Development of an automated flow injection chemiluminescence immunoassay for human immunoglobulin G

The development of a competitive solid phase flow-injection immunoassay with on-column chemiluminescence detection is described. The immunoreactor column consists of a transparent teflon tubing packed with immobilized antibodies. It is placed in front of the photomultiplier tube. The entire assay takes place in this immunoreactor cell. The assay is performed by injection of a mixture of sample HIgG and acridinium labeled HIgG followed by hydrogen peroxide to initiate the chemiluminescence reaction. One assay cycle including regeneration takes 7 min. The system is fully automated and controlled by computer to ensure exact timing resulting in a precision of 1 to 3%, dependent on concentration. The detection limit was 7 fmol. The method was applied to serum samples without any sample clean-up. The average recovery from spiked serum samples was 103%.Dedicated to Professor Dr. Dr. h.c. mult. J.F.K. Huber on the occasion of his 70 birthday     

流动注射化学发光免疫分析研究I. 血清中乙型肝炎表面抗原的测定

我们首次以键合有抗体的多孔玻璃作为固相免疫分析的免疫反应器, 以化学发光作为最终检测手段, 建立了一种新的、高效率的免疫分析技术-流动注射化学发光免疫分析技术。实验表明: 采用该技术可使单次测定时间从ELISA(Enzyme-linked Immunosorbent Assay)法的二十多小时降至二十分钟, 且所有操作均可在微机控制下自动完成。用该方法对人血清中乙型肝炎表面抗原的测定结果与ELISA法所得结果一致, 对同一样品连续九次测定的相对标准偏差为7.2%。因此, 该方法具有自动化程度高、分析速度快、稳定性好的优点。     

Automated stand-alone flow injection immunoanalysis system for the determination of cephalexin in milk

A fully automated stand-alone flow injection immunoanalysis (FIIA) device for the determination of cephalexin in milk is developed with a main focus on the investigation of the influence of the sample matrix. The system is based on principles of flow-through immunoassays and on sequential addition of the assay components to an immunoreactor. Protein G is immobilised on the surface of the immunoreactor serving as affinity matrix for the polyclonal anti-cephalexin antibodies. A cephalexin-alkaline phosphatase conjugate is mixed with the analyte-containing sample and binds in a competitve manner to the corresponding antibodies in the immunoreactor. After substrate addition enzymatically generated p-aminophenol is detected at a carbon electrode at +150 mV vs. Ag/AgCl. One assay cycle takes 16 min including regeneration of the immunoreactor. The large excess of protein G allows for more than 150 regenerations without significant loss of signal height. Due to the high specificity of the anti-cephalexin antibodies, other beta-lactam antibiotics like penicillin, amoxicillin and cloxacillin do not interfere in the measurements, even when added at 10 mg l-1. To deactivate alkaline phosphatase present in milk, samples are heat-treated for 3 min prior to measurements. Cephalexin recoveries from two milk samples are 90 and 110%. The detection limit in milk is 1 microgram l-1 (mean relative standard deviation of 3%), less than the maximum residue level of 4 micrograms per kg milk fixed for some beta-lactam antibiotics in the European Union. The device is suitable for fast quantitative data generation from consecutively measured samples and thus adds to analytical screening methods.     

Sequential injection immunoassay for environmental measurements

Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis.     

Flow-Injection immunoassays with acridinium ester-based chemiluminescence detection

A type of flow-injection immunoassay has been developed that makes use of acridinium ester-labelled antibodies for detection. The entire assay, including detection, takes place in a transparent 20-μl immunoreactor containing antibodies covalently immobilized to a rigid beaded support, Pierce Trisacryl GF-2000. An assay for mouse IgG in which the immunoreactor is prepared by the immobilization of anti-mouse IgG is presented as an example. A two-site immunoassay is accomplished by the consecutive injection of the sample, acridinium ester-labelled antibodies and alkaline hydrogen peroxide to initiate chemiluminescence. The light emission is collected directly from the transparent immunoreactor, which is then regenerated in preparation for the next sample injection. The detection sensitivity and time required per sample are dependent on the assay flow-rate and for times of 10, 12 and 18 min per sample the limits of detection are 500, 200 and 50 amol of mouse IgG, respectively. The precision of replicate measurements has a relative standard deviation of 3–4%, and the same 20- μl immunoreactor can be used for at least 1 week with an inter-assay relative standard deviation of 5.9%.     

Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL⁻¹) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL⁻¹.     

Sequential injection technique for automation of complex analytical procedures: fluorometric assay of factor thirteen

A non-segmented flow method is used to automate an analysis which involves five different reagents. The fluorometric assay for factor thirteen (FXIII) is performed by the sequential injection analysis (SIA) technique. Two reactions take place in a single fine SIA system to produce the final fluorescing product. Because of its mechanical simplicity and versatility, the sequential injection (SI) technique is shown to be an attractive tool for automation of a complex analytical procedure. In addition to collecting quantitative and kinetic information, the SI system is illustratively used for optimization of the analysis and for obtaining validation information.     

反相流动注射胶束增敏化学发光分析法测定硝苯地平

基于胶束介质中硝苯地平对碱性鲁米诺-过氧化氢化学发光体系的增敏作用,结合反相流动注射技术,建立了流动注射化学发光分析法测定硝苯地平的新方法.硝苯地平浓度在3.5×10-10～4.0×10-8 g·mL-1范围内时,化学发光强度与硝苯地平的浓度呈良好的线性关系,其相对标准偏差为1.4%(n=11,c=3.5×10-9 g...     

Liposome-based flow injection enzyme immunoassay for theophylline

Preliminary results are presented on the development of a sensitive, quantitative immunoassay based on a regenerable, flow injection analysis system incorporating a double-amplification approach. The double amplification is achieved by means of liposome-encapsulated peroxidase enzyme molecules which are released subsequent to a competitive immunological reaction with analyte molecules for immobilized antibodies. The released peroxidase enzymatically cleaves, from an organofluorine substrate, fluoride ions which are then potentiometrically measured. The entire process is carried out in a flow injection analysis system. The competition between the analyte molecules (theophylline) and theophylline-derivatized liposomes for immobilized antibody sites in flow-through immunoreactor column results in unbound liposomes being carried downstream where they are ruptured in the presence of hydrogen peroxide andp-fluorophenol. The peroxidase molecules released react enzymatically to produce fluoride ions which are measured with an ion-selective electrode. The immunoreactor column is then regenerated with a chaotropic agent and the next sample or calibration solution is injected. By means of column regeneration and calibration, accurate quantitation can be achieved; a feature missing from conventional batch-type immunoassays. By means of this liposome/enzyme double-amplification approach, theophylline was determined over a range of concentrations from 0.2 to 4000 ng/ml. The detection limit of 200 pg/ml corresponds to about 100 femtomole of theophylline measured in the 100 l sample injected.Dedicated to Professor W. Simon on the occasion of his 60th birthday     

A chemiluminescence flow injection system for nitrite ion determination

A chemiluminescence system is described for the determination of nitrite ion based on new designs for an ozone generator, liquid-gas separator and chemiluminescence reaction cell. The method is based on the gas-phase chemiluminescence reaction between ozone and nitric oxide, which is generated from the reduction of nitrite with iodide in sulfuric acid solution. The efficiency of the system was evaluated by investigation of the analytical performance characteristics of the system for nitrite determination in batch and flow injection procedures. Under optimal conditions, the chemiluminescence response of the system was linear against the nitrite concentration over the range 1 to 1 × 10⁴ ng ml^?1 in the batch procedure and 10 to 5 × 10³ ng ml^?1 in the flow injection procedure, with detection limits of 1 and 10 ng ml^?1, respectively. The method is highly selective and allows for the determination of nitrite in the presence of high concentrations of several cationic, anionic and nitrogen containing species. It has been successfully applied to the analysis of nitrite in natural water and soil extracts.     

Determination of salbutamol using on-line solid-phase extraction and sequential injection analysis. Comparison of chemiluminescence and fluorescence detection

Determination of salbutamol using sequential injection analysis (SIA) with chemiluminescence and fluorescence detection has been devised. The chemiluminescence signal was emitted during the oxidation of salbutamol by potassium permanganate in sulfuric acid medium. Sodium polyphosphate was used as chemiluminescence enhancer. The fluorescence signal (excitation wavelength 230 nm) was also measured in sulfuric acid medium. Both detection techniques were compared with respect to the application of the methods to the determination of salbutamol in biological materials. The sample pre-treatment takes place directly in the SIA system, when salbutamol is adsorbed on the solid-phase (Baker-carboxylic acid) microcolumn integrated into the system. Sulfuric acid serves both as the reagent and the eluent. The lab-made SIA system consisted of a 2.5-mL Cavro syringe pump, ten-port Vici Valco selection valve and Spectra-Physics FS 970 fluorescence detector, which was lab-modified for chemiluminescence detection. The system was controlled by a PC using originally compiled LabVIEW-supported software. Concentrations, volumes of reagents and flow rates were optimised by a simplex method. Salbutamol was determined in the linear range 0.05-10 microg mL(-1) (RSD 1.53%), with the detection limit (3 sigma) 0.03 microg mL(-1) and sample throughput of 42 samples per hour with chemiluminescence detection in standard solutions. The fluorescence detection enabled the determination of salbutamol in standard solutions in the linear range 0.5-100 microg mL(-1) (RSD 2.69%), with the detection limit 0.2 microg mL(-1) and sample throughput of 24 h(-1). The proposed methods were applied to the determination of salbutamol in human serum and urine. However, serum is a very complicated matrix and the SIA-SPE analysis did not provide satisfactory results. It was possible to determine salbutamol in human urine using this technique. Better recovery was achieved with fluorescence detection.     

On-line monitoring of glucose and penicillin by sequential injection analysis

A sequential injection analysis (SIA) system has been developed for on-line monitoring of glucose and penicillin during cultivations of the filamentous fungus Penicillium chrysogenum. The SIA system consists of a peristaltic pump, an injection valve, two piston pumps, two multi-position valves and a detector. The glucose analyzer is based on an enzymatic reaction using glucose oxidase, which converts glucose to glucono-lactone with formation of hydrogen peroxide and subsequent detection of H₂O₂ by a chemiluminescence reaction involving luminol. The penicillin analysis is based on formation of penicilloic acid by penicillinase. Penicilloic acid is detected either by a chemiluminescence method or by a decolorization method. In the chemiluminescence method the penicilloic acid is quantified by its quenching effect on the chemiluminescence signal obtained when luminol reacts with iodine. In the decolorization method the penicilloic acid is detected spectrophotometrically by the decrease in the absorbance of an iodine-starch complex.     

A new analytical procedure for assay of lysozyme in human tear and saliva with immobilized reagents in flow injection chemiluminescence system.

A novel analytical procedure based on chemiluminescence (CL) detection was described for the determination of lysozyme at ng ml(-1) level by using controlled-reagent-release technology in a flow injection system. The analytical reagents involved in the CL reaction, including luminol and periodate, were both immobilized on the anion-exchange resins in the flow injection system. Through water injection, luminol and periodate were eluted from the anion-exchange column to generate the chemiluminescence, which was inhibited in the presence of lysozyme. By measuring the decrease of CL intensity, one could analyze the lysozyme quantitatively. The decrement of CL emission was linear over the logarithm of lysozyme concentration in the range of 30-1000 ng ml(-1). A typical analytical procedure, including sampling and washing, could be performed in 0.5 min at a flow rate of 2.0 ml min(-1), giving a throughput of 120 h(-1), with a relative standard deviation of less than 3.0%. The proposed method was applied successfully to the determination of lysozyme in human tear and saliva samples, and the recovery was from 92.0% to 105.7%.     

Sequential injection chemiluminescence immunoassay for anionic surfactants using magnetic microbeads immobilized with an antibody

A rapid and sensitive immunoassay for the determination of linear alkylbenzene sulfonates (LAS) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a neodymium magnet. Magnetic beads, to which an anti-LAS monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by means of a neodymium magnet and adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-LAS monoclonal antibody on the magnetic beads and the LAS sample and horseradish peroxidase (HRP)-labeled LAS, and was based on the subsequent chemiluminscence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The anti-LAS antibody was immobilized on the beads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the neodymium magnet, an LAS solution containing HRP-labeled LAS at constant concentration and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the flow cell by collecting the emitted light with a lens. A typical sigmoid calibration curve was obtained, when the logarithm of the concentration of LAS was plotted against the chemiluminescence intensity using various concentrations of standard LAS samples (0-500 ppb) under optimum conditions. The time required for analysis is less than 15 min.     

Flow injection electrochemical enzyme immunoassay for theophylline using a protein A immunoreactor and p-aminophenyl phosphate-p-aminophenol as the detection system.

A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V versus the saturated calomel electrode. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.     

An overview on flow methods for the chemiluminescence determination of phosphorus

A review on the flow analysis of phosphorus with chemiluminescence detection is presented. A brief discussion of the chemiluminescence principles and applications is given. Particular emphasis is devoted to coupling different flow techniques (flow injection, sequential injection, multicommutation, multisyringe flow injection, multi-pumping) to chemiluminescence detection. Enzymatic and non-enzymatic methods, mostly applied to environmental samples, are summarized and compared in terms of application range, detection limits, flow configuration, repeatability and sampling rate.     

Accelerated micro-sequential injection in lab-on-valve format, applied to enzymatic assays

The assay cycle of sequential injection (SI) analysis has been greatly accelerated by simultaneously processing two sample injections within the same manifold. This is achieved through micro-miniaturization of the SI system using the lab-on-valve format (LOV), and by optimizing the assay protocol for stopped-flow reaction rate measurements. The approach has been tested on enzymatic assays of glucose and ethanol, but it is, in principle, applicable to all SI reagent-based assays. The average assay time for a single run has been shortened from 200 s to approximately 30 s. Assays were carried out at 22 degree C and 37 degree C using commercially available reagent kits. For glucose, at 22 degree C, the calibration had a linear response (r(2)= 0.9999) for the concentration range of 100-1000 ppm. At 37 degree C, the calibration was linear (r(2)= 0.9996) for 100-600 ppm glucose, but was a second order polynomial curve (r(2)= 0.9996) for 100-1000 ppm. For ethanol, at both 22 degree C and 37 degree C, the calibrations were linear for the concentration range of 50-250 ppm. The r(2) at two temperatures was 0.9994 and 0.9995, respectively. For both the glucose and ethanol assays, the relative standard deviations were below 3%. Factors affecting sampling frequency are discussed. In this work, sequential injection is shown, for the first time, to achieve sampling frequency comparable to flow injection (FI), while retaining the advantages of small reagent consumption (typically microlitres per assay), minimized waste production, full automation of assay protocols, and zero carryover.     

A Model On-line Flow Injection Fluorescence Immunoassay Using a Protein a Immunoreactor and Lucifer Yellow

Abstract

A heterogeneous on-line fluorescence immunoassay has been developed for a model analyte (transferrin) using a flow injection analysis system containing a controlled pore glass protein A immunoreactor. Lucifer yellow VS (LYVS) a 4-aminonaphthalimide with a large stokes shift and pH independence was the fluorophore. For each assay the antibody-protein A reaction takes place at near neutral pH, and the complexes are eluted at acid pH. Transferrin levels in human serum has been determined by this method, and good within assay variations have been achieved.     

Simple determination of betamethasone and chloramphenicol in a pharmaceutical preparation using a short monolithic column coupled to a sequential injection system

This contribution describes the use of a new separation method based on a reversed-phase sequential injection chromatography (SIC) technique for simultaneous determination of chloramphenicol and betamethasone in pharmaceutical eye drops. A short monolithic column coupled with a sequential injection analysis (SIA) system enabled separation of two compounds in one step. A Chromolith Flash RP-18e, 25 x 4.6 mm column with a 5 mm precolumn (Merck, Germany) and a FIA1ab 3000 system (USA) with a 6-port selection valve and 5 mL syringe were used for sequential injection chromatographic separations in this study. The mobile phase used was acetonitrile-water (30:80, v/v), flow rate 0.48 mL/min; UV detection was at two wavelengths, i.e., 241 and 278 nm (absorption maxima of betamethasone and chloramphenicol, respectively). The basic validation parameters showed good results: linearity of determination for both compounds including internal standard (propylparaben) >0.999; repeatability of determination (RSD) in the range 0.8-1.7% at two different concentration levels, and detection limits in the range 0.5-1.0 mg/mL. The chromatographic resolution between compound peaks was greater than 2.1 and the analysis time was less than 8 min under optimal conditions. The developed sequential injection chromatography method was compared with the HPLC method and was found to be applicable for routine analysis of active compounds in pharmaceutical preparations.     

Determination of Cr(III) in urine, blood serum and hair using flow injection chemiluminescence analysis

A flow injection (FI) chemiluminescence method for the determination of Cr(III) in blood serum, urine and hair samples is reported. It is based on the chromium-catalyzed light emission from the luminol oxidation by hydrogen peroxide. The apparatus consists of an FI system with a flow cell formed by a coiled transparent tube suitable for chemiluminescence detection. The specificity of the method is achieved in presence of EDTA. The detection limit under optimum conditions is 0.01 μg L^–1 of Cr(III). Precision and accuracy were evaluated by determining Cr(III) concentrations in urine standards from the National Institute of Standard and Technology (NIST).