody (MAb) C31B9. Therefore, the newly expressed scFv protein was applied to quantitative ELISA to determine the total paeoniflorin (PF) and albiflorin (Alb) concentrations in peony root samples. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 to 25 μg/mL. The results obtained by ELISA employing both the recombinant scFv and the original MAbC31B9 showed a reasonably good agreement with each other.
Key words
Single chain variable fragment (scFv) - paeoniflorin - albiflorin - enzyme-linked immunosorbent assay (ELISA)
Introduction
Immunoassays using antibodies as detection agents for low molecular weight compounds have become an alternative to ”classical” analytical techniques [1]. However, the traditional hybridoma technique for the preparation of MAbs somehow has limitations regarding the yield of low-affinity MAbs when an animal is immunized with an unsuitable immunogen. Furthermore, the maintenance and storage of the hybridomas is highly labor intensive and it also requires a high level of expertise. Instead of immortalizing B cells of immunized animals in hybridomas for MAb production, a recent DNA recombinant technique has made it possible to immortalize variable regions of heavy (VH) and light (VL) chain antibody genes into bacteria, and to express recombinant single chain variable-fragment (scFv) antibodies - which consist of variable regions of VH and VL linked via a short peptide spacer [2], [3]. Once the generation of recombinant antibodies in bacteria is established, it is much easier and quicker to produce such antibodies than with the use of hybridomas. The maintenance of bacterial stock is also simpler and cheaper. The cloned and assembled V genes from the MAbs help to improve affinity by mutagenesis [4], [5] or are expressed in different hosts [6] and plants [7], [8]. Furthermore, the selection and screening of recombinant antibodies against haptens from naive libraries enable us to avoid the use of animals, thus allowing us to overcome the low immune responses generated by poor immunogens [9], [10].
Since determination of the concentrations of PF and Alb in peony root [Paeonia lactiflora Pallas (Paeoniaceae)] samples and in traditional Chinese medicines prescribed with peony roots is important for both the quality control and standardization of pharmacological activity, we previously reported the production of MAbs using hybridoma C31B9 and its application to quantitative ELISA for PF and Alb in crude drugs and traditional Chinese medicines [11]. In the present study, starting with the hybridroma C31B9, we attempted to directly clone the VL and VH genes from the cDNA resources of hybridoma C31B9 and to assemble VL and VH using splicing by overlap extension (SOE)-PCR via a (Gly4Ser)3 linker DNA. Constructed scFv were cloned into expression vectors and then they were expressed in E. coli. The recombinant scFv proteins were characterized and applied to quantitative ELISA as an alternative of MAbs.
#Materials and Methods
#Chemicals and immunochemicals
PF and Alb were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). PF-HSA was synthesized according to the method described in our previous report [11].
#Construction of scFv gene and its expression in E. coli
VH and VL genes of hybridoma C31B9 were assembled by SOE-PCR and then were cloned into a pET28a expression vector (Novagen, EMD Biosciences Inc., Darmstadt, Germany) in order to generate a pET28/scFv plasmid. pET28/scFv was transformed into E. coli BL21 (DE3) by the CaCl2 method and expressed scFv proteins by chemical induction. The detailed protocol is presented in the Supporting Information.
#Characterization of refolded scFv protein
Protein samples were resolved by SDS-PAGE according to the method of Laemmli [12] using 12.5 % acrylamide gel (Bio-Rad Laboratories, CA, USA) of 0.75 mm at 30 mA. Coomassie Brilliant Blue was also used to visualize the proteins.
Direct ELISA was performed to determine the reactivities of scFv proteins to PF-HSA conjugates. A 96-well immunoplate (Nunc, Roskilde, Denmark) was coated with 2 μg/mL PF-HSA in 50 mM carbonate buffer, pH 9.6 (100 μL/well), and blocked with 5 % skimmed milk (w/v) in PBS (SPBS) (300 μL/well) for 1 h. Washing was carried out three times with PBS containing 0.05 % Tween-20 (TPBS) between each step. The plate was reacted with various concentrations of refolded scFv proteins, 100 μL/well, and then the scFv proteins were combined with 100 μL anti-T7-Tag antibody horseradish peroxidase (HRP) conjugate (Novagen, 2,000-fold dilution) in TPBS for 1 h. After washing the plate three times with TPBS, 100 μL of substrate solution containing 0.3 mg/mL 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in 100 mM citrate buffer (pH 4.0) containing 0.003 % H2O2 were added to each well, and then the plates were incubated for 15 min. The absorbance at 405 nm was measured using a microplate reader (Immunomini NJ-2300, Nunc, Roskilde, Denmark). All reactions were carried out at 37 °C. Competitive ELISA was performed in the same manner as direct ELISA except for one step. Various concentrations of PF or related compounds dissolved in 10 % methanol were co-cultured with equal volumes of scFv solutions for 1 h.
The cross-reactivities of PF and related compounds were determined according to Wailer and Zenk’s equation [13].
The ELISA method of Friguet et al. [14] was used to estimate the dissociation constant (KD) of refolded scFv in solution. The detailed protocol is presented in the Supporting Information.
#Analytical protocol for the detection of PF and Alb based on refolded scFv
The standard curve of PF in competitive ELISA was performed in the same manner as that for direct ELISA described as above except for one step. Various concentrations of standard PF dissolved in 10 % methanol were co-cultured with equal volumes of scFv solution for 1 h. By plotting A/Ao (A is the absorbance with PF present and Ao is the absorbance without PF) versus the logarithm of the PF concentration, an S-shaped curve could be obtained. In addition, the converted data could also be fitted by a linear regression. When samples were incubated with the same scFv solutions as standard PF, we could calculate the concentrations of PF and Alb in the samples using the linearized standard curve. Various peony samples were prepared in the same manner previous as described in our report [11].
#Results and Discussion
We cloned and sequenced the VH, VL regions of a MAb from cDNA of hybridoma C31B9. The cloned VH and VL nucleotides and the deduced amino acid sequences linked with a (Gly4Ser)3 linker are shown in the Supporting Information. The scFv gene was constructed in the VH-linker-VL orientation by SOE-PCR. This constructed gene product was then cloned directly into the pET28a expression vector, thus resulting in the formation of plasmid pET28/scFv. Finally, full sequencing confirmed that no PCR mutation had occurred.
The BL21 strain (DE3) harboring pET28/scFv was used to determine the cytoplasmic expression of recombinant scFv maintaining an N-terminal T7-Tag and His-Tag. After IPTG treatment of E. coli cells overnight at 37 °C, the recombinant proteins appeared in the insoluble fractions of the total cell extracts as revealed by a SDS-PAGE analysis (Fig. [2], left). Although the overall expression level was high, the recombinant proteins were mostly expressed as inclusion bodies in the cytoplasm. The purification of scFv from inclusion bodies under denaturing conditions was carried out on a metal-chelate column pre-packed with His-Bind Resin. Fig. [2] (right) shows the purified recombinant proteins which demonstrated a molecular weight of about 30 kDa and this finding closely correlated with the theoretical values of constructed scFv (295.393 Da). The purification of scFv from inclusion bodies under denaturing conditions yielded 5.9 mg proteins per 100 mL of cell culture. After refolding using a stepwise dialysis system [15], the total recovery yield of scFv was 1.89 mg per 100 mL of cell culture. In our study, the yield of scFv was similar to the average yields (2.5 - 10 mg/L) previously reported in published articles using comparable expression and purification methods [16], [17].
It is crucial to confirm whether the specificity of the expressed scFv to PF and related compounds is the same as that of a MAb C31B9. When preparing a MAb C31B9, we had screened the supernatant containing MAbs in each well of the culture plates with a direct ELISA using immunoplate-adsorbed PF-HSA and HSA in order to select useful hybridomas secreting a MAb showing a specific reactivity to a moiety descended from PF, and not to BSA. Furthermore, the competitive ELISA using an immunoplate-adsorbed PF-HSA had been carried out to obtain the hybidoma secreting a MAb showing specific reactivity to PF as free antigen. The above-mentioned process with both directive and competitive ELISAs was exploited to validate the specific reactivity of the prepared and refolded scFv to PF.
The reactivity of the refolded scFv to PF-HSA conjugates and its binding activity to free PF and Alb are shown in Fig. [3]. The reactivity response curve was drawn by plotting absorbance against the logarithm of scFv protein concentrations in direct ELISA. The concentrations of the proteins positively correlated with the absorbance values in a logistical manner.
As we described in our previous work, the mother MAb C31B9 showed reactivity to both PF and Alb [11]. Next, a competitive ELISA was applied to estimate the binding activity of recombinant scFv to either free PF or Alb. scFv proteins were incubated with serially double diluted concentrations of free PF or Alb on an immunoplate. Any scFv proteins binding to free PF or Alb were washed out. The unbound scFv proteins that bound to immobilized PF-HSA conjugates were incubated with T7-Tag antibody HRP conjugates and then were treated with ABTS substrate. The absorbance of the bound scFv proteins to immobilized antigens decreased as opposed to a rise in the co-incubated PF or Alb concentrations. The inhibition ratio was described by A/Ao (A is the absorbance with PF or Alb, Ao is the absorbance without PF or Alb). When the inhibition ratio against the logarithm of PF or Alb concentrations was plotted, an inhibition curve was obtained as indicated in Fig. [4]. The dose-dependent binding of PF or Alb to scFv could be observed around 1.56 - 25 μg/mL, and the half-inhibition dose was either 4.18 μg/mL or 6.07 μg/mL, respectively, according to a linear regression analysis of the inhibition curve.
The cross reactivities of scFv in comparison to mother MAb C31B9 are estimated and shown in Table [1]. The competitive binding activities between scFv and free PF or Alb were almost the same as those of the mother MAb; meanwhile, the cross reactivities of scFv with oxypaeoniflorin and benzoylpaeoniflorin were also identical to those for MAbs. In addition, the cross-reactivity of scFv with other compounds was found to be insignificant. These results indicated that the scFv proteins generated by bacteria possessed the major properties of the mother MAb.
The ELISA method by Friguet et al. is a well accepted method for estimating the affinity values for antibodies in solution [14]. This method is equally available for antibodies against both small (hapten) and large molecular weight antigens without labeling of either antibodies or antigens. In brief, a fixed concentration of scFv proteins and a range of concentrations of PF were mixed and then allowed to reach equilibrium. Next, the concentrations of free scFv in the mixtures were measured by ELISA. We found that when the coated PF-HSA concentration was 2 μg/mL in the ELISA, the amount of scFv captured by the solid phase represented only a small fraction of the free scFv, and this amount should not perturb the antibody-antigen binding equilibrium. From the linear component of the calibration curve of absorbance versus antibody concentrations, we decided on a final concentration of 15 μg/mL for scFv for the antigen-antibody equilibrium experiment. Various concentrations of PF were incubated with scFv overnight at room temperature until they reached equilibrium. Free scFv amounts in the incubation mixture were determined by ELISA as described above. The KD of scFv in solution was (2.24 ± 0.01) × 10 - 6 M from the typical Scatchard plot among five repeat experiments.
Since the refolded scFv produced from bacteria possessed similar characteristics to its mother MAb C31B9, a competitive binding assay was established based on scFv binding to either free PF or PF-HSA conjugates adsorbed onto an immunoplate. Using PF as a standard compound, the full linear range of the assay was extended from 0.78 - 25 μg/mL as indicated in Fig. [5]. Inter-assay and intra-assay variations ranged between 2 % to 10 % and 2 % to 18 %, respectively. Next, we performed scFv based ELISA to analyze the PF and Alb concentrations in the samples (Table [2]). The minor PF related compounds of peony root such as oxypaeoniflorin and benzoylpaeoniflorin with the pinane skeleton really do react with the Mab C31B9 and the prepared scFv shown in Table [1]. It was proposed that these PF related compounds might influence immunoassays using the MAb C31B9 and also using the prepared scFv. The total concentrations of PF and Alb in the peony root samples using ELISA employing scFv are shown in Table [2] and the data obtained by an ELISA employing MAb C31B9 are shown together. The findings show a good correlation with each other (Fig. [6]). From this result, even though these PF related compounds are contained in peony root samples, the total concentrations of PF and Alb determined by ELISA employing the MAb C31B9 and the scFv were in a reasonable agreement with that by the HPLC method as previously reported by us [11]. Probably, this immunoassay using the scFv could be also available for the quantitative analysis of the total PF and Alb concentrations in peony root samples since the concentration of the other PF-related compounds such as oxypaeoniflorin and benzoylpaeoniflorin in samples might be low. However, we definitely realize that it should first be elucidated whether specific or unspecific cross-reactivities of the mother MAb C31B9 and the scFv against other natural products like phenylpropanoids, glycosides or polyphenols could interfere with the ELISAs using these probes in order to validate the robustness of these immunoassays in further investigations.
In conclusion, the expression, purification and refolding of the functional scFv against PF were successfully achieved in this study. The expression of scFv proteins in E. coli is a low-cost method for achieving a high production of recombinant functional antibodies. The recombinant scFv antibody displayed cross-reactivities similar to its mother MAb and it has been applied, for the first time, to a quantitative ELISA to investigate the total PF and Alb concentrations in peony root samples. The results showed a good correlation with the data obtained by the ELISA employing its mother MAb C31B9 and indicated that this technique is a powerful means for altering MAbs in an established quantitative ELISA.
Fig. 1 PF and its related compounds.
Fig. 2 Expression of scFv in BL21 (harboring pET28/scFv) and its purification. An analysis was performed using 12.5 % SDS-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue. Left: lane 1, Dalton marker VII L (kDa); lanes 2, and 3, total cells before and after 18 h of IPTG induction; lanes 4 and 5, soluble fraction and insoluble fraction after IPTG induction. Right: lane 1, Dalton marker VII L(kDa); lane 2: purified and refolded scFv.
Fig. 3 Reactivities of purified and refolded scFv to PF-HSA conjugates. To examine the reactivities of scFv, various concentrations of protein samples were added to each well of a 96-well immunoplate coated with PF-HSA (2 μg/mL).
Fig. 4 Specific binding activities of scFv to free PF and Alb in competitive ELISA. Various concentrations of PF or Alb were incubated with scFv in a 96-well immunoplate precoated with PF-HSA (2 μg/mL). A/Ao, Ao is the absorbance with no PF or Alb present, and A is the absorbance with PF or Alb present. The data shown are the representative findings of three individual experiments.
Fig. 5 The standard curve of PF using scFv in competitive ELISA was obtained by plotting A/Ao vs. logarithm of the PF concentration. The insert shows the linearized calibration curve using the same experimental data. Various concentrations of PF were incubated with scFv in a 96-well immunoplate with PF-HSA (2 μg/mL). Error bars = ± standard deviation, A/Ao, Ao is the absorbance with no PF present, and A is the absorbance with PF present. The data represents the means of five replicates.
Fig. 6 Correlation curve of the results obtained from ELISA employing scFv and MAb.
| Compound | Cross Reactivity (%) | |
| MAb | scFv | |
| Paeoniflorin Albiflorin Oxypaeoniflorin Benzoylpaeoniflorin |
100.0 143.7 5.2 29.4 |
100.0 145.2 0.4 16.7 |
| (1R)-endo-(+)-Fenchol | < 0.008 | < 0.008 |
| DL-Camphene | < 0.008 | < 0.008 |
| D-Camphor | < 0.008 | < 0.008 |
| (-)-α-Pinene | < 0.008 | < 0.008 |
| DL-Borneol | < 0.008 | < 0.008 |
| L-Menthol | < 0.008 | < 0.008 |
| Kojic acid | < 0.008 | < 0.008 |
| Paclitaxel | < 0.008 | < 0.008 |
| Thymol | < 0.008 | < 0.008 |
| Benzoic acid | < 0.008 | < 0.008 |
| Cross reactivities were determined according to Weiler’s equation [13]. | ||
| Paeoniae Radix (Place of cultivation)a |
Total concentrations of PF and Alb (%) | |
| ELISA (scFv) | ELISA (MAb)b | |
| 1) Nara, Japan | 3.72 ± 0.37 | 4.32 ± 0.09 |
| 2) Hokkaido, Japan | 3.23 ± 0.07 | 3.51 ± 0.14 |
| 3) Niigata, Japan | 2.68 ± 0.15 | 3.06 ± 0.09 |
| 4) Sichuan, China | 2.90 ± 0.01 | 3.69 ± 0.02 |
| 5) Anhui, China | 0.33 ± 0.01 | 0.53 ± 0.01 |
| a Samples were purchased from Nakai Koushindo Pharmacy (Kobe, Japan) and were classified according to their places of cultivation. | ||
| b Data are from Lu et al. [11]. | ||
Acknowledgements
This study was supported in part by a Grant-in-Aid (No. 08457586 for Y.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the research fund of Kyushu University Foundation, and the research fund of the Japan Kampo Medicine Manufacturers Association from the Japan Kampo Medicine Manufacturers.
- Supporting Information for this article is available online at
- Supporting Information .
References
- 1 Fitzpatrick J, Fanning L, Hearty S, Leonard P, Manning B M, Quinn J G. et al . Applications and recent developments in the use of antibodies for analysis. Anal Lett. 2000; 33 2563-609
- 2 Laroche Y, Demaeyer M, Stassen J M, Gansemans Y, Demarsin E, Matthyssens G. et al . Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal-antibody specific for human fibrin fragment D-dimer. J Biol Chem. 1991; 266 16 343-9
- 3 Worn A, Pluckthun A. Different equilibrium stability of ScFv fragments: identification, classification, and improvement by protein engineering. Biochemistry. 1999; 38 8739-50
- 4 Coia G, Hudson P J, Irving R A. Protein affinity maturation in vivo using E. coli mutator cells. J Immunol Methods. 2001; 251 187-93
- 5 Winkler K, Kramer A, Kuttner G, Seifert M, Scholz C, Wessner H. et al . Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody. J Immunol. 2000; 165 4505-14
- 6 Robin S, Petrov K, Dintinger T, Kujumdzieva A, Tellier C, Dion M. Comparison of three microbial hosts for the expression of an active catalytic scFv. Mol Immunol. 2003; 39 729-38
- 7 Eto J, Suzuki Y, Ohkawa H, Yamaguchi I. Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants. FEBS Lett. 2003; 550 179-84
- 8 Putalun W, Taura F, Qing W, Matsushita H, Tanaka H, Shoyama Y. Anti-solamargine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants. Plant Cell Rep. 2003; 22 344-9
- 9 Vaughan T J, Williams A J, Pritchard K, Osbourn J K, Pope A R, Earnshaw J C. et al . Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol. 1996; 14 309-14
- 10 Moghaddam A, Borgen T SJ, Kausmally L, Simonsen B, Marvik O J, Brekke O H. et al . Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J Immunol Methods. 2003; 280 139-55
- 11 Lu Z H, Morinaga O, Tanaka H, Shoyama Y. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. Biol Pharm Bull. 2003; 26 862-6
- 12 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 680-5
- 13 Weiler E W, Zenk M H. Radioimmunoassay for determination of digoxin and related compounds in Digitalis lanata . Phytochemistry. 1976; 15 1537-45
- 14 Friguet B, Chaffotte A F, Djavadi-Ohaniance L, Goldberg M E. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 1985; 77 305-19
- 15 Tsumoto K, Shinoki K, Kondo H, Uchikawa M, Juji T, Kumagai I. Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent-application to a human single-chain Fv fragment. J Immunol Methods. 1998; 219 119-29
- 16 Kim S H, Schindler D G, Lindner A B, Tawfik D S, Eshhar Z. Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies. Mol Immunol. 1997; 34 891-906
- 17 Ren X J, Gao S J, You D L, Huang H L, Liu Z, Mu Y. et al . Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency. Biochem J. 2001; 359 369-74
Hiroyuki Tanaka
Department of Medicinal Resources Regulation
Graduate School of Pharmaceutical Sciences
Kyushu University
3-1-1 Maidashi
Higashi-ku
Fukuoka 812-8582
Japan
Phone: +81-92-642-6668
Fax: +81-92-642-6668
Email: htanaka@phar.kyushu-u.ac.jp
References
- 1 Fitzpatrick J, Fanning L, Hearty S, Leonard P, Manning B M, Quinn J G. et al . Applications and recent developments in the use of antibodies for analysis. Anal Lett. 2000; 33 2563-609
- 2 Laroche Y, Demaeyer M, Stassen J M, Gansemans Y, Demarsin E, Matthyssens G. et al . Characterization of a recombinant single-chain molecule comprising the variable domains of a monoclonal-antibody specific for human fibrin fragment D-dimer. J Biol Chem. 1991; 266 16 343-9
- 3 Worn A, Pluckthun A. Different equilibrium stability of ScFv fragments: identification, classification, and improvement by protein engineering. Biochemistry. 1999; 38 8739-50
- 4 Coia G, Hudson P J, Irving R A. Protein affinity maturation in vivo using E. coli mutator cells. J Immunol Methods. 2001; 251 187-93
- 5 Winkler K, Kramer A, Kuttner G, Seifert M, Scholz C, Wessner H. et al . Changing the antigen binding specificity by single point mutations of an anti-p24 (HIV-1) antibody. J Immunol. 2000; 165 4505-14
- 6 Robin S, Petrov K, Dintinger T, Kujumdzieva A, Tellier C, Dion M. Comparison of three microbial hosts for the expression of an active catalytic scFv. Mol Immunol. 2003; 39 729-38
- 7 Eto J, Suzuki Y, Ohkawa H, Yamaguchi I. Anti-herbicide single-chain antibody expression confers herbicide tolerance in transgenic plants. FEBS Lett. 2003; 550 179-84
- 8 Putalun W, Taura F, Qing W, Matsushita H, Tanaka H, Shoyama Y. Anti-solamargine glycoside single-chain Fv antibody stimulates biosynthesis of solasodine glycoside in plants. Plant Cell Rep. 2003; 22 344-9
- 9 Vaughan T J, Williams A J, Pritchard K, Osbourn J K, Pope A R, Earnshaw J C. et al . Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat Biotechnol. 1996; 14 309-14
- 10 Moghaddam A, Borgen T SJ, Kausmally L, Simonsen B, Marvik O J, Brekke O H. et al . Identification of scFv antibody fragments that specifically recognise the heroin metabolite 6-monoacetylmorphine but not morphine. J Immunol Methods. 2003; 280 139-55
- 11 Lu Z H, Morinaga O, Tanaka H, Shoyama Y. A quantitative ELISA using monoclonal antibody to survey paeoniflorin and albiflorin in crude drugs and traditional Chinese herbal medicines. Biol Pharm Bull. 2003; 26 862-6
- 12 Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 680-5
- 13 Weiler E W, Zenk M H. Radioimmunoassay for determination of digoxin and related compounds in Digitalis lanata . Phytochemistry. 1976; 15 1537-45
- 14 Friguet B, Chaffotte A F, Djavadi-Ohaniance L, Goldberg M E. Measurements of the true affinity constant in solution of antigen-antibody complexes by enzyme-linked immunosorbent assay. J Immunol Methods. 1985; 77 305-19
- 15 Tsumoto K, Shinoki K, Kondo H, Uchikawa M, Juji T, Kumagai I. Highly efficient recovery of functional single-chain Fv fragments from inclusion bodies overexpressed in Escherichia coli by controlled introduction of oxidizing reagent-application to a human single-chain Fv fragment. J Immunol Methods. 1998; 219 119-29
- 16 Kim S H, Schindler D G, Lindner A B, Tawfik D S, Eshhar Z. Expression and characterization of recombinant single-chain Fv and Fv fragments derived from a set of catalytic antibodies. Mol Immunol. 1997; 34 891-906
- 17 Ren X J, Gao S J, You D L, Huang H L, Liu Z, Mu Y. et al . Cloning and expression of a single-chain catalytic antibody that acts as a glutathione peroxidase mimic with high catalytic efficiency. Biochem J. 2001; 359 369-74
Hiroyuki Tanaka
Department of Medicinal Resources Regulation
Graduate School of Pharmaceutical Sciences
Kyushu University
3-1-1 Maidashi
Higashi-ku
Fukuoka 812-8582
Japan
Phone: +81-92-642-6668
Fax: +81-92-642-6668
Email: htanaka@phar.kyushu-u.ac.jp
Fig. 1 PF and its related compounds.
Fig. 2 Expression of scFv in BL21 (harboring pET28/scFv) and its purification. An analysis was performed using 12.5 % SDS-polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue. Left: lane 1, Dalton marker VII L (kDa); lanes 2, and 3, total cells before and after 18 h of IPTG induction; lanes 4 and 5, soluble fraction and insoluble fraction after IPTG induction. Right: lane 1, Dalton marker VII L(kDa); lane 2: purified and refolded scFv.
Fig. 3 Reactivities of purified and refolded scFv to PF-HSA conjugates. To examine the reactivities of scFv, various concentrations of protein samples were added to each well of a 96-well immunoplate coated with PF-HSA (2 μg/mL).
Fig. 4 Specific binding activities of scFv to free PF and Alb in competitive ELISA. Various concentrations of PF or Alb were incubated with scFv in a 96-well immunoplate precoated with PF-HSA (2 μg/mL). A/Ao, Ao is the absorbance with no PF or Alb present, and A is the absorbance with PF or Alb present. The data shown are the representative findings of three individual experiments.
Fig. 5 The standard curve of PF using scFv in competitive ELISA was obtained by plotting A/Ao vs. logarithm of the PF concentration. The insert shows the linearized calibration curve using the same experimental data. Various concentrations of PF were incubated with scFv in a 96-well immunoplate with PF-HSA (2 μg/mL). Error bars = ± standard deviation, A/Ao, Ao is the absorbance with no PF present, and A is the absorbance with PF present. The data represents the means of five replicates.
Fig. 6 Correlation curve of the results obtained from ELISA employing scFv and MAb.
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