Fluorescent Immunosorbent Assay Research Articles

Fluorescent immunoassay for chloramphenicol based on the label-free polyadenine-mediated spherical nucleic acids triggered signal amplification.

A fluorescent immunosorbent assay incorporating signal amplification away from the surface of spherical nucleic acid (SNA) was developed for the detection of chloramphenicol (CAP). Through the conjugation of antibodies and poly-adenine (polyA) DNA onto the surface of gold nanoparticles (AuNPs), the fabrication of the nano-immunoprobe was achieved in a more straightforward and cost-effective manner. Moreover, a strategy utilizing the hybridization chain reaction (HCR) in the amplification step was devised, with particular attention given to the enzyme inhibition associated with SNA. The results demonstrated good performance on CAP detection with a linear range of 0.01-5ng/L with a detection limit of 0.005ng/L. The significance of this work mainly lies in the polyA-SNA-based immunoprobe and the thoughtful design to prevent enzyme inhibition.

Enzyme‐free photothermally amplified fluorescent immunosorbent assay (PAFISA) for sensitive cytokine quantification

AbstractCytokine monitoring has attracted great attention due to its significance in the diagnosis and treatment of many diseases, such as tumors, microbial infections, and immunological diseases. Enzyme‐linked immunosorbent assay (ELISA) is one of the most popular methods in cytokine detection, ascribing to the lavish signal amplification methods in the ELISA platform. In addition to classical enzymes, other signal amplifiers such as fluorescent probes, artificial nano‐enzymes, and photothermal reagents have been applied to reduce the detection limit and produce more sensitive ELISA kits. Due to the accumulative effect of heat, photothermal reagents are promising materials in the signal amplification of ELISA. However, the lack of efficient photothermal generation material at an aggregate scale may delay the further development of this area. In this contribution, based on an efficient organic photothermal aggregate material, an enzyme‐free photothermally amplified fluorescent immunosorbent assay system consisting of an assay microfluidic chip and detecting platform was developed. The photothermal nanoparticles with highly efficient photothermal conversion by harvesting energy via excited‐state intramolecular motions and enlarging molar absorptivity were successfully prepared. The detection concentration at 50 pg/mL of interleukin‐2 was achieved, realizing a signal improvement of detection limits by 20‐fold compared to that of previously reported photothermal ELISA. The microscopic imaging integrated with plane sweeping technology provided high spatial resolution and precision, indicating the potential of achieving high throughput profiling at the microscale. Moreover, as an alternative excitation source, light‐emitting diode not only provided a more affordable and miniaturized detection system but also revealed the great feasibility of intramolecular motion‐induced photothermy nanoparticles for biological analyses.

Detection of quinocetone in animal feed by quantum dots‐based indirect competitive fluorescent immunosorbent assay

AbstractA sensitive monoclonal antibody against quinocetone (QCT) was produced. Subsequently, the quantum dots‐based indirect competitive fluorescent immunosorbent assay (ic‐FLISA) for the rapid detection of QCT in animal feed was developed. The 50% inhibitory concentration (IC50) was 4.39 ng/ml, the limit of detection was 0.252 ng/ml, and the detection ranged from 0.515 to 37.150 ng/ml. The recoveries of the spiked QCT in the animal feed ranged from 96.48 to 102.52% and the coefficient of variation ranged from 2.20 to 6.59%. The established ic‐FLISA has high sensitivity which can provide an ideal tool to detect QCT in animal feed.

Quantum dots‐based fluorescence immunoassay for detection of tiamulin in pork

AbstractTiamulin (TML) is a kind of diterpenoid veterinary antibiotic. If abused, it will cause residues in edible tissues of animals and endanger human health. Thus, a facile, sensitive, and accurate method is essential for TML detection. In this study, TML complete antigen was synthesized by oximation and active ester method, and monoclonal antibodies with high specificity and sensitivity were prepared. ZnCdSe/ZnS quantum dots (QDs) coupled with goat anti‐mouse IgG were used as a fluorescence detection probe to establish indirect competitive fluorescent immunosorbent assay (icFLISA) for the determination of TML in pork. The 50% inhibitory concentration (IC50) of the icFLISA was 0.069 ng/ml, and the limits of detection (LOD) were 0.309 pg/ml. The recovery of the spiked pork samples for the icFLISA ranged from 85.6 to 96.8%, and the coefficient of variation (CV) was less than 9.4%. The results showed that the icFLISA based on ZnCdSe/ZnS QDs was more sensitive than the traditional indirect competitive enzyme‐linked immunosorbent assay for the detection of TML in edible animal tissues.

IgG Subclass Analysis in Patients with Chagas Disease 4Years After Benznidazole Treatment.

In humans, Trypanosoma cruzi infection is controlled by a complex immune response. Immunoglobulin G (IgG) is important for opsonizing blood trypomastigotes, activating the classic complement pathway, and reducing parasitemia. The trypanocidal activity of benznidazole is recognized, but its effects on the prevention and progression of Chagas disease is not well understood OBJECTIVE: We aimed to evaluate the levels of total IgG and cross-specific IgG subclasses in patients with chronic Chagas disease of different clinical forms before and after 4years of benznidazole treatment. Eight individuals with the indeterminate form and nine with the cardiac form who completed the treatment protocol were evaluated. The levels of total IgG and IgG1, IgG2, IgG3, and IgG4 isotypes were quantified in the serum of each individual using the fluorescent immunosorbent assay. The results are expressed as relative fluorescence unit. Patients with chronic Chagas disease presented decreased levels of total IgG at 48months after benznidazole treatment. Increased IgG1 and decreased IgG3 levels were observed in patients with the cardiac form and those with exacerbated clinical forms. In addition, a decrease in the IgG3/IgG1 ratio was observed in individuals with the cardiac form of Chagas disease. Benznidazole administration in the chronic phase differentially changes IgG subclasses in patients with cardiac and indeterminate forms, and monitoring the IgG3 level may indicate the possible prognosis to the cardiac form or worsening of the already established clinical form.

On-Site Determination of Classical Swine Fever Virus (CSFV) by a Fluorescent Microsphere-Based Lateral Flow Immunoassay Strip (FM-LFIAs) Based on Monoclonal Antibodies

Classical swine fever (CSF) is a highly infectious and lethal multisystem disease caused by the classical swine fever virus (CSFV). This virus has resulted in huge economic losses in the porcine industry. Rapid detection of CSFV is needed in order to prevent and control the early transmission of CSFV. In this study, nine hybridoma cell lines that secrete monoclonal antibodies against CSFV (anti-CSFV-mAb) were prepared using the cell fusion and cell surface fluorescent immunosorbent assay (CSFIA). A rapid, sensitive, and specific fluorescent microsphere-based lateral flow immunoassay strip (FM-LFIAs) based on the use of paired CSFV-mAbs was developed. The sensitivity of the FM-LFIAs for CSFV detection was 5.28 ng/mL with a linear range from 9.77 to 625 ng/mL. During clinical sample assays, the FM-LFIAs results were consistent with the measurements obtained using the reverse transcription – polymerase chain reaction (RT-PCR), highlighting the practical application of FM-LFIAs for the on-site detection of CSFV.

DLISA: A DNAzyme-Based ELISA for Protein Enzyme-Free Immunoassay of Multiple Analytes.

A DNAzyme-based ELISA, termed DLISA, was developed as a novel protein enzyme-free, triply amplified platform, combining a catalytic and molecular beacon (CAMB) system with a cation exchange reaction for ultrasensitive multiplex fluorescent immunosorbent assay. Classical ELISA, which employs protein enzymes as biocatalysts to afford amplified signals, suffers from poor stability caused by the irreversible denaturation of these enzymes under harsh conditions, such as heat and acidity. Compared with proteins, nucleic acids are more stable and adaptable, and they can be easily produced using a commercial DNA synthesizer. Moreover, the catalytic and cleavage activities of DNAzyme can be achieved in solution; thus, no enzyme immobilization is needed for detection. Taken together, these attributes suggest that a DNAzyme-based ELISA detection approach will be more robust than current ELISA assays. Importantly, the proposed triply amplified DLISA immunoassay method shows ultrasensitive detection of such targets as human IgG with a detection limit of 2 fg/mL (3 × 10(-17) M), which is well within the range of many important disease biomarkers. DLISA can also be used to construct a sensing array for simultaneous multiplexed detection. With these merits, this high-throughput, stable, simple, sensitive, and low-cost multiplex fluorescence immunoassay shows promise for applications in clinical diagnosis.

Abstract 532: GRK4 Palmitoylation is Necessary for Membrane Association and Dopamine-1 Receptor Activity

The renal dopaminergic system regulates a large percentage of renal sodium excretion under sodium loaded conditions; however, not much is known about intracellular dopaminergic regulatory mechanisms. The expression of the principal sodium regulatory dopaminergic receptor (the D1R) is negatively modulated by the G protein linked receptor kinase type 4 (GRK4) through phosphorylation while the D1R is expressed in the plasma membrane. Therefore, we hypothesized that mechanisms associated with GRK4 membrane localization might be associated with its activity. Using human renal proximal tubule cell lines (RPTC), we previously demonstrated that GRK4 is palmitoylated using 17-octadecynoic acid (17-ODYA) and click chemistry and that fenoldopam (FEN, D1R agonist) decreased palmitoylation only in normal cAMP-coupled RPTC. Here we expand on those studies by showing that GRK4 palmitoylation was inhibited by 2-bromopalmitate (2BP, 100 μmol/L, palmitoylation inhibitor) which shifted GRK4 expression from the membrane to the cytoplasm. We used two different techniques to show loss of GRK4 membrane association when GRK4 palmitoylation was inhibited. The first technique involved classic cell fractionation using ultracentrifugation and western blot identification of GRK4 in membrane protein fractions (VEH 0.174±0.023 RFU, 2BP 0.091±0.013 RFU, P<0.05, N=3) The second technique utilized a novel membrane-associated fluorescent linked immunosorbent assay (mFLISA) to demonstrate the decrease in GRK4 membrane association (VEH 13,568±664 RFU, 2BP 5,155±974 RFU, P<0.05, N=12). Additionally, we showed that inhibiting GRK4 palmitoylation ledto D1-like plasma membrane recruitment using a non-cell permeable fluorescent D1-like antagonist bodipy630 SKF83566 (VEH 58,242±3858 RFU, 2BP 80,153±4061 RFU, P<0.01, N=6). In summary, we demonstrated a novel D1R regulatory mechanism through GRK4 palmitoylation, affecting its localization and activity.

Novel multiplex fluorescent immunoassays based on quantum dot nanolabels for mycotoxins determination

The aim of this manuscript was the development of easy-to-operate quantum dots (QDs)-based immunochemical techniques for simultaneous screening of several mycotoxins in cereals. Two different approaches for multiplex fluorescent immunosorbent assay (FLISA) were employed. In the first approach a multiwell plate in which the different wells express a different mycotoxin (deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1) was considered as a multiplex because each sample was pretreated once and then will be distributed over a series of wells within the same plate (single-analyte multiplex, SAM). The entire assay allows the simultaneous determination of all compounds. For the double-analyte multiplex (DAM) two different specific antibodies were co-immobilized in one single well. Zearalenone and aflatoxin B1 were simultaneously determined, provided their conjugates are labeled with QDs which are fluorescent in different parts of the spectrum, by scanning the assay outcome at two different wavelengths. The limits of detection (LOD) for the simultaneous determination of deoxynivalenol, zearalenone, aflatoxin B1, T2-toxin and fumonisin B1 by SAM FLISA were 3.2, 0.6, 0.2, 10 and 0.4µgkg−1, respectively, while for the DAM FLISA they were 1.8 and 1µgkg−1 for zearalenone and aflatoxin B1, respectively. SAM FLISA principle was also presented in a qualitative on-site format and tested for on-site multiplex determination of four mycotoxins in cereals. The achieved cut-off values of 500, 100, 2 and 100µgkg−1 for deoxynivalenol, zearalenone, aflatoxin B1 and T2-toxin respectively. For simplification of multiassay results' evaluation the conjugates with QDs of different colors were used.

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.

Barakol-induced apoptosis in P19 cells through generation of reactive oxygen species and activation of caspase-9

Ethnopharmacological relevance Barakol, an anxiolytic agent isolated from Senna siamea leaves which has been traditionally used for producing natural sleep, has been described as toxic to patients. Aim of the study The aim of current study was to investigate the molecular mechanism of barakol-induced toxicity in mouse embryonal carcinoma P19 cell model. Materials and methods XTT assay was used to determine cell viability in P19 cells treated with barakol. Apoptotic cells were detected by Hoechst 33342 staining. Intracellular reactive oxygen species (ROS) generation was analyzed by flow cytometry using a fluorescent dye, DCFH-DA. Detection of apoptotic protein expression in P19 cells was performed by Western blot analysis. Caspase-9 activity was measured using a fluorescent immunosorbent enzyme assay kit. Results Treatment with barakol decreased cell viability in a concentration- and time-dependent manner with an IC 50 value of 1.5 mM in 24-h treated cells. A Hoechst 33342 assay revealed that barakol cytotoxicity was due to a significant increase in the number of apoptotic cells. Different scavengers to characterize ROS were utilized and revealed that hydroxyl radicals played a major role in ROS-induced apoptosis in barakol-treated cells. Western blot analysis demonstrated that barakol-induced apoptosis was mediated by the increase in expression ratio of Bax/Bcl-2. Furthermore, increase in caspase-9 activity after exposure to barakol for 24 h was also observed. Pretreatment of cells with N-acetyl- l-cysteine (NAC) attenuated intracellular ROS generation, the Bax/Bcl-2 protein expression, and apoptosis. Conclusions The mechanism of barakol-mediated toxicity in P19 cells is mainly associated with the ROS generation, followed by the imbalance of the Bax/Bcl-2 ratio, and caspase-9 activation leading to apoptotic cell death. Pretreatment of cells with NAC could antagonize the toxicity produced by barakol.

Detection of ricin and other ribosome-inactivating proteins by an immuno-polymerase chain reaction assay

Ribosome-inactivating proteins (RIPs) are plant proteins with enzymatic activity, classified as type 1 (single chain) or type 2 (two chains). They are identified as rRNA N-glycosidases (EC 3.2.2.22) and cause an irreversible inhibition of protein synthesis. Among type 2 RIPs, there are potent toxins (ricin is the best known) that are considered as potential biological weapons. The development of a fast and sensitive method for the detection of biological agents is an important tool to prevent or deal with the consequences of intoxication. In this article, we describe a very sensitive immuno-polymerase chain reaction (IPCR) assay for the detection of RIPs—a type 1 RIP (dianthin) and a type 2 RIP (ricin)—that combines the specificity of immunological analysis with the exponential amplification of PCR. The limit of detection (LOD) of the technique was compared with the LODs of the conventional immunological methods enzyme-linked immunosorbent assay (ELISA) and fluorescent immunosorbent assay (FIA). The LOD of IPCR was more than 1 million times lower than that of ELISA, allowing the detection of 10 fg/ml of dianthin and ricin. The possibility to detect ricin in human serum was also investigated, and a similar sensitivity was observed (10 fg/ml). IPCR appears to be the most sensitive method for the detection of ricin and other RIPs.

Elution characteristics of tobramycin from polycaprolactone in a rabbit model.

This study investigated the elution characteristics of tobramycin from polycaprolactone, a bioabsorbable polymer, in a rabbit model. Sixty rabbits were divided into two groups. Group 1 had polycaprolactone rods impregnated with 6% tobramycin surgically implanted into the proximal femoral intramedullary canal. Group 2 received polymethylmethacrylate rods of like size, shape, and antibiotic concentration. Serum and urine samples were obtained, and tobramycin levels were determined via fluorescent immunosorbent assay. Rabbits were sacrificed as long as 56 days after surgery. Local bone tobramycin concentration was determined using the agar diffusion method. Polycaprolactone delivered a significantly higher peak bone concentration of tobramycin (22.4 microg/mL) than did polymethylmethacrylate (13.59 microg/mL). Polycaprolactone also had a more gradual decrease in local tobramycin concentration than did polymethylmethacrylate. Neither polycaprolactone nor polymethylmethacrylate yielded consistently detectable (> 0.1 microg/mL) serum tobramycin levels. Urine concentrations mirrored those seen in bone, with polycaprolactone achieving significantly higher tobramycin concentrations than did polymethylmethacrylate. Polycaprolactone had superior elution characteristics compared with polymethylmethacrylate in this lapine model, suggesting that polycaprolactone might be a promising local antibiotic delivery vehicle for the treatment of osteomyelitis.

Serum osteocalcin levels of normal children in Taiwan.

Osteocalcin is mainly secreted by osteoblasts, and then diffuses into blood which can be detected by several experimental methods. This study determined the osteocalcin level by solid phase fluorescent immunosorbent assay (Pharmacia CAP; Sweden), a well-established and accurate laboratory method for determining the minor concentration of substances in blood. A total of 332 healthy children were enrolled in the study, including 176 boys and 156 girls ranging in age from one to fifteen years. It was concluded that (1) quantitative osteocalcin (OCs) value varies between 30.2 to 41.0 ng/ml; (2) there is an incremental tendency in both sexes until puberty; (3) girls generally have a higher osteocalcin level than boys before mid-puberty (2-12 years old). The differences are statistically significant.