Capture Of Molecules Research Articles

Droplet-Based Immunosensor for Simultaneous Immunoassays of Multiplex Histidine-Tagged Proteins.

This paper presents a droplet-based immunoassay chip allowing each droplet to be positioned in a passive droplet-positioning cavern under continuous flow. In addition, the chip surface can immobilize any kind of histidine-tagged capture agents for performing simultaneous multiplex immunoassays. Distinct families of monodispersed droplets were generated since a diaphragm, which is a thin elastomeric flap film suspended from the top of the main channel, forms a double T junction for shearing the aqueous liquids by the carrier flow. These two types of monodispersed droplets traverse the main channel to the downstream detection area and enter the passive positioning caverns for further immunoassay. A layer of Ni-Co film was coated on the substrate by electrodeposition in order to immobilize the multiplex histidine-tagged capture molecules. In this study, the tumor suppressor protein p53 and the extracellular signal-related kinase 1 (ERK1) were used as the capture agents. Then, both histidine-tagged proteins p53 and ERK1 were immobilized by the Ni-Co layer in a microarray format for subsequent immunoassay and fluorescence detection. The experimental results show that the detected fluorescence intensity is proportioned to the concentration of the encapsulated content in a small droplet. This proposed droplet-based immunoassay chip can immobilize multiplex histidine-tagged proteins, irrelevant to the species of proteins, to carry out simultaneous immunoassays and allow the operation sequence to be conducted automatically through the manipulation of droplets.

Aptamer-based SERS biosensor for whole cell analytical detection of E. coli O157:H7

Infectious outbreaks caused by foodborne pathogens such as E. coli O157:H7 are still imposing a heavy burden for global food safety, causing acute illnesses and significant industrial impact worldwide. Despite the growth of biosensors as a research field, continuous innovation on detection strategies, novel materials and enhanced limits of detection, most of the platforms developed at the laboratory scale never will get to meet the market.The use of aptamers as capture biomolecules has been proposed as a promising alternative to overcome the harsh environmental conditions of industrial manufacturing processes, and to enhance the performance under real, complex, conditions. In this work, we present the feasibility of using aptameric DNA sequences, covalently conjugated to 4-aminothiophenol-gold nanoparticle complexes for the sensitive and highly specific detection of E. coli O157:H7 via surface enhanced Raman spectroscopy (SERS) analysis. Low concentrations of E. coli O157:H7 were detected and quantified within 20 min in both pure culture (∼101 CFU mL−1) and ground beef samples (∼102 CFU mL−1). The SERS intensity response showed a strong negative linear correlation (r2 = 0.995) with increasing concentrations of E. coli O157:H7 (ranging from 102 to 106 CFU mL−1). High specificity was achieved at genus (L. monocytogenes, S. aureus S. typhimurium) species (E. coli B1201) and serotype (E. coli O55:H7) level, demonstrating with 95% of confidence that the interferent microorganisms tested generated a Raman signal response not significantly different from the background (p = 0.786).This work evaluates the incorporation of aptameric DNA sequences as bio capture molecules exclusively. The successful performance presented using non-modified citrate reduced GNPs, is promising for potential low-cost, high-throughput applications. The findings might be applied simultaneously to the detection of a wide variety of foodborne pathogens in a multiplexed fashion employing unique Raman probes and strain-specific aptamer sequences.

Shedding Light into the Subcutis: A Mass Spectrometry Based Immunocapture Assay Enabling Full Characterization of Therapeutic Antibodies after Injection in Vivo.

An understanding of what happens to therapeutic antibodies in vivo after subcutaneous injection is of high interest. Therefore, we applied the open flow microperfusion technique to extract interstitial fluid from the subcutaneous tissue. In order to analyze those biological samples, a specific and sensitive workflow was required. In this study, we present a complete workflow that enables full characterization of therapeutic antibodies after subcutaneous injection. Compared to classical pharmacokinetic approaches where only a limited number of peptides are detected, our workflow provides full sequence coverage and even enables the identification of potential quality attributes. The efficiency to purify therapeutic antibodies from biological matrixes of two different antibody capture molecules and two types of magnetic beads was compared. Furthermore, several desalting protocols were tested in the development of a miniaturized peptide map procedure. The best results were achieved using a commercial anti-human capture mAb fragment in combination with streptavidin coated magnetic beads, providing capture efficiencies of 90-100%. The optimized peptide map protocol that requires <1 μg of mAb includes two desalting steps and showed sequence coverages of 95-100%. The final method was successfully used for analysis of interstitial fluid and serum samples after a subcutaneous injection of a therapeutic antibody into a domestic pig.

Preparation of DNA-functionalized surfaces for simultaneous homeotropic orientation of liquid crystals and optical recognition of analytes: application to the determination of progesterone.

The work describes a simplified method for the preparation of liquid crystal (LC) bioassay using DNA-based capture molecules and having lower detection limits. The capture DNA probes of the stem-loop structure were immobilized on the surface of a glass slide. A homeotropic orientation of LC molecules can be obtained with the proper surface coverage of capture DNA probes. In the presence of analytes (specifically shown here for the progesterone as a model analyte), the molecular binding between capture DNA probes and progesterone opens the loop of the capture DNA probes. The opened sequence is then amenable to hybridization with a reporter DNA probe that is immobilized on gold nanoparticles. This changes the surface microstructure, disrupts the orientation of LC molecules, and results in an enhanced optical response, expressed as the average grey value of the images. This new kind of surface treatment for simultaneous recognition of target molecules and homeotropic anchoring of LCs reduces the number of preparation steps and makes the process of LC bioassay easier. This method has a detection limit as low as 0.1pmol·L-1 of progesterone. Graphical abstract Schematic presentation of the liquid crystal-based DNAassay. DMOAP: Dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride; TEA: Triethoxsilylbutyraldehyde; 5CB: 4-cyano-4'-pentylbiphenyl; P4: progesterone.

Robust Smartphone Assisted Biosensing Based on Asymmetric Nanofluidic Grating Interferometry.

Point-of-care systems enable fast therapy decisions on site without the need of any healthcare infrastructure. In addition to the sensitive detection, stable measurement by inexperienced persons outside of laboratory facilities is indispensable. A particular challenge in field applications is to reduce interference from environmental factors, such as temperature, to acceptable levels without sacrificing simplicity. Here, we present a smartphone-based point-of-care sensor. The method uses an optofluidic grating composed of alternating detection and reference channels arranged as a reflective phase grating. Biomolecules adsorbing to the detection channel alter the optical path length, while the parallel reference channels enable a direct common mode rejection within a single measurement. The optical setup is integrated in a compact design of a mobile readout device and the usability is ensured by a smartphone application. Our results show that different ambient temperatures do not have any influence on the signal. In a proof-of concept experiment we measured the accumulation of specific molecules in functionalized detection channels in real-time and without the need of any labeling. Therefore, the channel walls have been modified with biotin as capture molecules and the specific binding of streptavidin was detected. A mobile, reliable and robust point-of-care device has been realized by combining an inherently differential measurement concept with a smartphone-based, mobile readout device.

Immunoglobulin-like Domain of HsFcμR as a Capture Molecule for Detection of Crimean-Congo Hemorrhagic Fever Virus- and Zika Virus-Specific IgM Antibodies.

The cellular surface molecule HsTOSO/FAIM3/HsFcμR has been identified as an IgM-specific Fc receptor expressed on lymphocytes. Here, we show that its extracellular immunoglobulin-like domain (HsFcμR-Igl) specifically binds to IgM/antigen immune complexes (ICs) and exploit this property for the development of novel detection systems for IgM antibodies directed against Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV). His-tagged HsFcμR-Igl was expressed in Escherichia coli and purified by affinity chromatography, oxidative refolding, and size-exclusion chromatography. Specific binding of HsFcμR-Igl to IgM/antigen ICs was confirmed, and 2 prototypic ELISAs for the detection of anti-CCHFV and anti-ZIKV IgM antibodies were developed. Thereby, patient sera and virus-specific recombinant antigens directly labeled with horseradish peroxidase (HRP) were coincubated on HsFcμR-Igl-coated ELISA plates. Bound ICs were quantified by measuring turnover of a chromogenic HRP substrate. Assay validation was performed using paired serum samples from 15 Kosovar patients with a PCR-confirmed CCHFV infection and 28 Brazilian patients with a PCR-confirmed ZIKV infection, along with a panel of a priori CCHFV/ZIKV-IgM-negative serum samples. Both ELISAs were highly reproducible. Sensitivity and specificity were comparable with or even exceeded in-house gold standard testing and commercial kits. Furthermore, latex beads coated with HsFcμR-Igl aggregated upon coincubation with an IgM-positive serum and HRP-labeled antigen but not with either component alone, revealing a potential for use of HsFcμR-Igl as a capture molecule in aggregation-based rapid tests. Recombinant HsFcμR-Igl is a versatile capture molecule for IgM/antigen ICs of human and animal origin and can be applied for the development of both plate- and bead-based serological tests.

Aptamer as capture agent in enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of Aflatoxin B1

Aflatoxin B1 (AFB1), is one of the most toxic mycotoxins found to contaminate various food commodities like cereals, dried fruits, tree nuts, spices and crude vegetable oils. In spite of considerable progress in analytical techniques, there is still a need to develop rapid and highly sensitive detection platforms for AFB1. In this study, AFB1 specific aptamer was used as a capture molecule to develop an enzyme-linked apta-sorbent assay (ELASA) for ultrasensitive detection of AFB1. Under optimized conditions, the assay had a linear detection range from 1 μg to 1 pg with a limit of detection (LOD) of 1 pg/mL in buffer. Conventional ELISA with AFB1 hapten as the capture agent (LOD = 10 pg/mL) was also carried out to compare the results with the present method. Recovery studies in food samples like dried red chillies, groundnut and pepper using both the methods was found to be in the range of 88.49–106.4% at 10 ng/mL and 87.4% to 95.8% at 5 ng/mL for ELASA and 76.56–127.68% at 10 ng/mL and 82–101.2% at 5 ng/mL for ELISA. Higher detection (10 fold) and better recovery using ELASA suggest that the method could offer an early, ultrasensitive, high-throughput, qualitative and semi-quantitative detection of AFB1 in contaminated food samples.

The effects of antigen size, binding site valency, and flexibility on fab-antigen binding near solid surfaces.

Next generation antibody microarray devices have the potential to outperform current molecular detection methods and realize new applications in medicine, scientific research, and national defense. However, antibody microarrays, or arrays of antibody fragments ("fabs"), continue to evade mainstream use in part due to persistent reliability problems despite improvements to substrate design and protein immobilization strategies. Other factors could be disrupting microarray performance, including effects resulting from antigen characteristics. Target molecules embody a wide range of sizes, shapes, number of epitopes, epitope accessibility, and other physical and chemical properties. As a result, it may not be ideal for microarray designs to utilize the same substrate or immobilization strategy for all of the capture molecules. This study investigates how three antigen properties, such as size, binding site valency, and molecular flexibility, affect fab binding. The work uses an advanced, experimentally validated, coarse-grain model and umbrella sampling to calculate the free energy of ligand binding and how this energy landscape is different on the surface compared to in the bulk. The results confirm that large antigens interact differently with immobilized fabs compared to smaller antigens. Analysis of the results shows that despite these differences, tethering fabs in an upright orientation on hydrophilic surfaces is the best configuration for antibody microarrays.

Reduced graphene-oxide transducers for biosensing applications beyond the Debye-screening limit

In the field of label-free biosensing, various transducer materials and strategies are under investigation to overcome the Debye-screening limitation of charged biomolecules. We demonstrate an in-line, impedimetric aptasensor with reduced graphene-oxide (rGO) thin films as transducers to detect prostate specific antigens (PSA) in a physiological buffer solution. Unlike classical electrochemical impedance spectroscopy (EIS), this direct, label-free and fully-electronic biosensor approach does not need any redox markers. As specific capture molecules, short anti-PSA aptamers ensured a close binding of the target molecules to the transducer surfaces. Results showed a limit of detection smaller than 33 pM of PSA and a wide detection range from 0.033 to 330 nM fully covering the clinically relevant range of PSA (0.115–0.290 nM). This promising performance can be attributed to the bipolar electronic transport characteristics of the ultra-thin rGO layers similar to pristine graphene. The attachment of target biomolecules to the films changes the resistance of the rGO thin films. Such an in-line EIS configuration with rGO thin films opens promising prospects for biosensing beyond the Debye-screening limitation, which is a major challenge for conventional semiconductor field-effect devices towards clinical applications.

Magnetic force assisted electrochemical sensor for the detection of thrombin with aptamer-antibody sandwich formation

A magnetic force assisted electrochemical aptamer-antibody sandwich assay (MESA) was developed for the detection of thrombin as a model protein in serum samples. The MESA using the formation of sandwich complexes on the electrochemical sensor probe for reaction and the removal of unbound bioconjugates from the sensor surface without washing are controlled by a magnetic field. Thrombin was determined by the cathodic currents of a toluidine blue O (TBO) attached with thrombin antibody modified magnetic nanoparticle (MNP) at the sensor surface. To detect thrombin in a serum sample, we applied a thrombin-specific aptamer as the capture molecule bound to the functionalized conducting polymer layer (poly-(2,2´:5´,5″-terthiophene-3´-p-benzoic acid) (pTBA)), and streptavidin and starch coated-MNP was conjugated with biotinylated thrombin antibodies (Ab) and TBO as the bioconjugate (MNP@Ab-TBO). The characterization of MNP@Ab-TBO and sensor probe was performed using voltammetry, impedance spectroscopy, XPS, and UV–VIS spectroscopy. The experimental conditions were optimized in terms of pH, binding time, removal time of unbound bioconjugates, and applied potential. The dynamic ranges of thrombin were from 1.0 to 500 nM with detection limit of 0.49 ( ± 0.06) nM. The recovery test demonstrates the reliability of the proposed sensing system for a handheld device.

Current Conjugation Methods for Immunosensors.

Recent advances in the development of immunosensors using polymeric nanomaterials and nanoparticles have enabled a wide range of new functions and applications in diagnostic and prognostic research. One fundamental challenge that all immunosensors must overcome is to provide the specificity of target molecular recognition by immobilizing antibodies, antibody fragments, and/or other peptides or oligonucleotide molecules that are capable of antigen recognition on a compact device surface. This review presents progress in the application of immobilization strategies including the classical adsorption process, affinity attachment, random cross-linking and specific covalent linking. The choice of immobilization methods and its impact on biosensor performance in terms of capture molecule loading, orientation, stability and capture efficiency are also discussed in this review.

Applications of a novel biodetection system to saliva using protein fingerprints with data processing

A fundamental method has been developed focusing on a facile and rapid examination of periodontal disease. Periodontal disease is an oral disease thought to affect 80% of adults, and early detection with treatment is desirable for the improvement of the quality of life. Unfortunately conventional methods are not consistent as the disease is caused by a number of undefined bacteria and detection relies on the skills of the dentist. Thus an objective detection system is required. We have performed an experiment on saliva using a novel biodetection system, designated PepTenChip®. A disease model for saliva was prepared using a specimen from a healthy subject and a mixture of hemoglobin (f-Hb) and lactate dehydrogenase (LDH), which is used as a periodontal disease marker protein with healthy saliva. PepTenChip® is a peptide microarray in which fluorescent labelled structured peptides are immobilized on a novel amorphous carbon substrate. Since the peptides used as capture molecules are fluorescently labelled, labeling of analytes is not necessary. The fluorescence intensity change before and after application of analytes are detected rather than the ON/OFF detection common to conventional microarrays using a set of antigen–antibody. The fluorescence intensity value changes according to the concentration of captured protein allowing the generation of protein fingerprint (PFP) and dendrograms. The present method does not rely on a “one to one” interaction, unlike conventional biodetection, and advantages can be envisaged in the case of an undefined or unknown cause of disease. The statistical analyses, such as multivariate analyses, allow classification of the type of proteins added in saliva as mimetics of disease. PepTenChip® system is useful and convenient for examination of periodontal disease in health care.

Threshold-Based Quantification in a Multiline Lateral Flow Assay via Computationally Designed Capture Efficiency

Lateral flow assays (LFAs) are widely used for yes/no detection of analytes, but they are not well-suited for quantification. We show that the sensitivity of the test line in a lateral flow assay can be tuned to appear at a specific sample concentration by varying the density of capture molecules at the test line and that when test lines tuned for different responses are combined into a single test strip, lines appear at specific thresholds of sample concentration. We also developed a model based on mass-action kinetics that accurately described test line signal and shape over a wide matrix of capture molecules and sample concentrations in single-line strips. The model was used to design a three-line test strip with lines designed to appear at logarithmically spaced sample concentrations, and the experiments showed a remarkable match to predictions. The response of this "graded ladder bar" format is due to the effect of test line concentration on capture efficiency at each test line, not on sample depletion effects, and the effect is maintained whether a system is under kinetic or equilibrium control. These features enable design of nonlinear responses (logarithmic here) and suggest robustness for different systems. Thus, the graded ladder bar format could be a useful tool for applications requiring quantification of sample concentrations over a wide dynamic range.

Protein‐Glycan Interactions: Binding of Different Norovirus VLPs to a Panel of Histo‐Blood Group Antigens

Human noroviruses, highly contagious viruses are the major cause of most epidemic outbreaks of gastroenteritis.1 Our long-term goals are to develop point-of-care diagnostics for noroviruses; these are unavailable at the present time. PCR tests require trained personnel, equipment and are expensive. Antibody ELISAs tests are not very sensitive and are cross-reactive. There is a need to develop inexpensive, user-friendly rapid diagnostics for noroviruses. Noroviruses consist of five genogroups; genogroup GI, GII and GIV infect humans. The virion is composed of 90 dimers of a capsid protein VP1, which binds to human Histo-Blood Group Antigens (HBGAs).1 Therefore, we can detect noroviruses using HBGAs as capture molecules. However, the molecular level binding of noroviruses with HBGAs is not very well understood. As a first step towards our long-term goals of developing point-of-care diagnostics, we studied the interactions of VLPs (virus-like particles) with synthetic HBGAs. We used VLPs because noroviruses are difficult to grow in cell culture.2 Therefore, the protein was expressed in baculovirus, which spontaneously forms VLPs. 3 HBGAs are glycans that contain multiple carbohydrate moieties.4 In the ABH antigen system, there are four major types I–IV. We synthesized a panel of biotinylated synthetic ABH type I–IV glycans using chemical methods. The biotinylated glycans were attached to streptavidin coated sensor chips. These chips were used to study the binding affinities of VLPs from different genogroups using surface plasmon resonance (SPR). We used SPR because this is a highly sensitive label free system. Therefore, we do not have to label the VLPs; labeling VLPs may perturb the binding event. We found that VLPs from different genogroups bound with varying binding affinities to the synthetic glycans, suggesting that a library of glycans could be used in microarray format to yield a “fingerprint pattern of recognition” for each strain. The binding studies and the analysis of the biophysical studies will be the focus of this presentation. Support or Funding Information NIH (R01-AI089450) A panel of biotinylated bivalent HBGAs that have been reported as binding ligands for human noroviruses were synthesized using chemical methods. SPR studies show that VLPs from different genogroups bound with varying binding affinities to the synthetic glycans. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exosome-Modified Tissue Engineered Blood Vessel for Endothelial Progenitor Cell Capture and Targeted siRNA Delivery.

Instability and poor targeting causes the long-term patency of RNA-modified tissue engineering blood vessels (TEBVs) remaining unsatisfactory. RNA can be enriched in exosome and then delivered into targeted cells while whether exosome-modified TEBVs achieve RNA targeted delivery is unclear. Here, to promote the expression of klotho protein on the mesenchymal stem cell (MSC)-derived exosomes, klotho plasmids are first transfected into MSCs, and adenosine kinase (ADK) siRNA is then loaded into exosome (klotho/ADK siRNA-exosome) using electrotransfection. Flow chamber results show that klotho/ADK siRNA-exosome can effectively capture circulating endothelial progenitor cells (EPCs). Besides, the captured EPCs can endocytose this exosome, and then decompose it into klotho protein and ADK siRNA. Moreover, ADK siRNA promotes the paracrine of proangiogenic factors and adenosine from EPCs, which further facilitate proliferation and migration of endothelial cells. Based on polyethyleneimine-capped gold nanoparticles, exosome-modified TEBVs are constructed through layer-by-layer assembly. Animal experimental results show that klotho/ADK siRNA-exosome-modified TEBVs can maintain the patency up to one month, and good endothelialization is observed. In short, one exosome-modified TEBV is constructed, capture molecules on the surface of exosome capture the circulating EPCs, and the loaded RNA achieves its purpose of accurate treatment depending on the needs of patients.

Rationalizing and advancing the 3-MPBA SERS sandwich assay for rapid detection of bacteria in environmental and food matrices

Bacterial foodborne illness continues to be a pressing issue in our food supply. Rapid detection methods are needed for perishable foods due to their short shelf lives and significant contribution to foodborne illness. Previously, a sensitive and reliable surface-enhanced Raman spectroscopy (SERS) sandwich assay based on 3-mercaptophenylboronic acid (3-MBPA) as a capturer and indicator molecule was developed for rapid bacteria detection. In this study, we explored the advantages and constraints of this assay over the conventional aerobic plate count (APC) method and further developed methods for detection in real environmental and food matrices. The SERS sandwich assay was able to detect environmental bacteria in pond water and on spinach leaves at higher levels than the APC method. In addition, the SERS assay appeared to have higher sensitivity to quantify bacteria in the stationary phase. On the other hand, the APC method was more sensitive to cell viability. Finally, a method to detect bacteria in a challenging high-sugar juice matrix was developed to enhance bacteria capture. This study advanced the SERS technique for real applications in environment and food matrices.

Enhanced Quality Factor Label-free Biosensing with Micro-Cantilevers Integrated into Microfluidic Systems.

Microelectromechanical systems (MEMS) have enabled the development of a new generation of sensor platforms. Acoustic sensor operation in liquid, the native environment of biomolecules, causes, however, significant degradation of sensing performance due to viscous drag and relies on the availability of capture molecules to bind analytes of interest to the sensor surface. Here, we describe a strategy to interface MEMS sensors with microfluidic platforms through an aerosol spray. Our sensing platform comprises a microfluidic spray nozzle and a microcantilever array operated in dynamic mode within a closed loop oscillator. A solution containing the analyte is sprayed uniformly through picoliter droplets onto the microcantilever surface; the micrometer-scale drops evaporate rapidly and leave the solutes behind, adding to the mass of the cantilever. This sensing scheme results in a 50-fold increase in the quality factor compared to operation in liquid, yet allows the analytes to be introduced into the sensing system from a solution phase. It achieves a 370 femtogram limit of detection, and we demonstrate quantitative label-free analysis of inorganic salts and model proteins. These results demonstrate that the standard resolution limits of cantilever sensing in dynamic mode can be overcome with the integration of spray microfluidics with MEMS.

Dual Amplification Fluorescence Assay for Alpha Fetal Protein Utilizing Immunohybridization Chain Reaction and Metal-Enhanced Fluorescence of Carbon Nanodots.

As an emerging fascinating fluorescent nanomaterial, carbon nanodots (CDs) have attracted much attention owing of their unique properties such as small size, antiphotobleaching, and biocompatibility. However, its use in biomedical analysis is limited because of its low quantum yield. Herein, we constructed a dual amplification fluorescence sensor by incorporating immunohybridization chain reaction (immuno-HCR) and metal-enhanced fluorescence (MEF) of CDs for the detection of alpha fetal protein (AFP). The immunoplasmonic slide and detection antibodies-conjugated oligonucleotide initiator are served to capture and probe AFP molecules, respectively. Then, CD-tagged hairpin nucleic acids were introduced to trigger the HCR, in which the hairpin nucleic acid and oligonucleotide initiator are complementary. The interaction between CDs and the gold nanoisland film greatly improves the radiative decay rate, increases the quantum yield, and enhances the fluorescence emission of the CDs. Furthermore, the HCR provides secondary amplification of fluorescence intensity. Therefore, the MEF-capable immunohybridization reactions provide obvious advantages and result in exceptional sensitivity. In addition, the sandwich immunoassay method offers high specificity. The results show a wide linearity between the fluorescence intensity and AFP concentration over 5 orders of magnitude (0.0005-5 ng/mL), and the detection limit reaches as low as 94.3 fg/mL. This method offers advantages of high sensitivity and reliability, wide detection range, and versatile plasmonic chips, thus presenting an alternative for the technologies in biomedical analysis and clinical applications.

Development of a colorimetric nucleic acid-based lateral flow assay with non-biotinylated capture DNA

In recent years, many nucleic acid-based lateral flow assays (NALFAs) have been developed for rapid and simple detection of various analytes including DNA sequences. In a NALFA, target molecules are applied within a small volume of a rehydrating buffer. The analyte flows laterally to reach the capture molecules at where it forms a colorimetric signal. Usually, in NALFAs, capture molecules are modified for maximized adsorption on the surface. In most cases, the modification is a biotin. The biotinylated capture DNA is held at capture line by interaction with streptavidin. However, there is a demand on methods that permit utilizing unmodified capture molecules and allow a cost-effective development process. Here, we report on a biotin- and streptavidin-free model NALFA. We also present a systematic investigation on the effect of various rehydrating buffers’ composition and concentration. In addition, the impacts of a protein blocker, detergents and chaotropic and kosmotropic agents on the intensity of the signal over background were analyzed. It has been demonstrated that simultaneous presence of sodium dodecyl sulfate and bovine serum albumin doubles the intensity of visible bands in the presented unmodified NALFA. Finally, this paper presents an optimized cost-effective model system that can be adapted for hybridization-based NALFAs.

Development of an elisa for the diagnosis of reactive IgE antibodies anti-therapeutic horse sera

Hypersensitive diseases that involve IgE reactivity are important concern of public, especially those encompassing the potential pathogenesis from the administration of horse serum-based therapeutics such as antivenoms. A method for the definitive diagnosis of reactive IgE is important for identifying allergic patients to control severe collateral effects during planned and emergency application of immunotherapies when the allergy source cannot be avoided for treatment. To date, no tests have been developed to accompany the wide range of antivenoms produced from horse sera. The aim of this was to develop a cost-effective ELISA of high sensitivity and specificity to detect circulating patient IgE that binds horse IgG3, the most prevalent antibody class in passive antibody therapies. Horse IgG3 was purified in a single step on jacalin-Sepharose and absorbed to standard ELISA plates as the capture molecule for reactive human IgE. The direct performance evaluation with allergenic and non-allergenic patient, together with competitive peptides assays, showed high sensitivity and specificity to detect human IgE that recognized horse IgG3. The analytical sensitivity and ED50 were calculated to be 0.01 μg mL−1 and 0.052 μg mL−1, respectively. The intra- and inter-assay coefficient of variation ranged from 3.3 to 11.1% and 4.0–8.0%, respectively. The horse IgG3-based ELISA assay can detect reactive allergenic IgE at picomolar concentrations. The coefficient of variation suggests that it can be easily standardized between laboratories, provide rapid and can be applied to population surveillance. Patient management during treatment for envenomation would be greatly improved by a robust and reliable diagnostic test for preexisting allergies to mitigate life-threating consequences of hypersensitivity.