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Table of Contents
Year : 2017  |  Volume : 3  |  Issue : 3  |  Page : 18-27

Monoclonal antibody usage strategies for natural products in traditional chinese medicine

Department of Pharmacognosy, Faculty of Pharmaceutical Science, Nagasaki International University, Sasebo, Nagasaki 859-3298, Japan

Date of Submission19-Oct-2016
Date of Acceptance21-Jul-2017
Date of Web Publication24-Oct-2017

Correspondence Address:
Yukihiro Shoyama
Department of Pharmacognosy, Faculty of Pharmaceutical Science, Nagasaki International University, Sasebo, Nagasaki 859-3298
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/wjtcm.wjtcm_5_17

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Monoclonal antibodies (MAbs) against natural products with low-molecular weights have become an important tool when combined with other analytical systems. Eastern blotting involves a typical staining system wherein, for example, glycosides can be blotted to a membrane and cross-linked and stained using MAbs. An immunoaffinity column combined with a monoclonal antibody allows a one-step purification of hapten compounds or preparation of a knockout extract that removes only the target hapten molecules from a crude extract. Here, we discuss the application of these extracts. Single-chain variable fragment (scFv) proteins have led to novel assay systems such as fluobodies or antibodies coupled with green fluorescent protein for natural products. A typical novel application of a scFv gene would be in the context of plant breeding; this is designated “missile-type” molecular breeding, intended to increase hapten molecule concentrations in transgenic plants.

Keywords: Eastern blotting, immunoaffinity concentration, Knockout extract, monoclonal antibody, single-chain variable fragment

How to cite this article:
Shoyama Y. Monoclonal antibody usage strategies for natural products in traditional chinese medicine. World J Tradit Chin Med 2017;3:18-27

How to cite this URL:
Shoyama Y. Monoclonal antibody usage strategies for natural products in traditional chinese medicine. World J Tradit Chin Med [serial online] 2017 [cited 2019 Nov 17];3:18-27. Available from: http://www.wjtcm.net/text.asp?2017/3/3/18/217100

  Introduction Top

Because pharmacologically active compounds and/or marker components vary according to cultivation conditions, location, species differences, and harvest season, many countries have developed pharmacopoeia to ensure the quality control of natural products. Natural product and/or pharmacognostic analyses incorporate disciplines such as chemistry, biology, physics, and biochemistry. In addition, chromatographic analyses include techniques such as thin-layer chromatography (TLC), capillary electrochromatography,[1] and gas chromatography (GC) which may be coupled with mass spectrometry (GC-MS),[2] high-performance liquid chromatography (HPLC) coupled with MS (LC-MS),[3] and LC-MS-MS [4] coupled with an evaporative light scattering detector.[5]

As a result of the rapid development of molecular biosciences and their biotechnological applications, polyclonal antibodies were used for immunoassays until the 1970s, after which a shift occurred to the use of monoclonal antibodies (MAbs) against target molecules such as low molecular weight bioactive compounds; these MAbs have become an important tool [6] because their high specificity is advantageous. MAb-based immunoassays are used in a wide variety of analyses, such as receptor binding assays, enzyme assays,[7] and quantitative and/or qualitative analytical techniques both in vivo and in vitro. Regarding their uses in other field, at least 400 therapeutic MAbs are available. Although MAbs against natural small molecular products were first introduced in the 1980s, few existed except for those that targeted drugs such as morphine.[8] In the 1990s, increased preparation of MAbs against natural products such as terpenoids.[9],[10],[11],[12] alkaloids,[13],[14] plant saponins,[15],[16],[17],[18],[19] phenolics,[20],[21],[22],[23] and more recently, artemisinin,[24] daidzin,[25] puerarin,[26] and capsaicinoids [27] was reported, leading to the development of individual enzyme-linked immunosorbent assays (ELISAs) for the quality control of traditional Chinese medicine (TCM) agents.

Immunoaffinity purification is a highly specific technique that could potentially be used for the one-step isolation of a target molecule from complex mixtures such as cellular lysates.[28] For this technique, immunoaffinity columns are conjugated with MAbs and thus specifically bind and remove the target molecules. Several immunoaffinity columns, such as protein G affinity columns for use with MAbs expressed by hybridomas [29] and metal chelate affinity columns for single-chain variable fragment (scFv) proteins expressed by  Escherichia More Details coli,[30] have been commonly used to purify peptides and proteins. The use of an immunoaffinity column, compared to traditional cleanup techniques, could decrease the required amount of solvent and the number of purification steps, reduce the analysis time, and simplify the sample analysis compared to traditional cleanup techniques. Although immunoaffinity purification of higher molecule analytes such as peptides and proteins is widely used in both research and commercial proceedings, there are very few cases involving immunoaffinity purification of a small molecule compound. Therefore, this review will introduce and discuss the available information.

  Monoclonal Antibodies as Analytical Equipment Top

Determination of Hapten number in a Hapten-carrier protein conjugate

We will first discuss hapten number determination, as the confirmation of this factor in a synthesized antigen is the most important step in the first stage of MAb preparation. The synthesis of a hapten derived from an immune antigen, linker bridge, and conjugated carrier protein is necessary for the production of MAbs. Previously, however, no direct and appropriate methods for the determination of haptens conjugated with carrier protein existed in the absence of differential ultraviolet analysis, radiochemical, or chemical methods. Therefore, immunization through the injection of hapten-carrier protein conjugates was unreliable. A direct analytical method to assess hapten and carrier protein conjugates using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and an internal standard was initially introduced in the 1990s. Subsequently, all conjugates have been subjected to MALDI-TOF MS to confirm the hapten number before immunization. The first successful determination by MALDI-TOF MS involved forskolin, an activator of cyclic AMP,[31] followed by a component of marijuana [32] and opium alkaloids.[33] This methodology has recently become popular.[25]

[Figure 1] presents a co-MALDI-TOF MS of the major active marijuana component tetrahydrocannabinolic acid conjugated to bovine serum albumin (BSA), with BSA used as an internal standard. This spectrum shows only singly and doubly ionized molecule ions of the intact conjugate. The sharp peak at m/z 66465 is the [M + H] + of BSA. A smaller [M + H] + peak of the tetrahydrocannabinolic acid-BSA conjugate is visible at m/z 70581. The calculated molecular mass of the tetrahydrocannabinolic acid moiety is 4314. From this result, 12.7 molecules of tetrahydrocannabinolic acid were combined with BSA.[32] This method is also suitable for natural small molecule products, including glycosides.[16],[17],[19]
Figure 1: Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of a tetrahydrocannabinolic acid-bovine serum albumin conjugate[32]

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Preparation of monoclonal antibody and enzyme-linked immunosorbent assays

Glycosides of natural products were treated with a solution of NaIO4 to cleave sugar moieties and release aldehyde groups that could be combined with carrier proteins. The resulting synthesized antigen was injected into mice and used to prepare MAb as follows. A hybridoma that produced hapten-reactive MAb was obtained using standard procedures;[13] the resulting MAb was classified as an IgG isotype having light chain. The reactivity of this IgG MAb was tested using varying antibody titers and a dilution curve. Competitive ELISA is then used to determine the antibody concentration. In this assay, free MAb and a competing agent are bound to polystyrene microtiter plates precoated with hapten-human serum albumin (HSA). Under these conditions, the full measurement range of the assay is generally 5–200 ng/ml,[9] and the quantitative ELISA analysis results should agree well with the HPLC analysis results.[16] Cross-reactivity is the most important factor when determining the value of an antibody. Because ELISAs were established for phytochemical investigations involving crude plant extracts, assay specificity was confirmed by determining the cross-reactivity of the MAb with various related compounds. The resulting cross-reactivity data were examined using competitive ELISA and calculated using picomoles of hapten. In general, the MAb should react very weakly with only a small number of structurally related molecules and should not react with different types of compound. However, if a MAb exhibiting wide cross-reactivity is needed to detect all related compounds, a synthetic design and hybridoma selection will be necessary. For example, an anti-solamargine MAb has a very wide cross-reactivity for almost all solasodine glycosides [19] because these molecules share the same aglycone, solasodine, which forms the base material of a steroidal hormone.[34]

  Newly Developed Monoclonal Antibody Assay System Top

Time-resolved fluoroimmunoassay

The time-resolved fluoroimmunoassay (TRFIA) was initially developed in 1999.[35] This assay system uses lanthanide elements and related chelates as tracers with unique fluorescence properties. This technique is commonly used in the agriculture and food industries, laboratory medicine, forensic science, and environmental health studies.[36],[37],[38],[39],[40]

We will provide a brief description of an analysis of saikosaponin A in Bupleuri radix. First, a microplate coated with rabbit anti-mouse IgG was incubated with a crude extract of Bupleuri radix and a mouse anti-saikosaponin MAb;[17] a lanthanide-labeled saikosaponin A-HSA conjugate was used as the tracer. The developed competitive TRFIA exhibited a good fourth-order polynomial fitting from 0.01 to 10.0 μg/ml for a standard saikosaponin a sample, with a detection limit of 6 ng/ml.[40]

  Natural Product Staining Methods Top


Although MAb has recently become an indispensable tool for immunostaining in biochemical and biophysical investigations, few such investigations have involved small molecules. The incorporation of glycosides such as crocin, a saffron-derived molecule containing four glucoses and exhibiting various functions in cells, was confirmed in PC-12 cells using an anti-crocin MAb.[41] In another experiment, the authors evaluated the distribution of ginsenoside Rb1 in a fresh ginseng root slice by staining through eastern blotting,[42] and conducted a microscopic observation using an anti-ginsenoside Rb1 MAb.[43] [Figure 2] demonstrates ginsenoside Rb1 immunostaining in ginseng tissues, resulting in clear staining that was supported by gold particle staining and yielded good agreement with eastern blotting.[42] The immunostaining of aristolochic acid, which exhibits strong nephrotoxic activity (reviewed below), was investigated in murine kidney tissues.[44] Furthermore, the authors identified α-actinin-4 as a target protein of aristolochic acid [45] in human kidney cells through immunoprecipitation with an anti-aristolochic acid MAb, followed by LC-MS analysis after digestion.[46]
Figure 2: Histochemical staining of ginsenoside Rb1 in ginseng tissues using an anti-ginsenoside Rb1 monoclonal antibody[43]

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Eastern blotting

Southern, northern, and western blotting are well-known techniques for the detection of DNA, RNA, and protein molecules, respectively. However, all of these methods involve substances with larger molecular structures. In contrast, Taki et al.[47] and Towbin et al.[48] successfully blotted smaller molecular compounds such as glycosphingolipids and phospholipids from TLC to membranes, using a technique called far-eastern blotting. The author and collaborators have developed a similar method to detect glycosides in natural products. The initial success of this method, which was described as western blotting in 1997, involved a glycoalkaloid contained in Solanum species.[49] We later applied this method to detect glycyrrhizin, the most important constituent of licorice, and coined the term eastern blotting in 2001;[18] subsequently, we have applied eastern blotting to natural products such as saikosaponin A,[17] sennoside A and B,[50] and ginsenosides.[51],[52] Eastern blotting differs from far-eastern blotting in that the former method involves separation of the functions of the above glycoside into an epitope (aglycone part) and membrane-fixative (sugar moiety).[18] Although the sphingolipid-related starfish gangliosides, which were discussed in the context of far-eastern blotting, were blotted to membranes, this blotting was insufficient. Accordingly, we directly stained a TLC plate using anti-AG-2 MAb.[53] However, the name “eastern blotting” has remained under discussion since 2007.[54]

In brief, the procedure of eastern blotting is as follows. A developed, glycoside-bound TLC plate is covered with a polyvinylidene difluoride (PVDF) or polysulfone ether membrane; after adding a blotting solution, the plate is briefly heated. The blotted membrane is treated with a NaIO4 solution to cleave the sugar moieties and release protein-conjugated aldehyde groups as Schiff bases that will bind to the membrane and can be thus treated with corresponding MAbs, followed by a peroxidase-labeled goat anti-mouse IgG MAb. Finally, the PVDF membrane is exposed to substrate, as indicated in [Figure 3][18].
Figure 3: Scheme of eastern blotting for glycyrrhizin with an anti-glycyrrhizin monoclonal antibody[18]

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We have expanded our eastern blotting method to natural products with no linked sugar moieties, such as aristolochic acid [55] well known as nephrotoxicants cause of Balkan Endemic Nephropathy and Chinese Herb Nephropathy. [Figure 4] indicates a schema of hapten synthesis and staining for aristolochic acid.
Figure 4: Scheme of hapten synthesis and eastern blotting for aristolochic acid using an anti-aristolochic acid monoclonal antibody[55]

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Regarding the previously introduced case of solasodine glycosides, we present in [Figure 5] an eastern blot of solasodine glycosides to demonstrate that all solasodine glycosides can be stained.
Figure 5: Eastern blotting of solasodine glycosides using an anti-solamargine monoclonal antibody.[49] Solamargine (1), solasonine (2), khasianine (3), 3-O-β-D-glucopyranosyl solasodine (4), O-α-L-rhamnosyl-(1→2)-3-O-β-D-glucopyranosylsolasodine (5), 3-O-β-D-galacopyranosyl-solasodine (6), O-β-Dglucopyranosyl-(1→3)-3-O-β-D-galacopyranosyl-solasodine (7), solaverine I (8), solaverine II (9), 12-hydroxysolamargine (10) and 12- hydroxysolasonine (11), respectively

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Double eastern blotting

Double eastern blotting was also developed to evaluate ginsenosides using an anti-ginsenoside Rb1 MAb [15] and anti-ginsenoside Rg1 MAb.[16] [Figure 6] shows an example of the double eastern blotting of ginsenosides.[51],[52] Fingerprinting through double eastern blotting of various ginseng samples has yielded the following points. The Rf value reflects the number of sugars conjugated to a molecule. Pinkish spots indicate ginsenosides that possess the aglycone protopanaxtriol and exhibit hyperthymic activity, and blue spots indicate protopanaxadiol-type ginsenosides with depressive activity in the central nervous system.[57] Accordingly, several ginsenosides, including ginsenosides Rb1, Rc, and Rd, can be stained with the highly specific anti-ginsenoside Rb1 MAb.[15] Similar staining was observed with the anti-ginsenoside Rg1 MAb. These phenomena might be attributed to sugar modification consequent to the cleavage of sugar moieties, which leads to wide MAb cross-reactivity.[42]
Figure 6: Double eastern blotting of ginsenosides in Panax species using anti-ginsenosides Rb1 and Rg1 monoclonal antibodies.[42] Lanes I–VI indicate white ginseng, red ginseng, fibrous ginseng, Panax notoginseng, Panax quinquefolium, and Panax japonicus, respectively

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Recently, Fujii et al.[58] developed a double eastern blotting method to detect the glycosides glycyrrhizin (triterpene glycoside) and liquiritin (flavonoid glycoside), as shown in [Figure 7]. [Figure 7]a and [Figure 7]b show TLC plates stained with sulfuric acid and subjected to double eastern blotting, respectively. Regarding TLC, all compounds could be detected, including the individual aglycones of glycyrrhizin and liquiritin (glycyrrhetinic acid and liquiritigenin, respectively. This is a significant advantage for the detection of natural products. However, because TLC monitoring is not specific, fingerprinting patterns might be complicated. On the other hand, eastern blotting patterns are simple and specific to the antigenic molecule, as indicated in [Figure 7]b.
Figure 7: Double eastern blotting of the licorice compounds glycyrrhizin and liquiritin using anti-glycyrrhizin and anti-liquiritin monoclonal antibodies[58] (a): OH2SO4 staining, (b): double easter blotting, 1: glycyrrhizin, 2: glycyrrhetic acid, 3: liquiritin, 4: liquiritigenin

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  One-Step Purification on an Immunoaffinity Column Top

Immunoaffinity purification using commercially available MAbs and scFv proteins expressed in E. coli is commonly used to purify proteins and peptides. However, because MAbs against natural products are generally not available, these MAbs must be self-prepared. Purified MAb is then coupled to a gel (e.g., Affi-Gel Hz gel), which is used to prepare an immunoaffinity column. The column is washed with washing buffer, and the bound hapten is generally eluted with an elution buffer containing an organic solvent. After separation, each fraction is deionized and lyophilized to yield the pure hapten compound. For example, Yanagihara et al.[59] successfully obtained 45 μg of pure forskolin, a cyclic AMP activator that is currently used as a cardiac drug in Japan, from a crude extract of Coleus forskohlii root (10 mg) using an immunoaffinity column conjugated with anti-forskolin MAb and an elution buffer containing 40% methanol in phosphate-buffered saline. Moreover, because the ginseng root contains nearly a hundred dammarane-type saponins ginsenosides, as well as oleanane-type saponins, the isolation of individual saponins is quite tedious and requires repeated silica gel chromatography and/or preparative HPLC. An immunoaffinity column for ginsenoside Rb1 isolation uses an elution solvent comprising an acetic acid buffer with potassium isocyanate and 20% methanol, a lower concentration than that used to isolate forskolin.[60] Charged ginsenoside Rb1 was eluted with washing solution, repeatedly loaded, and finally isolated in a pure form. The antibody remained stable when exposed to the washing and eluting solvents, and the immunoaffinity column exhibited almost no decrease in capacity after more than 10 uses. Therefore, one-step purification of a crude natural product extract on an immunoaffinity column conjugated with an anti-target compound MAb can supply sufficient amounts of compound, on a bench and/or laboratory scale, for in vitro experiments because approximately 10 μg of antigen compound can be obtained per ml of gel. Furthermore, we have established several immunoaffinity columns using MAbs against solasodine glycosides [61] and glycyrrhizin.[62] Recently, immunoaffinity columns have been increasingly applied for the isolation of natural products such as isoflavones [63] and tetrodotoxin, of which very small amounts can be concentrated on an immunoaffinity column and detected using analytical devices.[64]

It became evident during immunoaffinity column purification that the washing solution contains all components except the antigenic molecule. Therefore, we will discuss this washing solution in the next section.

  Preparation of a Knockout Extract With an Immunoaffinity Column Top

The above-mentioned washing fraction, which is produced during one-step purification on an immunoaffinity column, is known as a knockout extract [65] because only the antigen molecule is eliminated, thus resembling the outcome of a knockout gene. In fact, an immunoaffinity column conjugated with anti-glycyrrhizin MAb could eliminate 99% of glycyrrhizin from a crude extract of licorice.[66] We accordingly named this washing fraction a glycyrrhizin-knockout extract. The capacity of our anti-glycyrrhizin immunoaffinity column to capture glycyrrhizin was nearly matched by the capacities of other columns to capture forskolin [59] and solasodine glycosides.[61] This result suggests that in solutions containing trace levels of glycyrrhizin that cannot be detected by ELISA, an immunoaffinity column could concentrate glycyrrhizin to a detectable level, as described above.

To investigate the profiles of our glycyrrhizin-knockout extract, we performed a TLC analysis and eastern blotting. Several spots corresponding to glycyrrhizin and other compounds were detected in a licorice extract. However, although the other spots were clearly detected in the glycyrrhizin-knockout extract, the spot corresponding to glycyrrhizin was completely absent. Furthermore, eastern blotting with an anti-glycyrrhizin MAb clearly detected glycyrrhizin in the licorice extract, but failed to detect this compound in the glycyrrhizin-knockout extract. These findings suggest that glycyrrhizin can be specifically eliminated from a licorice extract through separation on an anti-glycyrrhizin MAb immunoaffinity column.[66]

Inflammation leads to the upregulation of a range of enzymes and signaling mediators in affected tissues and cells. Nitric oxide (NO) is a highly reactive free radical involved in multiple physiological functions, including vasodilatation, neurotransmission, and inflammation.[67] During an inflammatory process, large amounts of NO are produced by inducible NO synthase (iNOS) in various cell types, including macrophages, in response to stimulation from inflammatory cytokines and/or bacterial lipopolysaccharide (LPS).[68] In certain inflammatory and autoimmune diseases, the overproduction of NO by iNOS triggers the pathogenic conditions of septic shock and organ destruction.[69],[70],[71] Therefore, blocking iNOS expression and thus inhibiting NO production might be a useful treatment option for a variety of inflammatory diseases. Uto et al.[68] found that a crude licorice extract could suppress NO release in LPS-treated mouse RAW264 macrophages. LPS evoked an increase in NO production, and this induction was blocked by treatment with licorice extract in a dose-dependent manner. The effects of licorice extract on iNOS protein and mRNA expression were surveyed, respectively, by western blotting and real-time PCR. LPS performed strong reduction of upregulations. To confirm the effect of glycyrrhizin in a crude licorice extract, a glycyrrhizin-knockout extract and combination of glycyrrhizin-knockout extract and glycyrrhizin were investigated. As shown in [Figure 8], the glycyrrhizin-knockout extract had a weak effect on NO production, compared with the licorice extract. However, the addition of glycyrrhizin to the glycyrrhizin-knockout extract restored the inhibition of NO production, suggesting a synergic effect between the glycyrrhizin-knockout extract and glycyrrhizin. This phenomenon was confirmed by western blotting analysis [Figure 8].[66]
Figure 8: Effect of licorice extract, glycyrrhizin-knockout extract, and co-treatment with glycyrrhizin-knockout extract and glycyrrhizin on nitric oxide production (a) and inducible nitric oxide synthase protein expression (b) in lipopolysaccharide-treated RAW264 macrophages.[66] Each bar represents the mean ± standard deviation of four separate experiments. *P < 0.05, **P < 0.01, *P < 0.001 compared with lipopolysaccharide alone

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Recently, Zhao et al.[72] successfully prepared a knockout baicalin extract from TCM. The use of this knockout extract system may increase because it is necessary to survey the role of a hapten component in a crude extract.

  Immunoaffinity Concentration Using an Affinity Column Top

Immunoaffinity enrichment is performed when a target protein content will be lower.[73],[74] However, a similar system has not yet been developed in the field of natural small molecule product research. Therefore, one example will be discussed here.

Panax japonicus, which differs morphologically from other Panax species, is distributed throughout Japan and China. Yahara et al. reported a lack of ginsenoside-Rbl expression in P. japonicus and isolated and structurally analyzed a group of oleanane-type saponins designated “chikusetsusaponins.”[75] Morita et al.[76] conducted a chemical analysis of saponins to investigate the varieties of P. japonicus. The findings suggest that ginsenoside-Rb1 might be present in trace levels. However, because the authors used ELISA to identify higher concentrations than those reported previously,[76] we used an immunoaffinity column conjugated with anti-ginsenoside Rb1 MAb for the immunoaffinity enrichment of ginsenoside-Rb1.

The crude rhizome extract of P. japonicus was loaded on an immunoaffinity column and washed with the washing solvent, followed by an elution solvent. [Figure 9] shows the H2 SO4 staining (A) and eastern blotting (B) profiles of the two fractions separated by the immunoaffinity column [Figure 9]. Fraction 1, which was eluted in washing solvent, exhibited many spots, including chikusetsusaponins, similar to the original extract of P. japonicus. However, fraction 2 contained higher concentration of the molecules in compound 1, although two additional bands were still detected through TLC. Compound 1 was clearly indicative of a dammarane saponin with a protopanaxadiol framework and three sugars; the Rf value of this molecule was similar to that of ginsenoside-Rd, suggesting that compound 1 was chikusetsusaponin III. We finally identified compound 1 as chikusetsusaponin III through a direct comparison with an authentic sample.[75]
Figure 9: Immunoaffinity concentration of ginsenosides in Panax japonicus using an immunoaffinity column conjugated with anti-ginsenoside Rb1 monoclonal antibody.[56] (a) Thin-layer chromatography plate stained with sulfuric acid, (b) eastern blotting with anti-ginsenoside Rb1 monoclonal antibody. Lanes I–IV are washing fractions. Lanes V and VI are eluting fractions. G-Rg1, G-Re, G-Rd, G-Rc, and G-Rb1 indicate ginsenoside Rg1, ginsenoside Re, ginsenoside Rd, ginsenoside Rc, and ginsenoside Rb1, respectively

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A clear unknown band, compound 2, appeared in fraction 5, which was eluted in elution solvent. Ginsenoside-Rb1 was not detected by eastern blotting or TLC, as indicated in [Figure 8][56]. However, compound 2 exhibited the same staining color as ginsenoside Rb1, suggesting that compound 2 also contains protopanaxadiol as an aglycone. Furthermore, when the Rf values of compound 2 and ginsenoside Rb1, which possesses four sugar moieties, were compared, the result indicated that compound 2 might possess five sugar moieties. These findings suggested that compound 2 might be chikusetsusaponin III-20-O-gentiobiose, or chikusetsusaponin IV, following a direct comparison with an authentic sample of chikusetsusaponin IV.[75] Finally, we concluded that P. japonicus rhizome contains no ginsenoside-Rb1, as previously reported.[75] It became evident that structurally related unknown compounds with MAb cross-reactivity can be separated, purified, and isolated on an immunoaffinity column, depending on the strength of affinity for the MAb. This newly developed purification system may introduce a new mode of MAb purification technology.

  Single-Chain Variable Fragment of Monoclonal Antibody and Applications for Analytical Methodology Top

Single-chain variable fragment

The current method of MAb production through hybridoma technology requires the use of animals, specialized cell culture facilities, and extensive commitments of time and labor. To overcome some of these obstacles, MAbs can be produced by expressing only the scFv. The scFv is a contiguous polypeptide containing both the heavy chain variable region, and the light chain variable region of an immunoglobulin, linked by a short flexible peptide. Although the scFv is approximately one-sixth the size of a full-length MAb, this smaller molecule retains the high affinity and specificity of the parental MAb. Fortunately, scFvs can be expressed in various hosts, including bacteria,[77] yeast,[78] insect cell,[79] and plants.[80] In addition, scFvs may have multiple applications in the fields of food, cosmetics, environmental industry, and plant breeding can be opened. For example, Putalun et al. successfully achieved missile-type molecule breeding using an anti-solamargine scFv gene.[81] The induction of scFv gene in the host plant, Solanum khasianum, stimulated antigen molecule biosynthesis. In this case, because scFv protein expression was confirmed in the host plant, it might function as a neutralizing antibody against hapten molecules in plant cells, leading to a 3-fold increase in solasodine glycoside, the hapten molecule, relative to the concentration in the wild-type plant,[81] Subsequent successful increases in the hapten molecule, plumbagin, which has antioxidant, anti-inflammatory, anticancer, antibacterial, and antifungal activities,[82] were reported in the transgenic host plant, Plumbago zeylanica, following the induction of a scFv against plumbagin,[83]


Green fluorescent protein (GFP) was first isolated from jellyfish by Shimomura et al.[84] in 1962, and has since been widely applied throughout the field of biochemistry. A chimeric protein of GFP with a scFv is called a fluobody [85] and can be used in qualitative and quantitative immunoassays such as fluorescence-linked immunosorbent assays (FLISAs),[86],[87],[88],[89] rather than standard ELISAs. In addition, recent advances in biotechnology have enabled the expression of GFP in various hosts, including bacteria, yeast, plants, animals, and even living cells, for multiple purposes across many fields.[90],[91],[92] Recently, Sakamoto et al. successfully designed a FLISA against natural products such as plumbagin and ginsenoside Re.[93],[94],[95] The use of fluobody-based assay systems might increase in the field of natural product research, given the evidence to suggest the increased adequacy, speed, simplicity, and sensitivity of this assay system, which eliminates unnecessary secondary antibody and labeled enzyme-substrate reactions.

  Conclusion Top

This review has discussed the applications of various MAbs according to specific affinities for hapten molecules in natural small molecule products. Here quantitative, highly specific, and reproducible analytical systems such as ELISA and double eastern blotting, which yields several types of information (e.g., sugar number and type of aglycones), facilitates the elucidation of structural features and biological activity. Immunoaffinity has expand beyond the limitation of one-step hapten compound isolation to include knockout extracts, which can be used to determine the true active compound in the crude extract. Moreover, the combination of several knockout extract in TCM may be able to solve the pharmacological activity of TCM. The importance of scFv proteins is increasing in it importance. For instance, fluobodies can be used as analytical tools. Furthermore, a new technology related to novel plant breeding named technologies using scFv gene, such as missile-type molecule breeding was discussed in this reviewed. Its importance might be increasing, because no success of direct stimulation system for the biosynthetic pathway of natural product having small molecule. Moreover, one possibility may be to elucidate the role of hapten molecule in plant cells.

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Conflicts of interest

There are no conflicts of interest.

  References Top

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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9]


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