RESUMEN
Verbascoside and isoverbascoside are two active phenylethanoid glycosides mainly found in plants of the order Lamiales. This study analyzes the verbascoside and isoverbascoside levels and the total phenolic contents in the water and ethanolic extracts of 20 medicinal plants of the order Lamiales commonly used in Thailand. The related bioactivities, including the antioxidant activity via the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reduction activity potential assays and anti-tyrosinase and -inflammatory activities via the cyclooxygenase and nitric oxide assays are also investigated. The extracts of several plant species, including Barleria prionitis, B. lupulina, Rhinacanthus nasutus, Orthosiphon aristatus, and Nicoteba betonica, exhibit high verbascoside and isoverbascoside content levels. The correlation analysis between the bioactive activities and the active compounds demonstrates a significant association between the verbascoside level in the water extracts and both the DPPH antioxidant activity and the nitric oxide level in the anti-inflammatory assays. This study provides the first report on the verbascoside and isoverbascoside quantification of several plant samples. The findings provide valuable insights for future research on lesser-studied plants possessing high verbascoside and isoverbascoside levels, which exhibit promising anti-inflammatory activities.
RESUMEN
To detect major soy isoflavone glycosides, namely daidzin (DZ) and genistin (GEN), novel open sandwich fluorescence-linked immunosorbent assay (os-FLISA) was developed by taking advantage of enhanced interactions between variable regions of heavy (VH) and light chain (VL) domains in the presence of an antigen. The VH and VL genes were expressed in Escherichia coli as a chimera protein with green fluorescence protein (AcGFP1) and maltose-binding protein (MBP), respectively. Comprehensive characterization of os-FLISA displayed nearly the same specificity as parental DZ- and GEN-specific monoclonal antibody, demonstrating the potential of the developed assay for detection of both DZ and GEN. Their detectable range in this system exhibited at 0.1-12.5 µg mL-1. Subsequent validation analysis revealed that os-FLISA was reliable and accurate system for detection of total soy isoflavone glycosides. Notably, this is the first FLISA based on an open sandwich system, which can be employed for the detection of small molecules.
Asunto(s)
Glycine max/química , Técnicas de Inmunoadsorción , Isoflavonas/análisis , Anticuerpos Monoclonales/genética , Escherichia coli/genética , Fluorescencia , Análisis de los Alimentos/métodos , Proteínas Fluorescentes Verdes/genética , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/genética , Isoflavonas/inmunología , Límite de Detección , Proteínas de Unión a Maltosa/genética , Proteínas Recombinantes/genética , Reproducibilidad de los ResultadosRESUMEN
In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.
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Acetatos/farmacología , Centella/efectos de los fármacos , Ciclopentanos/farmacología , Oxilipinas/farmacología , Raíces de Plantas/efectos de los fármacos , Triterpenos/metabolismo , Agrobacterium , Biotecnología , Centella/microbiología , Diploidia , Enfermedades de las Plantas/microbiología , Hojas de la Planta/efectos de los fármacos , Raíces de Plantas/microbiología , TetraploidíaRESUMEN
Derris scandens (Roxb.) Benth. is a medicinal plant used for treatment of musculoskeletal pain in Thai traditional medicines. Its stem contains active compound genistein-7-O-[α-rhamnopyranosyl-(1 to 6)-ß-glucopyranoside] (GTG) which is used as a biomarker for standardization of D. scandens extracts. As an alternative for rapid quantitation of GTG, a monoclonal antibody against GTG was prepared and applied for an indirect competitive enzyme-linked immunosorbent assay (ELISA) to determine GTG in plants and herbal products. The established method provided a quantification range of 0.31-10 µg/mL with a limit of detection of 0.29 µg/mL. The assay was validated for precision and accuracy by intra- and interassay variation analyses, recovery test, and comparison analysis between the amounts of GTG determined by ELISA and HPLC. The results exhibited that the developed ELISA is sensitive and effective for determination of GTG in D. scandens plant materials and herbal products.
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Anticuerpos Monoclonales/inmunología , Derris/química , Ensayo de Inmunoadsorción Enzimática/métodos , Genisteína/análisis , Extractos Vegetales/análisis , Extractos Vegetales/inmunología , Control de Calidad , Cromatografía Líquida de Alta Presión , Genisteína/análogos & derivados , Genisteína/inmunologíaRESUMEN
INTRODUCTION: Miroestrol is the potent phytoestrogen isolated from White Kwao Krua (Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, a Thai traditional medicinal plant. Nowadays, various health supplementary products featuring White Kwao Krua are available worldwide. A sensitive and rapid analytical method for quantification of miroestrol is necessary for quality control of these products. OBJECTIVES: To prepare a single-chain variable fragment (scFv) antibody specific to miroestrol and develop a scFv-based enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of miroestrol in plant materials and health supplementary products. METHODS: A gene encoding anti-miroestrol scFv antibody was constructed and expressed in Escherichia coli SHuffle T7 strain. Anti-miroestrol scFv antibody was characterised and applied to ELISA. The developed scFv-based ELISA method was validated for its sensitivity, specificity, accuracy and precision. RESULTS: Anti-miroestrol scFv antibody was highly specific to miroestrol. The scFv-based ELISA was applied to determine miroestrol in the range 0.06-7.81 µg/mL, with the limit of quantification of 0.06 µg/mL miroestrol. The accuracy of the assay was validated by its 95.08-103.99% recovery from the spiked miroestrol recovery experiment and in good correlation with the results from the monoclonal antibody-based ELISA. The relative standard deviation of the intra- and inter-assay were less than 6.0%. CONCLUSION: The developed scFv-based ELISA was sensitive, specific, accurate, and precise for determination of miroestrol and useful for quality control of P. candollei plant raw materials and supplementary products.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Pueraria/química , Anticuerpos de Cadena Única/inmunología , Esteroides/inmunología , Secuencia de Aminoácidos , Límite de Detección , Control de Calidad , Reproducibilidad de los ResultadosRESUMEN
Homoharringtonine (HHT), also known as omacetaxine, is a natural compound found in the genus Cephalotaxus and is a promising pharmaceutical drug used for the treatment of chronic or accelerated phase chronic myeloid leukemia. As a tool for the quantitative determination of HHT, a specific monoclonal antibody against HHT (MAb 6A1) was generated by conjugates prepared via sodium periodate-mediated oxidation. The developed indirect competitive enzyme-linked immunosorbent assay (icELISA) using MAb 6A1 was found to be highly specific and sensitive with a limit of detection for HHT of 48.8 ng/mL. Validation assays to evaluate precision and accuracy of the method were conducted by the use of intra- and inter-assay analysis, recovery test, and comparison analysis between the amounts of HHT determined by ELISA and high-performance liquid chromatography. These results revealed that the established icELISA using MAb 6A1 is specific, sensitive, and reliable enough to be applied to the qualitative analysis for HHT. Furthermore, the results of this study support the usefulness of sodium periodate as a reagent for the conjugation between Cephalotaxus alkaloids and proteins for producing specific antibodies.
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Ensayo de Inmunoadsorción Enzimática/métodos , Harringtoninas/análisis , Anticuerpos Monoclonales/inmunología , Cromatografía Líquida de Alta Presión , Harringtoninas/inmunología , Homoharringtonina , Reproducibilidad de los ResultadosRESUMEN
The peptide linker between variable domains of heavy (VH) and light (VL) chains is one of important factors that influence the characteristics of scFv, including binding activity and specificity against target antigen. The scFvs against daidzin (DZ-scFvs) with different linker lengths were constructed in the format of VH-(GGGGS)n-VL (n = 1, 3, 5, and 7). They were expressed in the hemolymph of silkworm larvae using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system, and their reactivity against daidzin and related compounds were evaluated using an indirect competitive enzyme-linked immunosorbent assay (icELISA), which is applicable for quantitative analysis of daidzin. The results showed that the reactivity of scFvs against daidzin was increased, whereas specificity slightly decreased when their peptide linker was lengthened. These results suggested that the linker length of DZ-scFvs contributes to its reactivity. In addition, the results emphasize that the linker length could control the reactivity of DZ-scFvs.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Isoflavonas/química , Larva/inmunología , Ingeniería de Proteínas , Secuencias de Aminoácidos , Animales , Especificidad de Anticuerpos , Bombyx/química , Bombyx/inmunología , ADN/genética , ADN/inmunología , Expresión Génica , Hemolinfa/inmunología , Cadenas Pesadas de Inmunoglobulina/biosíntesis , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/biosíntesis , Cadenas Ligeras de Inmunoglobulina/genética , Isoflavonas/inmunología , Larva/química , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Relación Estructura-ActividadRESUMEN
Daidzin (DZ) and genistin (GEN) are two major soy isoflavone glycosides isolated from soybeans. Soy products containing isoflavones have recently been widely accepted for commercial use. However, the Japanese Government has suggested that soy isoflavone intake should be limited because of their estrogenic effects due to their interactions with estrogen receptors. In this study, we established a one-step indirect competitive immunochromatographic assay (ICA) for rapid and sensitive detection of total isoflavone glycosides (DZ and GEN) using gold nanoparticles conjugated with a monoclonal antibody against DZ. This assay was able to be completed in 15min following the immersion of a test strip in an analyte solution. Furthermore, the limit of detection for the total amount of isoflavone glycosides was â¼125ngmL(-1). Considering that the major soy isoflavone glycosides found in soy products are DZ and GEN, this study demonstrates the potential use of ICA for the assessments of over consumption of isoflavones in soy supplements and foods, which would increase the safe dietary intake of soy products.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Cromatografía de Afinidad/métodos , Glycine max/química , Glicósidos/química , Oro Coloide/química , Isoflavonas/químicaRESUMEN
Soy isoflavones are known as major bioactive compounds in soybean (Glycine max), which is an indispensable food. Despite their utility, the consumption of isoflavones has recently been limited because they exhibit oestrogenic and topoisomerase II inhibitory effects. To assess their intake limitation, accurate, sensitive, and effective quantitative analyses are necessary. In this study, we produced the monoclonal antibody (MAb) against daidzin (DZ) and applied it to an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the simultaneous determination of DZ and genistin (GEN), which are known as two major soy isoflavone glycosides in soy products. Using the DZ-MAb, we developed a sensitive icELISA method, where the limit of detection for DZ and GEN was 1.95ng/ml. Several validation analyses revealed that the icELISA is sufficiently accurate and sensitive to be used to assess the overconsumption of soy isoflavones, which would lead to the safe dietary intake of soy products.
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Ensayo de Inmunoadsorción Enzimática/métodos , Glycine max/química , Glicósidos/análisis , Isoflavonas/análisis , Extractos Vegetales/análisis , Anticuerpos Monoclonales/análisis , Hidrólisis , Sensibilidad y EspecificidadRESUMEN
A single-chain variable fragment antibody (scFv) against plumbagin (PL) accumulated the PL production in the hairy roots of Plumbago zeylanica. Recombinant Agrobacterium rhizogenes (ATCC 15834) containing an scFv gene against PL (PL-scFv) were obtained through triparental mating and transformed into P. zeylanica to induce PL-scFv protein in the hairy roots. Up to 40 µg recombinant PL-scFv were expressed per milligram of soluble protein in transgenic P. zeylanica hairy root cultures. The mean PL content obtained from transgenic hairy roots (12.24 µg/100 mg dry weight) exhibited 2.2 times higher than those obtained from wild-type (5.48 µg/100 mg dry weight). The high correlation between the PL-scFv expression level and PL content of the recombinant plants suggested that the PL biosynthesis pathway had been modulated by the expression of PL-scFv protein in the hairy roots of P. zeylanica.
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Naftoquinonas/inmunología , Naftoquinonas/metabolismo , Anticuerpos de Cadena Única/genética , Agrobacterium/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Plumbaginaceae , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 µg mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.
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Bombyx/metabolismo , Técnica del Anticuerpo Fluorescente/métodos , Naftoquinonas/análisis , Anticuerpos de Cadena Única/metabolismo , Animales , Bombyx/crecimiento & desarrollo , Ensayo de Inmunoadsorción Enzimática/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Larva/genética , Larva/metabolismo , Nucleopoliedrovirus/genética , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunologíaRESUMEN
A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli (E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.
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Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente/métodos , Ginsenósidos/metabolismo , Proteínas Fluorescentes Verdes/química , Anticuerpos de Cadena Única/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/metabolismo , Espectrometría de FluorescenciaRESUMEN
A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 µg per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
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Ácido 2,4-Diclorofenoxiacético/inmunología , Bombyx/genética , Expresión Génica , Herbicidas/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genética , Ácido 2,4-Diclorofenoxiacético/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bombyx/crecimiento & desarrollo , Bombyx/metabolismo , Bombyx/virología , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Hemolinfa/química , Hemolinfa/metabolismo , Hemolinfa/virología , Herbicidas/análisis , Larva/genética , Larva/metabolismo , Larva/virología , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Anticuerpos de Cadena Única/aislamiento & purificación , Anticuerpos de Cadena Única/metabolismoRESUMEN
A single-chain variable fragment antibody (scFv) against ginsenoside Re (G-Re) was constructed and applied to an enzyme-linked immunosorbent assay (ELISA) for determining the total concentration of ginsenosides in various ginsengs. The variable heavy and light chain genes were cloned directly from the cDNA of the 4G10 hybridoma cell line and assembled by means of splicing by overlapping extension PCR (SOE-PCR) using specific primers designed to have flexible peptide (Gly(4)Ser)(3) between the variable heavy chain and light chain domains. The constructed scFv gene was ligated into the pET28a expression vector and transformed into E. coli BL21 (DE3). The recombinant scFv against G-Re (GRe-scFv) was expressed as a chimera protein containing the His6-tag at its N-termini, purified by immobilized metal ion affinity chromatography (IMAC), and refolded by a stepwise dialysis method. The yield of GRe-scFv after purification was 1.7 mg per liter of culture medium. Characterization of GRe-scFv revealed that it retained the characteristics of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10) which has wide cross-reactivity with 20(S)-protopanaxadiol- and 20(S)-protopanaxatriol-type ginsenosides. The detectable range for G-Re in ELISA using scFv antibody was 0.02-10 µg/ml. Based on validation analysis, the use of GRe-scFv in ELISA is a precise, accurate, and sensitive method. In light of the time-consuming and labor-intensive procedures for the preparation of MAb, speedy bacterial expression of GRe-scFv is a powerful alternative tool for producing MAb to use in ELISA for quantitative analysis of total ginsenoside concentrations.
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Ginsenósidos/análisis , Panax/química , Anticuerpos de Cadena Única/inmunología , Secuencia de Aminoácidos , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática/métodos , Ginsenósidos/inmunología , Datos de Secuencia Molecular , Alineación de Secuencia , Anticuerpos de Cadena Única/químicaRESUMEN
A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 microg/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.
Asunto(s)
Bombyx/genética , Clonación Molecular/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Ginsenósidos/análisis , Anticuerpos de Cadena Única/biosíntesis , Animales , Baculoviridae/genética , Ensayo de Inmunoadsorción Enzimática/normas , Vectores Genéticos , Ginsenósidos/inmunología , Nucleopoliedrovirus/genética , Proteínas Recombinantes , Anticuerpos de Cadena Única/genéticaRESUMEN
A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.
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Ensayo de Inmunoadsorción Enzimática/métodos , Técnica del Anticuerpo Fluorescente Directa/métodos , Inmunoadsorbentes/análisis , Naftoquinonas/inmunología , Anticuerpos de Cadena Única/análisis , Reacciones Cruzadas , Escherichia coli/genética , Escherichia coli/metabolismo , Límite de Detección , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/metabolismoRESUMEN
Pueraria candollei (White Kwao Khuer) is a medicinal plant containing puerarin, daidzin, genistin, daidzein, and genistein as major isoflavonoids used for its rejuvenating and estrogenic effects. In order to analyze these compounds, a single enzyme-linked immunosorbent assay (ELISA) for total isoflavonoids was developed using anti-puerarin and anti-daidzin polyclonal antibodies (PAbs). The range for calibration of isoflavonoids by ELISA was 0.05-6.25 microg/mL. Total isoflavonoid concentrations in P. candollei samples determined by the newly developed assay system showed good agreement with those analyzed by HPLC. Based on validation analysis, this analytical method by ELISA is a precise, accurate, and sensitive method for the determination of total isoflavonoids in P. candollei.
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Anticuerpos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Flavonoides/análisis , Isoflavonas/inmunología , Pueraria/química , Animales , Cromatografía Líquida de Alta Presión , Femenino , ConejosRESUMEN
We constructed a single-chain variable fragment (scFv) antibody against plumbagin (PL) with improved specific binding to PL. Variable heavy- and light-chain genes were cloned directly from the cDNA of hybridoma cell line 3A3 and assembled using the splice-overlap extension polymerase chain reaction (SOE-PCR) with specific primers including flexible peptide (Gly(4)Ser)(3) linker primers. The constructed scFv gene was ligated into the pET28a expression vector and transformed into Escherichia coli BL21 (DE3). The denatured protein expressed as inclusion bodies in E. coli was solubilized, purified, and refolded by a stepwise dialysis. Intriguingly, the refolded scFv against PL displayed higher PL-binding specificity than that of its parental monoclonal antibody, MAb 3A3, which suggests the possibility of improving the function by constructing the scFv antibody. These notable properties of the recombinant antibody against PL made it possible to develop an enzyme-linked immunosorbent assay (ELISA) for reliable determination of PL.