Development of Indirect Competitive ELISA for Lithospermic Acid B of Salvia miltiorrhiza with Its Specific Antibodies Generated via Artificial Oil Bodies

Lithospermic acid B (LSB), the major water-soluble ingredient of Salvia miltiorrhiza (Danshen), has been shown to be an active ingredient responsible for the therapeutic effects of this traditional Chinese herb used to treat cardiac disorders. This study aimed to develop an indirect competitive enzyme linked immunosorbent assay (ELISA) for the detection of LSB. Firstly, LSB was chemically conjugated to a modified oil-body protein, lysine-enriched caleosin, recombinantly expressed in Escherichia coli. Antibodies against LSB (Ab-LSB) were successfully generated by immunizing hens with artificial oil bodies constituted with the LSB-conjugated caleosin. Western blotting showed that Ab-LSB specifically recognized LSB, but not the carrier protein, lysine-enriched caleosin. To detect LSB via indirect competitive ELISA, LSB was conjugated with bovine serum albumin (LSB-BSA) and coated on a microplate. The binding between Ab-LSB and LSB-BSA on the microplate was competed dose-dependently in the presence of free LSB with a concentration ranging from 5 to 5 × 104 ng/mL. The IC50 value was approximately determined to be 120 ng/mL for LSB regardless of its complex with a metal ion of Na+, K+ or Mg2+.

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Prevention and Therapeutic Effects and Mechanisms of Tanshinone IIA Sodium Sulfonate on Acute Liver Injury Mice Model

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/4097398 ◽

2016 ◽

Vol 2016 ◽

pp. 1-8

Author(s):

Lunjie Lu ◽

Jun Zhou ◽

Jingying Zhang ◽

Jun Che ◽

Yang Jiao ◽

...

Keyword(s):

Liver Injury ◽

Acute Liver Injury ◽

Salvia Miltiorrhiza ◽

Water Soluble ◽

Tanshinone Iia ◽

Therapeutic Effects ◽

Mice Model ◽

Sodium Sulfonate ◽

Pharmacologically Active ◽

Magnesium Isoglycyrrhizinate

Tanshinone IIA sodium sulfonate (TSS) is a water-soluble derivative of tanshinone IIA, which is the main pharmacologically active component of Salvia miltiorrhiza. This study aimed to verify the preventive and therapeutic effects of TSS and its combined therapeutic effects with magnesium isoglycyrrhizinate (MI) in D-galactosamine- (D-Gal-) induced acute liver injury (ALI) in mice. The potential regulatory mechanisms of TSS on ALI were also examined. Our results may provide a basis for the development of novel therapeutics for ALI.

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Detection of total bile acids in biological samples using an indirect competitive ELISA based on four monoclonal antibodies

Analytical Methods ◽

10.1039/c6ay03243e ◽

2017 ◽

Vol 9 (4) ◽

pp. 625-633 ◽

Cited By ~ 1

Author(s):

Shuchen Liu ◽

Yue Zhang ◽

Baoping Qu ◽

Gaofeng Qin ◽

Jinjun Cheng ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Clinical Practice ◽

Biological Samples ◽

Competitive Elisa ◽

Routine Clinical Practice ◽

Enzyme Linked Immunosorbent Assay ◽

Indirect Competitive Elisa ◽

Different Types ◽

Total Bile Acids

We investigated a newly developed indirect competitive enzyme-linked immunosorbent assay for the determination of 5 major components of TBA, which works efficiently in different types of biological samples, and may be suitable for routine clinical practice.

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The Use of Chicken Igy in a Double Antibody Sandwich Elisa for the Quantification of Melittin in Bee Venom and Bee Venom Melittin Content in Cosmetics

Journal of Apicultural Science ◽

10.1515/jas-2015-0011 ◽

2015 ◽

Vol 59 (1) ◽

pp. 97-107

Author(s):

Lindsey Y. K. Suh ◽

Tayabaa Kartoon ◽

Naiyana Gujral ◽

Youngmee Yoon ◽

Joo Won Suh ◽

...

Keyword(s):

Sandwich Elisa ◽

Competitive Elisa ◽

Bee Venom ◽

Raw Material ◽

Enzyme Linked Immunosorbent Assay ◽

Western Blot Assay ◽

Detection Systems ◽

Indirect Competitive Elisa ◽

Double Antibody ◽

Das Elisa

Abstract Two enzyme-linked immunosorbent assay (ELISA) - based detection systems: indirect competitive ELISA and biotinylated double antibody sandwich ELISA (DAS-ELISA) were developed to determine the melittin concentration in honeybee (Apis mellifera) venom and the melittin concentration in cosmetics which contain bee venom. The indirect competitive ELISA employed chicken anti-melittin IgY. The biotinylated DAS-ELISA employed anti-melittin monoclonal antibody (MAb) and biotinylated anti-melittin IgY. To produce anti-melittin IgY; Sigma melittin was emulsified with Freund‘s incomplete adjuvant and immunised to Leghorn laying chickens intramuscularly at four different sites (50 μg/mL, 0.25 mL per site) of the breast muscles. After 5 to 8 weeks of the immunisation, anti-melittin IgY was extracted and analysed by ELISA. The anti-melittin IgY antibody produced was highly specific to melittin and did not cross-react with other bee venom proteins, as examined by ELISA and a western-blot assay. Indirect competitive ELISA demonstrated a higher range of melittin detection (2.5 to 80 μg/mL). Double antibody sandwich ELISA using MAb as the capture antibody and biotinylated polyclonal IgY as the detection antibody, provided a lower range of detection (2.5 - 40 ng/mL), which has a 1000 times higher sensitivity than that of indirect competitive ELISA. Therefore, indirect competitive ELISA is a useful tool to measure the concentration of melittin in bee venom as a raw material. Biotinylated DAS-ELISA, on the other hand, is more suitable for nanoscale quantification of melittin in commercial products.

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Chinese Herbal Tanshinol Prevents Osteoporosis via Coordinately Promoting Osteogenesis and Inhibiting Osteoclastogenesis

10.21203/rs.3.rs-139701/v1 ◽

2021 ◽

Author(s):

tailin wu ◽

jianchao wang ◽

jianzhou luo ◽

haitao lin ◽

fei wang ◽

...

Keyword(s):

Bone Marrow ◽

Oral Delivery ◽

Salvia Miltiorrhiza ◽

Chinese Herb ◽

Water Soluble ◽

Control Group ◽

Group Model ◽

Three Dimension ◽

Mineral Density ◽

Water Soluble Compound

Abstract Background: Osteoporosis severely affects patients’ life quality due to increased risks of fragility fractures. Tanshinol is a primary water-soluble compound purified from the Chinese herb Salvia Miltiorrhiza, which exhibits potent antioxidant and anti-inflammatory properties. However, whether Tanshinol functions in preventing and protecting osteoporosis remains unknown. Thus, current study proposed to systematically investigate the protective effects and the underlying mechanisms of Tanshinol in bone marrow mesenchymal stem cells (BM-MSCs) and ovariectomized (OVX) mice model. Materials and methods: Different concentrations of Tanshinol were given to induce differentiation of BM-MSCs respectively, and detected the expression of key markers of osteoclast and osteogenesis. The C57BL/6 mice were divided into control group, model group, low (80 mg/kg×day, i.g), medium (160 mg/kg×day, i.g) and high (240 mg/kg×day, i.g) concentrations Tanshinol groups. After 6 weeks treatment of Tanshinol, mice distal femurs were taken to measure bone mineral density (BMD) and three dimension parameters.Results: In the present study, we for the first time showed that Tanshinol could promote osteogenesis in the mouse BM-MSCs, as seen from the increase of the osteogenic markers such as ALP activity and collagen I. Meanwhile, Tanshinol inhibited the RANKL induced osteoclastogenesis in the bone marrow monocytes (BMMs). Animal studies showed that oral delivery of Tanshinol could attenuate in ovariectomy induced osteoporosis. Molecularly, Tanshinol activated Wnt signal pathway in MSCs while inhibited Akt in BMMs, suggesting these pathways might be involved in the osteoporosis protective role of Tanshinol. Conclusions: our study has revealed a potential application of Salvia miltiorrhiza derivative Tanshinol in the treatment of osteoporosis.

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Monoclonal Antibody-Based Enzyme Linked Immunosorbent Assay of Aflatoxin B1, T-2 Toxin, and Ochratoxin A in Barley

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/73.1.71 ◽

1990 ◽

Vol 73 (1) ◽

pp. 71-76 ◽

Cited By ~ 3

Author(s):

Nannapaneni Ramakrishna ◽

John Lacey ◽

Alan A.G Candlish ◽

John E Smith ◽

Ian A Goodbrand

Keyword(s):

Monoclonal Antibodies ◽

Ochratoxin A ◽

Aflatoxin B1 ◽

Chloroform Extract ◽

Barley Grain ◽

Competitive Elisa ◽

Water Soluble ◽

Enzyme Linked Immunosorbent Assay ◽

Simple Liquid ◽

The Mean

Abstract Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1f 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile- 0.5% KCI-6% H2S04 (89 + 10 + 1 ) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonltrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCI buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1( 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, Inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were <12% for Bi and OA but as high as 17% forT2.

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Tanshinol Alleviates Osteoporosis and Myopathy in Glucocorticoid-Treated Rats

Planta Medica ◽

10.1055/s-0043-108761 ◽

2017 ◽

Vol 83 (16) ◽

pp. 1264-1273 ◽

Cited By ~ 4

Author(s):

Guanghua Chen ◽

Xinle Zhang ◽

Han Lin ◽

Guizhi Huang ◽

Yahui Chen ◽

...

Keyword(s):

Growth Factor ◽

Transforming Growth Factor ◽

Salvia Miltiorrhiza ◽

Healing Process ◽

Water Soluble ◽

Control Group ◽

Therapeutic Effects ◽

Mrna Levels ◽

Model Group ◽

Mineral Density

AbstractTanshinol is a major water-soluble active component of Salvia miltiorrhiza. In this study, we aimed to investigate whether tanshinol has potential therapeutic effects against glucocorticoid-induced osteoporosis and glucocorticoid-induced myopathy. Ninety-six female Sprague-Dawley rats were randomly assigned to five groups: a control group, a model group, and three model groups treated with 25 or 50 mg/kg of tanshinol, or calcitriol. All model groups received prednisone acetate for 90 days to induce glucocorticoid-induced osteoporosis. Afterwards, all animals underwent a surgical procedure to induce bone defects at the right proximal tibia. Prednisone treatment was stopped after surgery, but tanshinol or calcitriol treatment was continued to the endpoint. At the experimental endpoint, compared to the model group, 25 mg/kg tanshinol could significantly reverse glucocorticoid-induced loss of bone mineral density by 12.5 %, while enhancing mechanical bone strength, causing a significant 11 % increase in trabecular number, and reducing trabecular separation by 28 %. In addition, tanshinol improved the bone microarchitecture and prevented glucocorticoid-induced bone loss by promoting bone formation and inhibiting bone resorption. Moreover, results of bone defect repair and muscle weight measurements revealed that tanshinol accelerated the bone fracture healing process and attenuated muscle atrophy caused by glucocorticoid. Furthermore, qRT-PCR analysis showed a 1-fold upregulation in mRNA levels of transforming growth factor beta and roughly 6-fold increases in vascular endothelial growth factor mRNA expression in calluses from the tanshinol groups. Tanshinol also preserved muscular ubiquitin mRNA levels from glucocorticoid-induced elevation. These findings demonstrate the potential benefits of tanshinol against glucocorticoid-induced osteoporosis and glucocorticoid-induced myopathy, which warrants further investigation in future studies.

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Establishment and Application of a Method for the Determination of Ganoderic Acid A

Journal of Food Quality ◽

10.1155/2020/6621853 ◽

2020 ◽

Vol 2020 ◽

pp. 1-7

Author(s):

Shengyun Li ◽

Yaowu Yuan ◽

Chenchen Yu ◽

Hao Gao ◽

Jianxin Tan ◽

...

Keyword(s):

Competitive Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Response Relationship ◽

Dose Response Relationship ◽

Ganoderic Acid ◽

Second Stage ◽

Indirect Competitive Elisa ◽

Rabbit Igg ◽

Content Range

A method for the quantitative determination of ganoderic acid A was constructed using the principle of indirect competitive enzyme-linked immunosorbent assay (ELISA), and this method was used to determine the ganoderic A contents of Ganoderma lucidum samples in the market. The conjugate of ganoderic acid A and bovine serum albumin was used for four rounds of immunization on test rabbits to obtain rabbit antiganoderic acid A antibody IgG. The enzyme-labeled plate was coated with the conjugate of ganoderic acid A and ovalbumin. The first stage reaction in the indirect competitive ELISA was that the conjugate of ganoderic acid A in the sample competed with the conjugate coated on the enzyme-labeled plate to bind rabbit antibodies. The second stage reaction was the combination of goat anti-rabbit IgG–horseradish peroxidase and rabbit antiganoderic acid A antibody IgG. The results of the determination of ganoderic acid A standard by this method showed that the coefficient of variation of repeated wells in the group was <5%, the detection limit of ganoderic acid A was 0.6 μg/L, and ganoderic acid A had a substantial dose-response relationship in the content range of 0.9–72.9 μg/L (R2 = 0.994). This method was used to measure the ganoderic A content of 12 varieties of G. lucidum in the market and showed the obvious differences in the ganoderic acid A contents of the different varieties. This method is simple, fast, and of great importance to the quality control of Ganoderma products.

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Determination of Azadirachtin in Agricultural Matrixes and Commercial Formulations by Enzyme-Linked Immunosorbent Assay

Journal of AOAC International ◽

10.1093/jaoac/84.4.1001 ◽

2001 ◽

Vol 84 (4) ◽

pp. 1001-1010 ◽

Cited By ~ 8

Author(s):

Kalidindi Hemalatha ◽

Namburi B K Venugopal ◽

Beedu S Rao

Keyword(s):

Immunosorbent Assay ◽

Cross Reactivity ◽

Competitive Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Agricultural Commodities ◽

Indirect Competitive Elisa ◽

Capture Assay ◽

Ic50 Value ◽

Antibody Capture

Abstract An enzyme-linked immunosorbent assay (ELISA) was developed for azadirachtin (aza), a biopesticide from the neem tree (Azadirachta indica A. Juss). The immunogen was synthesized by epoxidation using the furan ring in the aza molecule. Rabbits were immunized with either bovine serum albumin (BSA)-azadirachtin or ovalbumin (OA)-azadirachtin conjugate. Evaluation of the antisera by antibody capture assay showed that the antibody titer of antisera raised against OA-aza was 1:30 000. An indirect competitive ELISA was developed with BSA-azadirachtin as coating antigen and aza-specific antibodies raised against OA-aza immunogen. The immunoassay showed an inhibitory concentration (IC50) value of 75 ppb, with a range of detection from 0.5 to 1000 ppb for azadirachtin [based on regression analysis, y = 85.87 (−18.89x); r2 = −0.97]. Cross-reactivity of the antibodies with 2 aza- derivatives (22,23-dihydro-23β-methoxy azadirachtin and 3-tigloylazadirachtol) was 33 and 29%, respectively. The indirect competitive ELISA was validated and evaluated by quantitating aza in spiked agricultural commodities and from neem formulations. Azadirachtin was spiked into 5 different agricultural commodities: tomato, brinjal, coffee, tea, and cotton seed at 500 and 1000 ppb and recovered at 62–100%. In samples drawn from 6 lots, the aza content in neem-seed kernels ranged from 0.1 to 0.15%; in commercial neem formulations the content ranged from 200 to 2000 ppm. The method developed may be applied to environmental monitoring of aza and quality assurance studies of aza-based commercial formulations.

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Quantitation of Aflatoxin B1-N7-Guanine Adduct in Urine by Enzyme-Linked Immunosorbent Assay Coupled with Immunoaffinity Chromatography

Journal of AOAC International ◽

10.1093/jaoac/80.5.1013 ◽

1997 ◽

Vol 80 (5) ◽

pp. 1013-1022 ◽

Cited By ~ 5

Author(s):

Tfflrumurthy Vidyasagar ◽

Vadapalli Vyjayanthi ◽

Nayak Sujatiia ◽

Beedu Sashidhar Rao ◽

Ramesh Venkataramana Bhat

Keyword(s):

Immunosorbent Assay ◽

Aflatoxin B1 ◽

Polyclonal Antibodies ◽

Immunoaffinity Chromatography ◽

Competitive Elisa ◽

Aflatoxin M1 ◽

Enzyme Linked Immunosorbent Assay ◽

Immunoaffinity Column ◽

Indirect Competitive Elisa

Abstract IndiaA specific and sensitive method to quantitate aflatoxin B1-N7-guanine adduct in urine samples by immunoaffinity chromatography coupled with indirect competitive enzyme-linked immunosorbent assay (ELISA) is reported. A novel in vitro method to synthesize an antigen (bovine serum albumin-guanine-aflatoxin B1) and its use to produce polyclonal antibodies specific to the hapten aflatoxin B-N7-guanine are discussed. An indirect competitive ELISA developed to quantitate aflatoxin adduct showed a 50% inhibition at 15.6 pmol aflatoxin B1-N7-guanine (y=66.73+ (-19.8)x, r=-0.997). Interference by aflatoxin B1 was less than 5%, and no interference by guanine, aflatoxin Gι and aflatoxin M1 was observed. An immunoaffinity column was also developed, by using these polyclonal antibodies, for single-step purification of aflatoxin B1-N7-gua-nine. The immunoaffinity column and the indirect competitive ELISA were evaluated and validated by quantitation of aflatoxin B1-N7-guanine in urine from rats dosed with aflatoxin B1 (1 mg/kg body weight). Spiking studies with standard aflatoxin B1-N7-guanine adduct at 2 and 4 μg/mL phosphate- buffered saline gave 96 and 100% recoveries, respectively, for immunoaffinity cleanup column. The method also was tested successfully for quantitating aflatoxin B1-N7-guanine adduct in spiked human urine. Recoveries of the adduct were 79-90%.

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Indirect competitive ELISA and colloidal gold-based immunochromatographic strip for amantadine detection in animal-derived foods

Analytical Methods ◽

10.1039/c8ay02806k ◽

2019 ◽

Vol 11 (15) ◽

pp. 2027-2032

Author(s):

Mingfei Pan ◽

Jingying Yang ◽

Shijie Li ◽

Guozhu Wang ◽

Junping Wang ◽

...

Keyword(s):

Immunosorbent Assay ◽

Colloidal Gold ◽

Antiviral Drug ◽

Competitive Elisa ◽

Enzyme Linked Immunosorbent Assay ◽

Immunochromatographic Strip ◽

Indirect Competitive Elisa

In this research, an indirect competitive enzyme-linked immunosorbent assay (ELISA) and colloidal gold-based immunochromatographic (GICG) strip were developed for the detection of the antiviral drug amantadine (AM) in animal-derived foods.

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