Covalent Immobilization of Antibodies through Tetrazine-TCO Reaction to Improve Sensitivity of ELISA Technique

Enzyme-linked immunosorbent assay (ELISA) is routinely used to detect biomolecules related to several diseases facilitating diagnosis and monitoring of these, as well as the possibility of decreasing their mortality rate. Several methods have been carried out to improve the ELISA sensitivity through antibodies immobilization on the microtiter plates. Here, we have developed a strategy of antibodies immobilization to improve the ELISA sensitivity increasing the antibody density surface through the tetrazine (Tz)-trans-cyclooctene (TCO) reaction. For this, we prepared surfaces with tetrazine groups while the captured antibody was conjugated with TCO. The tetrazine surfaces were prepared in two different ways: (1) from aminated plates and (2) from Tz-BSA-coated plates. The surfaces were evaluated using two sandwich ELISA models, one of them using the low-affinity antibody anti-c-myc as a capture antibody to detect the c-myc-GST-IL8h recombinant protein, and the other one to detect the carcinoembryonic human protein (CEA). The sensitivity increased in both surfaces treated with tetrazine in comparison with the standard unmodified surface. The c-myc-GST-IL8h detection was around 10-fold more sensible on both tetrazine surfaces, while CEA ELISA detection increased 12-fold on surfaces coated with Tz-BSA. In conclusion, we show that it is possible to improve the ELISA sensitivity using this immobilization system, where capture antibodies bond covalently to surfaces.

Study on Factors Affecting the Performance of a CRISPR/Cas-Assisted New Immunoassay: Detection of Salivary Insulin as an Example

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas is now playing a significant role in biosensing applications, especially when the trans-cleavage activity of several Cas effectors is discovered. Taking advantages of both CRISPR/Cas and the enzyme-linked immunosorbent assay (ELISA) in analytical and clinical investigations, CRISPR/Cas-powered ELISA has been successfully designed to detect a spectrum of analytes beyond nucleic acid. Herein, we developed a CRISPR/Cas12a-assisted new immunoassay (CANi) for detection of salivary insulin as an example. Specifically, factors (antibody selection, temperature, and assay time) affecting the CRISPR/Cas-based ELISA system’s performance were investigated. It was observed that the concentration of blocking solution, selection of the capture antibody pairs, and the sequences of triggering ssDNA and guiding RNA affected this immunoassay sensitivity. In contrast, the preincubation of CRISPR/Cas12a working solution and pre-mixture of detection antibody with anti-IgG–ssDNA did not show influence on the performance of CANi for the detection of insulin. Under optimized conditions, the sensitivity for detection of salivary insulin was 10 fg/ml with a linear range from 10 fg/ml to 1 ng/ml.

A Novel Microfluidic Chip for Fast, Sensitive Quantification of Plasma Extracellular Vesicles as Biomarkers in Patients With Osteosarcoma

Plasma circulating extracellular vesicle (EV) has emerged as a promising biomarker for diagnosis and prognosis of various epithelial tumors. However, fast and efficient capture of EVs with microfluidic chip in sarcoma remains to be established. Herein, we reported a ZnO-nanorods integrated (ZNI) microfluidic chip, where EV capture antibody was uniformly grafted to the surface of the ZnO-nanorods of the chip to enhance the plasma turbulence formation and the capture efficiency at the micro-scale. Based on osteosarcoma (OS) cell line, we demonstrated that a combination of CD81 and CD63 antibody on ZNI chip yielded the greatest amount of total EVs, with an extra sensitive limit of detection (LOD) of ~104 particles mL-1. Furthermore, the addition of fluorescent labeling of Vimentin (VIM), a previously reported sarcoma cell surface biomarker, could enabled the dual visualization of total plasma EVs and VIM-positive EVs from OS patients’ plasma. Based on our ZNI chip, we found that the amount of plasma total EVs was significantly different between OS and healthy donors (1562 a.u. versus 639 a.u., p< 0.05), but not between metastatic and nonmetastatic OS (p> 0.05). Interestingly, patients with metastatic disease had a significantly greater amount of VIM-positive EVs (1411 a.u. versus 231 a.u.., p< 0.05) and increased VIM-positive/total EVs ratio (0.943 versus 0.211, p< 0.05) in comparison with the nonmetastatic counterpart. Therefore, our ZNI microfluidic chip has great potential for the fast quantification of plasma EVs, and the microfluidic-based quantification of total and VIM-positive EVs might serve as a promising biomarker for the diagnosis and surveillance in OS patients.

Generation and characterization of monoclonal antibodies against mature hepcidin and its application to neutralization and quantitative alteration assay

ABSTRACT Hepcidin regulates the quantity of ferroportin (FPN) on cellular membrane. In our cell assay expressing ferroportin labeled with green fluorescence, FPN was internalized and degraded only after treatment with hepcidin-25, not hepcidin-22 or hepcidin-20, leading to accumulation of cellular iron. Thus we generated murine monoclonal antibodies (mAbs) against hepcidin-25, and then characterized and validated their functions. Among them, several mAbs showed a neutralizing activity that may prevent ferroportin internalization induced by hepcidin-25. To measure hepcidin level in various fluids, mAbs specific for human and rat hepcidin-25 were selected. As for rat, a sandwich ELISA developed using clone rHN1 as capture antibody and biotinylated clone mHW1 as a detection reagent had high sensitivity, allowing for the detection of 1-100 ng/mL of hepcidin-25. Rat hepcidin-25 level in plasma was measured at an average concentration of 63.0 ng/mL in healthy condition, and at 218.2 ng/mL after stimulation of lipopolysaccharide.

Magnetic mesoporous CoFe2O4 labels reacted with TMB for use in a sandwiched photothermal immunoassay for thyroglobulin

An innovative photothermal immunoassay with a sandwich-type immunoreaction mode was designed for the sensitive screening of thyroglobulin on the capture antibody-coated microtiter plates by using a handheld digital thermometer as...

Microfluidic-Based Detection of AML-Specific Biomarkers Using the Example of Promyelocyte Leukemia

A microfluidic assay for the detection of promyelocytic leukemia (PML)-retinoic acid receptor α (RARα) fusion protein was developed. This microfluidic-based system can be used for rapid personalized differential diagnosis of acute promyelocyte leukemia (APL) with the aim of early initiation of individualized therapy. The fusion protein PML-RARα occurs in 95% of acute promyelocytic leukemia cases and is considered as diagnostically relevant. The fusion protein is formed as a result of translocation t(15,17) and is detected in the laboratory by fluorescence in situ hybridization (FISH) or reverse transcriptase polymerase chain reaction (RT-PCR). Diagnostic methods require many laboratory steps with specialized staff. The developed microfluidic assay includes a sandwich enzyme-linked immunosorbent assay (ELISA) system for PML-RARα on surface of magnetic microparticles in a microfluidic chip. A rapid detection of PML-RARα in cell lysates is achieved in less than one hour. A biotinylated PML-antibody on the surface of magnetic streptavidin coated microparticles is used as capture antibody. The bound translocation product is detected by a RARα antibody conjugated with horseradish peroxidase and the substrate QuantaRed. The analysis is performed in microfluidic channels which involves automated liquid processing with stringent washing and short incubation times. The results of the developed assay show that cell lysates of PML-RARα-positive cells (NB-4) can be clearly distinguished from PML-RARα-negative cells (HL-60, MV4-11).

Microcentrifuge Tubes as Disposable Immunoelectrochemical Cells for the On-Site Determination of GFAP, Biomarker of Hemorrhagic Stroke

Stroke is the leading cause of mortality worldwide. Differentiating patients with intracerebral hemorrhage (ICH) or ischemic stroke in the first hours of symptoms onset is of paramount importance to the optimal management of patients. Current diagnosis of acute stroke relies on neuroimaging techniques that provide valuable information but not always are readily available. In this context, the development of analytical tools capable of a rapid and on-site differentiation between the types of stroke is an important challenge with great socio-economic benefits. Glial fibrillary acidic protein (GFAP) is considered one of the ICH biomarkers in patients with symptoms of acute stroke. In this work, a simple electroanalytical device for the analysis of GFAP was developed combining stainless-steel pins and a microcentrifuge tube. The sandwich immunoassay for the determination of GFAP was carried out inside the microcentrifuge tube immobilizing the capture antibody on the bottom of the tube. The three stainless-steel pins acting as electrodes were inserted in the cap in such a way that, when the immunoassay is finished, the tube is turned bottom up allowing the electrochemical detection in the same tube.

Development of a specific immunoassay to selectively measure active tryptase in airway samples

Aim: Tryptase is a tetrameric trypsin-like serine protease contained within the secretory granules of mast cells and is an important mediator of allergic inflammatory responses in respiratory diseases. Detection of active tryptase in the airway may provide important information about asthma and other respiratory diseases. Materials & Methods: An activity based probe has been incorported within an immunoassay to allow for measurement of active tryptase in human tissues. Results: A specific Simoa immunoassay to measure active tryptase in nasosorption samples was developed and qualified using an activity-based probe label and a specific antitryptase capture antibody. Conclusion: The assay was capable of measuring active tryptase in human samples, which will enable evaluation of the role of tryptase proteolytic activity in human disease.