Orientation of capture antibodies on gold nanoparticles to improve the sensitivity of ELISA-based medical devices.

The sensitivity of ELISA-based devices strongly depends on the right orientation of antibodies on the sensor surface. The aim of this work was to increase the analytical performance of a commercial ELISA-based medical device (VIDAS®), thanks to the specific orientation of antibodies on gold nanostructured disposables. For this purpose, fPSA VIDAS® assay was used as model and the disposable providing the antigen binding surface (SPR®) was functionalized with gold nanostructures coated with monovalent half-fragment antibodies (reduced IgG, rIgG).

A method to obtain purified free light chain monomers and dimers from urine samples of patients with multiple myeloma.

Antibody light chains are synthesized in excess by plasma cells, and this excess can be secreted into biological fluids as dimers or monomers in various proportions. Structural differences between monomers or dimers of free light chains (FLC) can affect their biological functions and possibly their pathogenicity. They also may exhibit differential immune reactivity, perhaps explaining discrepant quantifications when measured by different immunoreagents.

Data about performances of whole and monovalent half-fragments antibodies in immunosorbent assays.

The data here presented are related to the research article entitled "Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies" [1] aimed to compare the performance in ELISA of whole antibodies and their corresponding monovalent half-fragments obtained by reduction. Half-fragment antibodies represent an interesting method to orient antibodies in high-sensitive immunoassays taking advantage of the free sulfhydryl groups of the hinge region [2], [3], [4] that allow their oriented binding on maleimide functionalized microplates. Data here presented describe the contribution of both chemical reduction and orientation on the antigen binding capacity of whole and half-fragments antibodies.

Sensitivity and reproducibility enhancement in enzyme immunosorbent assays based on half fragment antibodies.

The free sulfhydryl groups of the hinge region of monovalent antibody fragments (rIgG) allow the orientation of rIgG on functionalized surfaces in immunosensors. To evaluate the contribution of reduction and orientation on signal enhancement we compared the performance of whole antibodies and their rIgG in ELISA performed on polystyrene or maleimide-functionalized microplates. Monoclonal anti-horseradish peroxidase (anti-HRP) and monoclonal anti-fPSA antibodies (1 mg/mL) were reduced with 2-mercaptoethylamine (53 mM).

Free light chain UV quantification compared with immunochemical measurement: How dimers and monomers may influence the results.

Serum κ and λ free light chain (FLC) levels are important for the management of plasma cell disorders. Immunochemical measurements on automated platforms with different reagents occasionally return different results that make them not interchangeable. The reasons for this behaviour are not clear and it is not known which result is the most accurate.

Inter-assay variability in automated serum free light chain assays and their use in the clinical laboratory.

Serum κ and λ free light chain levels are markers of plasma cell proliferation, and their measurements have been included in recent guidelines by the International Myeloma Working Group for the management of patients with plasma cellular dyscrasias. Five diagnostic methods for the immunochemical quantification of serum free light chains (FLC) are available, three based on polyclonal antibodies (Freelite, The Binding Site; FLC ELISA κ and λ, Sebia; human κ and λ FLC, Diazyme Laboratories) and two on monoclonal antibodies (N Latex FLC, Siemens Healthineers; Seralite, Sebia). Several studies have shown that these methods cannot be used interchangeably for the follow-up of patients because measured κ and λ FLC concentrations may differ significantly, especially at high levels.

Dabrafenib and Trametinib prolong coagulation through the inhibition of tissue factor in BRAF mutated melanoma cells in vitro.

Background: Neoplastic cells promote a hypercoagulable state by the expression of cell surface proteins, such as tissue factor. In BRAF mutated melanoma patients upon BRAF inhibitors, a hypercoagulable state correlates with prognosis, while a down-regulation of the hemostatic parameters is observed in patients responders as compared to non responders. The present study was intended to better clarify the strict relationship between coagulation mediators and target therapy in melanoma.

Different immunoreactivity of monomers and dimers makes automated free light chains assays not equivalent.

Background The automated immunochemical serum free light chains (FLC) assays, Freelite (a polyclonal antiserum) and N Latex FLC (a mixture of monoclonal antibodies), are not interchangeable, as they may provide different results on a same sample. This study was aimed to establish if the calibrators contain FLC oligomers, and if different reactivity against monomers and dimers contributes to the discrepancy. Methods Gel filtration chromatography fractions of the calibrators were subjected to a Western blot (WB) and analyzed by each reagent.

Effects of Zuccagnia punctata extracts and their flavonoids on the function and expression of ABCB1/P-glycoprotein multidrug transporter.

Ethnopharmacological Relevance: Zuccagnia punctata extracts (ZpE) are used in ethnomedicine as antimicrobial and anti-inflammatory drugs. The pharmacological properties of ZpE and their polyphenolic components suggest that they may be used as potential modulators on the P-glycoprotein (P-gp) multidrug transporter. P-gp is well known for its role in the acquired drug resistance by tumors following chemotherapy, causing a low drug bioavailability by extruding them out of the cells.

In vitro modulation of ABCB1/P-glycoprotein expression by polyphenols from Mangifera indica.

Many plant compounds are able to modulate the activity and/or the expression of the major multidrug transporter ABCB1/P-glycoprotein (P-gp). In this study, mango (Mangifera indica L.) stem bark extract (MSBE), its main polyphenol mangiferin and the mangiferin aglycone derivative norathyriol, as well as catechin, gallic acid and quercetin, were investigated for their potential ability to influence ABCB1 gene and P-gp expression in HK-2 cells, a proximal tubule line constitutively expressing this transporter.

In vitro effects of Mangifera indica and polyphenols derived on ABCB1/P-glycoprotein activity.

Many plant-derived compounds, including polyphenols, are able to affect the function of MDR-1/P-glycoprotein (P-gp ABCB1) multidrug transporter, leading to potential herb-drug interactions. This study evaluated the effects of mango (Mangifera indica L.) stem bark extract, MSBE, and related phenols on P-gp activity in both the HK-2 proximal tubule cell line, constitutively expressing P-gp, and in a Caco-2 cell sub-line selected by resistance to vincristine (Caco-2/VCR) and overexpressing P-gp.

Effects of Devil's Claw (Harpagophytum procumbens) on the multidrug transporter ABCB1/P-glycoprotein.

Unlabelled: Devil's Claw (Harpagophytum procumbens) a plant native to Southern Africa, has historically been used in traditional medicine to treat a wide range of diseases and currently is widely employed as anti-inflammatory and pain-relieving natural remedy in Europe and other parts of the world.

Aim Of The Study: Little is known about possible herb-drug interactions arising from effects of Devil's Claw on the major drug metabolizing enzymes or transporters. This study evaluated in vitro the effects of Devil's Claw on the multidrug transporter ABCB1/P-glycoprotein.

Albumin influences expression and function of the membrane transporter P-glycoprotein in HK-2 human proximal tubular cells.

Background: In proximal tubular cells exposed to albumin genes encoding membrane transporters were found to be up-regulated or down-regulated. P-glyco-protein (Pgp) is an efflux pump which transports a variety of compounds outside the cell. In the kidney, Pgp is located mainly on the luminal side of proximal tubular cells.

P-Glycoprotein inhibitory activity of lipophilic constituents of Echinacea pallida roots in a human proximal tubular cell line.

The N-hexane root extracts from Echinacea pallida, Echinacea angustifolia and Echinacea purpurea were evaluated for inhibition of the multidrug transporter P-glycoprotein (Pgp) activity, the product of the ABCB1 gene, involved in cancer multidrug resistance (MDR) and in herb-drug or drug-drug interactions. The biological assay was performed using the human proximal tubule HK-2 cell line that constitutively expresses ABCB1. The N-hexane extracts of all three species reduced the efflux of the Pgp probe calcein-AM from HK-2 cells two-fold in a concentration-dependent manner, and E.

Modulation of P-glycoprotein activity by cannabinoid molecules in HK-2 renal cells.

1. Endogenous and synthetic cannabinoid molecules have been investigated as possible MDR-1/P-glycoprotein (P-gp) modulators in HK-2-immortalized renal cells, using calcein acetoxymethylester (calcein-AM) as a P-gp substrate. 2.

Effects of grapefruit juice on the multidrug transporter P-glycoprotein in the human proximal tubular cell line HK-2.

The multidrug transporter MDR-1 P-glycoprotein (Pgp) has been recently pointed out as an important mechanism underlying chemical interaction between drugs and many commonly ingested substances, including grapefruit juice (GFJ). Modulation of intestinal Pgp dependent transport by GFJ may lead to changes in bioavailability of drugs that are substrates of Pgp itself, by affecting their presystemic clearance. Since other cellular sites expressing Pgp and devoted to drug disposition, like kidney proximal tubules, could be involved in these pharmacokinetic interactions, we investigated the effect of GFJ on the expression and activity of Pgp in the human immortalized tubular cell line HK-2.

Influence of different chemicals on MDR-1 P-glycoprotein expression and activity in the HK-2 proximal tubular cell line.

P-glycoprotein (Pgp), the MDR-encoded membrane transporter, is physiologically expressed in normal tissues with excretory functions, including kidney proximal tubules. In a preliminary report we have shown that HK-2, an immortalized cell line from normal human proximal tubule, expresses a functional Pgp and may be considered a valuable model for in vitro investigations on the Pgp role(s) in human renal pathophysiology. The present investigation was designed to further characterize the properties of HK-2 Pgp by exploring its responsiveness to a variety of exogenous or endogenous modulators.