Clinical Performance of Immunonephelometric Assay and Chemiluminescent Immunoassay for Detection of IgG Subclasses in Chinese.

BACKGROUND: Detection of IgG subclasses (IgGSc) is vital for the diagnosis and management of disease, especially IgG4-related diseases (IgG4-RD). This study aimed to evaluate the performances of the chemiluminescent immunoassay (CLIA) for detecting IgGSc and diagnosing IgG4-RD by IgGSc. METHODS: A total of 40 individuals with IgG4-RD, 40 with primary Sjogren's syndrome (pSS), and 40 healthy controls (HCs) were enrolled. Serum samples were collected for the simultaneous detection of IgG1, IgG2, IgG3, and IgG4 by the Siemens immunonephelometric assay and the CLIA. The correlation analysis was performed, and diagnostic value was analyzed by the receiver operating characteristic (ROC) curve. RESULTS: Patients with IgG4-RD had higher IgG4 (p < 0.001) and lower IgG1 (p < 0.001) than those with pSS, and HC. The results by the Siemens immunonephelometric assay and the CLIA showed a strong correlation in detecting IgG1, IgG2, IgG3, and IgG4 (r = 0.937, r = 0.847, r = 0.871, r = 0.990, all p < 0.001, respectively). The sum of IgG1, IgG2, IgG3, and IgG4 using two assays strongly correlated with total IgG by the IMMAGE 800 (r = 0.866, r = 0.811, both p < 0.001, respectively). For discriminating IgG4-RD from pSS and HC, no significant differences were observed in CLIA IgG4 and Siemens immunonephelometric assay IgG4 (z = 0.138, p = 0.891), which provided the area under the curves (AUCs) of 0.951 (p < 0.001) and 0.950 (p < 0.001), respectively. The AUCs of CLIA IgG1 and Siemens immunonephelometric assay IgG1 in distinguishing pSS from IgG4-RD and HC were 0.761 (p < 0.001) and 0.765 (p < 0.001), respectively, with no significant differences (z = 0.228, p = 0.820). CONCLUSIONS: The CLIA and the Siemens immunonephelometric assay appeared to have good consistency with comparable diagnostic value in detecting IgGSc, especially IgG4, and IgG1 that can accurately identify IgG4-RD or pSS in clinical practice.

High-throughput nephelometry methodology for qualitative determination of aqueous solubility of chemical libraries.

The purpose of the protocol reported in this work is the solubility profiling of large chemical libraries using nephelometry. This technique allows the qualitative classification of compounds as highly, moderately, or poorly water-soluble. The described methodology is not intended to yield quantitative solubility values of the studied compounds but can be used as a primary solubility assessment of large chemical libraries, to guide hit prioritization after High Throughput Screening (HTS) campaigns.

Efficacy of antifungal agents against fungal spores: An in vitro study using microplate laser nephelometry and an artificially infected 3D skin model.

Dermal fungal infections seem to have increased over recent years. There is further a shift from anthropophilic dermatophytes to a growing prevalence of zoophilic species and the emergence of resistant strains. New antifungals are needed to combat these fungi and their resting spores. This study aimed to investigate the sporicidal effects of sertaconazole nitrate using microplate laser nephelometry against the microconidia of Trichophyton, chlamydospores of Epidermophyton, blastospores of Candida, and conidia of the mold Scopulariopsis brevicaulis. The results obtained were compared with those from ciclopirox olamine and terbinafine. The sporicidal activity was further determined using infected three-dimensional full skin models to determine the antifungal effects in the presence of human cells. Sertaconazole nitrate inhibited the growth of dermatophytes, molds, and yeasts. Ciclopirox olamine also had good antifungal activity, although higher concentrations were needed compared to sertaconazole nitrate. Terbinafine was highly effective against most dermatophytes, but higher concentrations were required to kill the resistant strain Trichophyton indotineae. Sertaconazole nitrate, ciclopirox olamine, and terbinafine had no negative effects on full skin models. Sertaconazole nitrate reduced the growth of fungal and yeast spores over 72 h. Ciclopirox olamine and terbinafine also inhibited the growth of dermatophytes and molds but had significantly lower effects on the yeast. Sertaconazole nitrate might have advantages over the commonly used antifungals ciclopirox olamine and terbinafine in combating resting spores, which persist in the tissues, and thus in the therapy of recurring dermatomycoses.

Resazurin rapid screening for antibacterial activities of organic and inorganic nanoparticles: Potential, limitations and precautions.

Nanoparticles have been used as antibacterial agents in several products. To optimize their effectiveness, synthesis processes and particle modifications have been developed, creating the need for a rapid screening method to investigate their potencies. Owing to the opacity and insolubility of nanoparticles, a classical method to determine antibacterial activity-such as the minimum inhibitory concentration (MIC), which relies on turbidimetry-might not apply to them. In this study, we demonstrate the potential of a dye (resazurin)-based assay as an indicator of bacterial growth to rapidly screen the antibacterial activities of both organic and inorganic nanomaterials against both gram-negative (E. coli) and gram-positive (S. aureus) bacteria. The results indicate that the resazurin-based assay successfully determine the MIC of organic lipid nanocarriers, and several inorganic nanoparticles. However, the use of resazurin require a precaution for nanoparticles with photocatalytic properties, which may cause dye degradation at higher concentrations. In this study, resazurin bleaching was observed at approximately >50 mg/ml of TiO2. In summary, the modified MIC assay with resazurin can evaluate antibacterial activity of nanomaterials, whose turbidity interferer conventional MIC assay. This modification conserves an advantage of MICs assay which are simple and reliable. This would be useful for screening of antibacterial nanomaterials.

Rhamnolipids as Effective Green Agents in the Destabilisation of Dolomite Suspension.

In this paper, we describe an application of mono- and dirhamnolipid homologue mixtures of a biosurfactant as a green agent for destabilisation of a dolomite suspension. Properties of the biosurfactant solution were characterised using surface tension and aggregate measurements to prove aggregation of rhamnolipids at concentrations much lower than the critical micelle concentration. Based on this information, the adsorption process of biosurfactant molecules on the surface of the carbonate mineral dolomite was investigated, and the adsorption mechanism was proposed. The stability of the dolomite suspension after rhamnolipid adsorption was investigated by turbidimetry. The critical concentration of rhamnolipid at which destabilisation of the suspension occurred most effectively was found to be 50 mg·dm-3. By analysing backscattering profiles, solid-phase migration velocities were calculated. With different amounts of biomolecules, this parameter can be modified from 6.66 to 20.29 mm·h-1. Our study indicates that the dolomite suspension is destabilised by hydrophobic coagulation, which was proved by examining the wetting angle of the mineral surface using the captive bubble technique. The relatively low amount of biosurfactant used to destabilise the system indicates the potential application of this technology for water treatment or modification of the hydrophobicity of mineral surfaces in mineral engineering.

Multicommutated Flow Analysis System for Determination of Horseradish Peroxidase and Its Inhibitors.

A fully mechanized multicommutated flow analysis (MCFA) system dedicated to determining horseradish peroxidase (HRP) activity was developed. Detection was conducted using a flow-through optoelectronic detector-constructed of paired LEDs operating according to the paired emitter-detector diode (PEDD) principle. The PEDD-MCFA system is dedicated to monitoring the enzyme-catalyzed oxidation of p-phenylenediamine (pPD) by a hydrogen peroxide. Under optimized conditions, the presented bioanalytical system was characterized by a linear response range (33.47-200 U/L) with a detection limit at 10.54 U/L HRP activity and 1.66 mV·L/U sensitivity, relatively high throughput (12 signals recordings per hour), and acceptable precision (RSD below 6%). Additionally, the utility of the developed PEDD-MCFA system for the determination of HRP inhibitors allowing the detection of selected thiols at micromolar levels, is demonstrated. The practical utility of the flow system was illustrated by the analysis of some dietary supplements containing L-cysteine, N-acetylcysteine, and L-glutathione.

Common Pitfalls and Recommendations for Using a Turbidity Assay to Study Protein Phase Separation.

The turbidity assay is commonly exploited to study protein liquid-to-liquid phase separation (LLPS) or liquid-to-solid phase separation (LSPS) processes in biochemical analyses. Herein, we present common pitfalls of this assay caused by exceeding the detection linear range. We showed that aggregated proteins of high concentration and large particle size can lead to inaccurate quantification in multiple applications, including the optical density measurement, the thermal shift assay, and the dynamic light scattering experiment. Finally, we demonstrated that a simple sample dilution of insoluble aggregated protein (LSPS) samples or direct imaging of liquid droplets (LLPS) can address these issues and improve the accuracy of the turbidity assay.

Quantification of Complement Proteins with Special Reference to C1q: Multiplex Versus ELISA Versus Rocket Immunoelectrophoresis Versus Nephelometry.

Accurate determination of complement component C1q is hampered by the fact that C1q is an immune complex binding protein. Consequently, immunochemical techniques which rely on immune complex formation in fluid phase such as nephelometry and turbidimetry tend to give results which differ from those obtained by, for example, ELISA and other solid phase-based assays. In this chapter, we discuss the pros and cons of different techniques for the quantification of C1q and present a comprehensive protocol for a newly developed magnetic bead-based sandwich immunoassay which has replaced nephelometry in our complement diagnostic laboratory at the University Hospital in Uppsala.

Drop-of-blood acoustic tweezing technique for integrative turbidimetric and elastometric measurement of blood coagulation.

Many patients develop coagulation abnormalities due to chronic and hereditary disorders, infectious disease, blood loss, extracorporeal circulation, and oral anticoagulant misuse. These abnormalities lead to bleeding or thrombotic complications, the risk of which is assessed by coagulation analysis. Current coagulation tests pose safety concerns for neonates and small children due to large sample volume requirement and may be unreliable for patients with coagulopathy. This study introduces a containerless drop-of-blood method for coagulation analysis, termed "integrated quasi-static acoustic tweezing thromboelastometry" (i-QATT™), that addresses these needs. In i-QATT™, a single drop of blood is forced to levitate and deform by the acoustic radiation force. Coagulation-induced changes in drop turbidity and firmness are measured simultaneously at different instants. The parameters describing early, intermediate, and late stages of the coagulation process are evaluated from the resulting graphical outputs. i-QATT™ rapidly (<10 min) detected hyper- and hypo-coagulable states and identified single deficiency in coagulation factors VII, VIII, IX, X, and XIII. The linear relationship (r2 > 0.9) was established between fibrinogen concentration and two i-QATT™ parameters: maximum clot firmness and maximum fibrin level. Factor XIII activity was uniquely measured by the fibrin network formation time (r2 = 0.9). Reaction time, fibrin formation rate, and time to firm clot formation were linearly correlated with heparin concentration (r2 > 0.7). tPA-induced hyperfibrinolysis was detected in the clot firmness output at 10 min. i-QATT™ provides comprehensive coagulation analysis in point-of-care or laboratory settings, well suited to the needs of neonatal and pediatric patients and adult patients with anemia or blood collection issues.

Determination of a Beckman Coulter AU turbidometric method-specific caeruloplasmin reference interval.

BACKGROUND: Method-dependent variation of caeruloplasmin measurement is significant and necessitates the requirement for assay-specific reference intervals. Local determination becomes necessary in the absence of suitable published or manufacturer-quoted reference intervals. METHODS: Applicability of the Beckman Coulter AU-quoted reference interval was determined by assay of 20 surplus serum samples from patients attending the medical renal stones clinic at University Hospital Southampton. Subsequently, 60 additional samples were collected for local reference interval determination. Samples were analysed for caeruloplasmin using the Beckman Coulter AU turbidometric kit on an AU680 analyser. Outliers were removed, and non-parametric rank analysis of the results was performed. RESULTS: The Beckman Coulter-quoted reference interval of 200-600 mg/L was unsuitable with 40% of the verification samples falling below 200 mg/L. A caeruloplasmin reference interval of 150-320 mg/L was established. CONCLUSION: Users should be aware the quoted Beckman Coulter AU turbidometric reference interval may not be appropriate. We have established a method-specific adult reference interval for routine use.

Complex coacervation of pea albumin-pectin and ovalbumin-pectin assessed by isothermal titration calorimeter and turbidimetry.

BACKGROUND: This study investigates the complexation of a pea albumin-rich fraction and ovalbumin with pectin of different degrees of esterification (DE) and blockiness (DB) as a function of pH and biopolymer mixing ratio by turbidimetric titration and isothermal titration calorimetry (ITC). RESULTS: Turbidimetric analysis found maximum complexation occurred at a mixing ratio of 4:1 for pea albumin with high methoxy pectin, 8:1 for pea albumin with low methoxy pectin, and 8:1 for ovalbumin with low methoxy pectin. In the case of ovalbumin with high methoxy pectin, interactions were very weak. The pectin with high levels of esterification and blockiness displayed greater interactions with the pea albumin in both turbidimetry and ITC. However, low methoxy pectin imparted better interactions with ovalbumin and displayed higher optical density values than high methoxy pectin. CONCLUSIONS: The current study indicated that the different thermodynamic parameters of PA-pectin complexes can be tuned by controlling the structural characteristics (DB, DE, and d-galacturonic acid) of the pectin. © 2020 Society of Chemical Industry.

Analytical validation of a modified turbidimetric assay to screen sulphur oxidizing bacteria.

Conventional turbidimetric assay for sulphate determination was modified to 100 times lesser reaction volume on a convenient format using microtitre plate based platform, targeting routine microbiological applications to screen sulphur oxidizing bacteria (SOB) cultures. The modified assay was linear up to 1500 mg/L of sulphate concentration, which is about 37.5 times more than that of conventional assay. Upon regression analysis, linear equation y = 1.243× + 0.011 was obtained having R2 value of 0.998. The modified assay was fully validated in terms of precision, limit of detection (LOD), limit of quantification (LOQ), sensitivity, selectivity and robustness to assure the reliability during final applications. LOD and LOQ were found as 7.4 mg/L and 24.8 mg/L of sulphate concentration respectively. Further, accuracy of the assay over routine SOB screening media components was tested, and proved as reliable and suitable for the intended application.

Serum Free Light Chain Assay: Shift Toward a Higher κ/λ Ratio.

BACKGROUND: The analysis of serum free light chains (FLCs) is clinically relevant for the diagnosis and therapeutic management of clonal plasma cell disorders. This study compares the performance of monoclonal and polyclonal FLC κ and λ assays in clinical samples determined in a single academic center. METHODS: Serum FLCs were analyzed from 102 patients using the Freelite (Binding Site) and N Latex (Siemens) assays on the BN ProSpec System (Siemens). When available, data for protein electrophoresis, immunofixation, C-reactive protein, and estimated glomerular filtration rate (eGFR) were combined with FLC results to evaluate performance. RESULTS: Method evaluation showed acceptable imprecision and inaccuracy measures of <4.4% and 12.9%, respectively. Poor agreement between the methods was observed, including constant and proportional bias and poor correlation (Kendall τ, 0.671-0.901). The N Latex assay was not affected by the renal impairment estimated by eGFR, unlike the FLC κ/λ ratio results by the Freelite assay. With the Freelite assay, 98% of putative controls without monoclonal gammopathy (n = 42) showed a κ/λ ratio that was above the median of the standard diagnostic range or renal diagnostic range. A shift toward higher κ/λ ratios was also observed when retrospective data between 2011 and 2017 were compared. CONCLUSIONS: Unlike the Freelite assay, κ/λ ratios analyzed with the N Latex assay were not affected by renal failure. Both methods showed acceptable performances using nephelometry, but they were poorly correlated. A shift toward κ/λ ratios might impair the specificity of borderline increased κ/λ results. This should be considered when interpreting FLC κ and λ results.

Analytical validation of a quantitative method for therapeutic drug monitoring on the Alinity®c Abbott.

OBJECTIVE: The aim of this study was to evaluate the analytical performance of the Alinity®c Abbott compared to the Architect® immunoassay system for the determination of drugs having a narrow therapeutic index. METHODS: Valproic acid, amikacin, gentamicin, phenobarbital and vancomycin were analyzed using Particle-Enhanced Turbidimetric Inhibitor Immunoassay (Petinia), phenytoin and theophylline were analyzed using an immunoenzymatic method and a colorimetric method was performed to quantify lithium. The methods were validated according to the total error approach. Seven validation standards were analyzed in quintuplet during four days to establish the limits of the methods. Dilution integrity and interferences (hemolysis and high concentrations of bilirubin and lipids) were also tested. Depending on the analyte, the results obtained for twenty to forty patients on the Alinity® were compared to those obtained on the Architect®. RESULTS: The bias and the coefficients of variation for repeatability and for intermediate precision were lower than 15% for all drugs. Accuracy profiles were acceptable (acceptance limits fixed at 30%) in the validated ranges. The lower limits of quantification (LLOQ) were similar to those determined by Abbott except for gentamicin for which we determined a LLOQ at 1.22 mg/L while Abbott determined it at 0.5 mg/L. All assays diluted linear and analyte concentrations were not affected by interferences. Concentrations obtained for real samples on the Alinity®c are comparable to those obtained on the Architect®ci. CONCLUSIONS: The analytical validation of a method suitable for therapeutic drug monitoring of drugs on the Alinity®c meets the requirements of European Medicines Agency.

Coupled Turbidity and Spectroscopy Problems: A Simple Algorithm for Volumetric Analysis of Optically Thin or Dilute, In Vitro Bacterial Cultures in Various Media.

An approach binary spectronephelometry (BSN) to perform real-time simultaneous noninvasive in situ physical and chemical analysis of bacterial cultures in fluid media is described. We choose to characterize cultures of Escherichia coli (NC), Pseudomonas aeruginosa (PA), and Shewanella oneidensis (SO) in the specific case of complex media whose Raman spectrum cannot be unambiguously assigned. Nevertheless, organism number density and a measure of the chemical makeup of the fluid medium can be monitored noninvasively, simultaneously, and continuously, despite changing turbidity and medium chemistry. The method involves irradiating a culture in fluid medium in an appropriate vessel (in this case a standard 1 cm cuvette) using a near infrared laser and collecting all the backscattered light from the cuvette, i.e., the Rayleigh-Mie line and the inelastically emitted light which includes unresolved Raman scattered light and fluorescence. Complex "legacy" media contain materials of biological origin whose chemical composition cannot be fully delineated. We independently calibrate this approach to a commonly used reference, optical density at 600 nm (OD600) for characterizing the number density of organisms. We suggest that the total inelastically emitted light could be a measure of the chemical state of a biologically based medium, e.g., lysogeny broth (LB). This approach may be useful in a broad range of basic and applied studies and enterprises that utilize bacterial cultures in any medium or container that permits optical probing in the single scattering limit.

Use of High-Resolution Pressure Nephelometry To Measure Gas Vesicle Collapse as a Means of Determining Growth and Turgor Changes in Planktonic Cyanobacteria.

Previous work has demonstrated that the physical properties of intracellular bacterial gas vesicles (GVs) can be analyzed in vivo using pressure nephelometry. In analyzing the buoyant state of GV-containing cyanobacteria, hydrostatic pressure within a sample cell is increased in a stepwise manner, where the concomitant collapse of GVs due to pressure and the resultant decrease in suspended cells are detected by changes in nephelometric scattering. As the relative pressure at which GVs collapse is a function of turgor pressure and cellular osmotic gradients, pressure nephelometry is a powerful tool for assaying changes in metabolism that affect turgor, such as photosynthetic and osmoregulatory processes. We have developed an updated and automated pressure nephelometer that utilizes visible-infrared (Vis-IR) spectra to accurately quantify GV critical collapse pressure, critical collapse pressure distribution, and cell turgor pressure. Here, using the updated pressure nephelometer and axenic cultures of Microcystis aeruginosa PCC7806, we demonstrate that GV critical collapse pressure is stable during mid-exponential growth phase, introduce pressure-sensitive turbidity as a robust metric for the abundance of gas-vacuolate cyanobacteria, and demonstrate that pressure-sensitive turbidity is a more accurate proxy for abundance and growth than photopigment fluorescence. As cyanobacterium-dominated harmful algal bloom (cyanoHAB) formation is dependent on the constituent cells possessing gas vesicles, characterization of environmental cyanobacteria populations via pressure nephelometry is identified as an underutilized monitoring method. Applications of this instrument focus on physiological and ecological studies of cyanobacteria, for example, cyanoHAB dynamics and the drivers associated with cyanotoxin production in aquatic ecosystems.IMPORTANCE The increased prevalence of bloom-forming cyanobacteria and associated risk of exposure to cyanobacterial toxins through drinking water utilities and recreational waterways are growing public health concerns. Cost-effective, early-detection methodologies specific to cyanobacteria are crucial for mitigating these risks, with a gas vesicle-specific signal offering a number of benefits over photopigment fluorescence, including improved detection limits and discrimination against non-gas-vacuolate phototrophs. Here, we present a multiplexed instrument capable of quantifying the relative abundance of cyanobacteria based on the signal generated from the presence of intracellular gas vesicles specific to bloom-forming cyanobacteria. Additionally, as cell turgor can be measured in vivo via pressure nephelometry, the measurement furnishes information about the internal osmotic pressure of gas-vacuolate cyanobacteria, which relates to the metabolic state of the cell. Together these advances may improve routine waterway monitoring and the mitigation of human health threats due to cyanobacterial blooms.

Turbidimetry and Dielectric Spectroscopy as Process Analytical Technologies for Mammalian and Insect Cell Cultures.

The production of biopharmaceuticals in cell culture involves stringent controls to ensure product safety and quality. To meet these requirements, quality by design principles must be applied during the development of cell culture processes so that quality is built into the product by understanding the manufacturing process. One key aspect is process analytical technology, in which comprehensive online monitoring is used to identify and control critical process parameters that affect critical quality attributes such as the product titer and purity. The application of industry-ready technologies such as turbidimetry and dielectric spectroscopy provides a deeper understanding of biological processes within the bioreactor and allows the physiological status of the cells to be monitored on a continuous basis. This in turn enables selective and targeted process controls to respond in an appropriate manner to process disturbances. This chapter outlines the principles of online dielectric spectroscopy and turbidimetry for the measurement of optical density as applied to mammalian and insect cells cultivated in stirred-tank bioreactors either in suspension or as adherent cells on microcarriers.

Is it possible to shorten serum bactericidal testing?

Serum bactericidal test represents an alternative possibility for optimization of antibiotic treatment. The paper aimed to confirm non-inferiority of bactericidal testing using the broth dilution method according to the CLSI method (M21A) in comparison with turbidimetric and colorimetric modifications. We tested human blood sera (n = 76) of ten hematological patients, their blood was withdrawn prior to and during the course of antibiotic therapy. Testing employed the reference strain Escherichia coli ATCC 25922. The results of the modified turbidimetric method did not differ in a statistically significant way with the use of the wavelengths of 620 nm or 405 nm and the break-point <30% turbidity change after 24-hour incubation. The colorimetric method was also non-inferior from the CLSI method when resazurin was applied after 8-hour incubation and the results of subculture were read after 24-hour incubation. Both tested modifications can represent a shorter alternative to the CLSI reference method.