Fluorescence-linked Immunosorbent Assay Research Articles

Fluorescent immunosorbent assay for the detection of alpha lactalbumin in dairy products with monoclonal antibody bioconjugated with CdSe/ZnS quantum dots

In this work, a new method termed competitive fluorescence-linked immunosorbent assay (FLISA) was developed for specifically quantification of bovine α-lactalbumin (α-La) in dairy products. The monoclonal antibodies (mAbs) against α-La were produced through hybridoma technology, and the mAbs were covalently conjugated with the CdSe/ZnS quantum dots (QDs) using the crossing-linking reagents. Moreover, a competitive FLISA based on QD-mAb conjugates was established to detect α-La in dairy products. It was shown that there was a good linear relationship between inhibition efficiency, and logarithm of α-La concentration after the detection parameters were optimised in which the concentration of α-La varied from 0.1 to 1000ng/mL. The value of IC50 was 0.03μg/mL, and the FLISA method exhibited high sensitivity with the LOD at 0.1ng/mL. The developed FLISA has been successfully applied to determine α-La in commercial dairy products, providing more sensitive analysis compared with the ELISA method.

Monoclonal antibody-quantum dots CdTe conjugate-based fluoroimmunoassay for the determination of aflatoxin B1 in peanuts

A fluoroimmunoassay towards aflatoxin B1 (AFB1) was presented using quantum dots as the fluorescent label. The CdTe QDs were successfully linked to the monoclonal antibody against AFB1. Based on the conjugated complexes, a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) was developed for AFB1 detection. The 50% inhibition value (IC50) of the cFLISA was 0.149ng/mL in peanuts matrix. The method performance included the limit of detection (LOD) of 0.016ng/mL and considerable recoveries of 85–117% at three fortification levels (0.075, 0.15, and 0.3ng/g) from spiked AFB1 blank peanuts samples, along with coefficients of variation (CVs) below 10%. The cFLISA provided an alternative of rapid and sensitive detection for AFB1 and, moreover provided great potential for multiplexed mycotoxins determination simultaneously.

Fluidic Automation of Nitrate and Nitrite Bioassays in Whole Blood by Dissolvable-Film Based Centrifugo-Pneumatic Actuation

This paper demonstrates the full centrifugal microfluidic integration and automation of all liquid handling steps of a 7-step fluorescence-linked immunosorbent assay (FLISA) for quantifying nitrate and nitrite levels in whole blood within about 15 min. The assay protocol encompasses the extraction of metered plasma, the controlled release of sample and reagents (enzymes, co-factors and fluorescent labels), and incubation and detection steps. Flow control is implemented by a rotationally actuated dissolvable film (DF) valving scheme. In the valves, the burst pressure is primarily determined by the radial position, geometry and volume of the valve chamber and its inlet channel and can thus be individually tuned over an extraordinarily wide range of equivalent spin rates between 1,000 RPM and 5,500 RPM. Furthermore, the vapour barrier properties of the DF valves are investigated in this paper in order to further show the potential for commercially relevant on-board storage of liquid reagents during shelf-life of bioanalytical, ready-to-use discs.

Rapid and Sensitive Fluoroimmunoassay Based on Quantum Dots for Detection of Melamine in Milk

A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (icFLISA) method was developed for detection of melamine in milk based on secondary antibody (Ab2)-conjugated quantum dots (QDs). The sensitivity of icFLISA with a limit of detection (LOD) of melamine (3.88 ng mL−1) and 50% inhibition value (IC50) (31.58 ng mL−1), was much higher than that of icELISA. As a proof of principle, recovery tests involving four fortification levels (150, 80, 40, and 20 ngmL−1) in blank samples were also performed. The recoveries ranged from 80.85 to 110.54% with coefficient of variations (CVs) of 2.82–8.82%. When challenged with authentic samples, these results of icFLISA were consistent with that of icELISA, immuno-chromatographic assay and gas chromatography-single quadruple mass spectrometry (GC-MS)method, indicating that icFLISA can be considered as a new screening method for detection of melamine in milk.

Fluobodies against Bioactive Natural Products and their Application in Fluorescence-Linked Immunosorbent Assay

An enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody (MAb), Fab antibody, and single-chain variable fragment (scFv) antibody has become one of the most promising analytical methods owing to its rapidity, sensitivity, and reliability. Recently, a chimera of green fluorescent protein (GFP) with a scFv antibody, named fluobody, was proposed as a probe for an alternative immunosorbent assay; i.e., fluorescence-linked immunosorbent assay (FLISA). In this FLISA, an even more sensitive, simple, and rapid immunoassay can be performed by detecting the highly sensitive fluorophore of GFP that is genetically and directly fused to the scFv antibody. In addition, the time- and cost-consuming secondary antibody reaction and the following enzyme-substrate reaction, necessary for conventional ELISA, can be avoided, making it possible to complete the assay more rapidly. Focusing on naturally occurring bioactive products, fluobody recognizing 1,4-naphthoquinone, plumbagin and triterpenoid saponin, ginsenosides were successfully expressed in Escherichia coli (E. coli) and applied to FLISA. The construction, the expression, and the potential use of fluobody in quantitative/qualitative analysis of bioactive natural products are reviewed in this article.

High Sensitive Detection of Cry1Ab Protein Using a Quantum Dot-Based Fluorescence-Linked Immunosorbent Assay

Protein-based detection methods, enzyme-linked immunosorbent assay (ELISA) and lateral flow strip, have been widely used for rapid, spot, and sensitive detection of genetically modified organisms (GMOs). Herein, one novel quantum dot-based fluorescence-linked immunosorbent assay (QD-FLISA) was developed employing quantum dots (QDs) as the fluorescent marker for the detection of the Cry1Ab protein in MON810 maize. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-rabbit secondary antibody. The newly developed Cry1Ab QD-FLISA assay was highly specific to the Cry1Ab protein and had no cross-reactivity with other target proteins, such as Cry2Ab, Cry1F, and Cry3Bb. The quantified linearity was achieved in the value range of 0.05-5% (w/w). The limits of detection (LOD) and quantification (LOQ) of the QD-FLISA were 2.956 and 9.854 pg/mL, respectively, which were more sensitive than the conventional sandwich ELISA method. All of the results indicated that QD-FLISA was a highly specific and sensitive method for the monitoring of Cry1Ab in GMOs.

Single-Molecule Detection on a Protein-Array Assay Platform for the Exposure of a Tuberculosis Antigen

Based on a single-molecule sensitive fluorescence-linked immunosorbent assay, an analytical platform for the detection of lipoarabinomannan (LAM), a lipopolysaccharide marker of tuberculosis, was established that is about 3 orders of magnitude more sensitive than comparable current ELISA assays. No amplification step was required. Also, no particular sample preparation had to be done. Since individual binding events are detected, true quantification was possible simply by counting individual signals. Utilizing a total internal reflection configuration, unprocessed biological samples (human urine and plasma) to which LAM was added could be analyzed without the requirement of sample purification or washing steps during analysis. Samples containing about 600 antigen molecules per microliter produced a distinct signal. The methodology developed can be employed for any set of target molecules for which appropriate antibodies exist.

A chimera of green fluorescent protein with single chain variable fragment antibody against ginsenosides for fluorescence-linked immunosorbent assay

A chimera of green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon optimized for mammalian expression, with single-chain variable fragment (scFv) antibody against ginsenoside Re (GRe-scFv), named fluobody, has been successfully expressed in Escherichia coli ( E. coli) to develop simple, speedy, and sensitive fluorescence-linked immunosorbent assay (FLISA). Two chimera proteins were constructed to contain GRe-scFv at the C-terminus of AcGFP (C-fluobody) and at the N-terminus of AcGFP (N-fluobody). These fluobodies were then purified by ion metal affinity chromatography and refolded by stepwise dialysis. The characterization of both fluobodies revealed that C-fluobody was found to be appropriate probe for FLISA as compare with N-fluobody. Furthermore, improvement of limit of detection (LOD) was observed in FLISA using C-fluobody (10 ng/mL) due to its strong fluorescence intensity of AcGFP compared with conventional enzyme-linked immunosorbent assay (ELISA) using parental monoclonal antibody against ginsenoside Re (G-Re), MAb-4G10 (100 ng/mL). Since some steps required in ELISA can be avoided in this present FLISA, speedy and sensitive immunoassay also could be performed using fluobody instead of monoclonal antibody and scFv.

A fluorescent single domain antibody against plumbagin expressed in silkworm larvae for fluorescence-linked immunosorbent assay (FLISA)

A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 µg mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.

Simultaneous detection of multiple chemical residues in milk using broad-specificity antibodies in a hybrid immunosorbent assay

In this study, a novel immunoassay using 2 types of sensors (QDs and an enzyme) were simultaneously used for detecting multiple structurally different molecules in milk. The method integrates the fluorescence-linked immunosorbent assay (FLISA) using QD605 and QD655 as probes and an enzyme-linked immunosorbent assay (ELISA) using horseradish peroxidase (HRP) labeled secondary antibody. The FLISA was produced by anti-sulfonamide and anti-quinolone broad-specificity monoclonal antibodies (MAbs) for simultaneously detecting 6 sulfonamides and 11 quinolones. Combined with the FLISA, an ELISA was utilized for detecting melamine from the same milk samples. The cross-reactivity of the MAbs was retained while binding the QDs by using avidin and a secondary antibody as bridges. Milk samples were detected using this hybrid immunoassay, with limits of detection (LOD) of the quinolones (0.18 ng mL −1), sulfonamides (0.17 ng mL −1) and melamine (7.5 ng mL −1), respectively. The results demonstrated that the detection limits of the integrated methods were better than required and simplified the sample pretreatment process. The developed immunoassay is suitable for high-throughput screening of low-molecular weight contaminants.

A rapid and sensitive fluoroimmunoassay based on quantum dot for the detection of chlorpyrifos residue in drinking water

A rapid and sensitive indirect competitive fluorescence-linked immunosorbent assay (cFLISA) method based on quantum dots as the fluorescence label coupled with secondary antibody (Ab2) for the detection of chlorpyrifos in drinking water has been developed. The cFLISA method allowed for chlorpyrifos determination in a liner working range of 15.2–205.5 ng mL−1. The 50 % inhibition value (IC50) and the limit of detection (LOD) of the cFLISA were 50.2 ng mL−1 and 8.4 ng mL−1, while the IC50 and the LOD of the conventhional enzyme linked immunosorbent assay (ELISA) were 95.3 ng- mL−1 and 16.2 ng mL−1, respectively. When the concentrations of chlorpyrifos were 200, 100 and 50 ng mL−1, the recoveries ranged from 90.8 % to 108.2 % with a coefficient of variation (CV) of 7.5 %–15.2 %. In water sample analysis, the results of cFLISA were similar to those obtained from a cELISA and a high performance liquid chromatography (HPLC) method, while the detection time by cFLISA was reduced 0.5 h compared with ELISA. It showed that cFLISA could be used as a new screening method for the detection of pesticide residue.

A Fluoroimmunoassay Based on Quantum Dot−Streptavidin Conjugate for the Detection of Chlorpyrifos

A rapid and sensitive competitive fluorescence-linked immunosorbent assay (cFLISA) based on quantum dot-streptavidin conjugate (QDs-SA) was developed for the detection of chlorpyrifos in drinking water. The QDs-SA conjugate, which consists of 3-mercaptopropyl acid-stabilized CdTe nanoparticle QDs and streptavidin (SA) made through the active ester method, was employed to improve the sensitivity of QDs-SA-cFLISA. The 50% inhibition concentration (IC50) and the limit of detection (LOD) were 28.5 and 3.8 ng mL(-1), respectively. QDs-SA-cFLISA increased sensitivity 5.5-fold and reduced detection time by 1 h compared with conventional enzyme-linked immunosorbent assay (ELISA). With chlorpyrifos concentrations of 100, 50, and 20 ng mL(-1), recoveries ranged from 85.9% to 105.3% with coefficients of variation ranging from 6.3% to 13.5%. This study demonstrated that QDs-SA-cFLISA was more rapid and sensitive than conventional ELISA. Therefore, it can be used as a novel screening method for the detection of pesticide residues.

Development of sensitivity-improved fluorescence-linked immunosorbent assay using a fluorescent single-domain antibody against the bioactive naphthoquinone, plumbagin

A fluorescent single-domain antibody (fluobody), a fusion protein of a green fluorescent protein extracted from Aequorea coerulescens (AcGFP), a mutant that has been codon-optimized for mammalian expression, and a single-chain variable fragment antibody (scFv), against plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone; PL) was successfully constructed and expressed in Escherichia coli. The expressed fluobody was purified, refolded, and characterized to develop a speedy, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA) for the determination of PL. In this study, two kinds of fluobody containing PL-scFv at the N-terminus of AcGFP (N fluobody) or the C-terminus of AcGFP (C fluobody) were constructed with flexible amino acid linker (Gly(4)Ser)(2) between PL-scFv and AcGFP for comparative purposes. Characterization of the fluobodies revealed that the C fluobody has better properties as a probe for FLISA than the N fluobody because the fluorescence intensity of C fluobody was 18-fold higher than that of N fluobody. Moreover, C fluobody exhibited a fourfold-higher binding affinity than the N fluobody. More interestingly, the limit of detection for PL measurement in FLISA (24 ng mL(-1)) was improved to eightfold higher than that in conventional ELISA (0.2 microg mL(-1)), indicating that a sensitive immunoassay could be developed by using fluobody instead of monoclonal antibody or scFv.

Agonist Dose-dependent Phosphorylation by Protein Kinase A and G Protein-coupled Receptor Kinase Regulates β2 Adrenoceptor Coupling to Gi Proteins in Cardiomyocytes

Adrenoceptors receptors (ARs) play a pivotal role in regulating cardiovascular response to catecholamines during stress. beta(2)ARs, prototypical G protein-coupled receptors (GPCRs), expressed in animal hearts, display dual coupling to both G(s) and G(i) proteins to control the adenylyl cyclase-cAMP dependent protein kinase A (PKA) pathway to regulate contraction responses. Here, we showed that the beta(2)AR coupling to G(i) proteins was agonist dose-dependent and occurred only at high concentrations in mouse cardiac myocytes. Both the beta(2)AR-induced PKA activity, measured by fluorescence resonance energy transfer (FRET) imaging, and the increase in myocyte contraction rate displayed sensitivity to the G(i) inhibitor pertussis toxin (PTX). Further studies revealed that activated beta(2)ARs underwent PKA phosphorylation at a broad range of agonist concentrations. Disruption of the PKA phosphorylation sites on the beta(2)AR blocked receptor/G(i) coupling. However, a sufficient beta(2)AR/G(i) coupling was also dependent on the G protein-coupled receptor kinase (GRK)-mediated phosphorylation of the receptors, which only occurred at high concentrations of agonist (> or = 100 nm). Disruption of the GRK phosphorylation sites on the beta(2)AR blocked receptor internalization and coupling to G(i) proteins, probably by preventing the receptor's transportation to access G(i) proteins. Furthermore, neither PKA nor GRK site mutated receptors displayed sensitivity to the G(i)-specific inhibitor, G(i)CT. Together, our studies revealed distinct roles of PKA and GRK phosphorylation of the beta(2)AR for agonist dose-dependent coupling to G(i) proteins in cardiac myocytes, which may protect cells from overstimulation under high concentrations of catecholamines.

A novel quantum dot-based fluoroimmunoassay method for detection of Enrofloxacin residue in chicken muscle tissue

A rapid indirect competitive fluorescence-linked immunosorbent assay (cFLISA) based on quantum dots (QDs) as the fluorescent marker has been developed for the detection of Enrofloxacin (ENR) in chicken muscle tissue. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-mouse secondary antibody. The cFLISA method allowed for ENR determination in a liner working range of 1–100 ng mL −1 with the 50% inhibition value (IC 50) of 8.3 ng mL −1 and the limit of detection (LOD) of 2.5 ng mL −1. The recoveries for chicken muscle samples spiked with ENR at levels of 50–200 μg kg −1 ranged from 81% to 94% with coefficients of variation (CV) of 10–13%. In real chicken tissue sample analysis, the results of cFLISA were similar to those obtained from an indirect competitive enzyme-linked immunosorbent assay (cELISA) to a high performance liquid chromatography method (HPLC), which indicated that cFLISA is suitable as screening method for the monitoring of veterinary drug residues.

Characterization and application of quantum dot nanocrystal–monoclonal antibody conjugates for the determination of sulfamethazine in milk by fluoroimmunoassay

Quantum dot (Qdot) nanocrystals have been increasingly used as fluorescence labels in fluoroimmunoassays recently because of their excellent optical characteristics. In this paper, a new monoclonal antibody (MAb) against sulfamethazine (SMZ) was successfully produced and linked to Qdot nanocrystals by covalent coupling. The Qdot-MAb conjugates were characterized by SDS-PAGE and high-performance capillary electrophoresis (HPCE). An enzyme-linked immunosorbent assay (ELISA) method was utilized to evaluate the antigen-antibody binding affinity and then a novel direct competitive fluorescence-linked immunosorbent assay (cFLISA) for the detection of SMZ in milk by using Qdots as fluorescent labels was evaluated. The results showed that the 50% inhibition values (IC50) of the cFLISA were 4.3 ng/mL in milk and 5.2 ng/mL in PBS, and the limits of detection (LODs) were 0.6 ng/mL in milk and 0.4 ng/mL in PBS, respectively. The recoveries of SMZ from spiked milk samples at levels of 10-100 ng/mL ranged from 94 to 106%, with coefficients of variation (CVs) of 2.1-9.2%.

食物アレルゲンタンパク質の近赤外蛍光標識プローブによる検出

We aimed to detect the allergen proteins in food materials using recently developed near-infrared fluorescent probes. Sensitivities of this method were comparable to chemiluminescence detection methods, which are known to be sensitive. In addition, the sensitivities of this near-infrared fluorescent method were at least 10-50-times higher than those of the conventional visible fluorescent methods using Cy3 and Cy5 dyes. This method was effectively applicable to immunoblotting, dot-blotting and plate-assay (direct FLISA : fluorescence-linked immunosorbent assay) with ELISA plate. Allergen levels of the food sample were quantified by standard curves using standard allergen protein using the dot-blotting technique. This highly sensitive detection system also provided multiple detections of different allergens for different antibodies and dyes with distinct properties of wavelength. This enables high-throughput screening of characteristic allergen contents of target food materials, or cultivars. Generally, allergen proteins are recognized by patient's serum IgE. Therefore, we tried to detect patient's IgE-binding proteins, the putative allergens in foodstuffs. In this detection system, it was possible to detect IgE-binding proteins with sensitivity almost equivalent to a chemiluminescent detection system. Taken together, it was shown that this novel detection system was an effective technique for the sensitive detection and screening of food allergens.

Application of Quantum Dot−Antibody Conjugates for Detection of Sulfamethazine Residue in Chicken Muscle Tissue

A new indirect competitive fluorescence-linked immunosorbent assay (cFLISA) method for the detection of sulfamethazine (SM2) in chicken muscle tissue was demonstrated using quantum dots (QDs) as the fluorescence label coupled with secondary antibody. The sensitivity of the cFLISA was compared with that of the HPLC method. The 50% inhibition value (IC50) and the limit of detection (LOD) of the cFLISA were 7.7 and 1.0 ng/mL, respectively. When SM2 was spiked at levels of 50, 100, and 200 ng/g, recoveries ranged from 80.6% to 117.4%, with a coefficient of variation of 6.9-9.6%. In the incurred sample analysis, the amounts of SM2 quantified by cFLISA were similar to the results obtained by the HPLC method. This study shows that cFLISA could be used as a screening method in practice.

Development of Fluorescence-linked Immunosorbent Assay for High Throughput Screening of Interferon-γ

Human interferon-gamma (hIFN-gamma) is produced by lymphocytes and has a variety of biological properties. Measurement of hIFN-gamma is widely used for various immunological responses for allergic or autoimmune diseases. Enzyme-linked immunosorbent assay (ELISA) is an established immunoassay used to quantify cellular metabolites or cytokines. ELISA requires many incubation and wash steps and is not practically suitable for screening large numbers of samples. We have developed a fluorescence-linked immunosorbent assay (FLISA) method for the detection of hIFN-gamma. We measured the 50% inhibitory concentration (IC50) value of the hIFN-gamma production by interleukin (IL)-18 binding protein and anti-IL-18 monoclonal antibody. The IC50 described by FLISA was compared with that by ELISA. We developed a new system for measuring hIFN-gamma using Allophycocyanine (APC) fluorescent protein and compared it with the previous method using Cy5.5. The proposed FLISA had a smaller coefficient of variation than ELISA, and the means of coefficient of variation using the same samples measured by ELISA and FLISA were, respectively, 11.1% and 3.8%, suggesting that the edge effect often giving non-specific results may be smaller in FLISA than in ELISA. The improved FLISA system proposed is ideally suited for efficient measurements of hIFN-gamma. This homogeneous and multiplex method will be a powerful tool for high throughput screening for drug discovery research.

Development of a high-capacity homogeneous fluorescent assay for the measurement of leukotriene B 4

Current immunoassays for the measurement of leukotriene B 4 (LTB 4) typically utilize an enzyme-linked immunosorbent assay (ELISA) format that requires multiple incubations and washing steps and often expensive immunoassay kits. We have developed a bead-based, mix and read, indirect fluorescence-linked immunosorbent assay utilizing fluorometric microvolume assay technology (FMAT). The assay employs a monoclonal anti-LTB 4 antibody-coated onto goat antimouse antibody coupled polystyrene beads and an AlexaFluor-647-coupled LTB 4 ligand. Because the FMAT measurement is made only in the portion of the well volume containing the settled beads coated with AF647–LTB 4, the free label in the solution is not measured. Similarly, substances present in plasma that interfere with other immunoassays are largely ignored. The assay is robust (Z = 0.8; S/N = 250) and can be measured in the presence of relatively high concentrations of dimethyl sulfoxide or serum. It is inexpensive (<$0.10/assay) and amenable to robotics and has a sensitivity comparable to that of the most sensitive ELISA assays; the concentration of LTB 4 giving 50% inhibition (IC 50) was ca. 55 pg/ml. Cross-reactivity in the FMAT assay was comparable to that of the ELISA assay with significant cross-reactivity found only with 20-hydroxy LTB 4 and 12-epi LTB 4. Measurements of LTB 4 determined by FMAT were equivalent to those measured by standard ELISA in samples of ionophore-stimulated human neutrophils or whole blood.