An estimated quantitative lateral flow immunoassay for determination of artesunate using monoclonal antibody

There have been reports of fake artesunate (ART), which has led to deaths from untreated malaria in South East Asia. To rapidly screen for fake and adulterated ART products in the drug market, a lateral flow immunoassay (LFIA) based on a colloidal gold–monoclonal antibody probe for detection of ART within samples was developed. With this method, the calibration curve for ART was determined by the intensity ratio of the test and control bands at various ART concentrations. The linearity range was 12.5–200 μg/ml of ART. Samples were tested by the developed LFIA and can be calculated for ART contents. The levels of ART in the samples were also confirmed by enzyme-linked immunosorbent assay. The results of the two methods were in good conformance. The proposed LFIA was demonstrated to be a simple and rapid analytical method for detecting ART in the pharmaceutical formulation.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for detection of Sudan I in food samples

The use of Sudan I as an additive in food products has been prohibited in the European Union and many other countries. In this study, a highly sensitive and specific monoclonal antibody (mAb)-based indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of Sudan I in food samples was developed. The hapten derivative with a three-carbon-atom length of carboxylic spacer at the azobound para-position was synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugate was used as an immunogen, while the hapten-ovalbumin (OVA) conjugate was applied as a coating antigen. The mAb against Sudan I was produced by hybridoma technique and the corresponding ELISA was characterized in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed in concentrations of 0.1-100 ng mL⁻¹. The values of IC₅₀ for nine standard curves were in the range of 1.1-2.0 ng mL⁻¹ and the LOD at a signal-to-noise ratio of 3 (S/N = 3) was 0.07-0.14 ng mL⁻¹. The cross-reactivity values of the mAb with Sudan II, III and IV were 9.5%, 33.9% and 0.95%; no cross-reactivity was found with other six edible colorants: Lemon yellow, Bright blue, Indigotin, Kermes, Amarant and Sunset yellow, indicating the assay displays not only high sensitivity but also high specificity as well. The organic solvent effect on the assay was tested. It was observed that the ELISA was tolerated to 30% of methanol and 10% of acetonitrile without significant loss of IC₅₀ value. Six food samples were spiked with Sudan I and the methanolic extracts after appropriate dilution were analyzed by ELISA. Acceptable recovery rates of 88.2-110.5% and coefficients of variation of 2.5-17.4% were obtained. The ELISA for nine spiked samples was confirmed by high-performance liquid chromatography (HPLC) with a high correlation coefficient of 0.9840 (n = 9). The mAb-based ELISA proven to be a feasible quantitative/screening method for Sudan I analysis in food samples with the properties of high sensitivity, specificity, simplicity of sample pretreatment, high sample throughput and low expense.     

A new quality control method for lateral flow assay

We proposed a lateral flow assay (LFA) based on internal quality control microspheres to realize the accurate diagnosis of HbA1c in human body. This method can improve the precision and accuracy of HbA1c detection.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of glycyrrhizic acid

Glycyrrhizic acid (GL) is a major active compound of licorice. The specific monoclonal antibody (MAb) (designated as 8F₈A₈H₄2H₇) against GL was produced with the immunogen GL–BSA conjugate. The dissociation constant (K _d) value of the MAb was approximately 9.96×10⁻¹⁰ M. The cross reactivity of the MAb with glycyrrhetic acid was approximately 2.6%. The conventional indirect competitive enzyme-linked immunosorbent assay (icELISA) and simplified icELISA adapted with a modified procedure were established using the MAb. The IC₅₀ value and the detect range by the conventional icELISA were 1.1 ng mL⁻¹ and 0.2–5.1 ng mL⁻¹, respectively. The IC₅₀ value and the detect range by the simplified icELISA were 5.3 ng mL⁻¹ and 1.2–23.8 ng mL⁻¹, respectively. The two icELISA formats were used to analyze GL contents in the roots of wild licorice and different parts of cultivated licorice (Glycyrrhiza uralensis Fisch). The results obtained with the two icELISAs agreed well with those of the HPLC analysis. The correlation coefficient was more than 0.98 between HPLC and the two icELISAs. The two icELISAs were shown to be appropriate, simple, and effective for the quality control of raw licorice root materials.     

Detection of pork in heat-processed meat products by monoclonal antibody-based ELISA

An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody to a porcine thermal-stable muscle protein was developed for detection of pork in cooked meat products. The assay specifically detects porcine skeletal muscle, but not cardiac muscle, smooth muscle, blood, and nonmuscle organs. No cross-reactivity was observed with common food proteins. Validity of the assay was evaluated with laboratory formulated and commercial meat samples. The detection limit was determined as 0.5% (w/w) pork in heterologous meat mixtures. Overall, intra- and inter-assay coefficients of variation were 5.8 and 7.9%, respectively. The accuracy in analyzing market samples was 100% as verified by product labeling and confirmed by a commercial polycolonal antibody test kit.     

A monoclonal antibody-based ELISA for multiresidue determination of avermectins in milk

Due to the widespread use and potential toxicity of avermectins (AVMs), multi-residue monitoring of AVMs in edible tissues, especially in milk, has become increasingly important. With the aim of developing a broad-selective immunoassay for AVMs, a broad-specific monoclonal antibody (Mab) was raised. Based on this Mab, a homologous indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of AVMs in milk was developed. Under the optimized conditions, the IC?? values in assay buffer were estimated to be 3.05 ng/mL for abamectin, 13.10 ng/mL for ivermectin, 38.96 ng/mL for eprinomectin, 61.00 ng/mL for doramectin, 14.38 ng/mL for emamectin benzoate. Detection capability (CCβ) of the ELISA was less than 5 ng/mL and 2 ng/mL in milk samples prepared by simple dilution and solvent extraction, respectively. The optimized ELISA was used to quantify AVMs in milk samples spiked at different amounts. The mean recovery and coefficient of variation (CV) were 95.90% and 15.42%, respectively. The Mab-based ELISA achieved a great improvement in AVMs detection. Results proved this broad-selective ELISA would be useful for the multi-residue determination of AVMs in milk without purification process.     

Development and evaluation of a rapid lateral flow immunochromatographic strip assay for screening 19-nortestosterone

The lateral flow strip test for 19-nortestosterone is one kind of immunochromatographic assay. Nitrocellulose membrane was separately immobilized with goat anti-rabbit IgG (control line) and 19-NT-OVA conjugate (test line). Anti-19-NT polyclonal antibody labeled with colloidal gold particles acted as the detector reagent. The assay is qualitatively, not quantitatively, judged with positive or negative result. We tested the sensitivity of the strip using spiked swine urine, and each specimen was independently measured by LC/MS/MS. The sensitivity, measure by eye, was determined to be 200 ng/mL. The assay time was less than 15 min, and so suitable for on-site rapid test.     

Rapid and sensitive lateral flow immunoassay for influenza antigen using fluorescently-doped silica nanoparticles

We report on a lateral flow immunoassay (LFIA) for influenza A antigen using fluorescently-doped silica nanoparticles as reporters. The method is taking advantage of the high brightness and photostability of silica nanoparticles (doped with the dye Cy5) and the simplicity and rapidity of LFIA. The nucleoprotein of influenza A virion (one of its most abundant structural proteins) was used as a model to demonstrate a performance of the LFIA. Under optimized conditions and by using a portable strip reader, the fluorescence-based LFIA is capable of detecting a recombinant nucleoprotein as low as 250 ng?·?mL^-1 using a sample volume of 100 μL, within 30 min, and without interference by other proteins. The successful detection of the nucleoprotein in infected allantoic fluid demonstrated the functionality of the method. By comparison with a commercial influenza A test based on gold nanoparticles as reporters, the system provides an 8-fold better sensitivity.

Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (H_T) and the control line (H_C); the H_T/H_C ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0–1000 IU mL⁻¹) for AFP with a low limit of detection (0.1 IU mL⁻¹) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both <10%. Furthermore, in the analysis of human serum samples, excellent correlation (n = 284, r = 0.9860, p < 0.0001) was obtained between the proposed method and a commercially available CLIA kit. Results indicated that the Eu (III) chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.     

A monoclonal antibody-based immunosensor for detection of Sudan I using electrochemical impedance spectroscopy

Sudan I monoclonal antibodies (Mabs) were prepared by hybridoma technique and firstly used to develop a Sudan I immunosensor by immobilizing the Mabs on a gold electrode. o-Mercaptobenzoic acid (MBA) was covalently conjugated on the gold electrode to form a self-assembled monolayer (SAM). The immobilization of Sudan I Mabs to the SAM was carried out through a stable acyl amino ester intermediate generated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydrosuccinimide (NHS), which could condense antibodies reproducibly and densely on the SAM. The changes of the electrode behavior after each assembly step were investigated by cyclic voltammetric (CV) technique. The Sudan I concentration was measured through the increase of impedance values in the corresponding specific binding of Sudan I and Sudan I antibody. A linear relationship between the increased electron-transfer resistance (Ret) and the logarithmic value of Sudan I concentrations was found in the range of 0.05-50 ng mL⁻¹ with the detection limit of 0.03 ng mL⁻¹. Using hot chili as a model sample, acceptable recovery of 96.5-107.3% was obtained. The results were validated by conventional HPLC method with good correlation. The proposed method was proven to be a feasible quantitative method for Sudan I analysis with the properties of stability, highly sensitivity and selectivity.     

Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira

In recent years extensive numbers of molecular diagnostic methods have been developed to meet the need of point-of-care devices. Efforts have been made towards producing rapid, simple and inexpensive DNA tests, especially in the diagnostics field. We report on the development of a label-based lateral flow dipstick for the rapid and simple detection of multiplex loop-mediated isothermal amplification (m-LAMP) amplicons. A label-based m-LAMP lateral flow dipstick assay was developed for the simultaneous detection of target DNA template and a LAMP internal control. This biosensor operates through a label based system, in which probe-hybridization and the additional incubation step are eliminated. We demonstrated this m-LAMP assay by detecting pathogenic Leptospira, which causes the re-emerging disease Leptospirosis. The lateral flow dipstick was developed to detect of three targets, the LAMP target amplicon, the LAMP internal control amplicon and a chromatography control. Three lines appeared on the dipstick, indicating positive results for all representative pathogenic Leptospira species, whereas two lines appeared, indicating negative results, for other bacterial species. The specificity of this biosensor assay was 100% when it was tested with 13 representative pathogenic Leptospira species, 2 intermediate Leptospira species, 1 non-pathogenic Leptospira species and 28 other bacteria species. This study found that this DNA biosensor was able to detect DNA at concentrations as low as 3.95 × 10⁻¹ genomic equivalent ml⁻¹. An integrated m-LAMP and label-based lateral flow dipstick was successfully developed, promising simple and rapid visual detection in clinical diagnostics and serving as a point-of-care device.     

A simple and efficient method for potential point-of-care diagnosis of human papillomavirus genotypes: combination of isothermal recombinase polymerase amplification with lateral flow dipstick and reverse dot blot

Cervical cancer is the second most common cancer in the world’s woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1 h with a sensitivity of low to 100 fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.

     

Rapid PCR in a continuous flow device

Continuous flow polymerase chain reaction (CFPCR) devices are compact reactors suitable for microfabrication and the rapid amplification of target DNAs. For a given reactor design, the amplification time can be reduced simply by increasing the flow velocity through the isothermal zones of the device; for flow velocities near the design value, the PCR cocktail reaches thermal equilibrium at each zone quickly, so that near ideal temperature profiles can be obtained. However, at high flow velocities there are penalties of an increased pressure drop and a reduced residence time in each temperature zone for the DNA/reagent mixture, that potentially affect amplification efficiency. This study was carried out to evaluate the thermal and biochemical effects of high flow velocities in a spiral, 20 cycle CFPCR device. Finite element analysis (FEA) was used to determine the steady-state temperature distribution along the micro-channel and the temperature of the DNA/reagent mixture in each temperature zone as a function of linear velocity. The critical transition was between the denaturation (95 degrees C) and renaturation (55 degrees C-68 degrees C) zones; above 6 mm s(-1) the fluid in a passively-cooled channel could not be reduced to the desired temperature and the duration of the temperature transition between zones increased with increased velocity. The amplification performance of the CFPCR as a function of linear velocity was assessed using 500 and 997 base pair (bp) fragments from lambda-DNA. Amplifications at velocities ranging from 1 mm s(-1) to 20 mm s(-1) were investigated. The 500 bp fragment could be observed in a total reaction time of 1.7 min (5.2 s cycle(-1)) and the 997 bp fragment could be detected in 3.2 min (9.7 s cycle(-1)). The longer amplification time required for detection of the 997 bp fragment was due to the device being operated at its enzyme kinetic limit (i.e., Taq polymerase deoxynucleotide incorporation rate).     

Rapid evaluation of molecular integrals with ETOs

In this article, we discuss a way in which the theory of hyperspherical harmonics may be used for rapid evaluation of difficult molecular integrals when exponential‐type orbitals (ETOs) are used as a basis. One of us (J.E.A.) has implemented the method, and programs are available for general use. As a byproduct of this work, we are also able to evaluate generalized scattering factors for ETOs which allow first‐order density matrices to be measured experimentally using high‐quality X‐ray diffraction data. © 2015 Wiley Periodicals, Inc.     

Rapid liposome quality assessment using a lab-on-a-chip

Although liposomes have many outstanding features such as biocompatibility, biodegradability, low toxicity and structural diversity, and are successfully applied in many areas of chemistry and biotechnology, a lack of characterization standards and quality control tools are still inhibiting the translation of liposome technology into clinical routine. The greatest obstacle to clinical scale commercialization is the inability to ensure liposome formulation stability because small size variations or altered surface chemistries can significantly influence in vivo distribution and excretion kinetics that could in turn lead to unpredictable therapy outcomes. To enhance the product development process we have developed a microfluidic biochip containing embedded dielectric microsensors capable of providing quantitative results on formulation composition and stability based on the monitoring of the unique electric properties of liposomes. Computational fluid dynamic (CFD) simulations confirmed that microfluidics offer reproducible and well-defined measurement conditions where a moving liposome suspension within a microchannel behaves like a bulk material. Results of this study demonstrate the ability of microfluidics, in combination with dielectric spectroscopy and multivariate data analysis methods, to identify nine different liposomes. We also show that various liposome modifications such as membrane-bound surface proteins, lipid bilayer soluble drugs, as well as protein and dye encapsulations, can be detected in the absence of any labels or indicators. Since shelf-life stability of a liposome formulation is regarded of prime importance for regulatory approval and clinical application, we further provide a possible practical application of the developed liposome analysis platform as a high-throughput tool for industrial quality insurance purposes.     

Development and validation of a direct competitive monoclonal antibody-based immunoassay for the sensitive and selective analysis of the phytoregulator forchlorfenuron

Forchlorfenuron is a synthetic phytohormone with cytokinin-like activity used worldwide as a plant growth regulator to increase fruit size in a number of crops, mostly in kiwifruit and grape vines. A monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the determination of forchlorfenuron has been characterized and optimized. The selected immunoreagents afforded a highly selective assay with a limit of detection of 10 ng L(-1) in buffer. This direct competitive ELISA was validated in terms of trueness, precision, and robustness using both commercial juice and whole fruit samples. Recoveries from fortified kiwifruit juices and white and red musts were between 97 % and 131 %, with relative standard deviations below 16 %. When homogenized whole fruits were analysed after acetonitrile extraction, recoveries between 96 % and 113 % were found, with a limit of quantification of 5 μg kg(-1). The proposed immunoassay was validated by comparison with a reference chromatographic method using fruits from in-field treated grape and kiwifruit vines. Linear regression analysis of ELISA and HPLC-UV determinations showed an excellent correlation (r(2)=0.998), whereas analysis of the slope (0.99±0.01) and of the intercept (-1±3) clearly proved that the developed competitive immunoassay provided results that were statistically comparable to those obtained by the instrumental method for the analysis of forchlorfenuron in fruits at trace levels.