Micro-bead injection spectroscopy for label-free automated determination of immunoglobulin G in human serum

Abstract

Immunoglobulin G (IgG) represents the major fraction of antibodies in healthy adult human serum, and deviations from physiological levels are a generic marker of disease corresponding to different pathologies. Therefore, screening methods for IgG evaluation are a valuable aid to diagnostics. The present work proposes a rapid, automatic, and miniaturized method based on UV-vis micro-bead injection spectroscopy (μ-BIS) for the real-time determination of human serum IgG with label-free detection. Relying on attachment of IgG in rec-protein G immobilized in Sepharose 4B, a bioaffinity column is automatically assembled, where IgG is selectively retained and determined by on-column optical density measurement. A "dilution-and-shoot" approach (50 to 200 times) was implemented without further sample treatment because interferences were flushed out of the column upon sample loading, with minimization of carryover and cross-contamination by automatically discarding the sorbent (0.2 mg) after each determination. No interference from human serum albumin at 60 mg mL in undiluted sample was found. The method allowed IgG determination in the range 100-300 μg mL (corresponding to 5.0-60 mg mL in undiluted samples), with a detection limit of 33 μg mL (1.7 mg mL for samples, dilution factor of 50). RSD values were < 9.4 and < 11.7%, for intra and inter-assay precision, respectively, while recovery values for human serum spiked with IgG at high pathological levels were 97.8-101.4%. Comparison to commercial ELISA kit showed no significant difference for tested samples (n = 8). Moreover, time-to-result decreased from several hours to < 5 min and analysis cost decreased 10 times, showing the potential of the proposed approach as a point-of-care method. Graphical abstract Micro-Bead Injection Spectroscopy method for real time, automated and label-free determination of total serum human Immunoglobulin G (IgG). The method was designed for Lab-on-Valve (LOV) platforms using a miniaturised protein G bioaffinity separative approach. IgG are separated from serum matrix components upon quantification with low non-specific binding in less than 5 min.

AuNP array coated substrate for sensitive and homogeneous SERS-immunoassay detection of human immunoglobulin G

“…HIgG is the well-adopted serum marker to diagnose rheumatoid arthritis, liver disease and infectious diseases. Since the HIgG level in healthy human serum is usually in the range of 7–18 mg mL −1 , 7 the sample was 1000-fold diluted with PBS buffer (10 mM, pH 7.4) to match the quantitative detection range of this method (0.1–200 μg mL −1 ). Herein, the standard SERS-immunoassay process was performed to detect the diluted samples.…”

“…[1][2][3][4] The HIgG level in healthy adults ranges from 7 mg mL À1 to 18 mg mL À1 , but this value is usually altered associated with some diseases, such as rheumatoid arthritis, infectious, humoral immune deciency, and liver and metabolic diseases. [5][6][7] Therefore, the detection of HIgG in human serum is important for the early diagnosis and effective treatment of related diseases. 8 Immunoassay based on the specic interaction between an antigen and antibody is a powerful analytical method, which has become a popular tool for clinical diagnosis.…”

Lab-on-Valve Automated and Miniaturized Assessment of Nanoparticle Concentration Based on Light-Scattering

“…The configuration of the SI-LOV (MicroSIA, FIAlab instruments, Inc., Bellevue, WA, USA) system used for nanoparticles quantification (Figure S1) is described in Supporting Information. Optical detection and quantification of nanoparticles was performed in the LOV integrated detection unit (further details on Supporting Information) ,, by measuring the light attenuation (i.e., light lost due to scattering and/or absorbance events) when nanoparticles were present in the optical path.…”

“…LOV accommodates sampling, online dilution and mixing by flow reversal, and online detection as it integrates an optical detection unit, enabling several operations in a single setup. This system has successfully been employed to set simpler, faster and less-laborious quantification of small molecules, sorbent-based sample pretreatment, , and molecular recognition procedures. , Furthermore, considering the need of ensuring minimal stress and dilution during the analysis of organic NPs, the LOV platform emerges as a valuable alternative for NPs characterization as it offers (i) inert bore conduits ( ca . 1.0 mm), larger than conventional size-exclusion columns and ultrafiltration/dialysis membranes, (ii) low operation flow rates ( e.g ., 1 μL s –1 ), and (iii) short distance (10 mm) between the sampling port and the detection unit.…”

Antibody conjugation to carboxyl-modified microspheres through N-hydroxysuccinimide chemistry for automated immunoassay applications: A general procedure

“…Enzyme-linked immunosorbent assay (ELISA) and immunochromatography are selective and sensitive techniques based on solid phases for separation, extraction and possible pre-concentration of analytes from their respective matrix, prominent examples being the detection and quantification of pharmaceuticals in the aquatic environment [1] as well as in food-stuff [2] and of biomarkers in biological fluids [3]. In contrast to batch-wise procedures, such as microtiter plate-based platforms, automated methods reduce manual handling of reagents, increasing overall precision and decreasing time-to-result [4, 5]. In this domain, microparticles have been shown to be an adequate support for carrying out immunoassays in meso- and microfluidic systems.…”