Capillary Electrophoresis-based Immunoassay Research Articles

Development of β-conglycinin quantitative method in soybean foods using capillary electrophoresis-based immunoassay

Development of β-conglycinin quantitative method in soybean foods using capillary electrophoresis-based immunoassay

Abstract 1436: Determination of antibody's specificity towards phosphorylated protein targets with automated in-capillary enzyme treatment and immunoassay

Abstract Protein phosphorylation is a reversible reaction that is integral in numerous signaling cascades. Characterization of signaling cascades has been largely detected by immunoblotting with phospho-specific antibodies, which may or may not have enough specificity or affinity. Currently, a separate lysate without any phosphatase inhibitors or a separate blot is needed to determine an antibody's specificity. Here we describe a simple assay that leverages automation and quantitation with capillary electrophoresis-based immunoassay (CEIA) to assess the specificity of these antibodies with a single lysate preparation. In this study, three lysate models are used: K562 ± TNFα treatment, 50 ng/mL phorbol myristate acetate (PMA) differentiated THP-1 ± 1 μg/mL lipopolysaccharide (LPS) treatment, and cytotoxic T lymphocytes (CTL) ± 10 ng/mL PMA and 500 ng/mL ionomycin treatment. K562 cell lysates are commercially purchased whereas THP-1 lysates are generated in-house. For CTL cells, whole blood cells from a single donor are isolated and expanded with commercially available kits. Expanded CTL cells are then stimulated with PMA and ionomycin for 15 minutes. Untreated and treated lysate samples are separated and captured to the inner lumen of the capillary wall with UV activated crosslink chemistry. Cross-linked proteins are treated with lambda phosphatase for 1 hour followed by the immunoassay to investigate the specificity of antibodies against phosphorylated protein targets respective to each activated pathway using either chemiluminescent or fluorescent detection. Preliminary data suggest phospho-specific signal decreased >90% with no significant changes to the non-specific noise. The method described here eliminates the need for multiple lysate preparations or an additional blot to assess an antibody's specificity to a phosphorylated protein target. Citation Format: Daryl A. Taketa, Rainer Grant, Tufan Aydogdu. Determination of antibody's specificity towards phosphorylated protein targets with automated in-capillary enzyme treatment and immunoassay [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1436.

The stress susceptibility factor FKBP51 controls S-ketamine-evoked release of mBDNF in the prefrontal cortex of mice

We report here the involvement of the stress-responsive glucocorticoid receptor co-chaperone FKBP51 in the mechanism of in vivo secretion of mature BDNF (mBDNF). We used a novel method combining brain microdialysis with a capillary electrophoresis-based immunoassay, to examine mBDNF secretion in the medial prefrontal cortex (mPFC) in vivo in freely moving mice. By combining optogenetic, neurochemical (KCl-evoked depolarization), and transgenic (conditional BDNF knockout mice) means, we have shown that the increase in extracellular mBDNF in vivo is determined by neuronal activity. Withal, mBDNF secretion in the mPFC of mice was stimulated by a systemic administration of S-ketamine (10 or 50 mg/kg) or S-hydroxynorketamine (10 mg/kg).KCl- and S-ketamine-evoked mBDNF secretion was strongly dependent on the expression of FKBP51. Moreover, the inability of S-ketamine to evoke a transient secretion in mBDNF in the mPFC in FKBP51- knockout mice matched the lack of antidepressant-like effect of S-ketamine in the tail suspension test. Our data reveal a critical role of FKBP51 in mBDNF secretion and suggest the involvement of mBDNF in the realization of immediate stress-coping behavior induced by acute S-ketamine.

Capillary electrophoresis-based immunoassay with electrochemical detection as rapid method for determination of saxitoxin and decarbamoylsaxitoxin in shellfish samples

A simple and sensitive capillary electrophoresis (CE) based immunoassay (IA) with electrochemical (EC) detection method is presented for the assay of saxitoxin and decarbamoylsaxitoxin in shellfish samples. The method was based on competitive reactions between horseradish peroxidase (HRP)-labeled antigen (Ag*) and free antigen (Ag) with a limited amount of antibody (Ab). After incubation, the unbound Ag* and the bound enzyme-labeled complex (Ag*–Ab) were separated by CE and then the system of HRP catalyzing H2O2/o-aminophenol (OAP) reaction was adopted for EC detection. Using CEIA with EC detection, equilibrium of competitive reaction was reached in 30min, and the analytical results were further obtained within 6min. The linear range and the detection limit were 0.8–66.6ng/mL and 4.3–9.2μg/kg for saxitoxin, and 1.2–83.5ng/mL and 7.7–16.5μg/kg for decarbamoylsaxitoxin, respectively. The method was applied to quantitative analysis of saxitoxin and decarbamoylsaxitoxin in shellfish samples with rapid and simple pretreatment, and the results were consistent with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA) and the mouse bioassay. Recoveries of saxitoxin and decarbamoylsaxitoxin in shellfish samples were 92.5–107.6% and 94.8–113.3% for saxitoxin and decarbamoylsaxitoxin, respectively. The method is thus proposed as a fast and sensitive assay for the determination of saxitoxin and decarbamoylsaxitoxin in shellfish samples.

Development of a Capillary Electrophoresis-Based Enzyme Immunoassay with Electrochemical Detection for the Determination of Okadaic Acid and Dinophysistoxin2 in Shellfish Samples

A capillary electrophoresis-based enzyme immunoassay (CE-EIA) with electrochemical (EC) detection system was developed for the determination of two diarrheic shellfish poisoning (DSP) toxins okadaic acid (OA) and dinophysistoxin2 (DTX2). In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen (Ag*) and the bound enzyme-labeled complex (Ag*-Ab) were separated and then the system of HRP catalyzing H2O2/o-aminophenol (OAP) reaction was adopted. The limit of detection (S/N = 3) was determined to be 0.05 and 0.07 ng/mL for OA and DTX2, respectively. The total analysis time was less than 40 min. The developed CE-EIA with EC detection system was capable of quantitatively detecting OA and DTX2 contents in the tested contaminated samples, and the results were compared with the same samples analyzed through enzyme-linked immunosorbent assay (ELISA). Consistent results between CE-EIA with EC detection and ELISA were found in most of the tested samples. The proposed system appeared to be more sensitive and faster than ELISA for determination of OA and DTX2 in shellfish meat extracts. Real shellfish samples were validated in recovery test, and the recoveries tested by the proposed method were 91.7–108.3% and 95.2–112.5% for OA and DTX2, respectively. The CE-EIA with EC detection provides a valid and sensitive analytical approach, not previously available, for the determination of OA and DTX2 in shellfish samples.

Capillary Electrophoresis-Based Immunoassay for the Determination of Brevetoxin-B in Shellfish using Electrochemical Detection

Neurotoxic shellfish poisoning is a significant food-borne disease caused by potent cyclic polyether toxins (brevetoxins) that accumulate in the flesh of shellfish. Here we report a new procedure for brevetoxin analysis by capillary electrophoresis-based immunoassay (CE-IA) with electrochemical (EC) detection. In this method, after the competitive immunoreaction in liquid phase, the horseradish peroxidase (HRP)-labeled antigen and the bound enzyme-labeled complex were separated and then the system of HRP catalyzing H(2)O(2)-o-aminophenol reaction was adopted. The limits of detection (signal-to-noise = 3) was determined to be 0.1 ng/mL. Intra-day relative standard deviations (RSD, n = 5) for migration time and peak area were 3.6 and 4.5%, respectively. Inter-day RSD was 6.9 and 7.8% for migration time and peak area, respectively. The CE-IA with EC detection provides a sensitive analytical approach, not previously available, for the determination of brevetoxin-B in shellfish samples.

Determination of Nerve Growth Factor in Rat Spinal Cord by Capillary Electrophoresis-Based Immunoassay with a Laser Induced Fluorescence Detector

In order to study the relationship between spinal cord injury and the change of nerve growth factor (NGF), an analytical method for NGF by capillary electrophoresis-based immunoassay (CEIA) with laser-induced fluorescence (LIF) was developed. Having been dissolved in phosphate buffer solution and concentrated with vacuum freeze-drying, NGF in spinal cord of rat was allowed to react with NGF monoclonal antibody labeled with fluorescence isothiocyanate (FITC). Then the immuno-complex, FITC-labeled anti-NGF and FITC were separated and determined by LIF-CEIA using Kiton red as the internal standard. The linear range of the method was 2–30 ng mL−1 and the limit of detection was 0.35 ng mL−1. The relative standard derivations for relative migration time and relative fluorescence intensity ratio were 7.98% and 6.52%, respectively. The contents of NGF from spinal cord of rat were determined by both the proposed method and Western blotting. The results with the two methods agreed well. The spiked recoveries of the samples were 88.5–116.3%. The proposed method was rapid, precise and inexpensive.

Development of a Capillary Electrophoresis-Based Immunoassay with Laser-Induced Fluorescence for the Detection of Carbaryl in Rice Samples

A capillary electrophoresis-based competitive immunoassay (CEIA) with a laser-induced fluorescence (LIF) detector for the determination of carbaryl was developed. The method was based on the competitive reactions between fluorescently labeled carbaryl tracer (Ag*) and free carbaryl (Ag) with a limited amount of anticarbaryl antibody (Ab), and the relative amounts of each were separated and determined by capillary electrophoresis (CE) with an LIF detector. Using CEIA, equilibrium was reached in 30 min, and the analytical results were obtained within a further 8 min. The linear range and the detection limit for carbaryl were 0.16-50 ng/mL and 0.05 ng/mL, respectively. The sensitivity of this CEIA with an LIF detector was almost 14 times greater than that of ELISA, which used the same immuno-reagents. The method was also applied to the analysis of carbaryl in rice with rapid and simple sample pretreatment. The method is thus proposed as a fast and sensitive assay for the detection of carbaryl.

Capillary electrophoresis-based immunoassay for insulin antibodies with near-infrared laser induced fluorescence detection

A noncompetitive capillary electrophoresis (CE)-based immunoassay with near-infrared laser induced fluorescence detection (NIR-LIF) for insulin antibodies has been developed. In the assay, insulin was derivatized with a NIR fluorescent dye (NN382; LI-COR). Insulin antibodies were detected via the formation of an immunocomplex. Parameters affecting the separation such as pH, voltage and ionic strength were investigated. Furthermore, it was found that increasing the ramp time of the applied voltage improved the detection limit of the assay by an order of magnitude. The detection limit of the assay was 1.1 n M.

Capillary Electrophoresis Based Immunoassay for Monoclonal Antibody with Diode Laser Induced Fluorescence Detection

ABSTRACT A capillary electrophoresis based immunoassay (CEIA) for monoclonal antibody using diode laser induced fluorescence (LIF) detection was described. A direct assay for monoclonal anti-BSA in mouse serum was used as a model. BSA was labeled with Cy5 and used as the immunoreagent. The 635 nm line of a diode laser was used as the excitation source for LIF detection. The calibration curve for anti-BSA in mouse serum had a linear dynamic range of 4-40 nM. The concentration limit of detection (LOD) was 1.2 nM. Incubation time and CE conditions such as buffer concentration, pH and separation voltage were optimized, and the performances of different lasers as excitation sources were also compared.

Separation of bovine serum albumin and its monoclonal antibody from their immunocomplexes by sodium dodecyl sulfate–capillary gel electrophoresis and its application in capillary electrophoresis-based immunoassay

A non-competitive immunoassay was performed by sodium dodecyl sulfate–capillary gel electrophoresis with UV detection using bovine serum albumin (BSA) and monoclonal anti-BSA. BSA, anti-BSA and their immunocomplexes were well resolved under non-denaturing conditions. A linear calibration curve was obtained and can be used for the quantification of anti-BSA. The limit of detection of anti-BSA was 0.1 μ M under the present conditions. Compared with capillary zone electrophoresis, we believed that this method has the potential to be used as a more general format for performing capillary electrophoresis-based immunoassay of medium- and large-sized analytes.

Noncompetitive immunoassays using protein G affinity capillary chromatography and capillary electrophoresis with laser-induced fluorescence detection

A new and simple approach to perform immunoassay using protein G affinity capillary chromatography and laser-induced fluorescence detection was described. A noncompetitive assay for monoclonal anti-bovine serum albumin (BSA) was used to test the performance of the system. Fluorescein isothiocyanate labeled BSA was used as a tracer to determine anti-BSA in p M level. Capillaries with inner diameter of 150 μm were packed with recombinant protein G-bound perfusive support. The packed capillary was used to capture the immunocomplexes, which were subsequently desorbed by 100 m M glycine (pH 9.0). Open tube capillary electrophoresis-based immunoassay (CEIA) for anti-BSA was also performed. Using standard samples, calibration curves for anti-BSA was established in both assays. Compared with CEIA, this system improved the concentration sensitivity for about 100-fold due to the pre-concentration of immunocomplex in the protein G column, while the mass sensitivity was similar in the two methods.

Use of capillary electrophoresis-based competitive immunoassay for a large molecule

A systematic study on the optimization of capillary electrophoresis-based immunoassay (CEIA) was performed using bovine serum albumin (BSA) and monoclonal anti-BSA. The immunocomplex could not be resolved from free BSA or anti-BSA with UV detection. When fluorescein isothiocyanate-labeled BSA (FITC-BSA) was used as tracer, the free and bound FITC-BSA were well separated giving definite peaks with laser induced fluorescence detection. The factors affecting the separation of the free and bound FITC-BSA, including voltage, pH and ionic strength of the running buffer, were systematically analyzed. Competitive CEIAs were demonstrated in uncoated and coated capillaries with whole or Fab fragment of the antibody. The coefficient of variation for the quantification of BSA in coated capillary was less than that in uncoated capillary. This study demonstrated that competitive CEIA could be applied to quantify high-molecular-mass protein in biological fluids.

Capillary electrophoresis-based immunoassay to determine insulin content and insulin secretion from single islets of Langerhans

The development of a competitive immunoassay for insulin that is based on capillary zone electrophoresis with laser-induced fluorescence detection (CZE-LIF) is described. The assay uses fluorescein isothiocyanate-derivatized insulin (FITC-insulin) as a tracer and the Fab fragment of an anti-insulin monoclonal antibody as the immunoreagent. Reproducibility and reliability of the assay are improved when CZE capillaries are coated with a neutral hydrophilic polymer. The assay has an average relative standard deviation of 3.4% and a detection limit of 3 nM or about 6 fg injected. Using the assay, insulin content of single islets of Langerhans was determined to be 35 +/- 4 ng, which is in good agreement with literature values. The assay was also used to determine insulin secretion from single islets. During 5-min static incubations, 3 mM glucose elicited 0.8 +/- 0.2 ng of insulin release while 16 mM glucose elicited 3.2 +/- 0.5 ng of insulin release. These values are also in good agreement with previously reported results. These applications demonstrate the capability of rapidly determining a low level of protein in a complex biological microenvironment.

Capillary electrophoresis-based immunoassay for cortisol in serum.

A competitive immunoassay for cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anticortisol antibody and purified using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples.