Development and evaluation of an immunoassay for the quantification of N-acetylneuraminic acid (Neu5Ac) in foods and biosamples.

N-acetylneuraminic acid is an active ingredient in tonic foods and an important additive in foods and biopharmaceuticals. To address the limitations of existing methods of N-acetylneuraminic acid quantification, we developed an immunoassay based on antibodies induced in hens using artificial antigen, showing high sensitivity and specificity with no cross-reactivity with eight N-acetylneuraminic acid analogues. An IgY-based indirect competitive enzyme-linked immunosorbent assay showed a detection range of 1.14 to 70.08 ng/mL and a limit of detection of 0.57 ng/mL. In spiked samples, recoveries by the indirect competitive enzyme-linked immunosorbent assay ranged from 74.05% to 110.87% compared with HPLC (73.01% to 108.8%). Consistency between the indirect competitive enzyme-linked immunosorbent assay and HPLC was satisfactory (R2 = 0.9736), demonstrating this established immunoassay as a rapid and reliable approach for N-acetylneuraminic acid analysis. The assay described in this study provides an important method for the screening of N-acetylneuraminic acid in biological samples and foodstuffs.

Preparation of Anti-Zearalenone IgY and Development of an Indirect Competitive ELISA Method for the Measurement of Zearalenone in Post-Fermented Tea.

Post-fermented tea (PFT) is one of the most commonly consumed beverages worldwide. Rapid microbial growth and significant changes in the microbial composition of PFT during processing and storage pose a potential risk of contamination with mycotoxins such as zearalenone (ZEN). Screening for ZEN contamination in a simple, rapid, and inexpensive manner is required to ensure that PFT is safe for consumption. To monitor ZEN in PFT, ZEN was conjugated with bovine serum albumin to prepare egg yolk immunoglobulins (IgY). A specific indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on IgY was developed and validated. ZEN was extracted with acetonitrile and water (50:50, v/v) containing 5% acetic acid and purified using a mixture of primary and secondary amines and graphitized carbon black to remove matrix interference from the PFT samples. Under optimal conditions, the linear range of this assay was 13.8-508.9 ng mL-1, the limit of detection was 9.3 ng mL-1, and the half-maximal inhibitory concentration was 83.8 ng mL-1. Cross-reactivity was negligible, and the assay was specific for ZEN-related molecules. The recovery rate of ZEN in the control blanks of PFT samples spiked with a defined concentration of ZEN of 89.5% to 98.0%. The recovery and accuracy of the method were qualified for PFT matrices. No significant differences were evident between the results of the actual PFT samples analyzed by high-performance liquid chromatography and ic-ELISA. The collective data indicate that the developed ic-ELISA can be used for the rapid and simple detection of ZEN in PFT products.

A sensitive and specific enzyme-linked immunosorbent assay for the detection of pymetrozine in vegetable, cereal, and meat.

Pymetrozine is a neonicotinoid insecticide with high efficacy against aphids and planthoppers, and has been used worldwide. To monitor its residue in food, a highly specific and sensitive monoclonal antibody (McAb) was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to detect pymetrozine, with a 50% inhibition value (IC50) of 7.70 µg/L. The McAb showed little affinity for acetamiprid, hexazinone, metamitron, nitenpyram, metribuzin, and imidacloprid. The limits of detection (LOD) calculated from the analysis of broccoli, cabbage, wheat, maize, rice, chicken, fish, and crayfish samples were from 1.56 to 2.72 µg/kg and the average recoveries were from 81.25 to 103.19%. icELISA was confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that the optimised icELISA is a convenient and effective analytical tool for monitoring pymetrozine residues in food.

Monoclonal antibody based immunoassay: An alternative way for aquatic environmental selenium detection.

Environmental concerns about human health encouraged increasing methodological interest in selenium (Se), which is an essential non-metal trace element and varies within a narrow concentration range between essential and toxic. In this study, two types of long-armed Se haptens (Se-hapten-lc-NHS) were synthesized for the first time using active ester formalization. In producing monoclonal antibodies (mAbs), the derivatization of haptenized Se at para- (meta-) and ortho-sites showed different properties. Finally, a mAb derived from hybridoma 5A52 was confirmed to be capable of establishing an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). There was a successful quantitative determination of Se4+ with a detection range of 17 to 207 pmol mL-1 and a limit of detection of approximately 3.9 pmol mL-1. The mAb was found to be remarkably sensitive and specific, with no evidence of cross-reactivity with other ions. The assay was validated for four kinds of Se forms in water samples and showed satisfactory recoveries between 80 % and 108 %, with coefficients of variation of 2.1 %-11 %. The method proposed in our study offers a useful protocol for the rapid screening of Se and provides an alternative solution for the analysis of Se in aquatic environments.

Development of IgY-Based Indirect Competitive ELISA for the Detection of Fluoroquinolone Residues in Chicken and Pork Samples.

Fluoroquinolones (FQs) are among the antibiotics whose widespread use in farm-raised animals results in potentially harmful residues in the end products. Additionally, most Thai farmers use antibiotics. Amoxicillin and enrofloxacin were commonly used by pig farms, and hens were given enrofloxacin to prevent immunization side effects. Moreover, antibiotic overuse has harmed food safety in the long term, and the use of low-dose antibiotics causes bacterial resistance. Herein, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was used to make a fast, easy, sensitive, and cost-effective method for monitoring FQs residues. After immunizing hens with mixed multi-hapten ciprofloxacin-bovine serum albumin (CPFX-BSA) with norfloxacin-bovine serum albumin (NFX-BSA), the IgY antibody purified from egg yolk was used for the detection of FQs residues in chicken and pork samples. The efficiency of the IgY antibody showed excellent sensitivity, with 50% inhibitory concentration (IC50) of enrofloxacin at 0.05 µg/mL, far below the MRLs defined by the EU for muscle samples, which was not to exceed 100 µg/kg. The recovery range for chicken muscle samples spiked with ENFX concentrations of 1.00-0.01 µg/mL was 86.65-112.71%, similar to pork samples, which were 84.24-117.22.2%. This method has a lot of potential for analyzing fluoroquinolones in complex samples quickly, easily, and at a low cost on-site. The IgY-based ic ELISA was developed to detect ciprofloxacin (CPFX), norfloxacin (NFX), and enrofloxacin (ENFX) residues; it confirms that IgY could be a promising choice for the detection of antibiotic residues in food samples.

Development of a Highly Sensitive and Specific Monoclonal Antibody Based on Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Zearalenone in Food and Feed Samples.

Zearalenone (ZEN) contamination in food and feed is prevalent and has severe effects on humans and animals post-consumption. Therefore, a sensitive, specific, rapid, and reliable method for detecting a single residue of ZEN is necessary. This study aimed to establish a highly sensitive and specific ZEN monoclonal antibody (mAb) and an indirect competitive enzyme-linked immunosorbent assay (icELISA) for the detection of ZEN residues in food and feed. The immunogen ZEN-BSA was synthesized via the amino glutaraldehyde (AGA) and amino diazotization (AD) methods and identified using 1H nuclear magnetic resonance (1H NMR), a high-resolution mass spectrometer (HRMS), and an ultraviolet spectrometer (UV). The coating antigens ZEN-OVA were synthesized via the oxime active ester (OAE), formaldehyde (FA), 1,4-butanediol diglycidyl ether (BDE), AGA, and AD methods. These methods were used to screen the best antibody/antigen combination of a heterologous icELISA. Balb/c mice were immunized with a low ZEN-BSA dose at long intervals and multiple sites. Suitable cell fusion mice and positive hybridoma cell lines were screened using a homologous indirect non-competitive ELISA (inELISA) and an icELISA. The ZEN mAbs were prepared by inducing ascites in vivo. The immunological characteristics of ZEN mAbs were then assessed. The standard curves of the icELISA for ZEN were constructed under optimal experimental conditions, and the performance of the icELISA was validated. The two ZEN-BSA immunogens (conjugation ratios, 11.6:1 (AGA) and 9.2:1 (AD)) were successfully synthesized. Four hybridoma cell lines (2B6, 4D9, 1A10, and 4G8) were filtered, of which 2B6 had the best sensitivity and specificity. The mAb 2B6-based icELISA was then developed. The limit of detection (LOD), the 50% inhibitive concentration (IC50), and the linear working range (IC20 to IC80) values of the icELISA were 0.76 µg/L, 8.69 µg/L, and 0.92-82.24 µg/L, respectively. The cross-reactivity (CR) of the icELISA with the other five analogs of ZEN was below 5%. Three samples were spiked with different concentrations of ZEN and detected using the icELISA. The average intra-assay recoveries, inter-assay recoveries, intra-assay coefficients of variations (CVs), and inter-assay CVs were 93.48-99.48%, 94.18-96.13%, 12.55-12.98%, and 12.53-13.58%, respectively. The icELISA was used to detect ZEN in various samples. The results were confirmed using high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) (correlation coefficient, 0.984). The proposed icELISA was highly sensitive, specific, rapid, and reliable for the detection of ZEN in food and feed samples.

Electrofusion preparation of anti-triazophos monoclonal antibodies for development of an indirect competitive enzyme-linked immunosorbent assay.

Immunoassays have been widely used to detect small molecular contaminants due to the advantages of simplicity, high throughout and low-cost. Antibodies are essential reagents of immunoassays, their quality directly determines the characteristics of immunoassays. In this study, the monoclonal antibodies (mAbs) of triazophos were prepared by electrofusion, and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Under the optimal electrofusion conditions (cells treatment with pronase, the alternating electric field strength of 45 V cm-1, the direct current voltage of 3 kV), the fusion efficiency was 1.104 ± 0.063‱, which was improved more than 4-fold compared with the chemical fusion method (0.255 ± 0.089‱). Three hybrid cell lines that can stably secrete the anti-triazophos mAbs were obtained. The cell line 4G6F10 showed the highest sensitivity, which was used to generate mAb and develop an ic-ELISA. After optimization, the 50% inhibition concentration (IC50), limit of detection (LOD) and linear range (IC10-IC90) of the ic-ELISA were 0.32 ng mL-1, 0.08 ng mL-1 and 0.08-2.17 ng mL-1, respectively. There was no significant cross-reactivity with the analogues of triazophos. The average recoveries of triazophos in spiked samples were 77.5%-89.3% with the relative standard deviations of 0.1%-9.2%. In addition, the ic-ELISA showed good repeatability, reproducibility and accuracy for the analysis of apple samples spiked with triazophos.

[Rapid detection of aflatoxin B_1 in Chinese herbal medicines by indirect and direct competitive enzyme-linked immunosorbent assays: a comparative analysis].

The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and direct competitive enzyme-linked immunosorbent assay(dc-ELISA) were performed for the rapid detection of aflatoxin B_1(AFB_1) in Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen with self-made antigens and antibodies. Different extraction methods were investigated to reduce the matrix effects of different medicinal parts in Chinese herbal medicines. The sensitivity of dc-ELISA method was improved by optimizing the molar ratio of AFB_1 to horseradish peroxidase(HRP). In this study, the sensitivity(IC_(50)) of ic-ELISA and dc-ELISA was 0.046 and 0.023 ng·mL~(-1), with the limit of detection(LOD) of 0.007 and 0.004 ng·mL~(-1), respectively. The detection time was 3 h and 50 min for ic-ELISA and dc-ELISA, respectively. The recovery rates were within the range of 62.96%-104.4%, with RSDs of less than 10%. Confirmed by LC-MS/MS, three positive samples of Nelumbinis Semen were detected from 53 samples. Two ELISA methods established in this study were accurate, rapid and sensitive, and can be used for rapid screening of AFB_1 in Chinese herbal medicines such as Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen. In addition, the advantages and limitations of the two methods were compared and discussed, which can provide a reference for the testing institutions to choose the proper method.

Immunoassays for the rapid detection of pantothenic acid in pharmaceutical and food products.

Pharmaceutical and food products are fortified with pantothenic acid (PA) to address potential deficiency. Therefore, its fast, reliable, and accurate detection is of great importance to the quality control. Here, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a gold nanoparticle-based lateral flow immunoassay (LFIA) were established for the determination of PA based on an anti-PA monoclonal antibody (mAb). The ic-ELISA displayed a limit of detection (LOD) of 32.22 ng/mL, and the linear range was 64.44-628.84 ng/mL. Average recoveries of PA in fortified samples were 88.60-110.11% when using the ic-ELISA and a good correlation between the ic-ELISA and LC-MS/MS was obtained when analyzing samples. Furthermore, the developed LFIA strip showed a calculated LOD of 71.99, 115.80, and 240.12 ng/mL in B-complex Vitamin tablets, energy drink and infant milk powder samples, respectively. All the results demonstrated that both of these immunoassays are suitable for determining PA in pharmaceutical and food products.

Indirect competitive enzyme-linked immunosorbent assay based on a broad-spectrum monoclonal antibody for tropane alkaloids detection in pig urine, pork and cereal flours.

In this study, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) based on a broad-spectrum monoclonal antibody for tropane alkaloids (TAs) was established for the rapid screening of atropine, scopolamine, homatropine, apoatropine, anisodamine, anisodine and L-hyoscyamine residues in pig urine, pork and cereal flour samples through a simple sample preparation procedure. The half inhibitory concentrations of atropine, homatropine, L-hyoscyamine, apoatropine, scopolamine, anisodamine and anisodine were 0.05, 0.07, 0.14, 0.14, 0.24, 5.30 and 10.15 ng mL-1, respectivelyThe detection and quantitative limits of this method for TAs in samples were 0.18-73.18 and 0.44-74.77 µg kg-1. The spiked recoveries ranged from 69.88% to 147.93%, and the coefficient of variations were less than 14%. Good correlation (R2 = 0.9929) between the results of the ic-ELISA and the high performance liquid chromatography-tandem mass spectrometry support the reliability of the developed ic-ELISA method.

Rapid detection of aflatoxin B_1 in Chinese herbal medicines by indirect and direct competitive enzyme-linked immunosorbent assays: a comparative analysis / 中国中药杂志

The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and direct competitive enzyme-linked immunosorbent assay(dc-ELISA) were performed for the rapid detection of aflatoxin B_1(AFB_1) in Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen with self-made antigens and antibodies. Different extraction methods were investigated to reduce the matrix effects of different medicinal parts in Chinese herbal medicines. The sensitivity of dc-ELISA method was improved by optimizing the molar ratio of AFB_1 to horseradish peroxidase(HRP). In this study, the sensitivity(IC_(50)) of ic-ELISA and dc-ELISA was 0.046 and 0.023 ng·mL~(-1), with the limit of detection(LOD) of 0.007 and 0.004 ng·mL~(-1), respectively. The detection time was 3 h and 50 min for ic-ELISA and dc-ELISA, respectively. The recovery rates were within the range of 62.96%-104.4%, with RSDs of less than 10%. Confirmed by LC-MS/MS, three positive samples of Nelumbinis Semen were detected from 53 samples. Two ELISA methods established in this study were accurate, rapid and sensitive, and can be used for rapid screening of AFB_1 in Chinese herbal medicines such as Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen. In addition, the advantages and limitations of the two methods were compared and discussed, which can provide a reference for the testing institutions to choose the proper method.

Development of ELISA and chemiluminescence enzyme immunoassay for quantification of histamine in drug products and food samples.

Histamine (HA) is a biogenic amine associated with allergies and food poisoning. It is an important indicator of food freshness and quality. In recent years, a series of medical negligence cases have been reported to be related to the intravenous injection of antibiotics produced via fermentation with fish peptone due to HA contamination. To detect HA efficiently, mouse monoclonal antibody was developed. An enzyme-linked immunosorbent assay (ELISA) and a chemiluminescence enzyme immunoassay (CLEIA) were developed and compared with conventional HPLC analysis. Both immunoassays showed low cross-reactivity, low 50% inhibitive concentration (IC50; 1.2 µg/mL and 1.1 µg/mL), low limits of detection (LODs, IC10; 89.0 ng/mL and 73.4 ng/mL), and appreciable recoveries in spiked foods and drugs (from 73.4 to 131.0% and from 77.0 to 119.0%, espectively), demonstrating that the developed methods are sensitive, specific, fast, and reliable for HA detection in complicated real samples. Graphical abstract.

[Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection].

OBJECTIVE: To prepare monoclonal antibody against cotinine (COT) and to establish immunoassay for detecting COT in human urinary samples. METHODS: BALB/c mice were immunized with synthesized cotinine-bovine serum albumin (COT-BSA) to screen monoclonal antibody with technique of cell fusion. The monoclonal antibody was used for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic strip assay for the detection of COT in human urine. RESULTS: The monoclonal antibody against COT was identified by ic-ELISA with a 50%inhibitive concentration (IC50) value of 21 ng/mL; and it was also identified by colloidal gold immunochromatographic strip assay with a cut-off value of 100 ng/mL. For ic-ELISA, the range of detection was 0-100 ng/mL with a minimal limit of 0.1 ng/mL; the recovery of assay was 99.41%-117.98%, and the intra-assay and inter-assay coefficient variations were not higher than 15.31%and 15.07%, respectively. For colloidal gold immunochromatographic strip assay, the accuracy of stability and repeatability both were 100%. CONCLUSIONS: The ic-ELISA and colloidal gold immunochromatographic strip assay using the prepared monoclonal antibody against COT have been proved to be reliable for the rapid detection of COT in human urines, which may be used for monitoring of environmental tobacco smoke.

Visible and eco-friendly immunoassays for the detection of cyclopiazonic acid in maize and rice.

Cyclopiazonic acid (CPA) is an indole-tetramine mycotoxin commonly produced by Penicillium and Aspergillus and is widely found in agricultural products, fermented food, and feed. Food contaminated with CPA poses a substantial health risk to consumers. Therefore, eco-friendly immunoassays, including an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a lateral flow immunochromatographic strip (LFICS), were developed to monitor CPA in maize and rice samples. For this purpose, a monoclonal antibody (3H12) posed highly resistant to pH (5.6 to 9.6) and ethanol (≤50%) was generated by mouse immunization. Negative maize and rice samples or samples spiked with CPA were extracted with ethanol/0.01 M sodium borate buffer (4/1, v/v, pH 8.4). For ic-ELISA analysis, the limits of detection (LODs) were 0.48 and 0.28 ng/g for maize and rice samples, respectively. The recovery for spiked maize was 76.9% to 83.5% with the highest variable coefficient (CVmax ) being 9.32%. For spiked rice, the recovery was 85.3% to 105.1% with a CVmax of 8.56%. For LFICS analysis, the visible LODs were 2.5 and 1 ng/g and cutoff values were 5 and 2.5 ng/g for maize and rice samples, respectively. The LFICS method gave results within 5 to 10 min, providing an auxiliary analytical tool for the rapid, sensitive, and portable screening of the massive samples onsite.

Preparation monoclonal ß-type anti-idiotype antibody of zearalenone and development of green ELISA quantitative detecting technique.

Immunoassay has been widely used in the screening of mycotoxins, which may be hazardous to the operator or the environment. This study was to develop a green way to measure zearalenone (ZEN) with a monoclonal ß-type anti-idiotype antibody (Ab2ß) against ZEN in place of ZEN standard. Six monoclonal ß-type anti-idiotype antibodies were prepared. The 50% inhibitory concentration (IC50) value to ZEN of the six antibodies was between 34.45 ± 1.12-182.12 ± 15.40 nM. A green ELISA was then developed and validated. The quantitative conversion formula between ZEN and the monoclonal Ab2ß against ZEN was y = 0.092x0.722, R2 = 0.990. The working range was 2.63-100.64 ng ml-1. The recovery rate in spiked feed samples was from 82.15% to 102.79%, and the within-assay and between-assay coefficient variation (CV) level were less than 10.00%. A good correlation was obtained by high-performance liquid chromatography method (HPLC) to validate the developed method.

Polyclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for screening of paclobutrazol in fruits.

Paclobutrazol (PBZ) is a plant growth regulator (PGR) widely used in fruit and vegetable cultivation. However, due to the severe toxicity of PBZ, a sub-ppm level maximum residue limit (MRL) was established worldwide. Therefore, it is significant to propose a rapid, sensitive and high throughput screening method for monitoring the PBZ residues in foods. In this study, a simple and sensitive indirect competitive Enzyme-linked immunosorbent assay (icELISA) was established for PBZ detection in fruits basing polyclonal antibody. For both economy and pollution prevention, a microwave-solvent-free method was used to synthesize the PBZ hapten with high efficiency. The detection conditions, such as coating antigen concentration, antibody concentration, organic reagent concentration, ionic strength and pH, were optimized. Under the optimized conditions, this method showed high sensitivity and specificity. The detection range is 1.27-138.23 ng/mL, half-maximum inhibition concentration (IC50) is 13.26 ng/mL, and the IC20 was lower than the reported ELISAs for PBZ. Additionally, this method had high accuracy and precision. The recoveries were ranged from 88.78% to 96.80% in PBZ spiked apple samples with RSD below 4%. All the results showed that the polyclonal antibody based icELISA could be useful for PBZ screening in fruit samples.

Preparation of an anti-cotinine monoclonal antibody and its application in immunological detection / 浙江大学学报·医学版

OBJECTIVE@#To prepare monoclonal antibody against cotinine (COT) and to establish immunoassay for detecting COT in human urinary samples.@*METHODS@#BALB/c mice were immunized with synthesized cotinine-bovine serum albumin (COT-BSA) to screen monoclonal antibody with technique of cell fusion. The monoclonal antibody was used for the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatographic strip assay for the detection of COT in human urine.@*RESULTS@#The monoclonal antibody against COT was identified by ic-ELISA with a 50%inhibitive concentration (IC@*CONCLUSIONS@#The ic-ELISA and colloidal gold immunochromatographic strip assay using the prepared monoclonal antibody against COT have been proved to be reliable for the rapid detection of COT in human urines, which may be used for monitoring of environmental tobacco smoke.

Development of a broad-spectrum monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for the multi-residue detection of avermectins in edible animal tissues and milk.

Avermectins (AVMs) are a group of anti-parasitic agents that have been widely used in food-producing animals. To monitor the residue of the AVMs, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed with a simple sample preparation procedure. Conjugates of 4″-HS-IVM/AVM on three different proteins were used to raise a broad-spectrum monoclonal antibody (mAb), 6D4, that had IC50 values for avermectin, ivermectin, eprinomectin and emamectin of 7.2, 10.4, 19.8 and 20.8 µg L-1, respectively. The limit of detection and limit of quantitation of this method for AVMs in various matrix samples ranged from 0.5 to 5.4 µg L-1 and 1.0 to 10.3 µg L-1, respectively. The recoveries of the samples spiked with AVMs were in the range of 78.1-110.5% with coefficients of variation below 14%. The ic-ELISA was applied to monitor ivermectin in the milk samples, and the results showed a good correlation with that of HPLC-MS/MS.

Broad-spectrum monoclonal antibody and a sensitive multi-residue indirect competitive enzyme-linked immunosorbent assay for the antibacterial synergists in samples of animal origin.

To monitor the abuse of antibacterial synergists, a hapten, trimethoprim carboxylic derivative (TMPCOOH), was designed by using molecular modelling technology. A broad-spectrum monoclonal antibody (mAb) TMP/2G1 was prepared, for which the IC50 values of trimethoprim, diaveridine, aditoprim, baquiloprim, ormetoprim, and brodimoprim were 0.232, 0.527, 1.479, 4.354, 0.965, and 0.119 µg L-1, respectively. Based on the broad spectrum mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed to determine the residues of antibacterial synergists. The limit of detection regarding the developed ic-ELISA for antibacterial synergists ranged from 0.025 to 1.126 µg L-1 in milk, honey and edible animal tissues. The recoveries ranged from 81.4% to 107.7%, with a coefficient of variation less than 20%. A good correlation (R2 = 0.994) between the ic-ELISA and HPLC-MS/MS showed the reliability of the developed ic-ELISA.

Distribution kinetics of puerarin in rat hippocampus after acute local cerebral ischemia.

Puerarin, isolated from the roots of Pueraria lobata, is widely used for treating cerebral ischemia in China. The time- and dose-dependent distribution characteristics of puerarin in the ischemic hippocampus are unknown. In this study, puerarin concentration was determined by an indirect competitive ELISA using anti-puerarin monoclonal antibody. Area under the curve (AUC0-120 min) of puerarin (80 mg/kg) in the embolic hippocampus was higher than that in the normal hippocampus; the increase was significant only at 40 and 20 mg/kg. The maximum concentration (Cmax) of puerarin in the embolic hippocampus was higher than that in the normal hippocampus at all doses. The increase in both AUC0-120 min and Cmax was dose-dependent. Time to reach the maximum concentration (Tmax) of puerarin in the embolic and normal hippocampus was similar. Although the mean residence time in the embolic hippocampus differed from that in the normal hippocampus at 40 and 80 mg/kg, it was higher in the embolic hippocampus than in the normal hippocampus at 20 mg/kg. This is the first study to report that the time- and dose-dependent distribution characteristics of puerarin in the normal and embolic hippocampus after middle cerebral artery occlusion in rats dictate puerarin dose and duration to treat stroke.