Abstract
Due to the highly specific binding between an antibody and its target, superior analytical performances was obtained by immunoassays for phytochemical analysis over conventional chromatographic techniques. Here, we describe a simple method for producing a functional single-chain variable fragment (scFv) antibody against ganoderic acid A (GAA), a pharmacologically active metabolite from Ganoderma lingzhi. The Escherichia coli BL21(DE3) strain produced a large amount of anti-GAA scFv. However, in vitro refolding steps, which partially recovered the reactivity of the scFv, were required. Interestingly, the functional scFv was expressed as a soluble and active form in the cytoplasm of an engineered E. coli SHuffle® strain. Purified anti-GAA scFv, which yielded 2.56 mg from 1 L of culture medium, was obtained from simple and inexpensive procedures for expression and purification. The anti-GAA scFv-based indirect competitive enzyme-linked immunosorbent assay (icELISA) exhibited high sensitivity (linearity: 0.078–1.25 µg/mL) with precision (CV: ≤6.20%) and reliability (recovery: 100.1–101.8%) for GAA determination. In summary, the approach described here is an inexpensive, simple, and efficient expression system that extends the application of anti-GAA scFv-based immunoassays. In addition, when in vitro refolding steps can be skipped, the cost and complexity of scFv antibody production can be minimized.
Antibodies are the most frequently used biological agents for routine diagnostics, therapeutics, and studies in various fields. Their applications to chemical analysis provide various advantages such as binding specificity and methodological simplicity that extend beyond conventional chromatographic techniques. The subjects of target molecules for immunoassays have been expanded from large molecules (i.e., protein, peptide, and DNA) to small molecules (herbicides, natural products, and phytohormones). As such, many monoclonal antibodies (mAbs) against small molecules (haptens), such as amikacin,1) carbamazepine,2) aflatoxins,3) and daidzin,4,5) have been produced to develop mAb-based immunoassays for qualitative and quantitative analyses, which were shown to be simple and convenient analytical methods. Because the cost of recombinant antibodies (rAbs) produced using Escherichia coli is much lower than that of antibodies produced using hybridoma cells or other animal cell lines,6) immunoassays using E. coli-derived antibodies represent an economical approach for analytical applications. However, the E. coli-based production of rAbs against haptens is limited by an inability to retain rAb reactivity. Normally, refolding steps are required to recover the binding reactivity of the rAb, and the steps are determined by time-consuming trial-and-error procedures. The single-chain variable fragment (scFv) antibody is a simple and small arrangement of the functional rAb, in which the variable regions of the heavy (VH) and light (VL) chains are linked together using a flexible peptide linker. Using this simple configuration of a single domain, expression is not limited by the formation of disulfide bonds between the heavy and light chains, as observed in the expression of Fab and full-length immunoglobulin G (IgG). The reactivity of several scFv antibodies against a hapten is lost when the E. coli expression system is used and cannot fully be recovered through in vitro refolding.7,8) The quality of a recombinant antibody largely influences its analytical performance, such as its sensitivity and specificity in an immunoassay. The functional full-length IgG and scFv antibody against macromolecules has been successfully expressed in the cytoplasm of engineered SHuffle® T7 E. coli.9,10) Here, we aim to produce functional anti-ganoderic acid A (GAA, Fig. 1) scFv and to develop a corresponding indirect competitive enzyme-linked immunosorbent assay (icELISA) method that is inexpensive and reliable.
Previously, mAb-based icELISA was developed for the quantitative and qualitative analysis of GAA.11,12) The methods were simple and reliable for a GAA analysis of Ganoderma lingzhi (G. lingzhi belongs to family Ganodermataceae)-associated samples. A scientific review has indicated that the Ganoderma mushroom produces beneficial effects as an adjuvant agent for cancer treatment.13) In addition, G. lucidum also improves the quality of life [the International Prostate Symptom Score (IPSS)] of men who have lower urinary tract symptoms.14) To date, pharmacologically active compounds from the Ganoderma species have been found to be polysaccharides and triterpenes.15) Among the triterpenoids, GAA exhibits various benefits including the suppression of growth and invasive behavior of MDA-MB-231 cancer cells16) and sensitization of HepG2 cells to cisplatin.17) Although there is no evidence that a specific triterpenoid can function as a biomarker for standardization of Ganoderma-related products or samples, GAA is one of the predominant compounds in Ganoderma spp.,18) so the GAA content can be used to judge their quality,19) Due to market competition and consumer awareness, the GAA content in marketed G. lingzhi products should be presented for confidence in quality and quantity. A simple, reliable, and inexpensive analytical method is useful for standardization of raw materials and final products. To minimize the cost of a GAA quantitative analysis by immunoassay, anti-GAA scFv was constructed from hybridoma clone 12A that secreted mAb against GAA. First, anti-GAA scFv was expressed using the E. coli BL21(DE3) strain, and a large amount of target protein was expressed in the inactive form. Its reactivity could not be fully recovered by in vitro refolding. Therefore, an expression system using a commercially engineered E. coli SHuffle® strain was developed and optimized. As a result, anti-GAA scFv with higher reactivity was obtained, and it was applied to the icELISA development and validation. Anti-GAA scFv-based icELISA exhibited reliability for determination of GAA in G. lingzhi-associated samples.
Overall, this is a powerful and efficient system for the production of anti-GAA scFv. It may be applied to the expression of other anti-hapten scFvs whose reactivity cannot be recovered using the conventional E. coli strain with subsequent in vitro refolding. When the cost of immunoassays can be minimized, their applications can be extended.
MATERIALS AND METHODS
Chemical and Immunochemical ReagentsGAA (≥98%) and mouse serum albumin (MSA, ≥96%) were commercially obtained from Sigma-Aldrich Corp. The cation exchange resin (Toyopearl CM-650M) and immobilized metal affinity chromatography (IMAC) resin (cOmplete His-Tag Purification Resin) were purchased from Tosoh Co. (Tokyo, Japan) and Roche (Mannheim, Germany), respectively. Anti-T7 tag monoclonal antibody conjugated to horseradish peroxidase (HRP) was purchased from Novagen (Merck Millipore Corp., Darmstadt, Germany). E. coli SHuffle® T7 competent cell was commercially obtained from New England Biolabs (NEB). Enzymes and reagents for molecular cloning were purchased from Takara Bio Inc. (Kyoto, Japan). All other chemical and immunological reagents that have not been specified were commercial products for molecular biology experiments.
Construction of the Anti-GAA scFv Gene and Its Expression VectorsTotal RNA was prepared from hybridoma clone 12A that secreted mAb (IgG1 with kappa light chain) against GAA.11) The protocol for the RNA extraction and treatment is described in the Sepasol RNA I Super reagent manual (Nacalai Tesque, Kyoto, Japan). Then, cDNA was synthesized using the random hexamer primers of the First-Strand cDNA Synthesis Kit (Amersham Biosciences, Buckinghamshire, U.K.). Using the degenerate primers designed for the variable regions of the murine antibody,20) the genes encoding VH and VL of the anti-GAA mAb were amplified from cDNA by PCR (ExTaq DNA polymerase, Takara Bio Inc.). The thermocycler was set for initial denaturation at 94°C for 1 min followed by 30 cycles of 94°C for 1 min, 50°C for 1 min and 72°C for 1.5 min. The final extension was performed at 72°C for 7 min.
The PCR products of VH and VL were purified using the QIA quick PCR Purification Kit and then subcloned into the pGEM-T Easy vector (Promega) based on the TA-cloning method. The ligated products of pGEM-T-VH and pGEM-T-VL were transformed individually into the E. coli JM109 strain. Using the blue-white screening technique, colonies containing the recombinant pGEM-T vector with the expected VH or VL gene were identified. The selected vectors were extracted (AccuPrep® Plasmid Mini Extraction Kit, Bioneer, Korea), and the nucleotide sequence of the inserted gene was revealed using the BigDye® Terminator v1.1 Cycle Sequencing Kit.
When the genes encoding VH and VL were identified, specific primers for the construction of the anti-GAA scFv expression vector were designed (see Supplementary Material (SM), Table S1). The gene encoding anti-GAA scFv was constructed in the orientation of VH-(GGG GS)3-VL. The VH and VL genes were individually amplified by PCR (Prime Taq DNA Polymerase kit, GENET BIO Inc., Korea) using the P1, P2 and P3, P4 primer pairs, respectively (see SM, Fig. S1). Thermocycling consisted of initial denaturation at 95°C for 5 min followed by 30 cycles of 94°C for 30 s, 52°C for 30 s and 72°C for 60 s. The final extension was performed at 72°C for 5 min. The purified PCR products of VH and VL were linked into the scFv configuration [VH-(GGG GS)3-VL] by splicing through overlap extension (SOE)-PCR. Using the aforementioned thermocycling conditions with primers P1 and P4, the gene for anti-GAA scFv was constructed when the mixed VH and VL genes were used as the template. The amplified gene was digested with BamHI and HindIII and then subcloned into pET28a(+) to yield pET28a(+)/anti-GAA scFv.
To construct pET21b(+)/anti-GAA scFv (see SM, Fig. S1), primers P5 and P6 were used to amplify the anti-GAA gene by PCR (PrimeSTAR® HS premix, TaKaRa Bio Inc.). Thermocycling consisted of initial incubation at 98°C for 1 min followed by 25 cycles of 98°C for 10 s, 55°C for 5 s and 72°C for 60 s. The final extension was performed at 72°C for 5 min. The obtained product was digested and subcloned into pET21b(+). Both recombinant expression vectors of anti-GAA scFv were sequenced and then transformed into their E. coli expression hosts.
Expression of Anti-GAA scFv Using E. coli BL21(DE3)The E. coli BL21(DE3) cells harboring the pET28a(+)/anti-GAA scFv vector were initially cultured overnight (37°C, 120 rpm) in LB medium (50 µg/mL kanamycin). An aliquot of the culture (400 µL) was transferred to 40 mL of fresh LB medium (50 µg/mL kanamycin). The flask was shaken continuously (120 rpm, 37°C) until the optical density (OD600) of the culture reached 0.6, and expression of anti-GAA scFv was induced with 1 mM IPTG for 16 h (120 rpm, 37°C).
The cells were collected in a tube by centrifugation (8000 rpm, 5 min, and 4°C) and then washed with 40 mL of lysis buffer (50 mM Tris–HCl pH 7.0 containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 10% (v/v) glycerol). The cell pellet was resuspended in 16 mL of lysis buffer and lysed for 30 min with hen egg lysozyme (1 mg/mL). NaCl and Nonidet P-40 were added to the lysed cells to a final concentration of 50 mM and 0.1% (v/v), respectively. The treated cells were completely lysed by ultrasonication (750 W, 20 kHz). When the lysate was centrifuged (12000 rpm, 30 min, 4°C), the supernatant was collected and defined as the soluble protein fraction. The pellet (inclusion bodies or insoluble proteins) was dissolved in denaturing IMAC binding buffer (50 mM Tris–HCl pH 8.0 containing 8 M urea and 500 mM NaCl). The expression of anti-GAA scFv was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
Using the E. coli BL21(DE3) strain, anti-GAA scFv was mainly expressed as inactive protein in inclusion bodies. Therefore, the solubilized inclusion body fraction was subjected to a column containing the IMAC resin. Unbound protein were washed out with denaturing IMAC binding buffer containing 40 mM imidazole, and anti-GAA scFv was eluted with the same buffer containing 400 mM imidazole. The concentration and purity of the purified anti-GAA scFv was analyzed using the Bradford protein assay (Bio-Rad Laboratories)21) and SDS-PAGE, respectively.
Because E. coli BL21(DE3)-derived anti-GAA scFv was in the inactive form, its reactivity against GAA was recovered using the in vitro refolding method described previously.21) The purified anti-GAA scFv solution (1 mg/mL, 2 mL) was dialyzed against a denaturing buffer (50 mM Tris–HCl pH 8.0 containing 8 M urea, 0.25 M NaCl, and 5 mM 2-mercaptoethanol), in which disulfide bonds were completely reduced. Then, 2-mercaptoethanol was removed by dialysis against 50 mM Tris–HCl pH 8.0 buffer containing 8 M urea and 0.25 M NaCl. The anti-GAA scFv was added dropwise into stirred refolding buffer [40 mL, 50 mM Tris–HCl pH 8.0 containing 0.15 M NaCl, 1 mM EDTA, 0.5 M L-arginine or sucrose, 2 mM reduced glutathione, 1 mM oxidized glutathione, and 5% (v/v) glycerol]. The resulting solution was dialyzed against the same buffer an additional 12 h. The solution was dialyzed against a storage buffer [5% (v/v) glycerol in 10 mM phosphate-buffered saline (PBS)] and then concentrated using PEG 20000. All procedures were performed at 4°C. The concentration of the refolded protein was analyzed using the Bradford protein assay.
Expression of Anti-GAA scFv Using E. coli SHuffle®An engineered E. coli SHuffle® strain was applied to produce anti-GAA scFv in the active form in the cytoplasm. The E. coli cells harboring the pET21b(+)/anti-GAA scFv vector were cultured overnight (30°C, 120 rpm) in TB medium (100 µg/mL ampicillin). On the following morning, 400 µL of culture was added to a 300-mL flask containing 40 mL of TB medium (100 µg/mL ampicillin). The cells were cultured (30°C, 150 rpm) until the OD600 reached 0.6. An IPTG solution was added to the culture to adjust the final concentration to 1 mM, and the culture was continued for 16 h at different temperatures (16, 20, 25, 30°C) to induce expression of anti-GAA scFv. The induced cells were collected and treated using the same method described above for E. coli BL21(DE3). The reactivity of anti-GAA scFv in the fraction of soluble proteins was evaluated by icELISA.
To purify anti-GAA scFv, the soluble fraction of proteins extracted from the E. coli SHuffle® cells was dialyzed against the starting buffer [20 mM Tris–HCl pH 6.8 containing 10% (v/v) glycerol] and then subjected to a ready-to-use cation exchanger (HiTrap SP HP, 5×5 mL, GE Healthcare Life Sciences). After washing with the starting buffer, the bound proteins were eluted with the same buffer containing various concentrations of NaCl (0–600 mM). The fractions containing anti-GAA scFv were dialyzed against IMAC native binding buffer [50 mM Tris–HCl pH 7.4 containing 0.5 M NaCl, 10% (v/v) glycerol, and 0.1% Nonidet P-40] and subjected to the IMAC column. The non-target protein was washed with the same buffer containing 5 and 10 mM imidazole. Finally, the target anti-GAA scFv was eluted with eluting buffer [50 mM Tris–HCl pH 7.4 containing 0.5 M NaCl, 10% (v/v) glycerol, 0.1% Nonidet P-40, 250 mM imidazole].
Characterization of the Anti-GAA scFvPreliminary screening of anti-GAA scFv reactivity against different GAA-carrier protein conjugates and carrier proteins (GAA-ovalbumin, ovalbumin, GAA-methanesulfonic acid (MSA), MSA, GAA-bovine serum albumin, and bovine serum albumin) revealed that the anti-GAA scFv showed the highest reactivity against GAA-MSA, while it showed low reactivity against MSA. Therefore, GAA-MSA was used as coated antigen. Indirect ELISA and icELISA were applied to analyze the reactivity of anti-GAA scFv against immobilized GAA (GAA-MSA) and free GAA, respectively. Per the protocol for indirect ELISA, the GAA-MSA conjugate (2 µg/mL in 50 mM sodium carbonate buffer pH 9.6, 100 µL) prepared in our previous study12) was coated on the surface of a Nunc-immuno 96-well plate for 1 h. The unbound conjugate was washed out, and the unoccupied surface was blocked with proteins from skim milk [5% weight per volume (w/v) in PBS, 300 µL]. To evaluate the reactivity of scFv against immobilized GAA, different concentrations of anti-GAA scFv (100 µL) were applied and incubated for 1 h. Unbound scFvs were removed by washing with PBS containing 0.05% (v/v) Tween 20 (PBST), whereas bound scFvs were incubated with the anti-T7 antibody (HRP conjugate). One hour later, the plate was washed three times. The indirect ELISA signal was developed using a substrate solution [100 mM sodium citrate buffer (pH 4.0) containing 0.003% (v/v) H2O2 and 0.3 mg/mL of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 100 µL]. The absorbance was recorded (405 nm) after 20 min of incubation.
To evaluate the reactivity of anti-GAA scFv against free GAA, the procedure for icELISA was applied. The plate was coated and treated as described above. Next, the GAA solution (50 µL), which was prepared in 5% (v/v) methanol, was added followed by anti-GAA scFv (50 µL). In this step, anti-GAA scFv could bind to both immobilized and free GAA. One hour later, anti-GAA scFvs that bound free GAA were washed out, whereas anti-GAA scFvs that bound to the immobilized GAA were incubated with the anti-T7 antibody (HRP conjugate). Finally, the icELISA signal was developed and recorded. When anti-GAA scFv exhibited reactivity against the free antigen, the icELISA signal was decreased in a GAA-concentration dependent manner. All the incubation steps for both indirect ELISA and icELISA were performed at 37°C.
Validation and Application of Anti-GAA scFv-Based icELISAIn this study, the anti-GAA scFv was intended to be applied for GAA determination using the icELISA procedure. First, the sensitivity of the anti-GAA scFv-based icELISA was analyzed, where serial-diluted concentrations of GAA (0–40 µg/mL) were added as a competitor to the binding of immobilized GAA and anti-GAA scFv. The concentration of GAA that resulted in a 10% inhibitory effect of anti-GAA binding to immobilized GAA was set as the limit of detection (LOD). The range of the GAA concentration that exhibited a linear relationship with the signals of icELISA was defined as the calibration curve.
Variations within GAA detectable concentrations (0.039–1.25 µg/mL) were calculated and expressed as the percentage of the coefficient of variation (CV). The inhibitory variation between the wells (n=3) in a plate was defined as intraplate variation, whereas the variation between plates (n=3), in which triplicate absorbance were used from each plate, was assigned as the interplate variation.
An experiment of cross-reactivity (CR), which described the ratio of the binding reactivity between anti-GAA scFv and GAA and the reactivity of anti-GAA scFv against related compounds, was performed to evaluate the binding specificity of anti-GAA scFv. The percentage of CR was calculated using the following equation:
Using linear binding between anti-GAA scFv and GAA or the investigated compounds, the concentration that produced a 50% inhibitory effect between scFv and the immobilized GAA (IC
50) was calculated. Various structurally similar compounds to GAA were used in this experiment.
The validated anti-GAA scFv-based icELISA was applied to determine the GAA content in G. lingzhi samples. G. lingzhi was provided by Ken Sawai and Takeshi Sawai (Toyotanshien Co., Ltd., Sapporo, Japan). The samples were prepared, treated, and extracted as described previously.11) Various concentrations of ethanol were utilized for GAA extraction. The resultant residue that was obtained after evaporation was redissolved with 1.0 mL of methanol. Finally, the extracts were diluted with water to obtain a 5.0% (v/v) final concentration of methanol, and diluted extracts were analyzed directly by icELISA.
RESULTS AND DISCUSSION
Construction of the Anti-GAA scFv Gene and Expression VectorsThe PCR products of the genes encoding the VH and VL regions of anti-GAA mAb clone 12A were successfully amplified from cDNA and then identified and selected according to the CDR identification and numbering scheme methods described by Kabat and Chothia (http://www.bioinf.org.uk/abs/). The CDR regions of VH (345 bps) and VL (324 bps) were indicated as shown in Fig. 2. The nucleotide sequence of the anti-GAA scFv gene [VH-(GGG GS)3-VL] was assigned in DDBJ (accession number: LC222841). The gene was then subcloned into the pET28a(+) expression vector, in which the hexahistidine and T7 sequences were tagged onto the upstream end of the target gene. Then, the gene was amplified and subcloned into the pET21b(+) expression vector, in which the hexahistidine and T7 sequences was tagged onto the upstream and downstream ends, respectively. Both recombinant vectors were sequenced and transformed into expression strains of E. coli.
Expression of Anti-GAA scFv Using the E. coli BL21(DE3) StrainFirst, anti-GAA scFv was expressed using the E. coli BL21(DE3) system harboring recombinant pET28a(+)/anti-GAA scFv. Anti-GAA scFv was expressed after induction by IPTG (Fig. 3A). The results demonstrated that the target protein was expressed mainly in the insoluble fraction or inclusion bodies. Normally, conventional E. coli strains produce target scFv in the inactive form due to the reducing environment of the cytoplasm. However, a high purity of anti-GAA scFv (inactive form) can be obtained easily using a single IMAC column. A large amount of purified anti-GAA (5.68 mg) was obtained from 40 mL of culture medium.
Using a simple and rapid method for in vitro refolding, the target protein was refolded into a soluble form, which exhibited reactivity against immobilized GAA (GAA-MSA conjugate) and the free form of GAA. The refolded anti-GAA scFv reacted in a concentration-dependent manner to the immobilized GAA (see SM, Fig. S2a). Then, its reactivity against free GAA was evaluated by icELISA. The reactivity of anti-GAA scFv with free GAA was also observed to be dependent on the concentration of GAA, as indicated by the closed circle in Fig. 4a. The GAA concentration exhibiting linearity of binding with anti-GAA scFv that was in the range of 0.625–10.0 µg/mL, as shown by the closed circle in Fig. 4B. The reactivity of the refolded anti-GAA scFv was quite low compared to the parental mAb clone 12A [linearity: 6.10–195 ng/mL11)]. Because its reactivity against free GAA was quite low, its application for immunoassay was limited due to low sensitivity. These results implied that in vitro refolding was not efficient to recover the reactivity of the anti-GAA scFv, as the conformation and disulfide bonds may not have formed properly. Therefore, alternative expression systems needed to be considered to improve anti-GAA scFv reactivity. Typically, animal cell expression systems such as Chinese hamster ovary cells, the SP2/0 cell line, and the NS0 cell line or hybridoma are selected for antibody expression for therapeutic applications because these systems provide posttranslational modifications that promote proper antibody folding and N-glycosylation. For analytical applications of immunoassays, however, N-glycosylation is not required when it does not affect the reactivity of the antibody. E. coli Shuffle®, a strain that was engineered to product proteins containing disulfide bond(s), was used for anti-GAA scFv expression.
Expression of Anti-GAA scFv Using the E. coli SHuffle® StrainTemperature is an important factor that can affect the expression and folding of a target protein. Expression of anti-GAA scFv was induced with 1 mM IPTG at different temperatures (16, 20, 25, and 30°C). The reactivity of the expressed anti-GAA scFv was evaluated directly using the soluble protein (2 mg/mL) from the induced E. coli cells. The reactivity of the functional anti-GAA scFv against the immobilized GAA was highest when the temperature of expression was set to 20°C, at which the reactivity was almost twice as high as the antibodies expressed at 16 and 25°C (Fig. 5). Therefore, the temperature was set to 20°C for large-scale expression of anti-GAA scFv. For Fig. 3b, the E. coli cells induced for anti-GAA scFv expression at 20°C were fractionated into soluble and insoluble proteins. The target anti-GAA scFv was well expressed using this system. However, only a small portion of anti-GAA scFv was folded into the soluble form, and a major portion of anti-GAA scFv had accumulated into inclusion bodies. This implied that the expressed anti-GAA scFv was too large to be folded by the engineered enzymes in the cytoplasm of E. coli SHuffle®. The expressed anti-GAA scFv was thought to be in excess of the capacity of the folding enzymes, which resulted in the high accumulation in the inclusion bodies.
Using a cation exchanger, anti-GAA scFv-enriched fractions were obtained when the bound proteins were eluted with starting buffer containing 80–200 mM NaCl. These fractions were dialyzed against IMAC native binding buffer and subjected to an IMAC column. Finally, the target anti-GAA scFv was eluted (Fig. 3b), and 2.56 mg of the purified scFv was ultimately obtained from 1 L of culture.
Characterization of Anti-GAA scFvUsing the E. coli SHuffle® strain, the functional anti-GAA scFv was obtained by simple expression and purification. It reacted well with immobilized GAA in a concentration dependent manner (see SM, Fig. S2b). To obtain the absorbance at 405 nm of 1.0 in indirect ELISA, 2.84 µg/mL (E. coli BL21(DE3)) and 1.25 µg/mL (E. coli SHuffle®) of anti-GAA scFv were required, meaning that E. coli SHuffle® produced more reactive anti-GAA scFv against immobilized GAA than refolded anti-GAA scFv from E. coli BL21(DE3). In the indirect ELSIA using anti-GAA mAb, 0.5 µg/mL of the mAb was required to give the absorbance at 405 nm of 1.0. When these concentrations were compared, ELISA using anti-GAA mAb seems cost-effective. However, considering the production cost of anti-GAA mAb using hybridoma cell, anti-GAA scFv using E. coli systems is eventually much cheaper and effective.
Interestingly, the E. coli SHuffle® strain-derived anti-GAA scFv also exhibited higher reactivity against free GAA than the E. coli BL21(DE3) strain-derived refolded anti-GAA scFv (Fig. 4). Therefore, anti-GAA scFv from the E. coli SHuffle® expression system was used to develop icELISA for determination of GAA. With fixed concentration of immobilized GAA and anti-GAA scFv, serially diluted concentrations of free GAA were used. The anti-GAA scFv was competitively reacted with immobilized and free GAA, so the icELISA signal decreased as the concentration of GAA increased. Using E. coli SHuffle®-derived anti-GAA scFv, a linear correlation between the icELISA signals and GAA concentration was observed in the GAA concentration range of 78–1250 ng/mL, in which its reactivity was lower than parental mAb clone 12A (linearity: 6.10–195 ng/mL). This indicated that sensitivity of icELISA using this anti-GAA scFv is lower than the icELISA using its parental anti-GAA mAb. However, this lower sensitivity did not show any impact on performance of the icELISA using anti-GAA scFv because the concentration of GAA in the sample is quite high. However, E. coli SHuffle®-derived anti-GAA scFv exhibited more reactivity against GAA than E. coli BL21(DE3)-derived anti-GAA scFv (linearity: 0.625–10 µg/mL). These results suggested that intracellular folding mediated by folding enzymes was more powerful than in vitro refolding.
G. lingzhi consists of many triterpenoid compounds whose structures are related to GAA. Some of the available triterpenes were used to evaluate the binding specificity of anti-GAA scFv. Comparative binding reactivity of anti-GAA scFv against GAA and other related compounds was expressed as the percentage of CR. The results showed similar patterns of CR between anti-GAA scFv and the parental mAb (Table 1). However, an increased degree of CR was observed from the scFv. Due to different the conformations of scFv and the parental mAb, a flexibility of association between the variable domains may result in an increased degree of CR.
Table 1. Cross-Reactivity of Anti-GAA scFv against GAA Structure-Related Compounds
| Compounds | Cross-reactivity (%) |
|---|
| Anti-GAA scFv | Anti-GAA mAba) |
|---|
| Ganoderic acid A | 100.0 (IC50=330 ng/mL) | 100.0 (IC50=37.6 ng/mL) |
| Ganoderic acid AM1 | <0.660 | <0.1 |
| Ganoderic acid B | <0.660 | <0.1 |
| Ganoderic acid C1 | <0.660 | 0.15 |
| Ganoderic acid C2 | 1.31 | 0.42 |
| Ganoderic acid C6 | <0.660 | <0.1 |
| Ganoderic acid DM | <0.660 | 0.17 |
| Ganoderic acid E | <0.660 | <0.1 |
| Ganoderic acid H | 1.48 | <0.1 |
| Ganoderic acid K | <0.660 | 0.22 |
| Ganoderic acid LM2 | <0.660 | <0.1 |
| Ganoderic acid N | <0.660 | <0.1 |
| Ganoderic acid S | <0.660 | <0.1 |
| Ganoderic acid SZ | <0.660 | <0.1 |
| Ganoderic acid TN | <0.660 | <0.1 |
| Ganoderic acid T-Q | <0.660 | <0.1 |
| Ganoderic acid TR | 14.4 | 2.21 |
| Ganoderic acid Y | <0.660 | <0.1 |
| Ganoderic acid Z | <0.660 | <0.1 |
| Ganolucidic acid A | <0.660 | <0.1 |
| Ganoderenic acid A | 10.4 | 3.69 |
| Ganoderenic acid C | <0.660 | <0.1 |
| Ganoderenic acid D | <0.660 | 0.14 |
| Ganoderenic acid F | 3.82 | 1.07 |
| Ganoderenic acid H | <0.660 | <0.1 |
| Ganodermanondiol | <0.660 | <0.1 |
| Ganodermanontriol | <0.660 | <0.1 |
| Ganoderol A | <0.660 | <0.1 |
| Ganoderol B | <0.660 | <0.1 |
| Ganoderiol F | <0.660 | <0.1 |
| Lucialdehyde A | <0.660 | <0.1 |
| Lucialdehyde B | <0.660 | <0.1 |
a) The cross-reactivity of anti-GAA mAb was reported previously.11)
A linear correlation between the icELISA signals and GAA concentration was observed in the GAA concentration range of 0.078–1.25 µg/mL. Consequently, this range was set as the calibration cure for a quantitative analysis of GAA. Variations in binding between anti-GAA scFv and GAA were investigated and expressed as the CV (%) of the intraplate and interplate variations. Within the concentrations in the linearity range, the maximum CV was 4.07 and 6.20% for intraplate and interplate assays, respectively (see SM Table S2). Therefore, the icELISA is quite precise for GAA determination.
Known amounts of authentic GAA were added into diluted ethanol extracts of G. lingzhi, and the total content of GAA was analyzed. Finally, the proximity of the measured amount to the theoretical amount was calculated and expressed as the percentage of recovery. The non-spiked sample of diluted extract contained 6.787±0.52 µg of GAA. Recovery of GAA was close to 100%, as determined using the scFv-based icELISA from samples spiked with 2.5–7.5 µg of authentic GAA (Table 2). Therefore, these results reflected the reliable performance of anti-GAA scFv-based icELISA.
Table 2. The Recovery of GAA from
G. lingzhi Extract Spiked with Authentic GAA
| Spiked amount (µg) | Measured amount (µg) | Recovery (%) of GAA |
|---|
| 0 | 6.787±0.52 | — |
| 2.5 | 9.331±0.69 | 101.8 |
| 5 | 11.84±0.91 | 101.1 |
| 7.5 | 14.30±0.99 | 100.1 |
Anti-GAA scFv-based icELISA was used for a quantitative analysis of GAA in plant samples. These samples were prepared from distinct parts of G. lingzhi, including the pileus, stipe, and spore. Various concentrations of ethanol were used to extract GAA from these samples. Subsequently, the GAA contents in these samples were assessed using the validated anti-GAA scFv-based icELISA (Table 3). A similar content to the GAA content was extracted from the samples using 50–100% ethanol. However, lower efficiency of GAA extraction was observed when 0–25% ethanol was used to extract GAA from the samples. The decreased efficiency was observed clearly in the pileus sample, which contained a high amount of GAA. In all samples, the GAA content determined by anti-GAA scFv-based icELISA correlated to the content determined using the anti-GAA mAb-based icELISA and HPLC-UV methods. Because anti-GAA scFv exhibited a higher degree of CR against several GAA-related compounds than the parental mAb, the GAA contents analyzed using anti-GAA scFv were slightly higher than those analyzed using the anti-GAA mAb.
Table 3. Content of GAA in the
G. lingzhi Samples Determined by Anti-GAA scFv-Based icELISA
| Solvent for extraction | Part of G. lingzhi mushroom | Content of GAA [% (w/w) of dry weight] |
|---|
| Ethanol | Pileus | 3.879±0.289 |
| Stipe | 0.394±0.019 |
| Spore | 0.169±0.013 |
| 3 : 1 (v/v) of ethanol–water | Pileus | 3.835±0.130 |
| Stipe | 0.409±0.026 |
| Spore | 0.136±0.005 |
| 1 : 1 (v/v) of ethanol–water | Pileus | 3.860±0.130 |
| Stipe | 0.328±0.016 |
| Spore | 0.114±0.006 |
| 1 : 3 (v/v) of ethanol–water | Pileus | 2.553±0.201 |
| Stipe | 0.373±0.030 |
| Spore | 0.148±0.012 |
| Water | Pileus | 1.459±0.044 |
| Stipe | 0.210±0.017 |
| Spore | 0.176±0.016 |
CONCLUSION
The engineered E. coli SHuffle® system is useful for the production of functional anti-GAA scFv. A remarkably large amount of purified anti-GAA scFv was obtained using simple procedures of expression and purification. The binding reactivity and specificity against GAA was conserved when it was expressed in the form of scFv. The cost of E. coli SHuffle® expression system is much cheaper than its parental mAb production using mammalian hybridoma cell culture. When anti-GAA scFv was folded in cytoplasm compartment of E. coli SHuffle®, in vitro time- and cost-consuming refolding steps could be avoided. In addition, protocol of anti-GAA scFv-based icELISA is simple, and not need for expensive equipment and organic solvents as compared with HPLC system. The analytical validation of anti-GAA scFv-based icELISA demonstrated reliability for GAA determination. Therefore, this E. coli expression system is a cost-effective approach for GAA determination using the validated anti-GAA scFv-based icELISA.
Acknowledgments
We thank Mr. Ken Sawai and Mr. Takeshi Sawai (Toyotanshien Co., Ltd., Japan) for providing the G. lingzhi-involved samples. This work was supported by a Grant-in-Aid for Challenging Exploratory Research (26 660 147) of the Japan Society for the Promotion of Science (JSPS).
Conflict of Interest
The authors declare no conflict of interest.
Supplementary Materials
The online version of this article contains supplementary materials.
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