Simultaneous rapid detection of glycyrrhizin and sennoside A in Daiokanzoto samples by lateral flow immunoassay.

INTRODUCTION: Glycyrrhizin (GLY) and sennoside A (SA) are characteristic bioactive marker compounds of the Kampo medicine Daiokanzoto. Their accurate detection in blends of Rhei rhizoma and Glycyrrhizae radix of several species (4:1 or 4:2) is essential for quality control and to ensure therapeutic efficacy. A rapid, efficient assay can significantly facilitate their detection. OBJECTIVE: To establish a rapid qualitative assay for GLY and SA detection, a lateral flow immunoassay (LFA) was developed using specific monoclonal antibody (mAb) nanoparticles. METHODOLOGY: This assay harnesses the competitive binding of mAb nanoparticles to the immobilized analytes on test strips and free analytes in the samples. Two conjugates for detecting GLY and SA, GLY-bovine serum albumin and SA-human serum albumin, were separately immobilized on the test zones of LFA strips. The detection mechanism is reliant on the visual detection of color changes in the test zones. RESULTS: When GLY and SA were present in samples, they contended with the immobilized conjugates on the strip to bind with the mAb nanoparticles and produced distinct color patterns in the test zones. The limits of detection of the assay for GLY and SA were both 3.13 µg/mL. The capability of the LFA was substantiated using plant samples and Daiokanzoto, and its alignment with indirect competitive ELISA results was confirmed. CONCLUSION: The introduced LFA is a groundbreaking procedure that offers a rapid, straightforward, and sensitive method for simultaneously detecting GLY and SA in Daiokanzoto samples. It is instrumental in ensuring product quality.

Comparative stability and analytical performance of anti-miroestrol recombinant antibody in different cassettes.

Immunoassays are efficient for the phytochemical analysis of various matrices. However, producing an appropriate recombinant antibody for small molecules is challenging, resulting in costly analyses. In this study, we aimed to develop recombinant fragment antigen-binding (Fab) antibodies against miroestrol, a potent phytoestrogen marker of Pueraria candollei. Two expression cassettes of Fab were established for the production of active Fab antibodies using SHuffle® T7 Escherichia coli cells. The orientation of variable fragment heavy chain (VH) and variable fragment light chain (VL) in the expression vector constructs influences the reactivity, stability, and binding specificity of the resultant Fab. Stability testing of antibodies demonstrated that Fab is a more stable form of recombinant antibody than a single-chain variable fragment (ScFv) antibody in all conditions. Based on the obtained Fab, the ELISA specifically detected miroestrol in the range of 39.06-625.00 ng/mL. The intra- and inter-assay precisions were 0.74-2.98% and 6.57-9.76%, respectively. The recovery of authentic miroestrol spiked into samples was 106.70-110.14%, and the limit of detection was 11.07 ng/mL. The results for P. candollei roots and products determined using our developed ELISA with Fab antibody and an ELISA with anti-miroestrol monoclonal antibody (mAb) were consistent (R2 = 0.9758). The developed ELISA can be applied for the quality control of miroestrol derived from P. candollei. Therefore, the appropriate expression platform of Fab resulted in the stable binding specificity of the recombinant antibody and was applicable for immunoassays.Key points• ELISAs with Fab has higher sensitivity than that with ScFv.• Fab is more stable than ScFv.• Fab-based ELISA can be used for miroestrol determination of Pueraria candollei.

Construction of a monoclonal antibody against glabridin (2G4) and development of an enzyme-linked immunosorbent assay.

INTRODUCTION: Glabridin is a unique isoflavonoid found only in Glycyrrhiza glabra L. The pharmacological effects of glabridin are well established, especially for beauty- and wellness-related uses, such as antioxidant, anti-inflammatory, ultraviolet (UV) protection, and skin-lightening effects. Therefore, glabridin is often found in commercial products such as creams, lotions, and dietary supplements. OBJECTIVE: This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) using a glabridin-specific antibody. METHOD: Immunogen conjugation of glabridin-bovine serum albumin was performed via the Mannich reaction, and the resulting conjugates were injected into BALB/c mice. Subsequently, hybridomas were produced. An ELISA method for glabridin determination was developed and validated. RESULT: A highly specific antibody against glabridin was produced using clone 2G4. The assay range for the determination of glabridin was 0.28-7.02 µg/ml, with a detection limit of 0.16 µg/ml. The validation parameters in terms of accuracy and precision met the acceptable criteria. Standard curves of glabridin in various matrices were compared to evaluate the matrix effect on human serum using ELISA. Standard curves of the human serum and water matrix were obtained in the same manner, and the measurement range was 0.41-10.57 µg/ml. CONCLUSION: The developed ELISA method was used to quantify glabridin in plant materials and products with high sensitivity and specificity, and has potential applications in quantifying compounds in plant-derived products and human serum samples.

Quality assessment method for Chinpi, dried Citrus spp. peel and its derived Kampo medicines using specific monoclonal antibody against hesperidin.

INTRODUCTION: Hesperidin (hesperetin 7-rutinoside, HP), a flavonoid glycoside found in Citrus unshiu Marcowicz or Citrus reticulata Blanco (Rutaceae), has been reported to exert a variety of pharmacological effects. As the efficacies and qualities of their dried peel, Chinpi and its derived Kampo medicines can be evaluated by their HP contents, a method for HP detection must be developed. OBJECTIVES: To produce a specific monoclonal antibody against HP (mAb 5D12) to detect the HP contents in Japanese traditional medicines via indirect competitive enzyme-linked immunosorbent assay (icELISA). METHOD: BALB/c mice were immunised with many haptens of HP-bovine serum albumin (BSA) conjugates that were prepared using sodium periodate (NaIO4 ) to cause an immune response. In addition, conventional hybridoma techniques were utilised to generate mAb 5D12. RESULTS: The detection range of HP by the mAb 5D12-based icELISA was 1.56-25.0 ng/mL, with a detection limit of 1.12 ng/mL. The maximum coefficient of variation, as evaluated from the intra- and inter-assays, was <10.0%, and the percentages of recovery, as determined by the spike-recovery tests, were 105%-115%. Moreover, the HP content, which was obtained from the developed icELISA, correlated well with that obtained via high-performance liquid chromatography-ultraviolet (HPLC-UV). CONCLUSION: These validation analyses revealed that the established icELISA technique exhibited high precision and accuracy. Notably, this is the first report on the development of icELISA for the HP content-based quality control of Chinpi and its derived Kampo medicines.

Immunochromatographic assay for miroestrol and deoxymiroestrol, its cross-reactivity, and application in Pueraria mirifica (white Kwao Krua) analysis.

INTRODUCTION: Miroestrol (Mi) and deoxymiroestrol (Dmi) are trace, yet potent, phytooestrogens found in white Kwao Krua [Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham, PM]. However, the analysis of these substances is difficult because of complex matrix effects and their various analogues. In addition, alteration in the cross-reactivity of a gold nanoparticle (AuNP)-based immunochromatographic assay (ICA) resulting from the electrostatic adsorption between antibodies and AuNPs has not yet been evaluated. OBJECTIVES: This study aims to develop, characterise, and validate ICA with a monoclonal antibody exhibiting similar reactivity against Mi and Dmi (MD-mAb). MATERIALS AND METHODS: The ICA performance was validated for cross-reactivity and performance in comparison with those of indirect competitive enzyme-linked immunosorbent assays (icELISAs) with MD-mAb and mAb exhibiting specificity against Mi (Mi-mAb). RESULTS: The ICA showed a limit of detection (LOD) at 1 and 16 µg/mL for Mi and Dmi, respectively. The cross-reactivity of the ICA with Dmi was lower (6.25%) than that observed with the icELISA (120%). Cross-reactivity of ICA against other compounds of the PM was also correlated with those of icELISA; no false-positive/negative results were observed. The repeatability and reproducibility of the ICA were confirmed. The results obtained using ICA in samples of PM are correlated with the concentrations determined through icELISAs. CONCLUSION: An ICA with MD-mAb was constructed and validated. However, direct conjugation via the electrostatic adsorption of mAb-AuNPs was expected to alter the cross-reactivity of ICA, especially that of the analyte analogue Dmi.

Immunoaffinity separation of miroestrol and deoxymiroestrol from Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham using fragment antigen-binding antibody produced via Escherichia coli.

INTRODUCTION: Miroestrol and deoxymiroestrol are potent phytoestrogens and are oestrogen markers of Pueraria candollei var. mirifica. However, purifying these compounds is difficult because they only exist in trace amounts. OBJECTIVES: Active fragment antigen-binding (Fab) antibodies were produced via Escherichia coli SHuffle® T7 and used to selectively separate these compounds. MATERIALS AND METHODS: Two immunoaffinity separation approaches were developed, namely the immunoaffinity column (IAC) and a cell-based method. Group-specific Fab antibodies against miroestrol and deoxymiroestrol (anti-MD Fab) were used as biological binding reagents for selective separation. RESULTS: The Fab-based IAC effectively separated miroestrol and deoxymiroestrol (0.65 and 2.24 µg per 2 mL of resin, respectively) from P. mirifica root extract. When P. mirifica extract was added to E. coli cultures during Fab expression via a cell-based method, the target compound accumulated in intracellular compartments and, thus, were separated from E. coli cells after the removal of other compounds. A yield of 1.07 µg of miroestrol per gram of cell pellet weight was obtained. Miroestrol and deoxymiroestrol were successfully purified from P. mirifica extract using anti-MD Fab via the IAC and an intracellular cell-based method. CONCLUSION: The proposed methods can simplify the miroestrol and deoxymiroestrol extraction process and provide a basis for applications utilising recombinant antibodies to separate target compounds.

Harringtonine Ester Derivatives with Enhanced Antiproliferative Activities against HL-60 and HeLa Cells.

Harringtonine (HT), produced from Cephalotaxus species, is known to exhibit potent antiproliferative activity against myeloid leukemia cells by inhibiting protein synthesis. A previous study using acute promyelocytic leukemia (HL-60) cells raised the possibility that the C-5' methyl group of HT plays an important role in regulating leukemia cell line antiproliferative activity. In order to investigate the effect of hydrocarbon chains at C-5' on the resultant activity, the C-5' methyl group was replaced with various straight- and branched-chain hydrocarbons using the corresponding alcohols, and their antiproliferative activity against HL-60 and HeLa cells was investigated. As a result, 4'-n-heptyl-4'-demethylharringtonine (1f, n-heptyl derivative) showed the most potent cytotoxicity among the HT ester derivatives produced, with IC50 values of 9.4 nM and 0.4 µM for HL-60 and HeLa cells, respectively. Interestingly, the cytotoxicity of derivative 1f against HL-60 and HeLa cells respectively was ∼5 (IC50 = 50.5 nM) and ∼10 times (IC50 = 4.0 µM) those of HT and ∼2 (IC50 = 21.8 nM) and ∼4 times (IC50 = 1.7 µM) more than homoharringtonine (HHT). These results demonstrate the potential of the derivative 1f as a lead compound against leukemia.

Improvement in the binding specificity of anti-isomiroestrol antibodies by expression as fragments under oxidizing conditions inside the SHuffle T7 E. coli cytoplasm.

Sensitive and specific analysis of isomiroestrol (Iso) is required for the quality control of Pueraria candollei, a herb used to treat menopausal disorders. The anti-isomiroestrol monoclonal antibody (Iso-mAb) exhibits cross-reactivity with miroestrol and deoxymiroestrol, which impacts the analytical results. Here, the active and soluble forms of the single-chain variable fragment (Iso-scFv) and fragment antigen-binding (Iso-Fab) against Iso were expressed using Escherichia coli SHuffle® T7 to alter the binding specificity. The Iso-scFv format exhibited a higher binding activity than the Iso-Fab format. The reactivity of Iso-scFv towards Iso was comparable with that of the parental Iso-mAb. Remarkably, the binding specificity of the scFv structure was improved and cross-reactivity against analogs was reduced from 13.3-21.0% to ˂ 1%. The structure of recombinant antibodies affects the binding characteristics. Therefore, the immunoassays should improve specificity; these findings can be useful in agricultural processes and for quality monitoring of P. candollei-related materials.

From Nobeyama Radio Observatory to the international project ALMA -Evolution of millimeter and submillimeter wave astronomy in Japan.

The establishment of the Nobeyama Radio Observatory (NRO) in 1982 was an important event that greatly influenced the subsequent development of Japanese astronomy. The 45 m radio telescope and the Nobeyama Millimeter Array (NMA) pushed Japanese radio astronomy to the forefront of the world. As a plan beyond the Nobeyama telescopes, the Japanese radio astronomy community considered a large array to achieve unprecedented resolution at millimeter and submillimeter wavelengths under the project name of the Large Millimeter and Submillimeter Array (LMSA). After long and patient discussions and negotiations with the United States and Europe, the LMSA plan eventually led to the ALMA (Atacama Large Millimeter/submillimeter Array) as an international joint project, and the ALMA was inaugurated in 2013. This paper reviews the process from the establishment of the NRO to the realization of the ALMA, including planning of the LMSA, international negotiations, site survey, instrumental developments, and initial science results.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1-10 µM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton's tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI.

Plant-made antibody against miroestrol: a new platform for expression of full-length immunoglobulin G against small-molecule targets in immunoassays.

KEY MESSAGE: Plant expression platform is the new source of immunoglobulin G (IgG) toward small low-molecular-weight targets. The plant-made monoclonal antibody-based immunoassay exhibits comparable analytical performance with hybridoma antibody. Immunoassays for small molecules are efficiently applied for monitoring of serum therapeutic drug concentration, food toxins, environmental contamination, etc. Immunoglobulin G (IgG) is usually produced using hybridoma cells, which requires complicated procedures and expensive equipment. Plants can act as alternative and economic hosts for IgG production. However, the production of free hapten (low-molecular-weight target)-recognizing IgG from plants has not been successfully developed yet. The current study aimed at creating a plant platform as an affordable source of IgG for use in immunoassays and diagnostic tools. The functional IgG was expressed in Nicotiana benthamiana leaves infiltrated with Agrobacterium tumefaciens strain GV3101 with recombinant geminiviral vectors (pBY3R) occupying chimeric anti-miroestrol IgG genes. The appropriate assembly between heavy and light chains was achieved, and the yield of expression was 0.57 µg/g fresh N. benthamiana leaves. The binding characteristics of the IgG to miroestrol and binding specificity to related compounds, such as isomiroestrol and deoxymiroestrol, were similar to those of hybridoma-produced IgG (monoclonal antibody, mAb). The plant-based mAbs exhibited high sensitivity for miroestrol (IC50, 23.2 ± 2.1 ng/mL), precision (relative standard deviation ≤ 5.01%), and accuracy (97.8-103% recovery), as determined using quantitative enzyme-linked immunosorbent assay. The validated enzyme-linked immunosorbent assay was applicable to determine miroestrol in plant samples. Overall, the plant-produced functional IgG conserved the binding activity and specificity of the parent IgG derived from mammalian cells. Therefore, the plant expression system may be an efficient and affordable platform for the production of antibodies against low-molecular-weight targets in immunoassays.

Competitive immunochromatographic test strips for the rapid semi-quantitative analysis of the biologically active bitter glycoside, amarogentin.

Amarogentin (AG), a biologically active secoiridoid glycoside, is responsible for the efficacy of Gentianaceae based medications. Thus, qualitative and quantitative analyses of AG are of significance for batch to batch quality control purposes. By conjugating colloidal gold nanoparticles with the AG-specific monoclonal antibody, MAb 1E9, we were able to develop a single-step competitive immunochromatographic assay (ICA) for simple quantification of the AG content in plant samples. With a limit of detection of 250 ng/mL, the analytical results were obtained after immersing the ICA test strip in the detection mixture for 15 min. This new ICA is superior to conventional ICAs as it is considerably faster due to the speed with which the test strips can be produced and the omission of the time-consuming preparation phase that was previously required to make the fiber pad. Moreover, our ICA only needs a small amount of analyte (20 µL).The reliability of the reported test strip was confirmed by comparing its semi-quantitative results with those obtained via an indirect competitive enzyme-linked immunosorbent assay (icELISA). The positive correlation between these methods (R2 = 0.984) indicated that this new ICA could be applied for the semi-quantitative analysis of the AG content in plant samples.

Immunochromatographic assay for the detection of kwakhurin and its application for the identification of Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham.

INTRODUCTION: The plant Pueraria candollei var. mirifica (Airy Shaw & Suvat.) Niyomdham (PM), known by its common Thai name as white Kwao Krua, is sometimes misidentified because it presents similar botanical characteristics to those of Butea superba (red Kwao Krua). The phytochemicals in PM are phytoestrogens in the class of isoflavonoids, but Butea superba contains flavonoids that exhibit androgenic and antiestrogen effects. OBJECTIVES: This research aims to develop a simple analytical method for identification and to differentiate PM from red Kwao Krua and other Pueraria species. METHODS: A gold nanoparticle-based immunochromatographic assay (ICA) was developed for the detection of kwakhurin (Kwa), a unique compound found in PM. The parameters, including sensitivity, accuracy, precision, and specificity, were validated. All samples were analyzed using ICA and high-performance liquid chromatography with UV detector (HPLC-UV). The results of the two methods were compared for consistency checking. RESULTS: The cutoff limit of Kwa detection was 160 ng/mL, which was lower than in the HPLC-UV method. The repeatability and reproducibility of the ICA preparation and assembly showed high precision. The cross-reactivity to related isoflavonoids was less than 0.32%, which implied high specificity of the ICA for Kwa. Moreover, false-positive and false-negative results from other plant extracts were not observed. CONCLUSION: The developed ICA is applicable for distinguishing PM from red Kwao Krua and other Pueraria species. This simple analytical method can be applied for the identification of raw PM materials in the industrial and agricultural sectors.

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay to quantify swertiamarin and related compounds in Swertia japonica Makino.

INTRODUCTION: Swertia japonica Makino (S. japonica) has a long history of use as a folk medicine, and it is one of the three essential Japanese folk medicines. S.japonica has been reported to have various biological activities. The biologically active secoiridoid glycoside swertiamarin (SM) has been isolated from S. japonica. The efficacy of this plant is attributed to SM and related secoiridoid glycosides. To control the quality of S. japonica for medicinal use, a method for the determination of SM and other secoiridoid glycosides in the plant is needed. OBJECTIVE: To produce an anti-SM monoclonal antibody (MAb) and develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) for S. japonica standardisation and quality control. METHODOLOGY: SM was conjugated to cationised bovine serum albumin (cBSA), and the SM-cBSA conjugate was used to immunise BALB/c mice. Splenocytes from the immunised mice were then fused with SP2/0 myeloma cells to produce hybridoma cells that expressed anti-SM MAb. RESULTS: The developed icELISA was sufficiently sensitive and had a quantitative range of 0.78 to 12.5 µg/mL. Coefficients of variation below 10% indicated good repeatability. Recoveries in a spike and recovery assay ranged from 91.84% to 115.50%, which confirmed that the icELISA was accurate. The SM content measured using the icELISA was in agreement with the results of a high-performance liquid chromatography-ultraviolet (HPLC-UV) assay. CONCLUSION: The icELISA is suitable for the high-throughput analysis of SM and other secoiridoid glycosides in S. japonica. The method is fast, economical, and reliable for S. japonica quality control.

Molecular cloning and characterization of type III polyketide synthase from Plumbago zeylanica.

Two cDNAs encoding type Ш polyketide synthase (PKS1) and chalcone synthase (CHS, PKS2), were cloned from fresh leaves of Plumbago zeylanica L. (P. zeylanica). Their heterologous expression revealed that PKS1 catalyzed the formation of five α-pyrones from three to six acetate units by accepting acetyl-CoA and malonyl-CoA. In contrast, PKS2 catalyzed the formation of naringenin and bisnoryangonin by accepting p-coumaroyl-CoA and malonyl-CoA. Naringenin is thought to be involved in the biosynthesis of various bioactive flavonoids. PKS2 can be used to molecular breeding to enhance the production of these useful secondary metabolites via its overexpression.[Formula: see text].

Expression of actively soluble antigen-binding fragment (Fab) antibody and GFP fused Fab in the cytoplasm of the engineered Escherichia coli.

The expression of recombinant antibody fragments in the cytoplasmic space of Escherichia coli and the refolding process for restoring the structure and activity of such antibodies are not efficient. Herein, fragment antigen-binding (Fab) antibodies against miroestrol and deoxymiroestrol (MD-Fab) and their fusions with a green fluorescent protein (GFP) were expressed. The reactive MD-Fabs were successfully expressed as soluble and active forms in the cytoplasm of the SHuffle® T7 E. coli strain. Regarding the construct of MD-Fab alone, VH-CH1 could associate VL-CL into Fab in the oxidizing cytoplasm of the E. coli strain, and no additional in vitro refolding was needed. In the case of the fusions with GFP, when the C-terminus of VH-CH1 was linked with the N-terminus of GFP, the MD-Fab binding reactivity was retained, but the fluorescent activity of GFP interfered. When the C-terminus of GFP was linked to the N-terminus of VL-CL, the binding activity of MD-Fab was not observed. The constructed MD-Fabs had higher specificity toward deoxymiroestrol than the parental monoclonal antibody clone 12G11. In conclusion, MD-Fabs could be expressed using SHuffle® T7 E. coli cells. This process could be considered an economical, productive, and effective method to produce antibody fragments for immunoassay techniques.

ELISA for the Detection of the Prohibited Doping Agent Higenamine.

Higenamine is a natural benzyltetrahydroisoquinoline alkaloid produced by various plants. In the World Anti-Doping Agency report of 2020, higenamine is classified as a class S3 (selective and nonselective ß2-agonist) prohibited substance. To minimize the problems resulting from the misuse of higenamine-containing products as well as from the abuse of doping agents in sport, numerous higenamine-detection methods have been investigated. In the present study, a monoclonal antibody against the (S)-enantiomer of higenamine was successfully produced and applied in the indirect competitive ELISA to detect the content of (S)-higenamine in plant samples and related products. By immunizing BALB/c mice with higenamine-BSA, the aforementioned monoclonal antibody was produced even when the hapten number, which was the higenamine molecules conjugated to the BSA molecule, was relatively low (approximately 4). The MAb was characterized and utilized in the established icELISA assay with a detectable range of 7.81 - 125 ng/mL. The assay limit of detection (LOD) was 4.41 ng/mL, indicating higher sensitivity than the conventional HPLC-UV methods. Various validation processes demonstrated that icELISA was precise, with the maximum CV (%) of the intra- and inter-assays of 11.58% and 10.18%, respectively. Moreover, the assay was accurate, with the recovery rates of spiked (S)-higenamine ranging from 82% to 113%, and sufficiently reliable for the detection of (S)-higenamine in various kinds of samples. Notably, the present study describes the first immunoassay for (S)-higenamine.

An estimated quantitative lateral flow immunoassay for determination of artesunate using monoclonal antibody.

There have been reports of fake artesunate (ART), which has led to deaths from untreated malaria in South East Asia. To rapidly screen for fake and adulterated ART products in the drug market, a lateral flow immunoassay (LFIA) based on a colloidal gold-monoclonal antibody probe for detection of ART within samples was developed. With this method, the calibration curve for ART was determined by the intensity ratio of the test and control bands at various ART concentrations. The linearity range was 12.5-200 µg/ml of ART. Samples were tested by the developed LFIA and can be calculated for ART contents. The levels of ART in the samples were also confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good conformance. The proposed LFIA was demonstrated to be a simple and rapid analytical method for detecting ART in the pharmaceutical formulation.

Kwakhurin-magnetic particles conjugates enable fast enzyme immunoassay for the detection of kwakhurin in Pueraria candollei.

INTRODUCTION: Kwakhurin (Kwa) is one of the unique isoflavonoids produced in Pueraria candollei var. mirifica (P. candollei), which has long been used as folk medicine for rejuvenation in Thailand. Recently, the use of P. candollei-derived products has widely spread among Japanese women for cosmetic purposes. Correspondingly, there has been an increase in the number of reports regarding possible health hazards caused by estrogenic activity inherent to the plant; thus, the need for a detailed evaluation of the phytoestrogen content of P. candollei-derived products has gained a sense of urgency in recent years. OBJECTIVE: This study aims to develop a rapid enzyme immunoassay that can be applied to the quantitative analysis of Kwa in P. candollei and its derived products. MATERIAL AND METHOD: A rapid and sensitive immunoassay was developed with a combination of Kwa-specific monoclonal antibody (MAb 11F) and Kwa-magnetic particles (MPs) conjugates, which increased the surface area of the solid phase, resulting in a decrease in the immunoreaction time. RESULT: This novel MPs-based enzyme immunoassay (MPs-EIA) was used to determine Kwa concentration in the range from 2.44 to 78.1 ng/mL with a limit of detection of 1.90 ng/mL. Validation analyses revealed that the proposed MPs-EIA protocol was sufficiently precise and accurate for effective quantitative analysis of Kwa in P. candollei and its derived products.

Preincubation format for a sensitive immunochromatographic assay for monocrotaline, a toxic pyrrolizidine alkaloid.

INTRODUCTION: Monocrotaline (MCT), which is classified as a 1,2-dehydropyrrolizidine alkaloid (DHPA), is a toxic compound that is mainly produced by Crotalaria spp. MCT contamination in cereals and herbs leads to hepatitis, gastroenteritis, pulmonary vasculitis and hypertension, and different types of cancer. The current analytical methods for MCT are complicated and expensive using liquid chromatography equipped with mass spectrometry detection. OBJECTIVE: The aim of this study was to develop a simple and sensitive preincubation format for an immunochromatographic assay (PI-ICA) for MCT detection. METHODOLOGY: We conducted the PI-ICA via incubation of an MCT-containing sample with an anti-MCT monoclonal antibody conjugated with colloidal gold before strip dipping. We compared the PI-ICA detection sensitivity with that of the conventional ICA (Conv-ICA) format. RESULTS: The PI-ICA was sensitive with a limit of detection (LOD) of 0.61 ng/mL, which is a 16-fold improvement over the Conv-ICA format. These results indicated that the PI-ICA method exhibits high binding specificity for MCT and low cross-reactivity towards retronecine, retrorsine, senecionine and heliotrine. Sample solutions from plants containing MCT and related DHPAs produced positive results via PI-ICA analysis. CONCLUSIONS: The proposed PI-ICA system provides a highly sensitive method compared to Conv-ICA. In addition, the developed PI-ICA method is simple and highly effective for detecting MCT contamination.