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

2017 ◽  
Vol 9 (4) ◽  
pp. 625-633 ◽  
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.

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

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.

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

2001 ◽  
Vol 84 (4) ◽  
pp. 1001-1010 ◽  
Author(s):  
Kalidindi Hemalatha ◽  
Namburi B K Venugopal ◽  
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.

scholarly journals 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

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.

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

Molecules ◽  
2019 ◽  
Vol 24 (10) ◽  
pp. 1952 ◽  
Author(s):  
Yu-En Shih ◽  
Chao-Hsiang Chen ◽  
Nan-Hei Lin ◽  
Jason T.C. Tzen
Keyword(s):  
Metal Ion ◽  
Salvia Miltiorrhiza ◽  
Chinese Herb ◽  
Competitive Elisa ◽  
Water Soluble ◽  
Enzyme Linked Immunosorbent Assay ◽  
Therapeutic Effects ◽  
Oil Bodies ◽  
Body Protein ◽  
Indirect Competitive Elisa

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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