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.     

Multichannel homogeneous immunoassay for detection of 2,4,6-trinitrotoluene (TNT) using a microfabricated capillary array electrophoresis chip

A high-throughput homogeneous immunoassay for the sensitive detection of 2,4,6-trinitrotoluene (TNT) has been developed using radial capillary array electrophoresis microdevices. Samples consisting of equilibrium mixtures of anti-TNT antibody (Ab), fluorescein-labeled TNT, and various concentrations of unlabeled TNT were electrokinetically injected into 48 channels of a radial capillary array electrophoresis microchannel plate. The rapid electrophoretic separation allows us to analyze the equilibrium ratio formed by the competition between the labeled and the unlabeled TNT for Ab binding. The simultaneous parallel TNT separations facilitate determination of a calibration curve for the TNT assay, which has high sensitivity (LOD, 1 ng/mL) and a wide dynamic range (1-300 ng/mL).     

蛋白质样品的非竞争性毛细管电泳免疫分析激光诱导荧光检测

以牛血清白蛋白与其单克隆抗体为样品,初步研究了蛋白质的非竞争性毛细管电泳免疫分析方法。混合温育可以在２０ｍｉｎ内达到平衡,电泳分离在１２ｍｉｎ内完成。当示踪物浓度为３２０ｎｍｏｌ／Ｌ时,所得到的标准曲线的线性范围为８￣１５０ｎｍｏｌ／Ｌ,检出限为５ｎｍｏｌ／Ｌ,并考察了缓冲溶液的浓度、ｐＨ、分离电压等因素的影响。     

Encapsulation of fluorescent molecules by functionalized polymeric nanocontainers: investigation by confocal fluorescence imaging and fluorescence correlation spectroscopy

Nanocontainers (NCs) were prepared from amphiphilic triblock copolymers, having an average molecular weight of around 8000 g/mol, by using previously published preparation methods consisting of dispersing the polymer in an aqueous buffer solution containing molecules for encapsulation. A small molecular weight fluorophore, sulforhodamine B, as well as the fluorescent protein avidin labeled with Alexa 488 were encapsulated, and the resulting nanocontainers were characterized using fluorescence correlation spectroscopy (FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer size determination by FCS is very robust and compares well with results obtained from photon correlation spectroscopy: the measured diameters of the polymeric nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent molecules was determined by evaluating the molecular brightness of nanocontainers with an encapsulated fluorescently labeled protein (avidin-Alexa 488). Results indicate that the number of encapsulated avidin-Alexa 488 molecules corresponds well with the initial concentration of the fluorescently labeled protein and the encapsulated volume. A nanocontainer binding assay was developed using biotinylated fluorescently labeled nanocontainers. Binding of biotinylated nanocontainers to fluorescently labeled streptavidin was followed by fluorescence cross-correlation spectroscopy. The intrinsic dissociation constant, K(d), of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +/- 0.4 x 10(-8) M, and about 1921 +/- 357 molecules of labeled streptavidin are bound to each nanocontainer.     

Two-color electrophoretic immunoassay for simultaneous measurement of insulin and glucagon content in islets of Langerhans

A multianalyte CE competitive immunoassay using two-color detection was developed to measure insulin and glucagon in islets of Langerhans. Insulin was quantified with FITC-insulin (Ins*) and anti-insulin antibodies (Ins Ab) and glucagon was quantified with Cy5-glucagon (Glu*) and anti-glucagon antibodies (Glu Ab). A 3 mW Ar(+) laser at 488 nm and a 25 mW laser diode at 635 nm were used to excite FITC and Cy5, respectively. Fluorescence was split with a half-silvered mirror and passed through a 520 +/- 20 nm bandpass filter or a 663 nm longpass filter for the detection of insulin and glucagon, respectively. The two-color detection format enabled independent quantitation of both analytes even with concentrations of insulin immunoassay reagents 20-fold higher than glucagon reagents. Simultaneous calibration curves were generated and used to determine insulin and glucagon content in islets of Langerhans. Amounts of insulin and glucagon were 56.6 +/- 3.2 and 1.0 +/- 0.5 ng/islet, respectively. LODs were 7 nM insulin and 3 nM glucagon. The assay will be applicable to fast monitoring of multiple peptides secreted from islets of Langerhans and can be applied to other systems for the quantitation of multiple analytes with large differences in concentrations.     

Poly-N-Isopropylacrylamide Hydrogel Based Fluoroimmunoassay of Methyltestosterone

IntroductionMethyltestosterone(MT)isasynthesizedsteroid.Athletesusesyntheticsteroidstoincreasetheirsportgrade.This,fromtheviewofethics,violatedtheoriginalrulesofsport.Therefore,themonitoringofthedopeabusehasbecomemoreandmoreimportantworldwide.Sofar.themonitoringiscommonlycarriedoutbychromatographicseparationfollowedbymassspectrometricanalysis.Thetargetsamplesarenormallyurinel~2.Themonitoringbasedonbloodsampleismainlyusedasacomplementarymethodtoovercometheshortcomingfromtheanalysisusingurines…     

One-way multiplexed immunoassay strategy for simultaneous determination of multi-analytes by microchip electrophoresis

An integrated microchip electrophoresis (MCE) system with online immunoreaction and laser induced fluorescence (LIF) detection has been developed for simultaneous determination of multi-analytes. In this system, the multiplexed immunoreactions between multiple antibody-immobilized glass beads with analytes and respective fluorescently labeled antigens were performed in a sample reservoir. After online incubation, the immunoreaction solution was injected into a one-way separation channel, and free fluorescently labeled antigens were separated and detected in the separation channel. With the help of glass beads, the immunocomplex can not move into the separation channel, which simplifies the separation of fluorescently labeled antigens. With the use of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ) and theophylline (Th) as proof-of-principle analytes, the one-way multiplexed immunoassay could be completed within 20 min, resulting in a response curve over the range of 4.0-400 nM for each analyte. Detection limits (S/N = 3) for the drugs tested were in the range of 1.8 × 10(-9) to 2.5 × 10(-9) M. Compared with the conventional immunoassays, this assay is simple, rapid, sensitive and low cost, and provides an accurate procedure for a multiplex immunoassay. The present method has been applied for the simultaneous determination of PB, PHT, CBZ and Th in human serum, which showed a promise of automated clinical application.     

Continuous Immunoassay for Sulfamethazine by Surface Plasmon Resonance-Based Biosensor

Regeneration of the sensor chip surface is difficult in many surface plasmon resonance (SPR) biosensor assays. Improper regeneration will reduce life span of the sensor chip and decrease the quality of the data. Considering the advantages of reducing the regeneration frequency, a theoretically feasible continuous SPR biosensor immunoassay for sulfamethazine (SMT) was developed. In the continuous inhibitive immunoassay, the sensor chip surface is regenerated only once after a definite number of tests instead of every test. The SMT-bovine serum albumin (BSA) conjugate was covalently immobilized to a carboxymethyldextran modified gold film. The immobilization conditions of the antigen were studied and the working dilution of the antibody was optimized. The antibody was mixed with SMT of different concentrations prepared with PBS buffer to construct the calibration curve. The limit of detection was 0.5 ng mL^?1. The continuous SPR biosensor assay was proved to be simpler and more practical than a normal one.     

Competitive immunoassay by capillary electrophoresis with laser-induced fluorescence for the trace detection of chloramphenicol in animal-derived foods

A competitive immunoassay using CE with an LIF detector was developed for the detection of chloramphenicol (CAP). The method was based on the competitive reactions between fluorescently labeled CAP hapten and free CAP, with a limited amount of anti-CAP antibody. The poly(N-isopropylacrylamide) (pNIPA) hydrogel was added in the separation buffer as a dynamic modifier to reduce adsorption and enhance reproducibility. The linear range and LOD for CAP were 0.008-5 mug/L and 0.0016 mug/L, respectively. An ELISA using the same immuno-reagents was also developed for the analysis of CAP, with an LOD of 0.03 mug/L. The sensitivity of this CE immunoassay (CEIA)-LIF was almost 20 times greater than that of the ELISA. Using CEIA-LIF, equilibrium was reached in 15 min and the analytical results were obtained within 5 min by CE separation. Sample preparation for CEIA-LIF was not time-consuming and the matrix effect was easy to remove. An LOD of 0.1 mug/kg CAP in food matrices was easily achieved. This method is thus proposed as a fast and sensitive means of detecting trace amounts of CAP residues in animal-derived foods.     

A method for calculating <Superscript>16</Superscript>o/<Superscript>18</Superscript>o peptide ion ratios for the relative quantification of proteomes

A method is described for the identification and relative quantification of proteomes using accurate mass tags (AMT) generated by nLC-dual ESI-FT-ICR-MS on a 7T instrument in conjunction with stable isotope labeling using 16O/18O ratios. AMTs were used for putative peptide identification, followed by confirmation of peptide identity by tandem mass spectrometry. For a combined set of 58 tryptic peptides from bovine serum albumin (BSA) and human transferrin, a mean mass measurement accuracy of 1.9 ppm +/-0.94 ppm (CIM99%) was obtained. This subset of tryptic peptides was used to measure 16O/18O ratios of 0.36 +/- 0.09 (CIM99%) for BSA (micro = 0.33) and 1.48 +/- 0.47 (CIM99%) for transferrin (micro = 1.0) using a method for calculating 16O/18O ratios from overlapping isotopic multiplets arising from mixtures of 16O, 18O1, and 18O2 labeled C-termini. The model amino acid averagine was used to calculate a representative molecular formula for estimating and subtracting the contributions of naturally occurring isotopes solely as a function of peptide molecular weight. The method was tested against simulated composite 16O/18O spectra where peptide molecular weight, 16O/18O ratio, 18O1/18O2 ratios, and number of sulfur atoms were varied. Relative errors of 20% or less were incurred when the 16O/18O ratios were less than three, even for peptides where the number of sulfur atoms was over- or under-estimated. These data demonstrate that for biomarker discovery, it is advantageous to label the proteome representing the disease state with 18O; and the method is not sensitive to variations in 18O1/18O2 ratio. This approach allows a comprehensive differentiation of expression levels and tentative identification via AMTs, followed by targeted analysis of over- and under-expressed peptides using tandem mass spectrometry, for applications such as the discovery of disease biomarkers.     

Determination of metolcarb in food by capillary electrophoresis immunoassay with a laser-induced fluorescence detector

A capillary electrophoresis immunoassay (CEIA) was developed for the determination of trace metolcarb (MTMC) in food. The method was based on the competitive reactions between fluorescently labeled MTMC tracer and free MTMC with a limited amount of anti-MTMC antibody and the separation and determination by CE with LIF detector. A fluorescent reagent, FITC was labeled on MTMC to construct an immunofluorescent probe. CEIA experimental parameters such as the pH value and concentration of the running buffer and separation voltage as well as incubation time were systematically investigated. Under the optimized conditions, fluorescently labeled antigen and antibody bound could be well separated within 3 min using Na?B?O?/NaH?PO? buffer (20:10 mmol/L, pH 9.0) for background electrolyte, 20 kV for the separation voltage, and 20°C for the column temperature. The linear range of the method was 0.25-50.0 μg/L with LOD 0.07 μg/L. The RSD for relative migration time and relative fluorescence intensity ratio were 2.90% (intraday) and 4.73% (intraday), respectively. The proposed method has been applied to determine the residue of MTMC in food samples with the satisfactory recovery.     

Sensitive detection of tumor marker CA15-3 in human serum by capillary electrophoretic immunoassay with chemiluminescence detection

A new and sensitive non-competitive immunoassay (IA) for tumor marker carbohydrate antigen 15-3 (CA15-3) by CE coupling with ECL detection has been developed. This method is based on luminol-H(2)O(2 )reaction catalyzed by horseradish peroxidase (HRP). The optimum CE separation and CL detection conditions were investigated. After the non-competitive immunoreaction, the free HRP-labeled CA15-3 antibody (Ab*) and the bound Ab*-antigen (Ab*-Ag) complex were separated in a separation capillary and then catalyzed the CL reaction of luminol and H(2)O(2 )in a reaction capillary following the separation capillary. The calibration curve based on the peak areas of Ab*-Ag complex plotted against the concentrations of CA15-3 is in the range of 0-250 U/mL with a correlation coefficient of 0.9983 and the detection limit is 0.035 U/mL (S/N = 3). The response for five consecutive injections of 125 U/mL CA15-3 resulted in RSDs of 0.83% and 3.1% for the migration time and the peak area, respectively. The method was successfully used for the quantification of CA15-3 in human sera obtained from healthy persons and from patients with breast cancer.     

Isotope dilution gas chromatography/mass spectrometry method for determination of pyrethroids in apple juice

This paper presents the development of a highly precise and accurate analytical method for the determination of three matrix-bound pyrethroids, namely, cypermethrin, permethrin, and bifenthrin, using an isotope dilution gas chromatography/mass spectrometry technique. Identification of the analytes was confirmed under selective ion monitoring mode by the presence of two dominant ion fragments within specific time windows and matching of relative ion intensities of the ions concerned in samples and calibration standards. Quantitation was based on the measurement of concentration ratios of the natural and isotope analogues in the sample and calibration blends. Intraday and interday repeatabilities of replicate analyses of the pyethroids in an apple juice sample were below 0.5%. The expanded relative uncertainty ranged from 3 to 6%, which was significantly lower than the range obtained using internal or external calibration methods. As a labeled analogue is not available for bifenthrin, bifenthrin was determined using labeled cis-permethrin as the internal standard. The results were counterchecked by a gas chromatography-electron capture detection technique using PCB 209 as the internal standard. The method developed was applied to a recent pilot study organized by CCQM and the results were consistent with those of other participants.     

Fast quantitative characterisation of differential mobility responses

A chromatography-based method for producing mass flux-response surfaces for differential mobility spectrometers is described as a replacement for exponential dilution and mixing approaches. An exponential dilution or mixing experiment typically takes 150 min; while the exponential function in the Gaussian elution profile obtained from linear chromatography may be exploited in approximately 10 s. The approach was demonstrated with a gas chromatograph-mass spectrometer and the correlation of the calibration results to nominal on-column masses was within experimental error for 19 separate analyses. The method was then applied to a gas chromatographic (10.6 eV UV) differential mobility spectrometer. Mass fluxes in the range 10 pg s(-1) to 250 ng s(-1) were generated over the 5 s to 10 s associated with the elution of a chromatographic peak. The characterisations were repeated for a range of electrical field strengths from 10 kV cm(-1) to 30 kV cm(-1). Triplicate runs indicated that the approach was reproducible and that response surfaces could be generated rapidly from chromatographic data. The effects of trace impurities associated with the chromatographic eluent on the relationship between compensation voltage and electrical field strength was observed. This emphasised the importance of managing this aspect of the operation if reliable estimates of alpha functions for the compounds under study were to be obtained. Application of this approach to other detection systems with an 85% reduction in the analytical operations required to produce a reliable calibration function was also noted.     

Evaluation of a fluorescein-labeled estradiol derivative for use in affinity capillary electrophoresis

A fluorescein-labeled estradiol derivative was assessed for use in affinity capillary electrophoresis (ACE) in a competitive immunoassay format, in which the fluorescently labeled estradiol competed with unlabeled estradiol for a mouse anti-estradiol antibody. The preparation of the labeled estradiol produced a mixture of fluorescein-containing compounds that led to multiple peaks in the electropherogram and to which the antibody responded differently. Two of the components of the mixture, towards which the mouse antibody showed most affinity, were isolated using fraction collection via capillary electrophoresis (CE). The two fractions of the labeled estradiol products isolated by CE were characterized using mass spectrometric methods. The two active fluorescein-conjugated products differed in the carboxylate on the fluorescein moiety, one having a methyl group instead of the acidic hydrogen for the other. The estradiol antibody showed a stronger binding for the conjugate containing the methyl group, as determined from the estimated binding constants using Scatchard analysis. The isolated fractions of labeled estradiol were shown to be applicable to the ACE immunoassay method.     

Microchip-based amperometric immunoassays using redox tracers

A new chip-based electrochemical immunoassay protocol, based on the use of a ferrocene redox label, is described. Two reaction formats, based on direct (noncompetitive) and competitive modes of operation, were employed for illustrating the use of redox tracers in chip-based electrochemical immunoassays. The direct assay consisted of mixing the ferrocene-tagged antibody and the antigen analyte, a rapid electrophoretic separation of labeled free antibody and the labeled antigen/antibody complex, and a downstream anodic detection of the ferrocene tracer at gold-plated carbon screen-printed electrode detector. The competitive assay integrates precolumn reactions of the labeled antigen and the target antigen with the antibody with electrophoretic separation of the free and bound labeled antigens, along with amperometric detection of the redox tag. An internal standard has been used to normalize the peak area for the construction of calibration plots. Fundamental operating variables are examined and optimized. The use of a redox tracer offers the advantages of simplified protocol, wider linear range, higher stability, and higher separation efficiency compared to an analogous use of enzyme tags. The direct mouse-immunoglobulin G (IgG) assay and the competitive 3,3',5-triiodo-L-thyronine (T(3)) one were accomplished within less than 150 and 130 s (with field strengths of 256 and 192 V/cm), and offer minimum detectable concentrations of 2.5 x 10(-12) and 1 x1 0(-6) g/mL, respectively. Such use of redox labels for chip-based amperometric immunoassay protocols offers considerable promise for decentralized clinical or environmental testing.     

Sensitive and rapid chemiluminescence enzyme immunoassay for microcystin-LR in water samples

A highly sensitive, specific, simple, and rapid chemiluminescence enzyme immunoassay (CLEIA) was developed for the determination of microcystin-LR (MC-LR). Several physicochemical parameters such as the chemiluminescent assay mediums, the dilution ratio of MC-LR-OVA conjugate, monoclonal antibody concentration, and peroxidase labeled antibody concentration were studied and optimized. Under optimum conditions, calibration curve obtained for MC-LR had detection limits of 0.032 ± 0.003 μg L⁻¹, the 50% inhibition concentration (IC₅₀) was 0.20 ± 0.02 μg L⁻¹ and the quantitative detection range was 0.062-0.65 μg L⁻¹. The proposed methods was successfully applied to the monitoring of MC-LR in spiked water samples without significant effect of the matrix, and the recovery of MC-LR added to water samples at different concentrations ranged from 80% to 115% with the coefficients of variation (CVs) less than 9%. The LOD attained from the calibration curves and the results obtained for the real samples demonstrate the potential use of CLEIA as a screening tool for the analysis of MC-LR in environmental samples.     

Taking into account both preparation and injection in high-performance liquid chromatography linearity studies

As mentioned in International Conference on Harmonisation publications, linearity is a principal parameter in method validation. The most popular statistical tool used is linear least-squares regression. Contrary to what is still very often practiced, the correlation coefficient can in no way be considered as an indicator of the fit quality. There is in fact a test called the "lack-of-fit test" that enables one to answer the question, "Is the linear model adapted to the calibration curve?". However, this test can give erroneous conclusions when, at each level, several sources of variation for the response are influent. It often occurs in high-performance liquid chromatography, as shown in a following example, where the calibration curve is obtained from repeated injections of repeated dilutions of a parent solution. The lack-of-fit test rejected linearity, although it was at least questionable. In fact, the reason for a discrepancy of this kind lies in the presence of a double source of variation: injection and dilution. It is possible to overcome the problem by mixing a nested ANOVA with the standard least-square linear regression. As shown in an example, implementing this methodology for data processing allows one not only to carry out an unbiased lack-of-fit test but also give estimates of the dispersion introduced respectively by the preparation and the injection.     

Quantification of Cry1Ab in genetically modified maize leaves by liquid chromatography multiple reaction monitoring tandem mass spectrometry using 18O stable isotope dilution

Cry1Ab is one of the most common Bacillus thuringiensis (Bt) proteins in genetically modified crops, which exhibits strong resistance against insect pests. In the present study, a sensitive and precise liquid chromatography stable isotope dilution multiple reaction monitoring tandem mass spectrometry (LC-SID-MRM-MS) assay was developed and validated to quantify the amount of Cry1Ab expression in transgenic maize leaves. The measurement of protein was converted to measurement of unique peptides to Cry1Ab protein. Two peptides unique to Cry1Ab were synthesized and labeled in H(2)(18)O to generate (18)O stable isotope peptides as internal standards. The validated method obtained superior specificity and good linearity. And the inter- and intra-day precision and accuracy for all samples were satisfactory. The results demonstrated Cry1Ab protein was 31.7 ± 4.1 μg g(-1) dry weight in Bt-176 transgenic maize leaves. It proved that the novel LC-SID-MRM-MS method was sensitive and selective to quantify Cry1Ab in the crude extract without time-consuming pre-separation or purification procedures.     

Highly sensitive rapid chemiluminescent immunoassay using the DNAzyme label for signal amplification

A novel trace tag for chemiluminescent (CL) immunoassay was designed by using DNAzyme to functionalize antibody-labeled Au nanoparticles (AuNPs). The trace tag showed an excellent ability to catalyze the oxidation of luminol by hydrogen peroxide, leading to strong CL emission. By coupling the trace tag with a passive mixing accelerated immunoreaction system, a highly sensitive rapid flow-through CL immunoassay method was proposed. Using carcinoembryonic antigen (CEA) as a model analyte, the capture antibody for CEA was immobilized on paramagnetic microspheres, and DNAzyme-anti-CEA antibody functionalized AuNPs were prepared as trace tag. A three-dimensional helical glass tube kept at 37 °C in a water bath was used for passively mixing immunoreagents in a two-step sandwich immunoassay, with which each immunoreaction step could be finished within 150 s. With the help of a magnet, the immunocomplex could conveniently be separated from reactants. Compared with the horseradish peroxidase-based tag, the newly designed trace tag showed obvious signal amplification due to its strong catalytic ability and high loading ratio of DNAzyme on each AuNP. The proposed method showed a linear calibration range from 0.005 to 0.5 ng mL(-1) for CEA detection with a detection limit of 4.1 pg mL(-1) at a signal-to-noise ratio of 3 and acceptable detection reproducibility. The assay results of clinical serum samples were in acceptable agreement with the reference values. The designed immunoassay system with ultrahigh sensitivity provided a programmable and low-cost approach for high-throughput clinical application.