Fluorescence based immunochromatographic sensor for rapid and sensitive detection of tadalafil and comparison with a gold lateral flow immunoassay.

Tadalafil (TDL) is an illegal additive drug found in drinks and functional foods that could threaten public health. There was a great concern whether the adulteration occurred in coffee added with similar type of herbs. Here we have developed a rapid, simple, sensitive, and semi-quantitative lateral flow immunoassay (LFIA) based on gold and fluorescence labelled monoclonal antibody (mAb) for detection of TDL in coffee sample. Under optimal conditions, the cut off limits using gold nanoparticles labelled mAb (GLM) was found to be 250 ng/mL and 100 ng mL using fluorescent labelled mAb (FLM) in coffee samples. The coffee samples were spiked with TDL, and the LFIA with GLM gave average recoveries of 92-105.3% (intra-assay) and 96.6-105.9% (inter-assay), meanwhile with FLM gave recoveries 97.9-107.3% (intra-assay) and 98.3-108.9% (inter-assay). Results gave LFIA with FLM more sensitive than with GLM and all the test can be completed within 10 min, which would be an option for convenient and rapid assay of TDL detection.

Establishment of thrombin affinity column (TAC)-HPLC-MS/MS method for screening direct thrombin inhibitors from Radix Salviae Miltiorrhiae.

In order to develop an affinity HPLC method for screening direct thrombin inhibitors from Traditional Chinese Medicine (TCM), thrombin was immobilized on the glutaraldehyde-modified amino silica gel and was used as thrombin stationary phase. A thrombin affinity column (TAC) was made by packing the thrombin stationary phase into a bare column (2.0 * 1.0 mm, i.d.). The direct thrombin inhibitors could be screened through this TAC column. For the purpose of improvement of the discovery efficiency, a TAC-HPLC-MS/MS system was used to screen thrombin inhibitors from Radix Salviae Miltiorrhiae (RSM), a famous traditional Chinese medicine. After optimization of all the conditions, cryptotanshinone (Cry), dihydrotanshinone I (Dih-I) and tanshinone IIA (Tan-IIA) were screened out and identified as potential active components. The anticoagulant effects of these three compounds were tested by anticoagulant experiments in vitro. Furthermore, the interaction of three compounds with thrombin was studied by molecular docking. The result shows they have the potential to be used as preventive drugs. In short, this method can be used to screen anticoagulant drugs from traditional Chinese medicine, which provides convenience for screening anticoagulant drugs.

Flower-like gold nanoparticles-based immunochromatographic test strip for rapid simultaneous detection of fumonisin B1 and deoxynivalenol in Chinese traditional medicine.

A flower-like gold nanoparticles (FGN)-based immunochromatographic test strip (ICS) was developed and used for the first time for the rapid simultaneous detection of fumonisin B1 (FB1) and deoxynivalenol (DON) in Chinese traditional medicine. Several experimental conditions affecting the sensitivity of ICS have been investigated, including the type of FGN, the preparation conditions of FGN-monoclonal antibody (MAb) conjugates, and the process parameters of ICS. Under the optimal experimental conditions, the visual limit of detection was 5.0 ng/mL (corresponding to 50 µg/kg in Chinese traditional medicine samples) for both FB1 and DON, and detection can be completed within 5 min. In addition, the natural samples were analyzed using high-performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA), and the results of these methods showed good correlation with those obtained using ICS. The procedure using FGN-based simultaneous ICS was sensitive, rapid, and convenient for on-site detection of a large number of samples.

Development of Bispecific Antibody Derivatives for Cancer Immunotherapy.

Development of antibody-based immunotherapeutics has progressed from direct tumor-targeting, with antibodies such as rituximab, to blocking of immune checkpoints to reactivate antitumor immunity. In addition, bispecific antibodies/antibody fragments are also of great interest in cancer therapy, as these constructs have the ability to redirect immune effector cells to cancer targets and, thereby, enhance therapeutic efficacy. A number of bispecific antibody formats have been reported, with the first FDA-approved bispecific antibody being blinatumomab, a so-called bispecific T cell engager (BiTE), which redirects and potently activates T cell immune responses. Recently, we described an additional novel bispecific antibody derivative, termed RTX-CD47, which was designed to inhibit the innate immune checkpoint CD47-SIRPα only on -positive cancer cells. RTX-CD47 contains two antibody fragments in tandem and has monovalent binding specificity for CD47 and . Only upon dual binding to and CD47 RTX-CD47 blocks CD47 "Don't eat me" signaling. Here, we provide a detailed protocol for the construction and functional evaluation of such a bispecific antibody derivative.

A method using angiotensin converting enzyme immobilized on magnetic beads for inhibitor screening.

Angiotensin converting enzyme (ACE), fusing with FLAG tag, was overexpressed in human embryonic kidney 293T cells. This recombinant FLAG-tagged ACE was immobilized on anti-FLAG antibody coated magnetic beads by affinity method in crude cell lysate for the first time. The enzyme-immobilized magnetic beads (ACE-MB), without further cleavage procedure, were used directly to establish a cost-effective and reliable method for screening ACE inhibitors by coupling with fluorescence detection. The enzymatic activity of the ACE-MB was validated based on its Michaelian kinetic behavior towards hippuryl-histidyl-leucine by UHPLC-MS/MS method firstly. Then, several conditions were optimized including amount of magnetic beads, incubation temperature and time in the procedure of ACE immobilization and amount of ACE-MB in the microplate operation. Moreover, this screening assay was validated with Z' factors between 0.71 and 0.81 using four known ACE inhibitors (captopril, lisinopril, fosinopril and fosinoprilat). The developed method was applied for the screening of ACE inhibitors from a small compound library of 45 natural products. As a result, epiberberine and fangchinoline with certain ACE inhibitory activities were screened out in the assay and validated. The results demonstrate the usefulness of this screening method using ACE immobilized on magnetic beads and the advantage of great efficiency with respect to both time and reagents for screening ACE inhibitors.

Multiplexed immunochromatographic test strip for time-resolved chemiluminescent detection of pesticide residues using a bifunctional antibody.

A novel bifunctional antibody (BfAb) that could recognize methyl parathion and imidacloprid simultaneously was prepared by a hybrid hybridomas technique. Using the BfAb as the sole recognition reagent, a multiplexed immunochromatographic test strip based on a time-resolved chemiluminescence (CL) strategy was developed for quantitative detection of pesticide residues. Horseradish peroxidase (HRP) and alkaline phosphatase (ALP) were used as the CL probes to label the haptens of methyl parathion and imidacloprid, respectively. After the labeled haptens competed with methyl parathion and imidacloprid to bind with the BfAb immobilized on the test strip, the two CL reactions catalyzed by the enzymes were triggered simultaneously by coreactants injection. Due to the distinct CL kinetics characteristics of HRP and ALP, the signals for methyl parathion and imidacloprid detections were collected at 2.5s and 300s, respectively. The linear ranges for methyl parathion and imidacloprid were both 0.1-250ngmL-1, with detection limits of 0.058ngmL-1 (S/N=3). The whole assay process could be accomplished within 22min. The detection results for spiked traditional Chinese medicine samples demonstrated its application potential. The proposed method provided a low-cost, facile and rapid tool for multiplexed screening of pesticide residues using single antibody.

A fast and easy strategy for protein purification using "teabags".

Protein purification often involves affinity capture of proteins on stationary resin, alternatively proteins are captured on free flowing resin for subsequent separation from bulk fluid. Both methods require labour and time intensive separation of particulate matter from fluid. We present a method where affinity resin is contained within porous-walled containers, supporting clarification, product recovery, and concentration in a single step with minimal hands-on processing time, without significant investments in equipment.

An Efficient Protocol for Preparation of Gallic Acid from Terminalia bellirica (Gaertn.) Roxb by Combination of Macroporous Resin and Preparative High-Performance Liquid Chromatography.

In this article, macroporous resin column chromatography and preparative high-performance liquid chromatography were applied for preparation of gallic acid from Terminalia bellirica (Gaertn.) Roxb. In the first step, six kinds of resins were investigated by adsorption and desorption tests and AB-8 macroporous resin was selected for the enrichment of gallic acid. As a result, 20 g of gallic acid at a purity of 71% could be separated from 100 g of crude extract in which the content of gallic acid was 16.7% and the recovery of gallic acid reached 85.0%. In the second step, preparative high-performance liquid chromatography was selected to purify gallic acid. As a result, 640 mg of gallic acid at a purity of 99.1% was obtained from 1 g of sample in 35 min. The results demonstrated that macroporous resin coupled with preparative high-performance liquid chromatography was suitable for preparation of gallic acid from T. bellirica (Gaertn.) Roxb.

Multiarray on a test strip (MATS): rapid multiplex immunodetection of priority potato pathogens.

Multiarray on a test strip (MATS) was developed for the detection of eight important potato pathogens. The proposed assay combines the rapidity of immunochromatography with the high throughput of array techniques. The test zone of the immunochromatographic strip comprises ordered rows of spots containing antibodies specific for different potato pathogens. The assay benefits from the simplicity of immunochromatography; colored immune complexes form at the corresponding spots within the test zone. The presence and intensity of the coloration are used for identification of the target pathogens. The MATS was applied to the simultaneous detection of eight priority potato pathogens, characterized by the following limits of detection: 1 ng/mL for potato virus X and the ordinary type of potato virus Y, 10 ng/mL for potato virus M, 20 ng/mL for potato leaf roll virus, 40 ng/mL for necrotic-type potato virus Y, 100 ng/mL for potato virus S, 300 ng/mL for potato virus A, and 10(4) cells/mL for Clavibacter michiganensis subsp. sepedonicus. Analysis time was 15 min. The observed sensitivity of the MATS was comparable to the traditional enzyme-linked immunosorbent assay. The developed technique was tested on potato leaf extracts, and its efficiency for on-site control of the pathogens was confirmed in 100 % by commercial LFIA test strips. Graphical abstract Location of binding zones in the developed multiarray on a test strip (MATS) for simultaneous detection of eight pathogens.

Rapid lateral-flow immunoassay for the quantum dot-based detection of puerarin.

In this study, a rapid (within 10min) quantitative lateral-flow immunoassay using a quantum dots (QDs)-antibody probe was developed for the analysis of puerarin (PUE) in water and biological samples. The competitive immunoassay was based on anti-PUE monoclonal antibody conjugated with QDs (detection reagent). Secondary antibody was immobilized on one end of a nitrocellulose membrane (control line) and PUE-bovine serum albumin conjugate was immobilized on the other end (test line). In the quantitative experiment, the detection results were scanned using a membrane strip reader and a detection curve (regression equation: y=-0.11ln(x)+0.979, R(2)=0.9816) representing the averages of the scanned data was obtained. This curve was linear from 1 to 10µg/mL. The IC50 value was 75.58ng/mL and the qualitative detection limit of PUE was 5.8ng/mL. The recovery of PUE added to phosphate-buffered saline and biological samples was in the range of 97.38-116.56%. To our knowledge, this is the first report of the quantitative detection of a natural product by QDs-based immunochromatography, which represents a powerful tool for rapidly screening PUE in plant materials and other biological samples.

Membrane-based lateral flow immunochromatographic strip with nanoparticles as reporters for detection: A review.

Membrane-based lateral flow immunochromatographic strip (LFICS) is widely used in various fields because of its simplicity, rapidity (detection within 10min), and low cost. However, early designs of membrane-based LFICS for preliminary screening only provide qualitative ("yes/no" signal) or semi-quantitative results without quantitative information. These designs often suffer from low-signal intensity and poor sensitivity and are only capable of single analyte detection, not simultaneous multiple detections. The performance of existing techniques used for detection using LFICS has been considerably improved by incorporating different kinds of nanoparticles (NPs) as reporters. NPs can serve as alternative labels and improve analytical sensitivity or limit of detection of LFICS because of their unique properties, such as optical absorption, fluorescence spectra, and magnetic properties. The controlled manipulation of NPs allows simultaneous or multiple detections by using membrane-based LFICS. In this review, we discuss how colored (e.g., colloidal gold, carbon, and colloidal selenium NPs), luminescent (e.g., quantum dots, up-converting phosphor NPs, and dye-doped NPs), and magnetic NPs are integrated into membrane-based LFICS for the detection of target analytes. Gold NPs are also featured because of their wide applications. Different types and unique properties of NPs are briefly explained. This review focuses on examples of NP-based LFICS to illustrate novel concepts in various devices with potential applications as screening tools. This review also highlights the superiority of NP-based approaches over existing conventional strategies for clinical analysis, food safety, and environmental monitoring. This paper is concluded by a short section on future research trends regarding NP-based LFICS.

Use of specific polysaccharide-immobilized monodisperse poly(glycidyl methacrylate) core-silica shell microspheres for affinity purification of lectins.

Immobilization of polysaccharides (yeast mannan and gum arabic) on the macroporous poly(glycidyl methacrylate) monodisperse microspheres coated with silica (SiO2 )-containing amino groups on the surface was used to prepare affinity sorbents for lectin purification. The efficiency of isolating mannose specific Pisum sativum lectin was demonstrated on sorbent with immobilized yeast mannan and that of galactose specific Glycine hispida lectin on sorbent with immobilized gum arabic. The microspheres with immobilized polysaccharides can be used for selecting an affinity sorbent for purification of other mannose- and galactose-specific lectins. In contrast to yeast mannan, the gum arabic immobilized on the microspheres possesses much narrower specificity and is suitable for purification of only those galactose specific lectins which interact well with l-rhamnose or l-arabinose. The synthesized macroporous particles are capable of immobilizing 50 mg of polysaccharide per 1 g of the matrix, which is 10 times higher than the capacity of epoxy-activated Sepharose 6B. That makes it possible to obtain the same lectin quantity using a column of 10 times smaller volume. Another advantage of novel affinity sorbents comparing corresponding Sepharose gels is the possibility of sorbent drying after use.

Immunochromatographic strip for rapid detection of Cronobacter in powdered infant formula in combination with silica-coated magnetic nanoparticles separation and 16S rRNA probe.

Here we developed a sensitive, specific, and rapid immunochromatographic strip test for the detection of Cronobacter. Silica-coated magnetic nanoparticles were used to separate nucleic acid from Cronobacter lysate and eliminate the interference of food matrices successfully. A couple of 5'-end labeled probes, which was complementary to the 16S ribosomal DNA of Cronobacter, was used to hybridize with the nucleic acid. The hybrid product, labeled with digoxigenin on one side and biotin on the other side, was directly submitted to the immunochromatographic strip test and the anti-digoxigenin monoclonal antibody was immobilized on nitrocellulose membrane in the test line. The visualization was achieved by gold nanoparticles conjugated to streptavidin, and double red bands appearing in both test and control line indicated a positive result of the presence of Cronobacter in testing sample. The detection limit was 10(7) cfu mL(-1) in pure culture. After silica-coated magnetic nanoparticles treatment, the detection limit was 10(5) and 10(6) cfu mL(-1) in pure culture and powdered infant formula, respectively, and maintained stable even under the interference of 10(8) cfu mL(-1)Salmonella typhimurium. Furthermore, 100 positive powdered infant formula samples spiked 10(8) cfu mL(-1)Cronobacter and 20 negative samples with none bacteria were tested by the strip, and the sensitivity and specificity of the test were both as high as 100%. This approach showed promise for microbial detection concerning food safety or clinical diagnosis.

[New method for analyzing pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies].

Study on pharmacodynamic material basis of traditional Chinese medicines is one of the key issues for the modernization of traditional Chinese medicine. Having introduced the monoclonal antibody technology into the study on pharmacodynamic material basis of traditional Chinese medicines, the author prepared the immunoaffinity chromatography column by using monoclonal antibodies in active components of traditional Chinese medicines, so as to selectively knock out the component from herbs or traditional Chinese medicine compounds, while preserving all of the other components and keeping their amount and ratio unchanged. A comparative study on pharmacokinetics and pharmacodynamics was made to explicitly reveal the correlation between the component and the main purpose of traditional Chinese medicines and compounds. The analysis on pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies is a new method for study pharmacodynamic material basis in line with the characteristics of traditional Chinese medicines. Its results can not only help study material basis from a new perspective, but also help find the modern scientific significance in single herb or among compounds of traditional Chinese medicines.

Versatile nanocomposites in phosphoproteomics: a review.

Protein phosphorylation is one of the most important post-translational modifications. Phosphorylated peptides are present in low abundance in blood serum but play a vital role in regulatory mechanisms and may serve as casual factors in diseases. The enrichment and analysis of phosphorylated peptides directly from human serum and mapping the phosphorylation sites is a challenging task. Versatile nanocomposites of different materials have been synthesized using simple but efficient methodologies for their enrichment. The nanocomposites include magnetic, coated, embedded as well as chemically derivatized materials. Different base materials such as polymers, carbon based and metal oxides are used. The comparison of nanocomposites with respective nanoparticles provides sufficient facts about their efficiency in terms of loading capacity and capture efficiency. The cost for preparing them is low and they hold great promise to be used as chromatographic materials for phosphopeptide enrichment. This review gives an overview of different nanocomposites in phosphoproteomics, discussing the improved efficiency than the individual counterparts and highlighting their significance in phosphopeptide enrichment.

Combining human periodontal ligament cell membrane chromatography with online HPLC/MS for screening osteoplastic active compounds from Coptidis Rhizoma.

We have developed an online analytical method that combines human periodontal ligament cell membrane chromatography (hPDLC/CMC) with high-performance liquid chromatography and mass spectrometry (LC/MS) for recognizing and identifying osteoplastic active components from Coptidis Rhizoma. Retention fractions on hPDLC/CMC were enriched onto an enrichment column and the components were directly analyzed by combining a 10-port column switcher with an LC/MS system for separation and preliminary identification. Using simvastatin (SIM) as a positive control, berberine from Coptidis Rhizoma was identified as the active component which could act on the hPDLC. The MTT colorimetric assay, alkaline phosphatase (ALP) activity, and staining tests revealed that berberine could promote hPDLC growth, increase the secretion of ALP in the culture medium, and enhance the formation of mineralized nodule, thus it is a potential osteoplastic ingredient. This hPDLC/CMC-online-LC/MS method can be applied for screening active components acting on hPDLC from traditional Chinese medicines exemplified by Coptidis Rhizoma and will be of great utility in drug discovery using natural medicinal herbs as a source of leading compounds.

Enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino by column chromatography technique.

In present study, the performance and separation characteristics of nine macroporous resins for the enrichment and purification of gardenia yellow from Gardenia jasminoides var. radicans Makino have been evaluated. The adsorption and desorption properties of crude gardenia yellow solution on macroporous resins including HPD722, HPD100, HPD100A, HPD400, HPD400A, D101, AB-8, XAD-16, and NKA-9 have been compared. Then, HPD722 was chosen to purify gardenia yellow because of its strong adsorption and desorption abilities as well as high selectivity. Column packed with HPD722 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process of gardenia yellow. The optimal conditions were as follows: The crude gardenia yellow solution with concentration of 15 mg/mL was loaded in column packed with HPD722 resin at the flow rate of 1.0 mL/min, and the adsorbate-laden column was washed with 800 mL water, 600 mL 15% ethanol water solution respectively at the speed of 2.5 mL/min, then desorbed with 200 mL 80% ethanol water solution at the speed of 3.5 mL/min. The colority of the product obtained were up to 300. The method developed in this study provides a new approach for scale-up separation and purification of gardenia yellow from G. jasminoides var. radicans Makino.

Toward high-throughput drug screening on a chip-based parallel affinity separation platform.

High-throughput screening of compound libraries, including the study of fragments, has become one of the cornerstones in modern drug discovery research. During this process hits are defined that may be developed into valuable leads and eventually into possible drug candidates. In this paper, we have demonstrated that parallel zonal weak affinity chromatography in microcolumns on a chip offers a possible screening format for weakly binding ligands toward a protein target. We used albumin as a model system because this transport protein is well established as a binder (both weak and strong) for drug substances. Bovine serum albumin was immobilized on microparticulate diolsilica particles and then packed into a 24-channel cartridge, which served as the separation platform. Analysis of the obtained chromatograms yielded information about affinity even in the millimolar range. Employing this approach, thousands of substances can be screened in just a day. We feel confident that zonal affinity chromatography will provide a useful technology in the future for performing high-throughput screening.

Combination of solid-phase affinity capture on magnetic beads and mass spectrometry to study non-covalent interactions: example of minor groove binding drugs.

A simple and novel approach was developed to detect non-covalent interactions. It is based on combination of solid-phase affinity capture with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). One of the interacting molecules is bound to magnetic beads and is incubated with the target molecules in solution. The complex bound on the solid support is removed from the solution and transferred for MALDI analysis. Mass spectrometry is used only to detect the target compound, which is far more straightforward than detecting the intact non-covalent complex. To demonstrate the applicability of the method, an AT-rich oligonucleotide (5'-CCCCCAATTCCCCC-3') and its complementary biotinylated sequence (5'-biotin-GGGGGAATTGGGGG-3') were hybridized and immobilized to paramagnetic particles by streptavidin-biotin interaction. The immobilized duplex oligonucleotide was reacted with minor groove binding drugs, Netropsin, Distamycin A, Hoechst 33258 and 4',6-diamidino-2-phenylindole. The resulting DNA-drug complex bound to the particles was separated and analyzed by linear MALDI-TOFMS after washing. Drugs were selectively detected in the spectra. Relative binding strengths were also estimated using competitive complexation.

High-throughput screening for enzyme inhibitors using frontal affinity chromatography with liquid chromatography and mass spectrometry.

This work presents new frontal affinity chromatography (FAC) methodologies for high-throughput screening of compound libraries, designed to increase screening rates and improve sensitivity and ruggedness in performance. A FAC column constructed around the enzyme N-acetylglucosaminyltransferase V (GnT-V) was implemented in the identification of potential enzyme inhibitors from two libraries of trisaccharides. Effluent from the FAC column was fractionated, sequentially processed via LC/MS, and referenced to a similar analysis through a control FAC column lacking the enzyme. The resulting multidimensional data sets were compared across corresponding sample and control fractions to identify binders, in a semiautomated approach. A strong binder in the protonated form at m/z 795 was identified from the first library of 81 compounds, exhibiting an estimated Kd value of 0.3 microM. Other binders yielded Kd values ranging from 0.35 to 3.35 microM. To demonstrate the improvement in performance of this FAC-LC/MS approach over the conventional online FAC/MS approach, 15 compounds from this library were blended with a second library of 1000 synthetic trisaccharides and screened against GnT-V. All ligands in the 15-compound set were identified in this larger screen, and no ligands of greater affinity than compound 1 were found. Our results show that FAC-LC/MS is a reliable method for screening large compound libraries directly and useful for large-scale ligand discovery initiatives.