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Monoclonal Antibody Usage Strategies for Natural Products in Traditional Chinese Medicine

Yukihiro Shoyamaa aDepartment of Pharmacognosy, Faculty of Pharmaceutical Science, Nagasaki International University, Sasebo, Nagasaki 859‑3298, Japan

Abstract

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) have led to novel systems such as fluobodies or antibodies coupled with green fluorescent 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

Introduction and/or qualitative analytical techniques both in vivo and in vitro. Regarding their uses in other field, at least 400 Because pharmacologically active compounds and/or marker therapeutic MAbs are available. Although MAbs against components vary according to cultivation conditions, location, natural small molecular products were first introduced in the species differences, and harvest season, many countries have 1980s, few existed except for those that targeted drugs such developed pharmacopoeia to ensure the quality control of natural as morphine.[8] In the 1990s, increased preparation of MAbs products. Natural product and/or pharmacognostic analyses against natural products such as terpenoids.[9‑12] alkaloids,[13,14] incorporate disciplines such as chemistry, biology, physics, and plant saponins,[15‑19] phenolics,[20‑23] and more recently, . In addition, chromatographic analyses include artemisinin,[24] daidzin,[25] puerarin,[26] and capsaicinoids[27] techniques such as thin‑layer (TLC), capillary was reported, leading to the development of individual electrochromatography,[1] and (GC) enzyme‑linked immunosorbent assays (ELISAs) for the quality which may be coupled with mass spectrometry (GC‑MS),[2] control of traditional Chinese medicine (TCM) agents. high‑performance liquid chromatography (HPLC) coupled with MS (LC‑MS),[3] and LC‑MS‑MS[4] coupled with an Immunoaffinity purification is a highly specific technique that evaporative light scattering detector.[5] could potentially be used for the one‑step isolation of a target molecule from complex mixtures such as cellular lysates.[28] 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 Address for correspondence: Prof. Yukihiro Shoyama, Department of Pharmacognosy, Faculty of Pharmaceutical Science, occurred to the use of monoclonal antibodies (MAbs) against Nagasaki International University, Sasebo, Nagasaki 859‑3298, Japan. target molecules such as low molecular weight bioactive E‑mail: [email protected] 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 This is an open access article distributed under the terms of the Creative Commons Attribution‑NonCommercial‑ShareAlike 3.0 License, which allows others to remix, tweak, [7] as receptor binding assays, enzyme assays, and quantitative and build upon the work non‑commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Access this article online For reprints contact: [email protected] Quick Response Code: © 2017 World Journal of Traditional Chinese Medicine | Published by Wolters Kluwer ‑ Medknow Website: www.wjtcm.net Received: 19‑10‑2016, Accepted: 21-07-2017

DOI: How to cite this article: Shoyama Y. Monoclonal antibody usage strategies 10.4103/wjtcm.wjtcm_5_17 for natural products in traditional chinese medicine. World J Tradit Chin Med 2017;3:18-27.

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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 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 Figure 1: Matrix‑assisted laser desorption ionization‑time of flight mass and discuss the available information. spectrometry analysis of a tetrahydrocannabinolic acid‑bovine serum albumin conjugate[32]

Monoclonal Antibodies as Analytical Equipment Preparation of monoclonal antibody and enzyme‑linked Determination of Hapten number in a Hapten‑carrier immunosorbent assays protein conjugate Glycosides of natural products were treated with a solution We will first discuss hapten number determination, as the of NaIO4 to cleave sugar moieties and release aldehyde confirmation of this factor in a synthesized antigen is the groups that could be combined with carrier proteins. most important step in the first stage of MAb preparation. The resulting synthesized antigen was injected into mice The synthesis of a hapten derived from an immune antigen, and used to prepare MAb as follows. A hybridoma that linker bridge, and conjugated carrier protein is necessary produced hapten‑reactive MAb was obtained using standard for the production of MAbs. Previously, however, no direct procedures;[13] the resulting MAb was classified as an IgG and appropriate methods for the determination of haptens isotype having light chain. The reactivity of this IgG MAb conjugated with carrier protein existed in the absence of was tested using varying antibody titers and a dilution differential ultraviolet analysis, radiochemical, or chemical curve. Competitive ELISA is then used to determine the methods. Therefore, immunization through the injection of antibody concentration. In this assay, free MAb and a hapten‑carrier protein conjugates was unreliable. A direct competing agent are bound to polystyrene microtiter plates analytical method to assess hapten and carrier protein conjugates precoated with hapten‑human serum albumin (HSA). Under using matrix‑assisted laser desorption ionization‑time of these conditions, the full measurement range of the assay flight mass spectrometry (MALDI‑TOF MS), and an internal is generally 5–200 ng/ml,[9] and the quantitative ELISA standard was initially introduced in the 1990s. Subsequently, all analysis results should agree well with the HPLC analysis conjugates have been subjected to MALDI‑TOF MS to confirm results.[16] Cross‑reactivity is the most important factor when the hapten number before immunization. The first successful determining the value of an antibody. Because ELISAs were determination by MALDI‑TOF MS involved forskolin, an established for phytochemical investigations involving [31] activator of cyclic AMP, followed by a component of crude plant extracts, assay specificity was confirmed by [32] [33] marijuana and opium alkaloids. This methodology has determining the cross‑reactivity of the MAb with various [25] recently become popular. related compounds. The resulting cross‑reactivity data Figure 1 presents a co‑MALDI‑TOF MS of the major active were examined using competitive ELISA and calculated marijuana component tetrahydrocannabinolic acid conjugated using picomoles of hapten. In general, the MAb should to bovine serum albumin (BSA), with BSA used as an internal react very weakly with only a small number of structurally standard. This spectrum shows only singly and doubly related molecules and should not react with different ionized molecule ions of the intact conjugate. The sharp types of compound. However, if a MAb exhibiting wide peak at m/z 66465 is the [M + H] + of BSA. A smaller [M + H] cross‑reactivity is needed to detect all related compounds, a + peak of the tetrahydrocannabinolic acid‑BSA conjugate is synthetic design and hybridoma selection will be necessary. visible at m/z 70581. The calculated molecular mass of the For example, an anti‑solamargine MAb has a very wide tetrahydrocannabinolic acid moiety is 4314. From this result, cross‑reactivity for almost all solasodine glycosides[19] 12.7 molecules of tetrahydrocannabinolic acid were combined because these molecules share the same aglycone, with BSA.[32] This method is also suitable for natural small solasodine, which forms the base material of a steroidal molecule products, including glycosides.[16,17,19] hormone.[34]

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[44] Newly Developed Monoclonal Antibody Assay was investigated in murine kidney tissues. Furthermore, the authors identified α‑actinin‑4 as a target protein of aristolochic System acid[45] in human kidney cells through Time‑resolved fluoroimmunoassay with an anti‑aristolochic acid MAb, followed by LC‑MS The time‑resolved fluoroimmunoassay (TRFIA) was initially analysis after digestion.[46] developed in 1999.[35] This assay system uses lanthanide Eastern blotting elements and related chelates as tracers with unique Southern, northern, and western blotting are well‑known fluorescence properties. This technique is commonly used techniques for the detection of DNA, RNA, and protein in the agriculture and food industries, laboratory medicine, molecules, respectively. However, all of these methods involve forensic science, and environmental health studies.[36‑40] substances with larger molecular structures. In contrast, Taki We will provide a brief description of an analysis of et al.[47] and Towbin et al.[48] successfully blotted smaller saikosaponin A in Bupleuri radix. First, a microplate coated molecular compounds such as glycosphingolipids and with rabbit anti‑mouse IgG was incubated with a crude extract phospholipids from TLC to membranes, using a technique of Bupleuri radix and a mouse anti‑saikosaponin MAb;[17] a called far‑eastern blotting. The author and collaborators have lanthanide‑labeled saikosaponin A‑HSA conjugate was used developed a similar method to detect glycosides in natural as the tracer. The developed competitive TRFIA exhibited a products. The initial success of this method, which was good fourth‑order polynomial fitting from 0.01 to 10.0 μg/ml described as western blotting in 1997, involved a glycoalkaloid for a standard saikosaponin a sample, with a detection limit contained in Solanum species.[49] We later applied this method of 6 ng/ml.[40] to detect glycyrrhizin, the most important constituent of licorice, and coined the term eastern blotting in 2001;[18] Natural Product Staining Methods subsequently, we have applied eastern blotting to natural [17] [50] products such as saikosaponin A, sennoside A and B, and ginsenosides.[51,52] Eastern blotting differs from far‑eastern Although MAb has recently become an indispensable tool for blotting in that the former method involves separation of the immunostaining in biochemical and biophysical investigations, functions of the above glycoside into an (aglycone few such investigations have involved small molecules. The part) and membrane‑fixative (sugar moiety).[18] Although incorporation of glycosides such as crocin, a saffron‑derived the sphingolipid‑related starfish , which were molecule containing four glucoses and exhibiting various discussed in the context of far‑eastern blotting, were blotted functions in cells, was confirmed in PC‑12 cells using an to membranes, this blotting was insufficient. Accordingly, anti‑crocin MAb.[41] In another , the authors we directly stained a TLC plate using anti‑AG‑2 MAb.[53] evaluated the distribution of ginsenoside Rb1 in a fresh ginseng However, the name “eastern blotting” has remained under root slice by staining through eastern blotting,[42] and conducted discussion since 2007.[54] a microscopic observation using an anti‑ginsenoside Rb1 MAb. [43] Figure 2 demonstrates ginsenoside Rb1 immunostaining in In brief, the procedure of eastern blotting is as follows. ginseng tissues, resulting in clear staining that was supported A developed, glycoside‑bound TLC plate is covered with by gold particle staining and yielded good agreement with a polyvinylidene difluoride (PVDF) or polysulfone ether eastern blotting.[42] The immunostaining of aristolochic acid, membrane; after adding a blotting solution, the plate is briefly which exhibits strong nephrotoxic activity (reviewed below), 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 2: Histochemical staining of ginsenoside Rb1 in ginseng tissues Figure 3: Scheme of eastern blotting for glycyrrhizin with an using an anti‑ginsenoside Rb1 monoclonal antibody[43] anti‑glycyrrhizin monoclonal antibody[18]

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We have expanded our eastern blotting method to natural liquiritin (glycyrrhetinic acid and liquiritigenin, respectively. products with no linked sugar moieties, such as aristolochic This is a significant advantage for the detection of natural acid[55] well known as nephrotoxicants cause of Balkan products. However, because TLC monitoring is not specific, Endemic Nephropathy and Chinese Herb Nephropathy. fingerprinting patterns might be complicated. On the other Figure 4 indicates a schema of hapten synthesis and staining hand, eastern blotting patterns are simple and specific to the for aristolochic acid. antigenic molecule, as indicated in Figure 7b. Regarding the previously introduced case of solasodine glycosides, we present in Figure 5 an eastern of solasodine One‑step Purification on an Immunoaffinity glycosides to demonstrate that all solasodine glycosides can Column be stained. Immunoaffinity purification using commercially available Double eastern blotting MAbs and scFv proteins expressed in E. coli is commonly Double eastern blotting was also developed to evaluate used to purify proteins and peptides. However, because MAbs ginsenosides using an anti‑ginsenoside Rb1 MAb[15] and against natural products are generally not available, these anti‑ginsenoside Rg1 MAb.[16] Figure 6 shows an example of MAbs must be self‑prepared. Purified MAb is then coupled the double eastern blotting of ginsenosides.[51,52] Fingerprinting to a gel (e.g., Affi‑Gel Hz gel), which is used to prepare an through double eastern blotting of various ginseng samples immunoaffinity column. The column is washed with washing has yielded the following points. The Rf value reflects the buffer, and the bound hapten is generally eluted with an elution number of sugars conjugated to a molecule. Pinkish spots buffer containing an organic solvent. After separation, each indicate ginsenosides that possess the aglycone protopanaxtriol fraction is deionized and lyophilized to yield the pure hapten and exhibit hyperthymic activity, and blue spots indicate compound. For example, Yanagihara et al.[59] successfully protopanaxadiol‑type ginsenosides with depressive activity obtained 45 µg of pure forskolin, a cyclic AMP activator that is [57] in the central nervous system. Accordingly, several currently used as a cardiac drug in Japan, from a crude extract ginsenosides, including ginsenosides Rb1, Rc, and Rd, can be of Coleus forskohlii root (10 mg) using an immunoaffinity [15] stained with the highly specific anti‑ginsenoside Rb1 MAb. column conjugated with anti‑forskolin MAb and an elution Similar staining was observed with the anti‑ginsenoside Rg1 buffer containing 40% methanol in phosphate‑buffered MAb. These phenomena might be attributed to sugar saline. Moreover, because the ginseng root contains nearly a modification consequent to the cleavage of sugar moieties, hundred dammarane‑type saponins ginsenosides, as well as [42] which leads to wide MAb cross‑reactivity. oleanane‑type saponins, the isolation of individual saponins is Recently, Fujii et al.[58] developed a double eastern blotting quite tedious and requires repeated silica gel chromatography method to detect the glycosides glycyrrhizin (triterpene and/or preparative HPLC. An immunoaffinity column for glycoside) and liquiritin (flavonoid glycoside), as shown ginsenoside Rb1 isolation uses an elution solvent comprising in Figure 7. Figure 7a and 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

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), Figure 4: Scheme of hapten synthesis and eastern blotting for aristolochic solaverine II (9), 12-hydroxysolamargine (10) and 12- hydroxysolasonine acid using an anti‑aristolochic acid monoclonal antibody[55] (11), respectively

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a b Figure 7: Double eastern blotting of the licorice compounds glycyrrhizin and liquiritin using anti‑glycyrrhizin and anti‑liquiritin monoclonal [58] antibodies (a):0H2SO4 staining, (b): double easter blotting, 1:glycyrrhizin, 2: glycyrrhetic acid, 3: liquiritin, 4: liquiritigenin Figure 6: Double eastern blotting of ginsenosides in Panax species using anti‑ginsenosides Rb1 and Rg1 monoclonal antibodies.[42] Lanes I–VI named this washing fraction a glycyrrhizin‑knockout extract. indicate white ginseng, red ginseng, fibrous ginseng, Panax notoginseng, The capacity of our anti‑glycyrrhizin immunoaffinity column Panax quinquefolium, and Panax japonicus, respectively to capture glycyrrhizin was nearly matched by the capacities of other columns to capture forskolin[59] and solasodine an acetic acid buffer with potassium isocyanate and 20% glycosides.[61] This result suggests that in solutions containing methanol, a lower concentration than that used to isolate trace levels of glycyrrhizin that cannot be detected by ELISA, [60] forskolin. Charged ginsenoside Rb1 was eluted with an immunoaffinity column could concentrate glycyrrhizin to washing solution, repeatedly loaded, and finally isolated in a detectable level, as described above. a pure form. The antibody remained stable when exposed to the washing and eluting solvents, and the immunoaffinity To investigate the profiles of our glycyrrhizin‑knockout extract, column exhibited almost no decrease in capacity after more we performed a TLC analysis and eastern blotting. Several than 10 uses. Therefore, one‑step purification of a crude natural spots corresponding to glycyrrhizin and other compounds were detected in a licorice extract. However, although the other spots product extract on an immunoaffinity column conjugated were clearly detected in the glycyrrhizin‑knockout extract, the with an anti‑target compound MAb can supply sufficient spot corresponding to glycyrrhizin was completely absent. amounts of compound, on a bench and/or laboratory scale, for Furthermore, eastern blotting with an anti‑glycyrrhizin MAb in vitro because approximately 10 µg of antigen clearly detected glycyrrhizin in the licorice extract, but failed compound can be obtained per ml of gel. Furthermore, we to detect this compound in the glycyrrhizin‑knockout extract. have established several immunoaffinity columns using MAbs These findings suggest that glycyrrhizin can be specifically against solasodine glycosides[61] and glycyrrhizin.[62] Recently, eliminated from a licorice extract through separation on an immunoaffinity columns have been increasingly applied for anti‑glycyrrhizin MAb immunoaffinity column.[66] the isolation of natural products such as isoflavones[63] and tetrodotoxin, of which very small amounts can be concentrated Inflammation leads to the upregulation of a range of on an immunoaffinity column and detected using analytical enzymes and signaling mediators in affected tissues and devices.[64] cells. Nitric oxide (NO) is a highly reactive free radical involved in multiple physiological functions, including It became evident during immunoaffinity column purification vasodilatation, neurotransmission, and inflammation.[67] During that the washing solution contains all components except the an inflammatory process, large amounts of NO are produced by antigenic molecule. Therefore, we will discuss this washing inducible NO synthase (iNOS) in various cell types, including solution in the next section. macrophages, in response to stimulation from inflammatory cytokines and/or bacterial lipopolysaccharide (LPS).[68] Preparation of a Knockout Extract with an In certain inflammatory and autoimmune diseases, the overproduction of NO by iNOS triggers the pathogenic mmunoaffinity olumn I C conditions of septic shock and organ destruction.[69‑71] Therefore, The above‑mentioned washing fraction, which is produced blocking iNOS expression and thus inhibiting NO production during one‑step purification on an immunoaffinity column, might be a useful treatment option for a variety of inflammatory is known as a knockout extract[65] because only the antigen diseases. Uto et al.[68] found that a crude licorice extract molecule is eliminated, thus resembling the outcome of a could suppress NO release in LPS‑treated mouse RAW264 knockout gene. In fact, an immunoaffinity column conjugated macrophages. LPS evoked an increase in NO production, and with anti‑glycyrrhizin MAb could eliminate 99% of this induction was blocked by treatment with licorice extract in a glycyrrhizin from a crude extract of licorice.[66] We accordingly dose‑dependent manner. The effects of licorice extract on iNOS

22 World J Tradit Chin Med | Volume 3 | Issue 3 | July‑September 2017 Shoyama: Monoclonal antibody usage strategies protein and mRNA expression were surveyed, respectively, The crude rhizome extract of P. japonicus was loaded on by western blotting and real‑time PCR. LPS performed an immunoaffinity column and washed with the washing strong reduction of upregulations. To confirm the effect of solvent, followed by an elution solvent. Figure 9 shows glycyrrhizin in a crude licorice extract, a glycyrrhizin‑knockout the H2SO4 staining (A) and eastern blotting (B) profiles extract and combination of glycyrrhizin‑knockout extract of the two fractions separated by the immunoaffinity and glycyrrhizin were investigated. As shown in Figure 8, column [Figure 9]. Fraction 1, which was eluted in washing the glycyrrhizin‑knockout extract had a weak effect on NO solvent, exhibited many spots, including chikusetsusaponins, production, compared with the licorice extract. However, similar to the original extract of P. japonicus. However, the addition of glycyrrhizin to the glycyrrhizin‑knockout fraction 2 contained higher concentration of the molecules extract restored the inhibition of NO production, suggesting in compound 1, although two additional bands were still a synergic effect between the glycyrrhizin‑knockout extract detected through TLC. Compound 1 was clearly indicative and glycyrrhizin. This phenomenon was confirmed by western of a dammarane saponin with a protopanaxadiol framework blotting analysis [Figure 8].[66] and three sugars; the Rf value of this molecule was similar Recently, Zhao et al.[72] successfully prepared a knockout to that of ginsenoside‑Rd, suggesting that compound 1 was baicalin extract from TCM. The use of this knockout extract chikusetsusaponin III. We finally identified compound 1 as system may increase because it is necessary to survey the role chikusetsusaponin III through a direct comparison with an [75] of a hapten component in a crude extract. authentic sample. A clear unknown band, compound 2, appeared in fraction Immunoaffinity Concentration Using an Affinity 5, which was eluted in elution solvent. Ginsenoside‑Rb1 was not detected by eastern blotting or TLC, as indicated Column in Figure 8[56]. However, compound 2 exhibited the same Immunoaffinity enrichment is performed when a target protein staining color as ginsenoside Rb1, suggesting that compound [73,74] content will be lower. However, a similar system has not yet 2 also contains protopanaxadiol as an aglycone. Furthermore, been developed in the field of natural small molecule product when the Rf values of compound 2 and ginsenoside Rb1, research. Therefore, one example will be discussed here. which possesses four sugar moieties, were compared, the Panax japonicus, which differs morphologically from other result indicated that compound 2 might possess five sugar Panax species, is distributed throughout Japan and China. moieties. These findings suggested that compound 2 might be Yahara et al. reported a lack of ginsenoside‑Rbl expression in chikusetsusaponin III‑20‑O‑gentiobiose, or chikusetsusaponin P. japonicus and isolated and structurally analyzed a group of IV, following a direct comparison with an authentic sample oleanane‑type saponins designated “chikusetsusaponins.”[75] of chikusetsusaponin IV.[75] Finally, we concluded that Morita et al.[76] conducted a chemical analysis of saponins P. japonicus rhizome contains no ginsenoside‑Rb1, as to investigate the varieties of P. japonicus. The findings previously reported.[75] It became evident that structurally suggest that ginsenoside‑Rb1 might be present in trace levels. related unknown compounds with MAb cross‑reactivity can be However, because the authors used ELISA to identify higher separated, purified, and isolated on an immunoaffinity column, concentrations than those reported previously,[76] we used an depending on the strength of affinity for the MAb. This newly immunoaffinity column conjugated with anti‑ginsenoside Rb1 developed purification system may introduce a new mode of MAb for the immunoaffinity enrichment of ginsenoside‑Rb1. MAb purification technology.

a b 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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Fluobody 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),[86‑89] 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.[90‑92] Recently, Sakamoto et al. successfully designed a FLISA against natural products such as plumbagin and ginsenoside Re.[93‑95] The use of fluobody‑based assay systems might increase in the field a b of natural product research, given the evidence to suggest Figure 9: Immunoaffinity concentration of ginsenosides in Panax japonicus the increased adequacy, speed, simplicity, and sensitivity of using an immunoaffinity column conjugated with anti‑ginsenoside Rb1 this assay system, which eliminates unnecessary secondary [56] monoclonal antibody. (a) Thin‑layer chromatography plate stained with antibody and labeled enzyme‑substrate reactions. 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 Conclusion Rg1, ginsenoside Re, ginsenoside Rd, ginsenoside Rc, and ginsenoside This review has discussed the applications of various MAbs Rb1, respectively according to specific affinities for hapten molecules in natural small molecule products. Here quantitative, highly Single‑chain Variable Fragment of Monoclonal specific, and reproducible analytical systems such as ELISA Antibody and Applications for Analytical and double eastern blotting, which yields several types of information (e.g., sugar number and type of aglycones), Methodology facilitates the elucidation of structural features and biological Single‑chain variable fragment activity. Immunoaffinity has expand beyond the limitation The current method of MAb production through hybridoma of one‑step hapten compound isolation to include knockout technology requires the use of animals, specialized cell culture extracts, which can be used to determine the true active facilities, and extensive commitments of time and labor. To compound in the crude extract. Moreover, the combination overcome some of these obstacles, MAbs can be produced by of several knockout extract in TCM may be able to solve the expressing only the scFv. The scFv is a contiguous polypeptide pharmacological activity of TCM. The importance of scFv containing both the heavy chain variable region, and the light proteins is increasing in it importance. For instance, fluobodies chain variable region of an immunoglobulin, linked by a short can be used as analytical tools. Furthermore, a new technology flexible peptide. Although the scFv is approximately one‑sixth related to novel plant breeding named technologies using scFv the size of a full‑length MAb, this smaller molecule retains the gene, such as missile‑type molecule breeding was discussed high affinity and specificity of the parental MAb. Fortunately, in this reviewed. Its importance might be increasing, because scFvs can be expressed in various hosts, including bacteria,[77] no success of direct stimulation system for the biosynthetic yeast,[78] insect cell,[79] and plants.[80] In addition, scFvs may pathway of natural product having small molecule. Moreover, have multiple applications in the fields of food, cosmetics, one possibility may be to elucidate the role of hapten molecule environmental industry, and plant breeding can be opened. in plant cells. For example, Putalun et al. successfully achieved missile‑type Financial support and sponsorship [81] molecule breeding using an anti‑solamargine scFv gene. The Nil. induction of scFv gene in the host plant, Solanum khasianum, stimulated antigen molecule biosynthesis. In this case, because Conflicts of interest scFv protein expression was confirmed in the host plant, it might There are no conflicts of interest. function as a neutralizing antibody against hapten molecules in plant cells, leading to a 3‑fold increase in solasodine glycoside, References the hapten molecule, relative to the concentration in the 1. Lin X, Xue L, Zhang H, Zhu C. Determination of saikosaponins wild‑type plant,[81] Subsequent successful increases in the hapten a, c, and d in Bupleurum chinense DC from different areas by capillary molecule, plumbagin, which has antioxidant, anti‑inflammatory, zone . Anal Bioanal Chem 2005;382:1610‑5. anticancer, antibacterial, and antifungal activities,[82] were 2. Tor ER, Filigenzi MS, Puschner B. 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89. Sakamoto S, Shoyama Y, Tanaka H, Morimoto S. Application of green Kinjo J, et al. Development of sensitivity‑improved fluorescence‑linked fluorescent protein in immunoassays. Adv Biosci Biotechnol 2014;5:557‑63. immunosorbent assay using a fluorescent single‑domain antibody 90. Casey JL, Coley AM, Tilley LM, Foley M. Green fluorescent antibodies: against the bioactive naphthoquinone, plumbagin. Anal Bioanal Chem Novel in vitro tools. Protein Eng 2000;13:445‑52. 2010;396:2955‑63. 91. Peipp M, Saul D, Barbin K, Bruenke J, Zunino SJ, Niederweis M, et al. 94. Sakamoto S, Tanizaki Y, Pongkitwitoon B, Tanaka H, Morimoto S. Efficient eukaryotic expression of fluorescent scFv fusion proteins A chimera of green fluorescent protein with single chain variable directed against CD antigens for FACS applications. J Immunol fragment antibody against ginsenosides for fluorescence‑linked Methods 2004;285:265‑80. immunosorbent assay. Protein Expr Purif 2011;77:124‑30. 92. Naumann JM, Küttner G, Bureik M. Expression and secretion of 95. Sakamoto S, Pongkitwitoon B, Sasaki‑Tabata K, Putalun W, Maenaka K, a CB4‑1 scFv‑GFP fusion protein by fission yeast. Appl Biochem Tanaka H, et al. A fluorescent single domain antibody against plumbagin Biotechnol 2011;163:80‑9. expressed in silkworm larvae for fluorescence‑linked immunosorbent 93. Sakamoto S, Taura F, Pongkitwitoon B, Putalun W, Tsuchihashi R, assay (FLISA). Analyst 2011;136:2056‑63.

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