Surface Plasmon Resonance for Protease Detection by Integration of Homogeneous Reaction

The heterogeneous assays of proteases usually require the immobilization of peptide substrates on the solid surface for enzymatic hydrolysis reactions. However, immobilization of peptides on the solid surface may cause a steric hindrance to prevent the interaction between the substrate and the active center of protease, thus limiting the enzymatic cleavage of the peptide. In this work, we reported a heterogeneous surface plasmon resonance (SPR) method for protease detection by integration of homogeneous reaction. The sensitivity was enhanced by the signal amplification of streptavidin (SA)-conjugated immunoglobulin G (SA-IgG). Caspase-3 (Cas-3) was determined as the model. A peptide labeled with two biotin tags at the N- and C-terminals (bio-GDEVDGK-bio) was used as the substrate. In the absence of Cas-3, the substrate peptide was captured by neutravidin (NA)-covered SPR chip to facilitate the attachment of SA-IgG by the avidin-biotin interaction. However, once the peptide substrate was digested by Cas-3 in the aqueous phase, the products of bio-GDEVD and GK-bio would compete with the substrate to bond NA on the chip surface, thus limiting the attachment of SA-IgG. The method integrated the advantages of both heterogeneous and homogeneous assays and has been used to determine Cas-3 inhibitor and evaluate cell apoptosis with satisfactory results.

Evaluation of homogeneous proximity immunoassays for preclinical bioanalysis

Drug discovery is moving at a rapid pace and a fast turnaround of bioanalytical data is needed to sustain this pace. This article focuses on the evaluation of time-saving homogeneous proximity immunoassays such as Amplified Luminescent Proximity Homogeneous Assay, Time-Resolved Fluorescence Resonance Energy Transfer and Spatial Proximity Analyte Reagent Capture Luminescence as an alternative to industry popular platforms like mesoscale discovery (MSD) and Gyrolab®. Our evaluation showed that no one platform can be considered the best for all the parameters assessed. Homogeneous proximity platforms were found to be advantageous over MSD and Gyrolab for certain applications and are herein discussed. The factors affecting the performance of homogeneous assays and appropriate corrections are discussed. The homogeneous assays, due to their flexibility, hold a lot of untapped potential for the future of bioanalysis.

Verification of Low-Density Lipoprotein Cholesterol Levels Measured by Anion-Exchange High-Performance Liquid Chromatography in Comparison with Beta Quantification Reference Measurement Procedure

Background A new lipoprotein testing method based on anion-exchange HPLC (AEX-HPLC) was recently established. We verified the accuracy of LDL-C levels, a primary therapeutic target for the prevention of cardiovascular disease (CVD), measured by AEX-HPLC comparing with LDL-C levels measured by beta quantification-reference measurement procedure (BQ-RMP), homogenous assays, and calculation methods. Methods We compared LDL-C levels measured by AEX-HPLC (adLDL-Ch: LDL-Ch and IDL-Ch) and BQ-RMP using blood samples from 52 volunteers. AdLDL-Ch levels were also compared with those measurements by homogeneous assays and calculation methods (Friedewald equation, Martin equation, and Sampson equation) using blood samples from 411 participants with dyslipidemia and/or type 2 diabetes. Results The precision and accuracy of adLDL-Ch were verified by BQ-RMP. The mean percentage bias [bias (%)] for LDL-C was 1.2%, and the correlation was y = 0.990x + 3.361 (r = 0.990). These results met the acceptable range of accuracy prescribed by the National Cholesterol Education Program. Additionally, adLDL-Ch levels were correlated with LDL-C levels measured by the 2 homogeneous assays (r > 0.967) and the calculation methods (r > 0.939), in serum samples from patients with hypertriglyceridemia. Conclusions AEX-HPLC is a reliable method for measuring LDL-C levels for CVD risk in daily clinical laboratory analyses.

P5332Magic mirror on the wall, can we reach our LDL-C goal? Comparison of calculated LDL-C and direct measured LDL-C levels in atherogenic dyslipidemia condition

Background LDL-C represents the primary lipoprotein target for reducing cardiovascular risk. LDL-C can either be calculated or measured directly. Friedewald equation has certain limitations especially with high triglyceride and low LDL-C levels. Although a number of automated direct LDL-C assays are commercially available, non of them is considered to be equivalent to the “gold standard” of direct LDL-C, beta quantitation, a complex and expensive process that is unavailable in routine clinical practice. In atherogenic dyslipidemia condition (ADC) (triglycerides≥2.3 mmol/L and HDL-C<1.0 mmol/L) non-HDL-C and remnant cholesterol are proven additional risk factors. Purpose We compared one of the direct homogeneous assays with the widely used Friedewald's and the new Martin/Hopkins methods of estimation of LDL-C to see the differences in average LDL-C, remnant cholesterol and non-HDL-C levels and in availability of less than 1.8 mmol/L of LDL-C in atherogenic dyslipidemia condition. Methods We investigated 14 906 lipid profiles from fasting blood samples of Hungarian individuals with triglycerides <4.5 mmol/L. Total cholesterol (TC), HDL-C, triglycerides (TG) and direct LDL-C (D-LDL-C) were measured directly by the enzimatic assays. We also estimated calculated LDL-C by the Friedewald's formula (F-LDL-C) and by using the new Martin/Hopkins estimation (MH-LDL-C). We have now prepared first a variant of Martin/Hopkins table in mmol/L, in which the modified adjustable factors of 2.2 are included. We determined also non-HDL-C and remnant cholesterol (RC) as a difference of non-HDL-C and F-LDL-C (F-RC), MH-LDL-C (MH-RC), D-LDL-C (D-RC). Results In the investigated population 19.25% was F-LDL-C, 15.48% MH-LDL-C and 7.92% D-LDL-C below 1.8 mmol/L. ADC occurred at 8.12%. For ADC, when F-LDL-C<1.8 mmol/L (A), mean values for F-LDL-C, MH-LDL-C, D-LDL-C and non-HDL-C were 1.23±0.4; 1.65±0.39; 2.06±0.4 and 2.46±0.5 mmol/L respectively. These mean levels were 1.01±0.36; 1.4±0.3; 1.83±0.3 and 2.15±0.34 mmol/L for MH-LDL-C<1.8 mmol/L (B). For D-LDL-C<1.8 mmol/L (C), mean values were 0.79±0.35; 1.13±0.26; 1.54±0.19 and 1.83±0.25 mmol/L respectively. The average RC values (in mmol/L) for A were F-RC: 1.23±0.36; MH-RC: 0.81±0.18; D-RC: 0.4±0.17, for B 1.14±0.33; 0.74±0.14; 0.32±0.13, and for C 1.04±0.27; 0.70±0.1; 0.29±0.12 respectively. Conclusions The Friedewald equation tends to underestimate and the homogeneous enzimatic direct LDL-C assays to overestimate the LDL-C levels compared to the new, accurate, calculated LDL-C values in atherogenic dyslipidemia condition. Based on the data presented in our investigation we should like to propose that more realistic vasculo-protective lipid status can be attained if we calculate LDL-C using the Martin/Hopkins estimation.

Successive Optimization of a Homogeneous Immunoassay for Antibody Detection in Viral Infections

Although there are extensive literature reports on the use of gold nanoparticle (AuNP) based homogeneous assays for detection of biomolecules, very few experimental description and procedures involving their preparation are described. In this study, AuNPs conjugated to Bovine Serum Albumin or Envelope protein from Dengue II were developed as a homogeneous immunoassay for antibody detection. We report here optimization of key parameters to prepare an immunoassay like conjugation protein concentration, centrifugation time, electrolyte addition and assay temperature.&nbsp; We determined that saturating protein concentrations improved AuNPs surface coverage and uniformity of the assay and addition of sodium chloride improved sensitivity of the antibody detection method and assay stability.&nbsp; Furthermore, we showed that dynamic light scattering can be used to monitor changes in gold nanoparticles in the preparation and detection steps. Additionally, numerical simulations of the plasmonic optical response of AuNPs were carried out to scan for size-dependent response of the AuNPs. The AuNPs homogeneous immunoassay developed was further used in the detection of antibodies in vitro to detect Dengue virus infection.

Small Dense Low-Density Lipoprotein as Biomarker for Atherosclerotic Diseases

Low-density lipoprotein (LDL) plays a key role in the development and progression of atherosclerosis and cardiovascular disease. LDL consists of several subclasses of particles with different sizes and densities, including large buoyant (lb) and intermediate and small dense (sd) LDLs. It has been well documented that sdLDL has a greater atherogenic potential than that of other LDL subfractions and that sdLDL cholesterol (sdLDL-C) proportion is a better marker for prediction of cardiovascular disease than that of total LDL-C. Circulating sdLDL readily undergoes multiple atherogenic modifications in blood plasma, such as desialylation, glycation, and oxidation, that further increase its atherogenicity. Modified sdLDL is a potent inductor of inflammatory processes associated with cardiovascular disease. Several laboratory methods have been developed for separation of LDL subclasses, and the results obtained by different methods can not be directly compared in most cases. Recently, the development of homogeneous assays facilitated the LDL subfraction analysis making possible large clinical studies evaluating the significance of sdLDL in the development of cardiovascular disease. Further studies are needed to establish guidelines for sdLDL evaluation and correction in clinical practice.

A platform for the development of novel biosensors by configuring allosteric transcription factor recognition with amplified luminescent proximity homogeneous assays

Using isolated allosteric transcription factors as recognition elements, a versatile platform was established in vitro to develop sensitive biosensors for the detection of various chemicals.