Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.     

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site‐Specific Conjugation

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site‐specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.     

Development of ELISA and immunochromatographic assay for ofloxacin

Two rapid,sensitive and reliable immunoassay methods,namely competitive indirect enzyme-linked immunosorbent assay(CI- ELISA)and colloidal gold-based immunochromatographic assay(CGIA),were developed to detect ofloxacin(OFL).The linear range of the CI-ELISAwas from 0.5 to 128 ng/mL with a limit of detection(LOD)of 0.35 ng/mL.Good recoveries were obtained in analyzing simulated swine urine samples.The CGIA could accurately estimate OFL at concentrations as low as 10 ng/mL in less than 10 min,and test results were read visually without any instrument.     

Approaches for the detection and analysis of antidrug antibodies to biopharmaceuticals: A review

Antibody-based therapeutic agents and other biopharmaceuticals are now used in the treatment of many diseases. However, when these biopharmaceuticals are administrated to patients, an immune reaction may occur that can reduce the drug's efficacy and lead to adverse side-effects. The immunogenicity of biopharmaceuticals can be evaluated by detecting and measuring antibodies that have been produced against these drugs, or antidrug antibodies. Methods for antidrug antibody detection and analysis can be important during the selection of a therapeutic approach based on such drugs and is crucial when developing and testing new biopharmaceuticals. This review examines approaches that have been used for antidrug antibody detection, measurement, and characterization. Many of these approaches are based on immunoassays and antigen binding tests, including homogeneous mobility shift assays. Other techniques that have been used for the analysis of antidrug antibodies are capillary electrophoresis, reporter gene assays, surface plasmon resonance spectroscopy, and liquid chromatography-mass spectrometry. The general principles of each approach will be discussed, along with their recent applications with regards to antidrug antibody analysis.     

Chemically Programmable and Switchable CAR-T Therapy

Although macromolecules on cell surfaces are predominantly targeted and drugged with antibodies, they harbor pockets that are only accessible to small molecules and constitutes a rich subset of binding sites with immense potential diagnostic and therapeutic utility. Compared to antibodies, however, small molecules are disadvantaged by a less confined biodistribution, shorter circulatory half-life, and inability to communicate with the immune system. Presented herein is a method that endows small molecules with the ability to recruit and activate chimeric antigen receptor T cells (CAR-Ts). It is based on a CAR-T platform that uses a chemically programmed antibody fragment (cp-Fab) as on/off switch. In proof-of-concept studies, this cp-Fab/CAR-T system targeting folate binding proteins on the cell surface mediated potent and specific eradication of folate-receptor-expressing cancer cells in vitro and in vivo.     

Expression of Catalytic Domain of Protein Tyrosine Phosphatase 1B and Preparation of Its Polyclonal Antibody

This study focuses on the expression of human protein tyrosine phosphatase 1B(PTP1B) catalytic domain (△PTP1B) and preparation of polyclonal antibody against △PTP1B. △PTP1B gene was PCR amplified with the cDNA of human PTP1B as the template, and cloned into the pT7 expression vector. The recombinant pT7-△PTP1B was expressed in E. coli Rosetta( DE3 ) host cells and purified. The antiserum was prepared by immunizing rabbit with purified recombinant △PTP1B. The polyclonal antibody against △PTP1B was purified by PVDF immobilized antigen affinity chromatography. △PTP1B was correctly cloned, expressed, and purified as confirmed by PCR, DNA sequence ratio) and 0. 1 ng, respectively. This study provides an important basis for further studying the biological function of PTP1B and its relationship with human diseases.     

Amperometric Immunosensor for the Detection of 2,4-Dichlorophenoxyacetic Acid (2,4-D) in Water

ABSTRACT

An amperometric immunosensor for the determination of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in water has been developed using sequential injection analysis techniques. The system is based on a rapid competitive enzyme immunoassay employing an alkaline phosphatase-labeled monoclonal antibody directed against the herbicide and an immunoreactor with 2,4-D immobilized via bovine serum albumin either to Eupergit in a column or directly to the surface of a glass capillary. The detection limit of the immunosensor at 0.1 μg 2,4-D/l without enrichment of the analyte makes automatic measurements of 2,4-D in drinking and ground water feasible.     

Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding

The membrane‐proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER‐derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution‐based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three‐helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three‐helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid‐bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.     

Effect of hapten structures on specific and sensitive enzyme-linked immunosorbent assays for N-methylcarbamate insecticide metolcarb

Five different haptens of the N-methylcarbamate insecticide metolcarb were designed and synthesized. All of the haptens were conjugated with ovalbumin (OVA) for the coating antigen, and one hapten containing all of the structure of metolcarb was conjugated with bovine serum albumin (BSA) for the immunogen. Two polyclonal antisera were raised against the BSA conjugate, and ten antibody/coating conjugate combinations were selected for studies of assay sensitivity and specificity for metolcarb. A class-specific combination was found, with the I₅₀ of the assay ranged from 0.64 to 20.98 μg mL⁻¹ for seven tested N-methylcarbamate insecticides except for pirimicarb. Considering titer, I₅₀ and cross-reactivity of all combinations of antibody/coating conjugate, a competitive indirect enzyme-linked immunosorbent assay (ELISA) in a homologous system, whose limit of detection (LoD) reached 1.4 ng mL⁻¹, was presented. The results of competitive ELISAs indicated that coating hapten structure can significantly affect not only assay sensitivity but also its specificity.     

Development of an Enzyme Immunoassay for the Determination of the Herbicide Metsulfuron-Methyl Based on Chicken Egg Yolk Antibodies

Abstract

The development of an indirect competitive enzyme immunoassay for the sulfonylurea herbicide metsulfuron-methyl (MSM) is described. In contrast to traditional antibody generation in mammals, this extremely sensitive method is based on chicken egg yolk antibodies (IgY). They were raised in laying hens using an MSM-derivative-BSA hapten as immunogen. With a 1:10000 dilution of the antibody solution and a coating antigen (MSM-derivative-KLH) concentration of 10 μg L^?1 the IC₅₀ value achieved for the target analyte was 0.4 μg L^?1. The least detectable dose was established at 13 ng L^?1. Cross-reactivity was tested with 5 structurally related compounds, where only sulfometuron showed a significant binding. The ELISA was tested with spiked tap and surface water samples. This paper, for the first time, demonstrates the production of high-affinity IgY antibodies for a herbicide compound.