Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium

In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme‐linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti‐naringin monoclonal antibodies to CNBr‐activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products.     

Development of an enzyme‐linked immunosorbent assay and immunoaffinity chromatography for glycyrrhizic acid using an anti‐glycyrrhizic acid monoclonal antibody

In this work, a new monoclonal antibody specific for glycyrrhizic acid was prepared and characterized. A hybridoma secreting an anti‐glycyrrhizic acid monoclonal antibody was produced by fusing splenocytes from a mouse immunized against a glycyrrhizic acid–bovine serum albumin conjugate with the hypoxanthine–aminopterin–thymidine‐sensitive mouse myeloma cell line (Sp2/0‐Ag14). Subsequently, an indirect, competitive enzyme‐linked immunosorbent assay for glycyrrhizic acid was developed using the monoclonal antibody. In this assay, we detected an effective measuring range of 78.12–2500 ng/mL. Both intra‐assay and inter‐assay repeatability and precision were achieved, with relative standard deviations lower than 10%. In addition, glycyrrhizic acid levels in both formulated Chinese medicines and biological samples were determined with high sensitivity and efficiency. We then successfully developed a reliable immunoaffinity chromatography to separate glycyrrhizic acid completely from its parent medicine. These methods will contribute to further research investigations to better understand the interactions of glycyrrhizic acid with other drugs in the complex system of traditional Chinese medicine.     

Development and application of immunoaffinity column chromatography for atrazine in complex sample media

A rabbit antibody immunoaffinity (IA) column procedure was evaluated as a cleanup method for the determination of atrazine in soil, sediment, and food. Four IA columns were prepared by immobilizing a polyclonal rabbit anti-atrazine antibody solution to HiTrap Sepharose columns. Atrazine was bound to the IA columns when the loading solvents were either 100% water, 2% acetonitrile in water, or 10% methanol in phosphate buffered saline (PBS). Quantitative removal of atrazine from the IA columns was achieved with elution solvents of either 70% ethanol in water, 70% methanol in water, or 100% methanol. One control column was prepared using nonspecific rabbit IgG antibody. This control column did not retain any applied atrazine indicating atrazine did not bind indiscriminately to protein or the Sepharose support. The four IA columns showed reproducible coupling efficiency for the immobilization of the atrazine antibody and consistent binding and releasing of atrazine. The coupling efficiency (4.25 mg of antibody in 1 mL of resin bed) for the four IA columns ranged from 93 to 97% with an average of 96 ± 2% (2.1%). Recoveries of the 500, 50, and 5 ng mL⁻¹ atrazine standard solutions from the four IA columns were 107 ± 7% (6.5%), 122 ± 14% (12%), and 114 ± 9% (8.0%) respectively, based on enzyme-linked immunosorbent assay (ELISA) data. The maximum loading was approximately 700 ng of atrazine for each IA column (∼0.16 μg of atrazine per mg of antibody). The IA columns could withstand 100% methanol as the elution solvent and could be reused more than 50 times with no change in performance. The IA columns were challenged with soil, sediment, and duplicate-diet food samples and effectively removed interferences from these various matrices for subsequent gas chromatography/mass spectrometry (GC/MS) or ELISA analysis. The log-transformed ELISA and GC/MS data were significantly correlated for soil, sediment and food samples although the ELISA values were slightly higher than those obtained by GC/MS. The IA column cleanup procedure coupled with ELISA analysis could be used as an alternative effective analytical method for the determination of atrazine in complex sample media such as soil, sediment, and food samples.     

吡虫啉的酶联免疫吸附分析方法研究

通过碳二亚胺法将吡虫啉交联于牛血清蛋白(BSA)作为免疫抗原,混合酸酐法将吡虫啉交联于卵清蛋白(OVA)作为包被抗原,免疫Balb/c小鼠,采用B细胞杂交瘤技术,经免疫、融合、筛选、克隆,得到抗吡虫啉单克隆抗体,抗体亚类为IgG1,制备的单克隆抗体效价达1×107,确定了吡虫啉酶联免疫吸附分析方法(ELISA)的最佳工作条件,建立了定量测定吡虫啉的间接竞争ELISA方法。本方法的IC50为(15.12±1.28)μg/L,检出限为(1.76±0.02)μg/L。与其它吡虫啉结构类似物无交叉反应。批内相对标准偏差为4.5%;批间相对标准偏差5.1%,饮用水、重庆理工大学地下水和重庆市花溪河地表水平均添加回收率分别为102%,97%和85%。本研究建立了一种快速检测环境水中吡虫啉残留的方法。     

噻虫嗪单克隆抗体制备及酶联免疫吸附分析方法的建立

通过化学修饰合成了噻虫嗪人工半抗原,采用碳二亚胺法将该半抗原与牛血清蛋白(BSA)和卵清蛋白(OVA)偶联,成功制备了分子结合比合理的免疫原和包被原。经过免疫原免疫6周龄Balb/c小鼠、PEG介导免疫鼠脾细胞和骨髓瘤细胞的融合和阳性杂交瘤细胞的筛选和克隆化,获得了效价高达1∶6.4×105的抗噻虫嗪单克隆抗体,抗体亚类为IgG1型。优化了ELISA实验条件,建立了基于单克隆抗体技术的噻虫嗪残留间接竞争ELISA方法。本方法的抑制中浓度(IC50)为0.0255mg/L,检测灵敏度(IC20)为0.0022mg/L,检出限(IC10)为0.001mg/L。除噻虫胺外,该抗体与其它噻虫嗪结构类似物无交叉反应。以自来水为基质的噻虫嗪添加回收实验显示,0.01,0.5和10.0mg/L添加水平的回收率均大于75%,且各添加水平重复测定8次的相对标准偏差均小于8%,说明所建立的间接竞争ELISA准确度高,重复性好,适合水中噻虫嗪残留的检测。     

Bioanalysis of bevacizumab and infliximab by high-temperature reversed-phase liquid chromatography with fluorescence detection after immunoaffinity magnetic purification

This study presents two simple and rapid methods for the quantification of therapeutic mAbs based on LC. Two mAbs (bevacizumab and infliximab) in plasma samples were purified using magnetic beads immobilized with a commercially-available idiotype antibody for each mAb. Purified mAbs were separated with HT-RPLC and detected with their native fluorescence. Using immunoaffinity beads, each mAb was selectively purified and detected as a single peak in the chromatogram. The HT-RPLC achieved good separation for the mAbs with sharp peaks within 20 min. The calibration curves of the two mAbs ranged from 1 to 20 μg mL⁻¹ (bevacizumab) and 1–10 μg mL⁻¹ (infliximab), and they had strong correlation coefficients (r² > 0.998). The LOD of bevacizumab and infliximab was 0.07 and 0.15 μg mL⁻¹, and the LLOQ of bevacizumab and infliximab was 0.12 and 0.25 μg mL⁻¹, respectively. Thus, the sensitivities were sufficient for clinical analysis. Immunoaffinity purification with HT-RPLC produced a selective and accurate bioanalysis without an LC-MS/MS instrument. Both methods could become general-purpose analytical methods and complement the results obtained with conventional LBA.     

Novel approach to determine ghrelin analogs by liquid chromatography with mass spectrometry using a monolithic column

In our project, ghrelin analogs possessing enhanced stability and potential to significantly increase food intake were used. Three newly synthesized ghrelin analogs with fatty acid residues consisting of 8, 10, and 14 carbon atoms were studied. The main goal of this work was to develop a suitable analytical method for the determination of the stability of the novel ghrelin analogs in plasma. An appropriate liquid chromatography‐mass spectrometry method was developed and optimized. The results obtained were compared with the data measured by using a commercial enzyme‐linked immunosorbent assay kit, and a good correlation was found. A preparation strategy for plasma samples was optimized and consisted of simple dilution of the plasma samples followed by direct injection onto a very short monolithic column in combination with mass spectrometric detection. The developed analytical method was utilized for the determination of the stability of the prepared lipopeptides in plasma and for the quantification of the lipopeptides in a preliminary pharmacokinetic study. The feasibility of the developed separation method was clearly demonstrated. Accuracy and precision were within 80–120% and ±20% limits, respectively. Calibration curves were constructed in the range of 1–250 μg/mL.     

Determination of beta-conglycinin in soybean and soybean products using a sandwich enzyme-linked immunosorbent assay

Soybean protein has long been recognized as a source of dietary allergens for humans and animals with β-conglycinin being the major allergen. This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows for the detection of trace amount of β-conglycinin in soybean and soybean products. In the sandwich ELISA, mouse anti-β-conglycinin monoclonal antibody (Mab 5C5) was used as coating antibody, and rabbit anti-β-conglycinin polyclonal antibody (Pab) was used as secondary antibody. The assay showed high specificity for β-conglycinin with minimum cross-reactions with other soy proteins. The practical working range for the determination of β-conglycinin using the developed assay was 3–100 ng mL⁻¹ and the limit of determination (LOD) was 1.63 ng mL⁻¹. The recoveries of β-conglycinin in spiked soybean samples were between 88.1% and 106.6% with relative standard deviation less than 8.9% (intra-day) and 13.1% (inter-day). The developed method was used to analyze 469 soybean seed samples from different sources as well as five soybean products treated with different processing techniques. The data showed that the concentration of β-conglycinin decreased significantly after processing, especially for soybean protein isolation, where the concentration of β-conglycinin dropped to nearly zero. The assay provides a specific and sensitive method for the screening of β-conglycinin and allows for further investigation into hypersensitive mechanisms of soybean proteins and development of soybean processing techniques to reduce their negative effects.     

Sample clean-up by sol-gel immunoaffinity chromatography for determination of chloramphenicol in shrimp

The paper describes the characterisation and application of sol-gel columns prepared by entrapping anti-chloramphenicol (CAP) antibodies. Retention of CAP in the column was caused by specific interactions with the anti-CAP antibodies and not by non-specific adsorption to the sol-gel glass. After optimising important operation conditions, e.g. feeding medium, feeding flow-rate, elution medium, elution flow-rate and elution volume, the sol-gel columns were included in a clean-up procedure developed to determine CAP in shrimp. The selectivity of the columns was high enough to efficiently remove interfering matrix compounds. Due to the chromatographic conditions applied retention of cross-reacting substances in the immunoaffinity column did not pose a problem. CAP recovery of the analytical method was 68% with a relative standard deviation of 4% (n=4). In spite of applying highly complex shrimp extracts the columns could be used for clean-up of at least 12 samples. However, when detection of CAP is carried out with an UV detector the analytical method has a relatively poor sensitivity (LOD=1.8 ng/g, S/N=3). The most obvious way is to replace the UV detector by a detector based on an inherently more sensitive and selective detection principle, like a mass spectrometer.     

Online immunoaffinity assay-CE using magnetic nanobeads for the determination of anti-Helicobacter pylori IgG in human serum

About two-thirds of the world's population is infected with Helicobacter pylori (H. pylori). This Gram-negative bacterium is the most important etiological agent of chronic active type B gastritis and peptic ulcer diseases. Conventional methods such as gastric biopsy, ELISA and culture, require a long time for the determination of H. pylori infections. Moreover, the antibodies in human serum sample are capable to react immunologically with the purified H. pylori antigens immobilized on different kinds of support like magnetic nanobeads. In this study, we have developed an online immunoaffinity assay-CE to determine the concentration of anti-H. pylori IgG using magnetic nanobeads as a support of the immunological affinity ligands and an LIF as a detector. The separation was performed in 0.1 M glycine-HCl, pH 2, as the background electrolyte. The linear calibration curve to predict the concentration of H. pylori-specific immunoglobulin G antibodies in serum was produced within the range of 0.12-100 U/mL. The linear regression equation was i = 492.86+96.03 × C(anti-H. pylori), with the linear regression coefficient r(2) = 0.999. The LOD calculated by fluorescence detection procedure was of 0.06 U/mL. The whole assay was done in no more than 35 min and it was entirely automatized. The development of immunoaffinity assay-CE in this study demonstrates that there is a large possibility to introduce nanotechnology in several fields with significant advantages over the classic methodologies. Our proposition comprises the diagnosis and screening field.     

Use of biotin-streptavidin system for developing a viable, sensitive and specific antigen heterologous assay for hapten

The present study demonstrates improvement in sensitivity and specificity of hapten assay by using antigen heterology in conjunction with low molecular weight biotin label as compared to high molecular weight horseradish peroxidase (HRP) label. For generation of antiserum, cortisol-3-O-carboxylmethyl-oxime-bovine serum albumin (F-3-CMO-BSA) was used as immunogen whereas, for the preparation of primary label, corticosterone-3-carboxymethyl oxime (B-3-CMO) was coupled with biotinylcaproylhydrazide and HRP by employing N-hydroxysuccinimide mediated carbodiimide reaction. The data of the present study revealed that the antigen heterologous assay which employed high molecular weight HRP label showed 100% cross-reaction with corticosterone. On the contrary, when HRP was replaced with low molecular weight biotin label, less than 0.1% cross-reaction was observed with all analogous C₁₈, C₁₉, C₂₁ and C₂₇ steroids including corticosterone (0.2%). Moreover, the sensitivity of the later assay was 0.09 μg/dL, which is appreciable as compared to previously reported enzyme based assays. The recovery of the exogenously spiked serum pools lies in the range of 90.3-104.2%. The intra-assay and inter-assay coefficient of variation (CVs) ranged from 3.3% to 7.8% and 2.3% to 7.7%, respectively. The serum cortisol values obtained by this method correlated well with those obtained by radioimmunoassay; r = 0.9 (n = 50). The use of much stable biotin label in place of HRP has made the antigen heterologous enzyme linked immunosorbent assay (ELISA) of cortisol assay highly specific and sensitive.     

A micro-scale method employing surface plasmon resonance detection for the determination of conditions for immunoaffinity chromatography of proteins

Summary The BIAcore^TM Biosensor System utilizes a detection principle known as Surface Plasmon Resonance (SPR) which, in simple terms, detects changes in the refractive index of a solution in contact with a gold film. The gold film is surface modified with carboxymethylated dextran to produce a hydrophillic matrix onto which macromolecules may be covalently immobilized. In this respect, the BIAcore^TM is similar to any other insoluble matrix, such as a chromatographic support. The SPR detection principle, however, allows one to directly visualize the interaction under study, in real time and without the need for reporter molecules such as enzyme-labels. In addition, the very small sample requirements, automated robotics unit and ease of data analysis suggest the potential use of the BIAcore^TM instrumentation for assay development and possibly process development, especially where bio-specific interactions such as immunoaffinity chromatography are used as a step in the process. In this report, we demonstrate the use of the BIAcore^TM SPR detector for the micro-scale determination of conditions for immunoaffinity chromatography of soluble complementreceptor 1, sCR1.     

Generation and characterization of polyclonal antibodies against microcystins-Application to immunoassays and immunoaffinity sample preparation prior to analysis by liquid chromatography and UV detection

Polyclonal antibodies against microcystin-LR (MC-LR), a cyclic heptapeptide toxin, were generated in rabbits using MC-LR-BSA. An enzyme-linked immunosorbent assay (ELISA) was developed for the characterization of the antibodies and their potential use for analytical purposes. The concentration of MC-LR that inhibits 50% of antibody-antigen binding (IC₅₀) was 0.5 μg L⁻¹ for the indirect ELISA format and 0.9 μg L⁻¹ for the direct ELISA, using MC-LR-horseradish peroxidase conjugate. The limit of detection corresponding to IC₈₀ was found to be 0.06 μg L⁻¹, well below the Word Health Organization level for drinking water of 1 μg L⁻¹. The direct competitive ELISA was applied to water samples and was shown useful for screening purposes. The developed anti-microcystin antibodies were immobilized on solid supports for use in selective solid phase extraction (SPE) systems, prior to liquid chromatography (LC) quantification. An immunoaffinity cartridge (IAC), a Sepharose^®-based cartridge incorporating 2 mg of antibodies allowed the selective and quantitative recovery of a mixture of 0.2 μg of MCs showing potential use in sample preparation of real matrices. When applied to water and green algae samples, average recoveries from Sepharose^®-based cartridges were in the range of 86-113% for water samples and 85-92% for blue-green algae samples. Selectivity of the IAC clean-up was proven by comparison with non-specific solid phase extraction using octadecylsilica (ODS) sorbent. Results obtained using LC/UV after IAC clean-up agreed well with results obtained using liquid chromatography and mass spectrometry detection (LC/MS and LC/MS/MS) after SPE-C18 clean-up, allowing therefore to validate the resulting technique.     

Establishment of an immunoaffinity chromatography for simultaneously selective extraction of Sudan I, II, III and IV from food samples

The establishment of an immunoaffinity chromatography (IAC) for simultaneously selective extraction of four illegal colorants Sudan dyes (Sudan I, II, II and IV) from food samples was described. The IAC column was constructed by covalently coupling monoclonal antibody (mAb) against Sudan I to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. It was observed that IAC column was able to separately capture Sudan I, II, III and IV with maximum capacity of 295, 156, 184 and 173ng, respectively. The extraction conditions including loading, washing and eluting solutions were carefully optimized. Under optimal conditions, the extraction recoveries of the IAC column for Sudan I-IV at two different spiked concentrations were within 95.3-106.9%. After 50 times repeated usage, 64% of the maximum capacity was still remained. Six food samples randomly collected from local supermarket without spiking Sudan dyes were extracted with IAC column and detected by high performance liquid chromatography (HPLC). It was found that there was no detectable Sudan II, III and IV in all six food samples, but Sudan I with the content of 2.7-134.5ngg(-1) was detected in three food samples. To further verify the extraction efficiency, other three negative samples were spiked with Sudan I-IV at the concentrations of 20ngg(-1) and 50ngg(-1), which were then extracted with IAC column. The extraction recoveries and relative standard deviation (RSD) were 68.6-96.0% and 4.8-15.2%, respectively, demonstrating the feasibility of the prepared IAC column for Sudan dyes extraction.     

Usefulness of gold nanoparticles as labels for the determination of gliadins by immunoaffinity chromatography with light scattering detection

A simple and fast immunoaffinity method is proposed for the determination of gliadins for the first time using gold nanoparticles (AuNPs) as labels. The tracer used consists in a gliadin-AuNP conjugate prepared by the adsorption of gliadins onto the nanoparticle surface. Two AuNP sizes with diameters of 10 nm and 20 nm were assayed to compare the behaviour of the corresponding tracer in the assay. The method relies on the injection in a commercial Protein G column of a preincubated mixture containing gliadins, polyclonal anti-gliadin antibodies, and the gliadin-AuNP tracer. This approach allows the separation of free and bound tracer fractions without any additional elution step, and the direct measurement of the resonance light scattering intensity of the free tracer through the peak height of the immunochromatogram, which is proportional to the analyte concentration. The immunocolumn can be used up to 25 times without eluting and it can be regenerated for at least 20 times. The dynamic ranges of the calibration graphs and the detection limits are 0.5-15.0 and 1.5-15.0 μg mL⁻¹ gliadins, and 0.2 μg mL⁻¹ and 0.8 μg mL⁻¹ gliadins, using 20-nm and 10-nm Au-NPs as labels, respectively. The precision, expressed as relative standard deviation, ranges between 2.7% and 2.9% using 20-nm AuNPs and 4% and 6.1% for 10-nm AuNPs. The method has been applied to the determination of the prolamin fraction in beer samples, obtaining recovery values in the range 71.2% and 101.7%.     

Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples

The preparation and characterization of an immunoaffinity chromatography (IAC) column for the specific extraction and enrichment of trace contraceptive drug levonorgestrel (LNG) from water samples were described. The IAC column was constructed by covalently coupling specific polyclonal antibody against LNG to CNBr-activated Sepharose 4B and packed into a common solid phase extraction (SPE) cartridge. The extraction conditions including loading, washing and eluting solutions, as well as the effect of flow rate on the extraction were carefully optimized. Pure water, 5% of methanol and 50% of methanol were respectively selected as loading, washing and eluting solutions, while the flow rates in the loading, washing and eluting steps were selected to be 1.0, 2.0 and 0.5 mL min⁻¹, respectively. Under optimal conditions, the IAC column was characterized in terms of maximum capacity, extraction recovery and stability. It was found that, for IAC column packed with 0.2 g of solid support immobilized with antibody, the maximum capacity for LNG was about 260 ng. The extraction recoveries of the column for LNG at three different spiked concentrations were within 95.3-106.9%. After more than 35 times repeated usage, there was not significant loss of specific recognition. Using high performance liquid chromatography (HPLC) as an analytical tool, trace amount of LNG in the range of ng L⁻¹ was found in river water and wastewater samples after 600-fold enrichment, demonstrated the feasibility of the prepared IAC column for LNG extraction.     

Rapid separation of three glucosylated resveratrol analogues from the invasive plant Polygonum cuspidatum by high‐speed countercurrent chromatography

Three glucosylated resveratrol analogues (piceid, piceatannol glucoside, resveratroloside) were successfully isolated from the crude MeOH extract of the invasive plant species Polygonum cuspidatum by semi‐preparative high‐speed countercurrent chromatography with a two‐phase solvent system composed of cyclohexane‐ethyl acetate‐methanol‐water (1:5:1:5, v/v/v/v). Piceid (23 mg), resveratroloside (17 mg), piceatannol glucoside (15 mg) of purities over 80% were isolated from 500 mg crude MeOH extract in one step. Subsequent passage over a SPE column was used to quickly bring their purities to over 90%. The purities were determined by HPLC analysis and their structures were elucidated by proton nuclear magnetic resonance (¹H‐NMR), HMBC, ESI‐MS and HR‐MS.     

高效液相色谱法测定保健食品中的大豆异黄酮

建立了一种测定保健食品中大豆异黄酮的高效液相色谱分析方法,该方法可以使常见的大豆异黄酮6种主要成分大豆甙、黄豆甙、染料木甙、大豆甙元、黄豆黄素、染料木素得以分离和检测。采用乙腈-磷酸水溶液(pH2.8)作流动相,梯度洗脱,Venusil MP-C18色谱柱(150 mm×4.6 mmi.d.,5μm),流速为1.0 mL/min,紫外检测器,检测波长254 nm。结果表明各组分线性关系良好,相关系数R2为0.9991~0.9998,加标回收率在87%~106.9%,相对标准偏差均小于2%。检出限0.25~0.48μg/mL,该方法可同时测定大豆异黄酮的6种成分。     

Simple,rapid, and environmentally friendly method for the separation of isoflavones using ultra‐high performance supercritical fluid chromatography

Isoflavones are natural substances that exhibit hormone‐like pharmacological activities. The separation of isoflavones remains an analytical challenge because of their similar structures. We show that ultra‐high performance supercritical fluid chromatography can be an appropriate tool to achieve the fast separation of 12 common dietary isoflavones. Among the five tested columns the Torus DEA column was found to be the most effective column for the separation of these isoflavones. The impact of individual parameters on the retention time and separation factor was evaluated. These parameters were optimized to develop a simple, rapid, and green method for the separation of the 12 target analytes. It only took 12.91 min using gradient elution with methanol as an organic modifier and formic acid as an additive. These isoflavones were determined with limit of quantitation ranging from 0.10 to 0.50 μg/mL, which was sufficient for reliable determination of various matrixes.