Fluorometric Enzyme-linked Immunosorbent Assay Research Articles

IgE sensitivity to Malassezia pachydermatis and mite allergens in dogs with atopic dermatitis

In this study, dogs with atopic dermatitis were separated into non-food-induced atopic dermatitis (NFIAD) group (n = 15) and food-induced atopic dermatitis (FIAD) group (n = 37) based on an elimination diet test. IgE reactivity for crude Malassezia pachydermatis (M. pachydermatis) and house dust mites (HDM) allergen extracts was investigated in the two groups using fluorometric enzyme-linked immunosorbent assay (ELISA) and intradermal skin test (IDST). Nine (60%) of the 15 dogs in NFIAD group and 6 (16%) of the 37 dogs in FIAD group showed specific IgE for M. pachydermatis (Mann-Whitney U-test, P < 0.01). By immunoblotting analysis, the pooled serum samples from dogs with IgE for M. pachydermatis showed IgE reactivity for 50 kDa protein of M. pachydermatis. Twelve (80%) of the 15 dogs in NFIAD group and 8 (22%) of the 37 dogs in FIAD group showed specific IgE for HDM (Mann-Whitney U-test, P < 0.01). In addition, the dogs in NFIAD group significantly show a positive IDST to M. pachydermatis and HDM extracts compared with the dogs in FIAD group. The results suggest that dogs with NFIAD are at increased risk of becoming sensitized to the normal commensal organism M. pachydermatis compared with dogs with FIAD, perhaps co-sensitization occurred due to an HDM protease antigen’s, Der f 1 and/or Der p 1, proteolytic activity related epidermal skin barrier defects. Treatment to limit skin colonization may thus be especially important in NFIAD.

Alkaline Phosphatase-Triggered in Situ Formation of Silicon-Containing Nanoparticles for a Fluorometric and Colorimetric Dual-Channel Immunoassay

Enzyme-triggered in situ chromogenic and/or fluorogenic reactions under accessible conditions are significant for developing enzyme activity and related spectroscopic assays. Here, we describe a facile one-pot synthetic strategy to prepare silicon-containing nanoparticles with yellow-green fluorescence and orange-red color by mixing N-[3-(trimethoxysilyl)propyl]ethylenediamine and p-aminophenol (AP) in aqueous solution at a mild temperature. Encouraged by the AP-regulated simple synthetic procedure and the generation of AP from alkaline phosphatase (ALP)-catalyzed hydrolysis of 4-aminophenol phosphate (APP), a fluorometric and colorimetric dual-readout ALP activity assay can be rationally envisioned and developed by employing APP as the substrate. In the wake of the good analytical performance of such ALP activity assay and its successful combination with enzyme-linked immunosorbent assay (ELISA), corresponding fluorometric and colorimetric dual-readout ALP-based ELISA has been constructed for highly sensitive and quantitative determination of human prostate-specific antigen (PSA), the key biomarker of prostate cancer in human serum. The convincing performance in evaluating the PSA level in serologic tests unambiguously reveals the great potential of our proposed fluorometric and colorimetric dual-channel immunoassay in early clinical diagnosis by monitoring disease biomarkers.

Cancer-Associated Fibroblasts Accelerate Malignant Progression of Non-Small Cell Lung Cancer via Connexin 43-Formed Unidirectional Gap Junctional Intercellular Communication

Background/Aims: Gap junctions, which are assembled by connexins, can directly connect the cytoplasm of adjacent cells and enable gap junctional intercellular communication (GJIC) as well as metabolic coupling between neighboring cells. Here, we investigated the role of connexin 43 (Cx43) and its derived GJIC in the interplay between non-small cell lung cancer (NSCLC) cells and cancer-associated fibroblasts (CAFs). Methods: CAFs and NSCLC cells were co-cultured with direct contact and separated using flow cytometry. Glucose uptake, lactate production, and the expression and activity of PKM-2 and LDH-A in sorted CAFs were measured by a colorimetric assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Meanwhile, E-cadherin and N-cadherin expression and the migration and invasion of sorted NSCLC cells were detected by western blotting, wound width, and Transwell assays. Pyruvate, acetyl-CoA, and citric acid levels, ATP levels, and LDH-B and α-KG activity in sorted NSCLC cells were determined by a colorimetric or fluorometric assay and ELISA, respectively. Functional GJIC between cells and the subcellular location of connexins were detected by a “Parachute” assay and immunofluorescence. Levels of α-SMA, Cx43, and LDH-B in tissue from patients with NSCLC were determined by immunohistochemistry. Results: Cx43 accumulated in the plasma membrane, which favored the assembly of asymmetric unidirectional GJIC from CAFs to NSCLC cells. CAFs underwent increased aerobic glycolysis and promoted the epithelial-mesenchymal transition, migration, and invasion of NSCLC cells. In contrast, NSCLC cells experienced enhanced oxidative phosphorylation upon CAF stimulation, with an increase in ATP generation and thereby activation of the PI3K/Akt and MAPK/ERK pathways. Metabolic coupling between CAFs and NSCLC cells was under the strict control of Cx43-formed unidirectional GJIC. Patients with high tri-expression of α-SMA, Cx43, and LDH-B had the shortest overall survival and relapse-free survival compared with those with individual overexpression or high bi-expression. Conclusion: Cx43-formed unidirectional GJIC plays a critical role in mediating close metabolic cooperation between CAFs and NSCLC cells to support the malignant progression of NSCLC.

Rosuvastatin Inhibits Interleukin (IL)-8 and IL-6 Production in Human Coronary Artery Endothelial Cells Stimulated With Aggregatibacter actinomycetemcomitans Serotype b.

Rosuvastatin exhibits anti-inflammatory effects and reduces periodontal diseases and atherosclerosis; however, its role in regulating periodontopathogen-induced endothelial proinflammatory responses remains unclear. The purpose of this study is to determine whether rosuvastatin can reduce the proinflammatory response induced by Aggregatibacter actinomycetemcomitans (Aa) in human coronary artery endothelial cells (HCAECs). HCAECs were stimulated with purified Aa serotype b lipopolysaccharide (LPS) (Aa-LPS), heat-killed (HK) bacteria (Aa-HK), or live bacteria. Expression of Toll-like receptors and cellular adhesion molecules were evaluated by fluorometric enzyme-linked immunosorbent assay. Endothelial cell activation was evaluated by quantifying nuclear factor (NF)-kappa B-p65 and cytokine expression levels by quantitative polymerase chain reaction and flow cytometry. Effect of rosuvastatin in expression of the atheroprotective factor Krüppel-like factor 2 (KLF2) and cytokines were also studied using similar approaches. HCAECs showed increased interleukin (IL)-6, IL-8, intercellular adhesion molecule 1, and platelet endothelial cell adhesion molecule 1 expression when stimulated with Aa-LPS or Aa-HK. NF-κB-p65 activation was induced by all antigens. Aa-induced IL-6 and IL-8 production was inhibited by rosuvastatin, particularly at higher doses. Interestingly, reduced IL-6 and IL-8 levels were observed in HCAECs stimulated with Aa in the presence of higher concentrations of rosuvastatin. This anti-inflammatory effect correlated with a significant increase of rosuvastatin-induced KLF2. These results suggest Aa-induced proinflammatory endothelial responses are regulated by rosuvastatin in a mechanism that appears to involve KLF2 activation. Use of rosuvastatin to prevent cardiovascular disease may reduce risk of endothelial activation by bacterial antigens.

Quiescence Loosens Epigenetic Constraints in Bovine Somatic Cells and Improves Their Reprogramming into Totipotency.

Reprogramming by nuclear transfer (NT) cloning forces cells to lose their lineage-specific epigenetic marks and reacquire totipotency. This process often produces molecular anomalies that compromise clone development. We hypothesized that quiescence alters the epigenetic status of somatic NT donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) fibroblasts versus nonstarved mitotically selected (G1) controls. We show that G0 chromatin contains reduced levels of Polycomb group proteins EED, SUZ12, PHC1, and RING2, as well as histone variant H2A.Z. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay, we further show that G0 induced DNA and histone hypomethylation, specifically at H3K4me3, H3K9me2/3 and H3K27me3, but not H3K9me1. Collectively, these changes resulted in a more relaxed G0 chromatin state. Following NT, G0 donors developed into blastocysts that retained H3K9me3 hypomethylation, both in the inner cell mass and trophectoderm. G0 blastocysts from different cell types and cell lines developed significantly better into adult offspring. In conclusion, serum starvation induced epigenetic changes, specifically hypotrimethylation, that provide a mechanistic correlate for increased somatic cell reprogrammability.

When assay format matters: a case study on the evaluation of three assay formats to support a clinical pharmacokinetic study.

Data generated using various immunoassay methods are an integral part of the development of protein therapeutics. These assays are used in clinical and preclinical studies to establish the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics as well as to assess the immunogenicity properties of a therapeutic. PK assays measure therapeutic levels post-administration which is essential for understanding the effective dose and dose regimen for a therapeutic. Anti-OX40L is a fully humanized monoclonal antibody designed for the potential treatment of an autoimmune disease. The anti-OX40L human PK assay is required to be sensitive, robust, and precise. To address challenges due to assay sensitivity and reproducibility, as well as assay technology limitations, during development of the anti-OX40L human PK assay, three different assays, including an MSD-based electrochemiluminescence assay (ECLA), a fluorometric enzyme-linked immunosorbent assay (ELISA), and a colorimetric ELISA, were evaluated. The MSD-based assay was the most sensitive but posed risk of inter-well signal crosstalk. The fluorescence ELISA fell short on reproducibility. The colorimetric ELISA was ultimately chosen for supporting sample analysis. This paper presents characterization data obtained from each of these assay formats, challenges that were encountered in the development of the assay, and the rationale for selecting the ultimate assay format.

48 QUIESCENCE INDUCES LONG-TERM EPIGENETIC CHANGES IN BOVINE FIBROBLASTS THAT IMPROVE THEIR REPROGRAMMING INTO CLONED ANIMALS

Cloning by somatic cell nuclear transfer (SCNT) forces cells to lose their lineage-specific epigenetic marks and become totipotent again. This reprogramming process often results in epigenetic and transcriptional aberrations that compromise development. Development rates after SCNT can thus serve as a functional assay for genome-wide epigenetic reprogramming. Dolly the sheep, the first mammalian SCNT clone, was derived from a donor cell that was induced into quiescence by serum starvation. We hypothesized that quiescence alters the epigenetic status of donor cells and elevates their reprogrammability. To test this idea, we compared chromatin composition and cloning efficiency of serum-starved quiescent (G0) bovine adult male fibroblasts versus non-starved, diploid G1 controls. Mechanically synchronized G1 cells were generated by manual selection or mitotic shake-off and processed within 3 h post-mitosis. Based on morphological assessment and 5-ethyl-2′-deoxyuridine (EdU) incorporation during continuous labelling, &gt;93% of cells were captured in G1. Using quantitative confocal immunofluorescence microscopy and fluorometric enzyme-linked immunosorbent assay (ELISA), we show that G0 fibroblasts were significantly hypomethylated at lysines (K) of histone 3 (H3), specifically H3K4me3, H3K9me2, H3K9me3, and H3K27me3, but not H3K9me1. They were also significantly hypoacetylated at H3K9 and H4K5, hyperacetylated at H4K12, and unchanged at H4K16 positions. Furthermore, G0 cells significantly down-regulated the nuclear abundance of RNA polymerase II, histone variant H2A.Z, as well as polycomb group proteins EED, SUZ12, PHC1, and RING2. Following NT into metaphase-arrested oocytes, G0 chromatin condensed slower than that of G1 cells, indicating a more relaxed configuration. After 7 days of in vitro culture, H3K9me3, but not H4K4me3, H3K27me3, SUZ12, and RING2, remained hypomethylated in G0- versus G1-derived NT blastocysts, both in the inner cell mass and trophectoderm (730 v. 550 nuclei from 55 v. 42 G0 v. G1 blastocysts, respectively; n = 7 NT runs). Reduced H3K9me3 levels correlated with significantly increased mRNA abundance of the H3K9me3-specific histone demethylase KDM4B (or JMJD2B) in NT blastocysts. Expression of other pluripotency-related factors (NANOG, SOX2, STELLA, and IIFITM3), imprinted genes (SNRPN), and histone demethylases (KDM4A) was not affected in G0-derived blastocysts (32 G0 v. 55 G1 blastocysts; n = 4). Following NT, G0 donors developed significantly better into cloned blastocysts (175/382 = 46% v. 122/332 = 37% for G0 v. G1, respectively; n = 7, P &lt; 0.05). Likewise, after transfer into surrogate mothers, G0-derived blastocysts developed significantly better into live calves (5/18 = 28% v. 1/25 = 4% for G0 v. G1, respectively; n = 2, P &lt; 0.05). In conclusion, quiescence induced long-term epigenetic changes, specifically H3K9me3 hypomethylation, that correlated with increased donor cell reprogrammability. This research was supported by FRST C10X0303.

Elevated Urinary Levels of Vitamin D-Binding Protein in the Inhabitants of a Cadmium Polluted Area, Jinzu River Basin, Japan

Environmental cadmium (Cd) pollution and its effects on human health are still important issues. The most severe and representative manifestation of chronic Cd intoxication is Itai-itai disease, which is a syndrome that includes renal tubular dysfunction, osteomalacia, and generalized pain due to multiple bone fractures. The whole mechanism of how renal dysfunction relates to the development of bone lesions is unresolved. Vitamin D-binding protein (DBP) binds, transports and activates vitamin D, which plays a major role in calcium homeostasis and bone turnover. In this study, we measured urinary DBP levels and investigated their relationship to the markers of renal tubular dysfunction in the inhabitants of a Cd-polluted Jinzu River basin in Toyama Prefecture, Japan (Cd group). We also investigated age-matched subjects from an area known to have lower levels of Cd pollution (reference group). Urinary DBP was measured by a fluorometric enzyme-linked immunosorbent assay (ELISA), which was established in our laboratory. Significantly higher levels of urinary DBP were observed in the Cd group compared to the reference group. We observed significant positive correlations between urinary levels of DBP and renal tubular dysfunction markers in both groups. In the Cd group, urinary levels of DBP had a negative correlation with serum phosphate value. These results indicate that excretion of urinary DBP is increased after long-term Cd exposure and that the loss of DBP in urine may be linked to renal tubular dysfunction and possibly bone lesions in the inhabitants of Cd-polluted areas.

Development of the fluorometric ELISA method for determination of alpha1-microglobulinuria in a cadmium-polluted area in Japan.

To evaluate the usefulness of a newly developed fluorometric enzyme-linked immunosorbent assay (fluorometric ELISA) method for quantification of alpha1 -microglobulin (alpha1-m, protein HC) in an epidemiological study. Urinary alpha1-m in 37 female inhabitants in a cadmium-polluted area, including seven cases with Itai-itai disease, and ten inhabitants in a non-polluted area in Japan were examined. The alpha1-m was measured by both the fluorometric ELISA and a commercially available enzyme immunoassay (EIA) method to evaluate correlation of the two measurements. Concentration of beta2-microglobulin (beta2-m) was also determined in the same samples. The detection limit of this method was 3 ng/ml or less of alpha1-m. A significant, high positive correlation was obtained between the alpha1-m concentrations measured with the fluorometric ELISA and that of EIA (r = 0.95, P < 0.0001). A significant association was also shown between the alpha1-m and beta2-m concentrations in the urine samples. The concentrations of urinary alphal-m of the inhabitants in the cadmium-polluted area (mean: 6.21 mg/l, 95% confidence interval (95% CI): 4.06-9.50 mg/l) were significantly higher than those of the reference area (mean: 2.19 mg/l, 95% CI: 1.90-2.67 mg/l). The urinary alpha1-m level of the Itai-itai patients was shown to be highest at 39.63 mg/l (95% CI: 28.27 55.55 mg/l). When the cut-off value of 10 mg/l was employed, alpha1-m had a sensitivity of 100% and specificity of 100% for Itai-itai disease. These results suggest that this fluorometric ELISA is a useful tool to determine urinary alpha1-m in the epidemiological survey of renal tubular dysfunction, especially in the cadmium-polluted area of Japan.

Monoclonal antibody capture fluorometric enzyme linked immunosorbent assay for detection of porcine growth hormone in plasma

A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure growth hormone in swine plasma. Homogeneous pig growth hormone (pGH) was purified from pituitary gland, and used to raise monoclonal (mAb) and polyclonal antibodies. The mAb showing the highest reactivity was used as solid-phase antibody to capture pGH, while the rabbit biotin-labelled antibodies against pGH, followed by β-galactosidase-avidin-conjugate, were used for detection of mAb-bound pGH. The calibration graph was linear over the range 0.05–25 ng ml −1. Plasma did not interfere with the ability to measure pGH and the assay proved suitable for measuring pGH in plasma without any prior treatment of samples. Cross-reactivity and recovery tests confirmed the specificity of the method. The intra- and inter-assay coefficients of variation ranged between 7.2% and 9.7% and 12.1% and 18.7%, respectively. The analytical specificity of the method was validated by provocative tests for GH using the GH-releasing hexapeptide GHRP-6 in prepubertal gilts. The ELISA developed is sensitive, reproducible and suitable for measuring GH in pig plasma, and offers an alternative to radioimmunoassay.

A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor 165 in human plasma

A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure endogenous levels of vascular endothelial growth factor (VEGF 165) in human plasma. The ELISA can be performed in 10% human EDTA plasma, yielding a neat plasma sensitivity of 10 pg/ml or 0.2 pM. The recovery of recombinant human VEGF ( rhVEGF) added to human plasma ranges from 89 to 100%. The capture antibody depletes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coefficients of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18%, respectively. Characterization of the ELISA using plasmin derived VEGF variants suggests the assay is specific for the VEGF 165 isoform. The heterodimer, VEGF 165/110 quantitates similar to that of the intact VEGF 165 homodimer, however, the homodimers VEGF 121, VEGF 110 and the carboxy terminal domain (residues 111–165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal healthy individuals range from 20 to 141 pg/ml, with a mean of 42±22 pg/ml. There were no significant differences in VEGF levels between males and females. Circulating endogenous VEGF levels in cancer patients ranged from 32 to 418 pg/ml, averaging 129±17 pg/ml.

Specificity of an Enzyme-1 Inked Immunosorbent Assay for Dog Ige Antibody to Japanese Cedar (Cryptomeria Japonica) Pollen

ABSTRACT We developed a fluorometric enzyme-linked immunosorbent assay (ELISA) for allergen-specific IgE in dogs with the use of monoclonal anti-dog IgE; we assayed IgE antibody to Japanese cedar pollen in the sera of dogs with Japanese cedar pollinosis. To assess the specificity of this ELISA, a pooled serum sample from pollinosis dogs was subjected to gel chromatography. The peak of anti-pollen allergen IgE activity was different from the peaks of total IgA, IgG and IgM. When IgE antibody positive serum was heated at 56°C for 4 h, antibody activity was markedly reduced. Furthermore, polyclonal anti-dog IgA, IgG and IgM did not interfere with anti-pollen allergen IgE activity in the ELISA. From these results, this assay is considered to have a high specificity for dog IgE.

Fluorometric enzyme-linked immunosorbent assay for the measurement of IgE antibody to mite Dermatophagoides farinae.

We have developed a fluorometric enzyme-linked immunosorbent assay for measuring IgE antibody to Dermatophagoides farinae. Polystyrene microplates were coated with proteins extracted from the mites. The IgE antibody which attached to the solid-phase antigen was detected by anti-IgE antibody conjugated with beta-galactosidase. Four-methylumbelliferyl-beta-D-galactoside was used as the enzyme substrate and the fluorescence intensity of the reaction product was measured. The antibody levels determined by this method well correlated with those determined by the radioallergosorbent test (RAST). This method is simpler and less expensive to carry out than the RAST when dealing with a large number of serum specimens for seroepidemiological studies of asthma and nasal allergy.