Application Of Immunoassay Research Articles

Multiplex immunoassays using virus-tethered gold microspheres by DC impedance-based flow cytometry

Bead-based multiplex immunoassays for common use require enhanced sensitivity and effective prevention of non-specific adsorption, as well as miniaturization of the detection device. In this work, we have implemented virus-tethered gold microspheres for multiplex immunoassay applications, employing a DC impedance-based flow cytometer as a detection element. The advantages of virus-tethered gold microspheres, including excellent prevention of non-specific adsorption, are extended to signal enhancement arising from the large quantity of antibody loading on each virion, and to flexible movement of filamentous virus. Individual virus-tethered beads generate their own DC impedance and fluorescence signals, which are simultaneously detected by a chip-based microfluidic flow cytometer. This system successfully realized multiplex immunoassays involving four biomarkers: cardiac troponin I (cTnI), prostate specific antigen (PSA), creatine kinase MB (CK-MB), and myoglobin in undiluted human sera, elevating sensitivity by up to 5.7-fold compared to the beads without virus. Constructive integration between filamentous virus-tethered Au-layered microspheres and use of a microfluidic cytometer suggests a promising strategy for competitive multiplex immunoassay development based on suspension arrays.

Portable GMR Handheld Platform for the Detection of Influenza A Virus.

Influenza A virus (IAV) is a common respiratory pathogen infecting many hosts including humans, pigs (swine influenza virus or SIV), and birds (avian influenza virus or AIV). Monitoring swine and avian influenza viruses in the wild, farms, and live poultry markets is of great significance for human and veterinary public health. A portable, sensitive, and quantitative immunoassay device will be of high demand especially in the rural and resource-limited areas. We report herein our Z-Lab point-of-care (POC) device for sensitive and specific detection of swine influenza viruses with minimum sample handling and laboratory skill requirements. In the present study, a portable and quantitative immunoassay platform based on giant magnetoresistive (GMR) technology is used for the detection of IAV nucleoprotein (NP) and purified H3N2v. Z-Lab displays quantitative results in less than 10 min with sensitivities down to 15 ng/mL and 125 TCID50/mL for IAV nucleoprotein and purified H3N2v, respectively. This platform allows lab-testing to be performed outdoors and opens up the applications of immunoassays in nonclinical settings.

Current Advances in Quantum-Dots-Based Photoelectrochemical Immunoassays.

As a newly developed technique, photoelectrochemical (PEC) immunoassays have attracted great attention in recent years because of their low cost and desirable sensitivity. Because the detection signal originates from the photoelectric conversion of photoelectric materials, the appearance and application of quantum dots (QDs), which possess unique photophysical properties and regulated optoelectronic characteristics, has taken the development of PEC immunoassays to new heights. This review concisely introduces the general mechanism of QDs-based photoelectric conversion for immunoassays and summarizes the current advances in QD applications in immunoassays. Given that signal strategies and photoactive materials are the key elements in PEC biosensor systems, we comprehensively highlight the state-of-the-art signaling strategies and various applications of QDs in PEC immunoassays to introduce advances in QDs-based PEC immunoassays. Finally, challenges and future developmental trends are briefly discussed.

Study on the antigenicity of metallofullerenol: antibody production, characterization, and its enzyme immunoassay application.

With their intriguing structures and properties, metallofullerenols have attracted considerable attention in biological and medical applications. Due to the increasing biomedical interest, effective detection methods are important to monitor and control metallofullerenols. However, the detection of metallofullerenols becomes very difficult after polyhydroxylated modification due to the lack of detectable features. Antibody-based immunoassay methods have been important tools for detection and will better meet the needs of analysis of metallofullerenols. Thus, the antigenicity of metallofullerenol has been studied for the first time. In this study, no immune response was detected when metallofullerenol Gd@C82(OH)x was used as immunogen. However, the polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen, indicating that metallofullerenol can act as hapten. The specificity of the obtained antibody was investigated. It has been found that the hydroxyl groups on the surface of the carbon cage, the encapsulated metal, and the size and shape of the carbon cage did not affect the recognition specificity of the antibody. Based on the obtained antibody, an indirect competitive enzyme immunoassay was developed for the determination of metallofullerenol with detection limits of 18ng/mL in PBS. This enzyme immunoassay method was successfully used to detect metallofullerenol in serum. This work can provide an innovative way to determine metallofullerenols. Graphical abstract The polyclonal antibody against metallofullerenol was produced using metallofullerenol-KLH (keyhole limpet hemocyanin) as immunogen. Based on the obtained antibody, a competitive enzyme immunoassay was developed for the determination of metallofullerenol.

Robust enzyme-free electrochemical immunoassay of CEA enhanced by porous PdCu nanoparticles

The interferences of dissolved oxygen towards the electrochemical immunosensors occurred constantly, which will restrict the practical clinical application of electrochemical immunoassay. Herein, a new enzyme-free strategy has been proposed for ultrasensitive electrochemical immunoassay of carcinoembryonic antigen (CEA), which can completely avoid the interference of dissolved oxygen. Porous PdCu nanoparticles can catalyze aniline to produce polyaniline by the inherited peroxidase-like activity for generating electrochemical detection signal. More interesting, with the assistance of electropolymerization strategy, the electrochemical measurement signal could enhance sharply. Accordingly, we established a sandwich-type electrochemical immunosensor based on porous PdCu nanoparticles produced polyaniline with electropolymerization strategy for enzyme-free and dissolved oxygen interference-free detection of CEA, affording a satisfied performance with a broad linear detection range (0.1pg/mL∼10ng/mL) and low detection limit (0.08pg/mL). This method might be considered as a robust approach for clinical applications.

Clinical Application of Immunoassay for Identification of Staphylococcus aureus and Monitoring Treatment Response in Diabetic Foot Infection

Category: Basic Sciences/Biologics, Diabetes Introduction/Purpose: Currently, there is no sensitive and specific diagnostic tool for identifying the active pathogen in a polymicrobial environment such as diabetic foot infection (DFI). In addition, monitoring the success of antibiotic treatment is limited to clinical signs and nonspecific inflammatory markers. Consequently, surgeons are often forced to make interventional decisions without adequate prognostic information. While DFI are generally polymicrobial, about 50% prominently include Staphylococcus aureus (SA). We investigated applicability of utilizing a novel diagnostic immunoassay that measures the patient’s current production of anti-SA antibodies (IgG) to accurately diagnose SA and monitor its pathogenic activity. We hypothesize that 1) compared to standard culture, the immunoassay has a higher sensitivity to detect SA and 2) able monitor changes in pathogenic activity of SA in DFI. Methods: From July 2015 to August 2016, we enrolled 20 diabetic patients with DFU who displayed clinical symptoms and signs of infection which necessitated hospitalization and undertook initial foot salvage therapy (FST): irrigation and debridement followed by wet-to-dry dressings and 6 weeks of intravenous antibiotic treatment. At weeks 0, 4, 8 and 12, the infected DFUs samples were obtained for standard culture and 16 S rRNA microbiome analysis. Whole blood and serum samples were collected to measure the abundance of anti-SA IgG in the serum and in the in vitro secretions of antibody-secreting cells harvested from whole blood in “medium enriched for newly synthesized antibody”. Sensitivity and specificity for detection of SA were compared against the standard culture and microbiome analysis. Preliminary analyses compare the ability of the SA immunoassay to track therapy and its concordance with changes in the microbiome. Results: Of the 20 enrolled patients, 18 were available for at least partial follow-up and only four completed the entire sampling protocol. At the enrollment, 12 patients (60%) were identified positive for SA infection by at least one diagnostic method, while only 8 were diagnosed by standard culture. Six out of 10 SA-positive patients showed polymicrobial growth. The concordance rate for the presence or absence of SA was 85% between the immunoassay and microbiome, 70% between immunoassay and standard culture and 75% between microbiome and standard culture. Comparison of serial samples from the 7 subjects who were SA-positive by both the immunoassay and microbiome analysis demonstrated trends that the two novel assays provide complementary measures of therapeutic success. (Figure 1) Conclusion: Measurement of anti-SA antibodies showed higher sensitivity than standard culture and was able to monitor changes in pathogenic activity of SA in DFI undertaking salvage treatment. This novel immnoassay may serve as an important diagnostic and prognostic tools for monitoring SA infection in polymicrobial DFI. It provides important information for counseling patients of treatment response, prognosis, and determining to pursuit further foot salvage versus amputation. Future study will include expanding immunoassay measure other commonly found organisms in DFI.

Current Immunoassay Methods for the Rapid Detection of Aflatoxin in Milk and Dairy Products.

The presence of mycotoxins in foodstuff causes serious health problems to consumers and economically affects the food industry. Among the mycotoxins, aflatoxins are very toxic and highly carcinogenic contaminants which affect the safety of many foods, and therefore endanger human health. Aflatoxin M1 (AFM1 ) found in milk results from the biotransformation of aflatoxin B1 . Many efforts have been made to control the source of AFM1 from farmers to dairy product companies. However, AFM1 escapes ordinary methods of food treatment such as cooking, sterilization, and freezing, hence it appears in milk and dairy products. The presence of high levels of AFM1 constitutes an alarming threat as milk and dairy products contain essential nutrients for human health, especially for infants and children. For this reason, there is a pressing need for developing a fast and reliable screening method for detecting trace aflatoxins in food. Several analytical methods based on high-performance liquid chromatography (HPLC) and mass spectroscopy have been used for aflatoxin detection; however, they are expensive, time-consuming, and require many skills. Recently, immunoassay methods, including enzyme-linked immunosorbent assay (ELISA), immunosensors, and lateral flow immunoassay (LFIA), have been preferred for food analysis because of their improved qualities such as high sensitivity, simplicity, and capability of onsite monitoring. This paper reviews the new developments and applications of immunoassays for the rapid detection of AFM1 in milk.

Electrodeposition of Chitosan Composite Film and its Application for Protein Conjugation

Developing sensitive, reliable and simple methods for detection of protein biomarkers in complex biological matrixes is a major challenge in recent years. In most cases, immunoassays are performed in muti-well-plates in well-equipped labs, which is time consuming and expensive. There is great interest in transitioning immunoanalysis from the lab so the analytical capabilities of immunoanalysis can be employed to diagnose disease at the point-of-care and monitor the environment in the field. In immunoassay applications, antibodies are immobilized onto biointerface to capture specific biomarkers. To improve the sensitivity and reduce the size of immunosensor, immobilizing antibody at high density is essential. Here, we developed an easier method to conjugate the antibodies within the chitosan composite film and remove the non-specific binding with electrical field.

The β-lactoglobulin content of bovine milk: Development and application of a biosensor immunoassay

An optical biosensor immunoassay exploiting surface plasmon resonance is described for the quantification of β-lactoglobulin in milk. Samples were diluted with buffer, and the protein estimated from binding with a polyclonal antibody immobilised on the sensor surface. Analytical method performance characteristics including range, detection limit, precision and accuracy were determined and reported. The temporal variability in the β-lactoglobulin content of milk from pasture-fed cows during early lactation and across a production season was investigated. The content of β-lactoglobulin decreased from >10 mg mL−1 in early colostrum to <5 mg mL−1 in mature milk, and the β-lactoglobulin content of skim milk powder trended from 25 to 60 mg g−1 across a season. In view of its allergenicity, these data will improve understanding of the expression of innate β-lactoglobulin in the milk of pasture-grazed dairy herds, thereby providing information that is applicable to the formulation of bovine milk-based products.

Fluorescence and Scattering Light Cross Correlation Spectroscopy and Its Applications in Homogeneous Immunoassay.

In this work, we propose fluorescence and scattering light cross-correlation spectroscopy (FSCCS) based on laser confocal configuration using silver nanoparticle (SNPs) and Alexa Fluor 488 (Alexa) as probe pairs. FSCCS is a single molecule (particle) method, and its principle is similar to that of fluorescence cross-correlation spectroscopy (FCCS). We established the setup of FSCCS using single wavelength laser and developed an immunoassay model of FSCCS. The reliability and adaptability of FSCCS method were evaluated by homogeneous sandwich immunoassay mode. In the study, liver cancer biomarker alpha-fetoprotein (AFP) was used as an assay model, two different antibodies were labeled with SNPs and fluorophore Alexa Fluor 488, respectively. In the optimal conditions, the linear range of AFP covers 5 pM to 580 pM and the detection limit is 3.1 pM. This method was successfully applied for direct determination of AFP levels in human serum samples, and the obtained results were in good agreement with data obtained via ELISAs. The advantage of this method lies in its simplicity, attractive SNPs probes, high sensitivity and selectivity and high efficiency. We believe that FSCCS method exhibits promising potential applications in homogeneous bioassays and study on the molecular interaction and nanoparticle-molecule interaction.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations.

Pulse laser-induced generation of cluster codes from metal nanoparticles for immunoassay applications

In this work, we have developed an assay for the detection of proteins by functionalized nanomaterials coupled with laser-induced desorption/ionization mass spectrometry (LDI-MS) by monitoring the generation of metal cluster ions. We achieved selective detection of three proteins [thrombin, vascular endothelial growth factor-A165 (VEGF-A165), and platelet-derived growth factor-BB (PDGF-BB)] by modifying nanoparticles (NPs) of three different metals (Au, Ag, and Pt) with the corresponding aptamer or antibody in one assay. The Au, Ag, and Pt acted as metal bio-codes for the analysis of thrombin, VEGF-A165, and PDGF-BB, respectively, and a microporous cellulose acetate membrane (CAM) served as a medium for an in situ separation of target protein-bound and -unbound NPs. The functionalized metal nanoparticles bound to their specific proteins were subjected to LDI-MS on the CAM. The functional nanoparticles/CAM system can function as a signal transducer and amplifier by transforming the protein concentration in...

A microfluidic immunoassay system on a centrifugal platform

A lab-on-a-CD immunoassay system was designed, fabricated, and tested. The actuator for the centrifugal platform consists of a flyball governor and a group of spring plungers. The microfluidic structures were fabricated using replica processes with poly-dimethylsiloxane (PDMS) as structural material. For better control, the valves in the microfluidic system were designed to be mechanical-pinch ones and actuated. The burst frequencies of the micro-valves can be tuned independently by adjusting the heights and pre-stresses of corresponding spring plungers. For the research work reported in this paper, four sequential valves and one inward pump were integrated on a centrifugal microfluidic system to develop an automated immunoassay system. Mouse IgG was used as a model analyte to test the feasibility of using the proposed device for immunoassay applications. Sample solution of goat anti-mouse IgG conjugated with magnetic nanoparticles was used for magnetic trapping mechanism to eliminate the blocking process. Rabbit anti-mouse IgG labeled with fluorescent molecules was used as the signal reporter. The entire microfluidic assay process, including beads trapping, immunoassay reaction, washing and pumping was completed in a centrifugal platform. The detection sensitivity may also be improved by using higher quality monoclonal antibodies. This new immunoassay platform offers some significant advantages over those reported in the field: simple fabrication, robust control, easy integration, and lower cost. The system may also be modified for detection of multiple targets. The technology has great potential for applications in complex fluid handling needed in biological, chemical and medical application in Lab-on-a-CD systems.

Application of fluorescence polarization immunoassay for determination of carbamazepine in wastewater

Carbamazepine is an antiepileptic drug that can be used as a marker for the cleaning efficiency of wastewater treatment plants. Here, we present the optimization of a fast and easy on-site measurement system based on fluorescence polarization immunoassay and the successful application to wastewater. A new monoclonal highly specific anti-carbamazepine antibody was applied. The automated assay procedure takes 16 min and does not require sample preparation besides filtration. The recovery rates for carbamazepine in wastewater samples were between 60.8 and 104% with good intra- and inter-assay coefficients of variations (less than 15 and 10%, respectively). This automated assay enables for the on-site measurement of carbamazepine in wastewater treatment plants.

Comparison of various bismuth film electrodes in simultaneous electrochemical detection of heavy metals for application in quantum dot-linked immunoassays

Various electrode materials commonly used in electroanalysis were tested in preparation of respective bismuth film electrodes and compared in simultaneous electrochemical detection of heavy metals, simulating dissolved quantum dots used as labels of antibodies in quantum dot-linked immunosorbent assays. According to results obtained during voltammetric measurements, it can be concluded that selected commercial screen-printed electrodes showed many significant drawbacks which are described in detail. Contrary to the gold disk electrode, conventional glassy carbon electrode coated with thin bismuth film was applicable up to −1.5 V without the need to use very high content of bismuth in supporting electrolyte. Moreover, significant interactions among present metals were observed during their electrochemical stripping analysis, especially in presence of copper and silver.

Application of immunoassay in fast quality evaluation of traditional Chinese medicine

The quality evaluation of traditional Chinese medicine can be divided into two aspects, effectiveness and safety. The existing methods for evaluating the quality of traditional Chinese medicine(TCM) are mostly based on the testing instruments, which can not meet the practical needs of simple, rapid and on-site in production and life. Immunoassay, characterized by simple, rapid, sensitive, specific, low-cost and high-throughput, is widely used in the fields of clinical diagnosis, environmental pollution monitoring, food safety testing, and other fields. In recent years, immunoassay technology has been gradually applied in the field of quality control of TCM, involving quantitative detection of effective components of TCM, detection of harmful substances in TCM, and detection of exogenous pollutants in TCM. This paper summarizes the principle of the wide application of ELISA and colloidal gold immunostrip technology and its application in quality evaluation of TCM, this technique has a good application prospect in the field of rapid detection of the quality of TCM. In this paper, the principles of the widely used ELISA and colloidal gold immune test strip technology as well as their application in quality evaluation of TCM were reviewed, and the results showed that this technique had a good application prospect in the field of rapid detection of the quality of TCM.

Single-Molecule Counting Applied to Immunoassays

We investigated the sensitivity of single-molecule TIRF counting in diagnostic immunoassay applications. This work focused on using single-molecule techniques purely for detection, with the assay itself taking place on a separate platform with the detectable label being eluted for single-molecule measurement. Such an approach both limits the amount of background in the final measurement and sets up a universal detection procedure for a potentially wide variety of immunoassays. We took advantage of the low volume required for single-molecule measurements and demonstrated a sample reloading approach to further concentrate the sample on the single-molecule surface. SM reloading—for a biotin-streptavidin surface capture reaction—is a remarkably robust procedure, independent of starting concentration, and so can be used for assaying unknown samples. We tested model assays with single-molecule detection to substantiate this approach and demonstrate the potential of single-molecule counting for diagnostic applications.

Inductive Integrated Biosensor With Extended Operative Range for Detection of Magnetic Beads for Magnetic Immunoassay

Biosensors are prominent in several areas, such as medical diagnosis, food preparation, pharmaceutical industries, and clinical analysis; high performances are required in spite of attaining accuracy, sensitivity, low cost, easy handling, and portability, but the major parameter that is always representing a challenge for biosensors is specificity. High sensitivity and specificity can be obtained by combining appropriate transduction methods together with immunoassay techniques. In this paper, integrated inductive biosensors for the magnetic immunoassay process, which use magnetic beads as markers for biomolecules, are presented with potential applications to proteins and DNA measurements. The working principle and a dedicated fabrication technology, which also embeds thermal actuation and control, are described; in particular, an improved sensing architecture is proposed here, which allows one to expand the microsensor operative field toward very low bead concentrations. The sensor characterization results are presented together with the analytical model of the transduction principle; a detection limit of approximately 300 beads has been demonstrated. The microsensor is also capable of operating by measuring up to 450 000 beads with a good linearity. Therefore, the sensor architecture proposed here has been demonstrated for wide operating field with high resolution and good linearity; the results proposed confirm the suitability of the devices developed for magnetic immunoassay applications.

Influence of exposed magnetic nanoparticles and their application in chemiluminescence immunoassay

To a certain extent, uneven coating of nanoparticles may lead to exposure of their contents. In this work, a simulation system was established and exposed Fe3O4 magnetic nanoparticles (MNPs) were used to investigate the influence of the exposed case in a chemiluminescence immunoassay. Some factors such as the concentration of MNPs, coexistence of horseradish peroxidase (HRP), different particle sizes, and various treatment methods were investigated. It was confirmed that both the bodies of nanoparticles and metallic oxides on the exposed surface have associations with the detection signal. This conclusion may provide a new way to explain the deviation emerged in a chemiluminescence immunoassay. In addition, an exposed Fe3O4 MNPs based chemiluminescence immunoassay was developed to detect human chorionic gonadotropin (hCG). Carboxyl-functionalized MNPs were modified with anti-α-hCG antibody, and further used as solid-phase supports for sandwich-type immunoassay. The detection limit for hCG under optimal conditions was as low as 1.184ng/mL with a linear response range of 2–100ng/mL and a correlation coefficient of 0.989. The proposed method could be used for the determination of hCG in serum samples, which demonstrated their application potential in rapid and cost-effective detection of biological samples.

Reversion of gold nanoparticle aggregates for the detection of Cu2+ and its application in immunoassays.

A high concentration of copper is a hazardous element to organisms and human health. Although various strategies have been reported for the sensitive detection of copper, a facile and rapid detection of aqueous copper has seldom been addressed to date. Here, we present an easy and accessible colorimetric method to detect Cu2+ using the redispersion of cysteamine-modified gold nanoparticles (CA-AuNPs). Initially, CA caused the aggregation of AuNPs due to the electrostatic interaction and aggregated AuNPs can be regenerated in basic medium. The subsequent addition of Cu2+ to the CA-AuNP dispersion could effectively trigger the aggregation of CA-AuNPs, resulting from the coordination reactivity between the deprotonated CA and Cu2+. This strategy resulted in a detection limit (LOD) of 1.52 μM in drinking water, which is below the U.S. Environmental Protection Agency permissible limit (20 μM). To demonstrate the broad application of CA-AuNPs, we further applied this method to plasmonic immunoassays based on the competitive interaction of Cu2+ between CA-AuNPs and enzymes. The LOD of the Down syndrome biomarker hyperglycosylated human chorionic gonadotropin (H-hCG) was 0.125 mIU mL-1, which is better than that of commercial immunoassays. Importantly, the determination of H-hCG in serum indicates its applicability for the measurement of real samples. Our assay agrees well with the current immunoassay systems and thus it can easily be expanded to a more common sensing platform for different types of biotargets by changing the corresponding antibodies.