Developed Immunoassay Method Research Articles

Sensitive detection of HSP70 using a current-amplified biosensor based on antibody-loaded PS-AuNPs@Cys/Au modified ITO chip.

A biosensing electrochemical platform for heat shock protein 70 (HSP70) has been developed by integrating a three-electrode indium tin oxide (ITO) on a chip. The platform includes modifications to the reference electrode and working electrode for the detection of HSP70. The new platform is constructed by assembly of HSP70 antibody on PS-AuNPs@Cys/Au indium tin oxide (ITO) electrode to create a high HSP70 sensitive surface. The PS-AuNPs@Cys/Au indium tin oxide (ITO) electrode is obtained by immersing the ITO electrode into the PS-AuNPs@Cys solution and performing constant potential deposition at -1.4V (Ag/AgCl). The PS-AuNPs@Cys/Au film deposited on ITO glass provides a desirable substrate for the immobilization of the HSP70 antibody and improves the loading of antibody between PS-AuNPs@Cys/Au and the electrode resulting in a significant amplification. Under optimal conditions, the fabricated sensor demonstrates a linear range extending from 0.1 ng mL- 1 to 1000 ng mL- 1, with an impressive detection limit of 25.7 pg mL- 1 (S/N = 3). The developed immunoassay method successfully detected the HSP70 content in normal human blood samples and outperformed the ELISA method commonly used for clinical sample analysis.

Development of immunoassay based on a specific antibody for sensitive detection of nicosulfuron in environment

Nicosulfuron, one of the most widely used selective herbicides in corn field, can effectively control annual and perennial grass weeds, sedges, and some broadleaf weeds. The residual phytotoxicity of nicosulfuron in soil and water has become increasingly prominent. Therefore, an efficient method for detection of nicosulfuron was critical to ensure the sustainable and healthy development of agriculture and the ecological environment. In this paper, five nicosulfuron haptens which contained carboxyl group or aldehyde groups were designed and synthesized, and an indirect competitive immunoassay was developed for the first time. The assay showed an IC50 of 8.42 ng/mL and had negligible cross reactivities toward other sulfonylurea herbicides. In the spike and recovery studies, the recovery rate from soil samples was 95 %–104 %, and that of wheat roots was 92 %–98 %, which showed a good correlation with LC-MS analysis for nicosulfuron. The immunoassay was then used to quantify nicosulfuron concentration which could cause the obvious phytotoxic symptoms to wheat. Obvious symptoms of nicosulfuron phytotoxicity in wheat root was observed at the concentration of 0.068 ± 0.006 mg/kg (ELISA result) which was consistent with 0.072 ± 0.007 mg/kg obtained by LC-MS. The developed immunoassay method is an effective tool for environment contamination monitoring.

Enzyme-controllable just-in-time production system of copper hexacyanoferrate nanoparticles with oxidase-mimicking activity for highly sensitive colorimetric immunoassay

Nanozymes are a series of elaborately designed nanomaterials that can mimic the catalytic sites of natural enzymes for reactions. Bypassing the tedious design and preparation of nanomaterial, in this work, we report on a novel just-in-time production system of copper hexacyanoferrate nanoparticles (CHNPs), which act as an oxidase-mimicking nanozyme. This system can rapidly produce CHNPs nanozyme on demand by simply mixing Cu(II) with potassium hexacyanoferrate(III) (K3[Fe(CN)6]). It is found that once K3[Fe(CN)6] is reduced to K4[Fe(CN)6], the formation of CHNPs is inhibited. Therefore, the just-in-time production system of CHNPs was coupled with alkaline phosphatase (ALP) to construct an enzyme-controllable just-in-time production (ECJP) system, in which ALP could inhibit the production of by catalyzing the hydrolysis of ascorbic acid 2-phosphate (AAP) to generating ascorbic acid (AA). The ECJP system is then used to probe the activity of ALP by employing 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) as the chromogenic substrate, and a detection limit of 0.003 U L−1 was achieved. Moreover, by adapting ALP as the enzyme label, an ECJP system-based colorimetric immunoassay protocol was established for sensitive detection of aflatoxin B1 (AFB1), and a detection limit as low as 0.73 pg mL−1 was achieved. The developed immunoassay method is successfully applied to the detection of AFB1 in peanut samples. The operation of ECJP system is quite simple and the coupling of ALP with CHNPs nanozyme can arouse dual enzyme-like cascade signal amplification. So, we believe this work can offer a new perspective for the development of nanozymes-based biodetection methods and colorimetric immunoassay strategies.

Development of an ic-ELISA and Immunochromatographic Strip Assay for the Detection of Diacetoxyscirpenol in Rice.

Diacetoxyscirpenol (DAS) is a highly toxic type A trichothecene, a natural contaminant in food and animal feed, which is a serious hazard to human and animal health. An anti-DAS mAb, 3H10, with high sensitivity and specificity, was prepared, and an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a lateral-flow immunochromatographic strip (ICA strip) were developed for rapid testing of DAS in rice samples. The 50% inhibitory concentration and limit of detection of ic-ELISA were 5.97 and 0.78 ng/mL, respectively. The recovery of rice samples ranged from 99.4 to 110.7%, demonstrating that the analytical method can be used to detect rice samples. The cutoff value of the lateral-flow ICA strip to DAS was 500 ng/g. The developed immunoassay method can provide an effective method of initially detecting and screening DAS in food and feed samples.

Fluorescent and colorimetric immunoassay of nuclear matrix protein 22 enhanced by porous Pd nanoparticles

A modified ELISA realizing fluorescent and colorimetric immunoassay of nuclear matrix protein 22 (NMP 22) was developed based on porous Pd nanoparticles. The unique structure and excellent enzyme mimetic activity of porous Pd nanoparticles favor to oxidize o-phenylenediamine (OPD) into 2,3-phenazinediamine (oxOPD) by H2O2, producing colorimetric and fluorescence dual-readout signal for the detection of NMP 22. The developed immunoassay method will offer great potential in clinical research and diagnostic applications.

Levels of antimüllerian hormone in serum during the normal menstrual cycle

To determine whether levels of antimüllerian hormone (AMH) in serum vary during the normal menstrual cycle, using the most recently developed immunoassay method. Prospective cohort study. Local community. Women with normal menstrual cycles and between the ages of 18 and 45 years were recruited (n = 45). Blood samples were collected on 5 days within each cycle: two in the follicular phase and three after confirmed ovulation. Exclusion criteria were anovulatory cycles, incomplete sample collection, insufficient blood volume, or non-Caucasian ethnicity. None. Serum samples were tested for levels of AMH using a new immunoassay method (Ansh Labs). The effects of body mass index (BMI) and smoking on serum AMH levels were considered. Serum AMH levels varied significantly during the menstrual cycle, with the highest levels in the follicular phase. When the analysis was stratified by age, AMH variation during the menstrual cycle was significant only for women older than 30 years. Serum AMH levels were not significantly altered by BMI or smoking. The new AMH immunoassay revealed a follicular phase rise in serum levels, particularly in women over the age of 30 years. This is consistent with other reports finding an interaction of menstrual cycle variation in AMH and chronological age. Nonetheless, the extent of variation is small, and sampling on any day of the menstrual cycle is expected to adequately reflect ovarian reserve. NCT01337999.

Ultrasensitive electrochemical immunosensors for clinical immunoassay using gold nanoparticle coated multi-walled carbon nanotubes as labels and horseradish peroxidase as an enhancer

A novel electrochemical immunosensor for the ultrasensitive detection of α-fetoprotein (AFP) is described with the use of a gold nanoparticle/(3-mercaptopropyl) trimethoxysilane modified electrode as a desirable platform and gold nanoparticle functionalized multi-walled carbon nanotube (MWCNT) nanocomposite labeled secondary antibodies (Ab2) as a signal tag. At first, gold nanoparticles (AuNPs) were attached on the MWCNTs surface using poly(diallyldimethylammoniumchloride) (PDDA) as a linkage reagent to obtain AuNP–MWCNT nanocomposites. Subsequently, horseradish peroxidase (HRP) and thionine (Thi) labeled secondary antibodies (Thi-Ab2) were used to bind AuNP–MWCNT nanocomposites with a high load amount and good biological activity. Moreover, the immunosensor can provide amplified signals because of the increased surface area and biocompatibility of AuNP–MWCNT nanocomposites. On the other hand, the AuNP functionalized (3-mercaptopropyl) trimethoxysilanes (MPTS) were used for the biosensor platform to increase the surface area as well as improving the electronic transmission rate to capture a large amount of primary antibodies (Ab1). Thus, amplified signals can be obtained by an electrochemical sandwich immunoassay protocol in the presence of H2O2. The proposed sensing strategy provides a wide linear dynamic range from 0.01 to 50 ng mL−1 with a low detection limit of 3 pg mL−1 (S/N = 3). Significantly, the developed immunoassay method showed a good stability, selectivity and reproducibility.

Development of enzyme-linked immunosorbent assay for rapid determination of sildenafil in adulterated functional foods

To determine the sildenafil in adulterated functional foods, a rapid direct competitive enzyme-linked immunosorbent assay (ELISA) procedure employing a polyclonal antibody generated from sildenafil-KLH was established. The antibody showed high sensitivity and specificity in phosphate buffer, with an IC50 of 6±0.1 ng/mL and the limit of detection was 0.6±0.02 ng/mL. The matrix effect of the functional food samples was easily eliminated by using methanol and distilled water for extraction and dilution with PBS, without any clean-up procedure such as solid phase extraction or liquid–liquid extraction, much more simple and rapid than the other reports. For the validation of the established SIL–ELISA, the functional food sample spiked at three different concentrations, analysed by HPLC. The results showed good correlation between the data of ELISA and HPLC (r 2=0.9832). It approved that HPLC methods show the same results with ELISA, and the developed immunoassay method is considered reliable for detection of sildenafil.

Electrochemical Immunosensor for α‐Fetoprotein Based on Gold Nanoparticles/Graphene‐Prussian Blue

AbstractThe electrochemical immunosensor for α‐fetoprotein (AFP) was fabricated based on the platform of gold nanoparticles (GNP)/graphene (Gr)‐prussian blue (PB). By electrodeposition, GNP were modified on the surface of the prepared Gr‐PB. The anti‐AFP‐1,1′‐ferrocenedicarboxylic acid (FcDA) as label was directly immobilized on the platform of GNP/Gr‐PB. And after the immunoreactions, the formed complex inhibited the electron transfer and decreased the catalytic current of FcDA toward the reduction of H2O2. And in the range of 10–3200 pg·mL−1, the decreased current is linear with the concentration of AFP, with a detection limit of 3 pg·mL−1. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme linked immunosorbent assay (ELISA) method.

Poly(MAA-PEGMA)-coated superparamagnetic nanoparticles-based magnetic affinity immunoassay for detection of interleukin-6

Using poly(MAA-PEGMA)-coated superparamagnetic nanoparticles (PMNPs) as carrier, a rapid and sensitive immunoassay for quantitative determination of interleukin-6 (IL-6) has been developed for the first time. This protocol involved a sandwich format in which the IL-6 in samples was first captured by the primary monoclonal antibody (mAb) immobilized on the surface of PMNPs, and then recognized by the second monoclonal antibody labeled with horseradish peroxidase (HRP-mAb). The HRP-mAb dilution and immunoreaction time were examined and optimized. The linear range for IL-6 assay was 3.2–1000 pg mL−1, and the Limit Of Detection (LOD) was 1.0 pg mL−1. The developed immunoassay method showed high precision, high sensitivity, acceptable recovery and relatively good reliability, and could be used to detect serum IL-6 levels with consistent results in comparison with those obtained by the Enzyme-Linked Immunosorbent Assay (ELISA) method.

Magnetic beads-based electrochemical immunosensor for detection of pseudorabies virus antibody in swine serum

A novel magnetic electrochemical immunosensor has been developed for the detection of pseudorabies virus antibody in swine serum. The magnetic glass carbon electrode was fabricated to manipulate magnetic beads for the direct sensing applications. Magnetic beads were employed as the platforms for the immobilization and immunoreaction process, and gold nanoparticles were chosen as electroactive labels for the electrochemical detection. The parameters concerning the assay strategy were carefully investigated. Under the optimal conditions, the linear response range of pseudorabies virus antibody dilution ratio (standard positive serum) was 1:250 to 1:1000 with a detection limit of 1:1000. Finally, this developed immunoassay method was successfully applied in the detection of pseudorabies virus antibody in swine serum, and had a good diagnostic accordance in comparison with ELISA.

A newly developed immunoassay method based on optical measurement for Protein A detection

We describe the development of an immunoassay using an antibody–silver nanoparticle (Ab–AgNP) conjugate as a catalyst for the silver enhancement reaction. The immuno-reaction signals that were magnified by silver metal precipitation were quantified using a commercial flatbed scanner. Protein A from Staphylococcus aureus ( S. aureus), a common clinical pathogenic bacterium, was used in this research. The ease of infection of S. aureus necessitates the development of a fast detection method. The framework of the method described in this paper is based on the sandwich immunoassay and contains a 1st antibody (immunoglobulin G, IgG), an antigen (Protein A), and a 2nd antibody–colloidal silver conjugate (IgG–AgNPs). The silver enhancement reaction, a signal amplification method in which silver ions are reduced to metallic silver, is used to magnify the immuno-reaction signal. The change in signal, as visualized in grayscale, can be easily observed and analyzed by our optical scanning detection system. The relationship between antigen concentration and grayscale value is discussed. The detectable concentration limit for the antigen was found to be 1 ng/mL with 10 μg/mL of IgG and 300 μM of the IgG–AgNP conjugate. This immunoassay method provides the advantages of low cost, easy operation, and short detection time. Moreover, it has potential applications in clinical diagnoses.

Conductive carbon nanoparticles-based electrochemical immunosensor with enhanced sensitivity for α-fetoprotein using irregular-shaped gold nanoparticles-labeled enzyme-linked antibodies as signal improvement

A new electrochemical immunoassay protocol for sensitive detection of α-fetoprotein (AFP, as a model) is designed using carbon nanoparticles (CNPs)-functionalized biomimetic interface as immunosensing probe and irregular-shaped gold nanoparticles (ISNGs)-labeled horseradish peroxidase- anti-AFP conjugates (HRP- anti-AFP-ISNG) as trace label. The low-toxic and high-conductive CNPs provided a high capacity nanoparticulate immobilization surface and a facile pathway for electron transfer. In comparison with conventional label methods, i.e. spherical gold nanoparticles-labeled HRP- anti-AFP and HRP-labeled anti-AFP, the electrochemical immunosensor using HRP- anti-AFP-ISNGs as trace labels exhibited high bioelectrocatalytic response toward enzyme substrate and a wide dynamic range from 0.02 to 4.0 ng/mL with a low detection limit of 10 pg/mL toward AFP (at 3 σ). The developed immunoassay method showed good selectivity and acceptable reproducibility. Clinical serum samples with various AFP concentrations were evaluated by using the electrochemical immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA), respectively, and received in good accordance with results obtained from these two methods.

Enhanced conductometric immunoassay for hepatitis B surface antigen using double-codified nanogold particles as labels

A new conductometric immunoassay for hepatitis B surface antigen (HBsAg) was developed based bioelectrocatalytic reaction on a microcomb-type electrode by using double-codified nanogold particles as labels. This microcomb-type electrode was fabricated on an interdigitated transducer covered with a well-ordered anti-HBs/protein A/nanogold architecture. The double-codified nanogold particles were prepared by using nanogold-labeled anti-HBs antibodies conjugated with horseradish peroxidase (HRP). Sandwich-type immunoassay protocol was successfully introduced for the detection of HBsAg. The formation of the immunocomplex changed the direct electrical communication between the carried HRP and the electrode, and thus local conductivity variations could be assayed based on the bioelectrocatalytic reaction of the carried HRP in 0.01M PBS (pH 7.0) containing 60μM H2O2, 0.08M KI and 0.1M NaCl. Under optimized conditions, the linear range obtained by using HRP-conjugated anti-HBs as secondary antibodies was 1.5–450ng/mL HBsAg, while the assay sensitivity by using double-codified nanogold particles could be further increased to 0.01ng/mL with the linear range from 0.1 to 600ng/mL HBsAg. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real sample with consistent results in comparison with those obtained by the ELISA method.

Ultrasensitive Electrochemical Immunosensor for Clinical Immunoassay Using Thionine-Doped Magnetic Gold Nanospheres as Labels and Horseradish Peroxidase as Enhancer

A new signal amplification strategy based on thionine (TH)-doped magnetic gold nanospheres as labels and horseradish peroxidase (HRP) as enhancer holds promise to improve the sensitivity and detection limit of the immunoassay for carcinoembryonic antigen (CEA), as a model protein. This immunoassay system was fabricated on a carbon fiber microelectrode (CFME) covered with a well-ordered anti-CEA/protein A/nanogold architecture. The reverse micelle method was initially used for the preparation of TH-doped magnetic gold nanospheres (nanospheres), and the synthesized nanospheres were then labeled on HRP-bound anti-CEA as a secondary antibody (bionanospheres). Sandwich-type protocol was successfully introduced to develop a new high-efficiency electrochemical immunoassay with the labeled bionanospheres toward the reduction of H2O2. Under optimized conditions, the linear range of the proposed immunoassay without HRP as enhancer was 1.2-125 ng/mL CEA, whereas the assay sensitivity by using HRP as enhancer could be further increased to 0.01 ng/mL with the linear range from 0.01 to 160 ng/mL CEA. The developed immunoassay method showed good precision, high sensitivity, acceptable stability and reproducibility, and could be used for the detection of real samples with consistent results in comparison with those obtained by the enzyme-linked immunosorbent assay (ELISA) method.

Quartz crystal microbalance immunoassay for carcinoma antigen 125 based on gold nanowire-functionalized biomimetic interface

Gold nanowires with of designed length on a solid substrate have been proven as an efficiently immobilized affinity support for the detection of carcinoma antigen 125 (CA 125) in this study. The presence of gold nanowires provides a well-defined three-dimensional structure, and greatly amplifies the coverage of the anti-CA 125 protein on the probe surface. Moreover, the amount of anti-CA 125 varied with the change of the morphology of the probe, and achieved an optimal quartz crystal microbalance (QCM) response towards anti-CA 125 adsorption at the number of gold nanolayers of 5. The formed immune-probe exhibits good QCM responses for the detection of CA 125, and allows the detection of CA 125 at concentrations as low as 0.5 U ml(-1). The QCM immunosensor exhibited good precision, high sensitivity, acceptable stability, accuracy and reproducibility. The as-prepared immunosensors were used to analyze CA 125 in human serum specimens. Analytical results suggest that the developed immunoassay method is a promising alternative approach for detecting CA 125 in the clinical diagnosis. Compared with conventional ELISA, the proposed immunoassay system was simple and rapid without multiple labeling and separation steps. Importantly, the route provides an alternative approach to incorporate multiple gold nanolayers onto the solid matrix for biosensing applications.

Development of ELISA for the Determination of Transgenic Bt‐Cottons Using Antibodies against Cry1Ac Protein from Bacillus thuringiensis HD‐73

AbstractThe area cultivated with Bt‐cottons expressing Cry1Ac gene increases year by year in China and other countries. To evaluate any potential adverse impacts on the environment from the release of Bt (Bacillus thuringiensis) technology, the development of a method for easily detecting the activity of the Cry1Ac toxins is of particular interest. The aim of this study was to develop sandwich‐ELISA for the detection of Cry1Ac protein in Bt‐cotton tissues. A specific antibody was obtained from rabbits inoculated with Cry1Ac protein derived from Bt strain HD‐73 and a secondary antibody conjugated to HRP could combine the Bt Cry1Ac protein specifically. The limit of detection was 5 ng/mL and there were no cross‐reactions between the positive control of Cry1Ab/1Ac, Cry1C, Cry2A, Cry3Bb1 and Cry9C. Extracts of proteins from cotton leaves were used to evaluate the suitability of the assay. Tris‐borate buffer and sodium carbonate buffer were employed for the extraction of protein, the limit absorbance of detection was 0.134 and 0.449, respectively, and the latter produced a higher background. The results showed that cultivars GK‐12, GK‐22, insect‐resistant cotton, bivalent transgenic cotton and shiyuan 321 assayed positively and NON was the negative sample. The PCR method was used for the validation of the developed assay. Although both methods allowed the same results to be obtained, ELISA needed simple equipment and took less time. The developed immunoassay method is considered reliable for the detection of Bt Cry1Ac protein.