Determination of Immunoglobulin G by a Hemin–Manganese(IV) Oxide-Labeled Enzyme-linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) is reported for human immunoglobulin G based on synthesized hemin–MnO₂ nanocomposite as the label. Enhanced sensitivity was obtained due to the increased catalytic activity of the hemin–MnO₂ nanocomposite toward 3,3′,5,5′-tetramethylbenzidine compared to hemin and MnO₂ alone. The synthesized hemin–MnO₂ nanocomposite was characterized by transmission electron microscopy and its catalytic activity to 3,3′,5,5′-tetramethylbenzidine was investigated by ultraviolet–visible absorption spectroscopy. After assembly of the sandwich-type immunoassay in the 96 wells of the plate the hemin–MnO₂-based label catalyzed 3,3′,5,5′-tetramethylbenzidine into blue compounds that were monitored by a plate reader. The absorbance increased with the concentration of human immunoglobulin G. The immunoassay displayed high sensitivity, a long linear dynamic range, and good selectivity for human immunoglobulin G. The immunoassay was also used for the determination of human immunoglobulin G in serum with favorable results. The developed assay combines the high throughput and low cost of ELISA with the simplicity of nanocomposite labeling and is suitable for application in clinical diagnosis.     

Fluorescence‐based lateral flow assays for rapid oral fluid roadside detection of cannabis use

With the recent worldwide changes in the legalization of marijuana, there is a significant need for rapid, roadside screening test for driving under the influence of drugs. A robust, sensitive, lateral flow assay has been developed to detect recent use via oral‐fluid testing for Δ⁹‐tetrahydrocannabinol (THC). This proof‐of‐concept assay uses a fluorescent‐based immunoassay detection of polymeric beads, conjugated to antibodies against native THC. The fluorescent technique allows for significantly lower limits of detection and higher precision determination of recent marijuana use without the use of urine or blood sampling—thus allowing for roadside identification. Detection levels of 0.01 ng/mL were distinguished from background and the lower limit of quantification was determined to approach 1 ng/mL.     

Establishment and validation of a microfluidic capillary gel electrophoresis platform method for purity analysis of therapeutic monoclonal antibodies

Capillary and microfluidic chip electrophoresis technologies are heavily utilized for development, characterization, release, and stability testing of biopharmaceuticals. Within the biopharmaceutical industry, CE‐SDS and M‐CGE are commonly used for purity determination by separation and quantitation of size‐based variants. M‐CGE is used primarily as an R&D tool for product and process development, while cGMP release and stability testing applications are commonly reserved for CE‐SDS. This paper describes the establishment of an M‐CGE platform method to be used for R&D and cGMP applications, including release and stability testing, for monoclonal antibodies. The M‐CGE platform method enables testing for product development support and cGMP release and stability using the same method, and utilization of one CE technology for the entire lifecycle of a biopharmaceutical product. Critical method parameters were identified, and the analytical design space of those critical parameters was defined using design of experiments (DOE) studies. Once defined through DOE studies, the method design space was validated according to ICH Q2 (R1) guidelines. Additional molecules of the same validated class were verified for use in the method by experimental confirmation of accuracy, specificity, and stability indicating capabilities. The platform method model facilitates rapid utilization of the method in development and GMP testing environments, and eliminates the need for individual validations for assets of the same class entering early stage development.     

Cross‐Reactive Sensor Arrays for the Detection of Peptides in Aqueous Solution by Fluorescence Spectroscopy

A simple but powerful method for the sensing of peptides in aqueous solution has been developed. The transition‐metal complexes [PdCl₂(en)], [{RhCl₂Cp*}₂], and [{RuCl₂(p‐cymene)}₂] were combined with six different fluorescent dyes to build a cross‐reactive sensor array. The fluorescence response of the individual sensor units was based on competitive complexation reactions between the peptide analytes and the fluorescent dyes. The collective response of the sensor array in a time‐resolved fashion was used as an input for multivariate analyses. A sensor array comprised of only six metal–dye combinations was able to differentiate ten different dipeptides in buffered aqueous solution at a concentration of 50 μM . Furthermore, the cross‐reactive sensor could be used to obtain information about the identity and the quantity of the pharmacologically interesting dipeptides carnosine and homocarnosine in a complex biological matrix, such as deproteinized human blood serum. The sensor array was also able to sense longer peptides, which was demonstrated by differentiating mixtures of the nonapeptide bradykinin and the decapeptide kallidin.     

Analysis of microcystins by capillary zone electrophoresis coupling with electrospray ionization mass spectrometry

In this paper, a rapid and effective method based on capillary zone electrophoresis (CZE) coupled with electrospray ionization mass spectrometry (ESI-MS) was established for the trace analysis of microcystin (MC) isomers in crude algae sample. The experimental conditions including the composition, acidity and concentration of buffer, separation voltage, injection time, and MS detection parameters were investigated in detail. A capillary separation system was as follows: a uncoated fused-silica capillary tube (50 μm i.d. × 90 cm), 40 mmol L⁻¹ ammonium acetate solution (pH 9.86) as running buffer, 25 kV as separation voltage, 20 kV × 3 s water first and 20 kV × 20 s for sample injection. Mass analysis was performed in ESI source, with sheath gas temperature 150 °C, sheath gas pressure 10 psi, and sheath gas flow 6 L min⁻¹. And sheath liquid was 7.5 mmol L⁻¹ acetic acid in 50% isopropanol-water (3 μL min⁻¹). Protonation and ammonium adduct molecular ions m/z 506.9 (MC-LR) and 532.0 (MC-YR) were used for the quantification of MCs. Under these conditions, two MCs were baseline separated within 9 min, the calibration curves were obtained in the range of 0.11-10.0 μg mL⁻¹ and 0.16-10.5 μg mL⁻¹ for MC-LR and MC-YR, respectively. Meanwhile, limits of detection were 0.05 and 0.08 μg mL⁻¹ for MC-LR and MC-YR, respectively. The recoveries for the two MCs were in the range of 95.8-108%. The developed approach had been successfully applied to the analysis of MCs in crude algae samples.     

Microtiterplate phosphate assay based on luminescence quenching of a terbium complex amenable to decay time detection

We describe a terbium-ligand complex (TbL) for a microtiterplate assay for phosphate (P) in the 0.3-100 μmol L⁻¹ range based on luminescence quenching. As the pH optimum is at neutral pH (7.4) the probe is quenched by both, primary (H₂PO₄⁻) and secondary phosphate (HPO₄²⁻). The LOD is 110 nmol L⁻¹. A Stern-Volmer study revealed that quenching is mostly static. Due to the ms-decay time of TbL, the first luminescence lifetime assay for phosphate could also be developed. The lifetime-based calibration plot is linear between 0.5 and 5 μmol L⁻¹ of P. The effect of various surfactants on assay performance and a study on interferents are presented. The probe was successfully applied to determination of P in commercial plant fertilizers and validated against the molybdenum blue test. The probe is the most sensitive lanthanide-based probe for phosphate.     

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed.Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL⁻¹, the calibration curve was linear from 5.0 to 40 ng mL⁻¹ (R² = 0.952) with an IC₅₀ value of 18.2 ng mL⁻¹. In the extracts of 20 Chinese traditional drugs, the detection capability (CCβ) of vardenafil was 0.08 mg g⁻¹, the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue.The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.     

Orientation Control of Photo‐Immobilized Antibodies on the Surface of Azobenzene‐Containing Polymers by the Introduction of Functional Groups

In our photo‐induced immobilization technique for an antibody (IgG) using azopolymers, the introduction of COOH and NMe₂ into the azopolymers, which can introduce surface charges, strongly affected the immobilization properties such as the efficiency of immobilization and the activity of the immobilized IgG (i.e., the orientation of the immobilized IgG). The introduction of COOH promoted a more active orientation of the immobilized IgG. The orientation was determined during the adsorption process onto the azopolymer surface in solution before photo‐immobilization, and was maintained during the photo‐immobilization. The surface charge of the azopolymer appears to be an important factor for IgG orientation, which involves electrostatic interactions between its Fab and the azopolymer surface.

     

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.     

Kinetics of comet formation in single‐cell gel electrophoresis: Loops and fragments

We investigated the mechanisms of DNA exit during single‐cell gel electrophoresis (the comet assay) by measuring the kinetics of the comet tail formation. In the neutral comet assay, the rate of DNA exit was found to be dependent on the topological state of DNA, which was influenced by either ethidium bromide or a low radiation dose. The results clearly show that the comet tail is formed by extended DNA loops: the loop extension, being reversible when the DNA torsional constraint remains in the loops, is favored when the constraint is relaxed. The kinetics of the comet formation in the case of a high radiation dose points out that accumulation of the single‐strand breaks causes DNA fragmentation. In contrast to the neutral comet assay, the alkaline comet assay is not related to the chromatin loops. Our results imply that the alkaline treatment induces detachment of the loops from the nuclear matrix, and the comet tail is formed by ssDNA fragments, the ends of which are pulled out from the comet head by electric force. We suggest that the kinetic approach can be considered as an important improvement of the comet assay.