Enantioselective sequential-injection chemiluminescence immunoassays for 3,3′,5-triiodothyronine (T3) and thyroxine (T4)

This study deals with the development of enantioselective flow-through immunosensors for triiodothyronine (T₃) and tetraiodothyronine (thyroxine, T₄) on the basis of a competitive assay using enantioselective antibodies. The instrumental set-up is based on a simple sequential-injection system equipped with a chemiluminescence detector and an immunoreactor, which consists of a flow-cell packed with immobilized haptens. As haptens, 4-amino-l-phenylalanine (4-amino-l-Phe), 4-amino-d-Phe or l-T₃ were used. Antibodies directed against 4-amino-l- or d-Phe or l-T₃ were labeled with an acridinium ester. Three different approaches for immobilizing the haptens were investigated including simple adsorption on polystyrene, chemical binding to an activated methacrylate polymer and binding via the biotin-streptavidin binding (BSB) system. The latter approach showed the best results regarding repeatability and sensivity. Using biotinylated l-T₃ immobilized onto a streptavidin-derivatized trisacryl support and labeled anti-l-T₃ antibodies, a detection limit of 15.5 fmol/ml for l-T₃ was obtained. One assay cycle including regeneration takes only about 5 min. This approach was applied to detect l-T₃ in plasma samples without any sample pre-treatment. The average recovery from spiked plasma sample was about 93% with a R.S.D. below 5%.     

A biased solid-phase radioimmunoassay for thyroxine (T4) using T4-125I presaturated antibodies

An assay for thyroxine (T₄) is demonstrated using specific antibodies covalently coupled to controlled pore glass (CPG). In this assay immobilized antibodies saturated with labeled T₄ are employed in a preloaded unit tube configuration. These complexes are stable for long periods of time when stored in buffer. This concept results in a highly reproducible and sensitive assay for T₄ that requires a single pipetting step.     

Chromatographic competitive binding immunoassays: a comparison of the sequential and simultaneous injection methods

Two approaches for performing competitive binding immunoassays by HPLC and other flow-based systems are the simultaneous and sequential injection methods. Both these techniques make use of a column with a limited amount of antibody, onto which is injected a sample and a fixed amount of a labeled analyte analog. An indirect measure of the unlabeled analyte in the sample is then obtained by looking at the amount of analog in either the nonretained or retained peaks. In the simultaneous injection mode, the sample and labeled analog are applied at the same time to the column, while in the sequential mode the sample is injected first, followed by the analog. This results in a difference in the analytical characteristics of these two approaches. This study used chromatographic theory and previous data obtained for injections of human serum albumin (HSA) onto an anti-HSA antibody column to compare the response, detection limits, range, and sensitivity of these methods. Under equivalent conditions, it was found that the sequential method always provided the best lower limit of detection and sensitivity. However, the simultaneous mode had a broader dynamic range and higher upper limit of detection. From these observations, several guidelines were developed regarding the use and selection of such assays for new applications.     

Quantitative LC-ESI-MS Analysis for Pesticides in a Complex Environmental Matrix Using External and Internal Standards

Abstract

Quantitative liquid chromatography electrospray mass spectrometry (LC-MS) analysis for pesticides in a complex environmental matrix using external and internal standard calibration was investigated. Various approaches to introducing different internal standard compounds to address quantitative errors associated with signal suppression were also examined. The study involved the analysis of pesticides in wheat hay matrix samples using three kinds of internal standard compounds: deuterium labeled (D₃), carbon-13 (singly labeled), and structural analogs (derivatives) of the target analytes. Introduction of the internal standard by volumetric addition and direct post-column infusion were also studied and compared.

Isotopically labeled internal standards (i.e. D₃- ¹³C-) were found to be effective in correcting quantitative errors associated with signal suppression. The application of singly labeled ¹³C compounds may result in nonlinear calibration due to mass interference with the target analyte species. The interference may be compensated by using quadratic curve-fitting or subtraction of the interfering component. Although ineffective as volumetric internal standards, structural analogs can be effective in compensating for signal suppression when introduced into the LC effluent by continuous post-column infusion. Furthermore, the post-column introduction method allows the application of a single internal standard compound for the quantification of each analyte in a multi-component mixture.

The use of internal standards can be effectively incorporated into residue analysis development methods for pesticides in environmental matrices. High accuracy and reproducibility can be achieved while improving method efficiency by reducing the need for comprehensive sample clean-up.     

Determination of femtomole/milliliter concentrations of enprostil acid in human plasma using high-performance liquid chromatography-laser-induced fluorescence detection

This paper describes the use of multiple-column high-performance liquid chromatography (HPLC) combined with laser-induced fluorescence for the determination of femtomole/milliliter concentrations of enprostil acid, a prostaglandin analogue, in human plasma. The drug is isolated from plasma by phenyl solid-phase extraction and fluorescently labeled at its carboxyl functional group with a large excess of 2-bromoacetyl-6-methoxynaphthalene. A multi-column method using both normal- and reversed-phase chromatography is necessary to separate the labeled drug from the unreacted reagent. Post-column dilution of the mobile phase with water after the reversed-phase chromatography allows on-line concentration of the labeled analyte onto a guard column prior to the microbore HPLC. A loop guard column device provides a simple way to inject up to 1.0 ml of sample solution onto a microbore column without significantly reducing the column efficiency. A 325-nm He-Cd laser is used to excite the labeled drug, and fluorescence emission is monitored at 450 nm. Using this system, we are able to derivatize, detect, and quantify 5 pg of the prostaglandin analogue in 1.0 ml of plasma.     

Determination of Hydrocortisone in Cosmetics by CdSe/CdS Quantum Dots-Based Fluoroimmunoassay Using Magnetic Fe3O4/Au Nanoparticles as Solid Carriers

This work demonstrated the feasibility of detecting hydrocortisone in cosmetics using a novel CdSe/CdS quantum dots‐based competitive fluoroimmunoassay with magnetic core/shell Fe₃O₄/Au nanoparticles (MCFN) as solid carriers. Hydrocortisone antigen was labeled with the synthesized core/shell CdSe/CdS quantum dots (QDs) to form the antigen‐QDs conjugate. Meanwhile, hydrocortisone antibody was incubated with MCFN and the immobilized antibody was obtained. The immobilized antibody was then mixed sequentially with hydrocortisone and a slightly excess amount of the QDs‐labeled hydrocortisone antigen, allowing their competition for binding with the antibody immobilized on MCFN. The bound hydrocortisone and the antigen‐QDs conjugates on MCFN were removed subsequently after the mixture was applied to a magnetic force. The analyte concentration was obtained by measuring the fluorescence intensity of the unbound hydrocortisone antigen‐QDs conjugates. The proposed method was characterized by simplicity, rapidity, and high sensitivity with a wide linear working range of 0.5 to 15000 pg·mL^?1 and a low detection limit of 0.5 pg·mL^?1. The proposed method was successfully applied to the determination of hydrocortisone in cosmetics with satisfactory results.     

Spectral and fluorescent kinetics features of Nd3+ ion in Nb2O5, Ta2O5 and La2O3 mixed lithium zirconium silicate glasses

A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L⁻¹ HCl–0.10 mol L⁻¹ NaCl solution containing 0.10 mmol L⁻¹ Br₂. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL⁻¹ to 40 ng mL⁻¹ with a detection limit of 5.2 × 10⁻¹⁰ g mL⁻¹. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.     

High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.     

Detection and Separation of Free Amino Acid Enantiomers by Capillary Electrophoresis with a Chiral Crown Ether and Indirect Photometric Detection

18-Crown-6 tetracarboxylic acid (18C6H₄) has been successfully used as a chiral selector for capillary electrophoretic (CE), high-performance liquid chromatographic (HPLC), and gas chromatographic (GC) separation of the enantiomers of DL-amino compounds. We have previously used X-ray crystallographic analysis and HPLC with an immobilized 18C6H₄ chiral stationary phase to study chiral recognition by 18C6H₄ of several DL amino acids (DL-AA). In this study CE was used for chiral recognition of several DL-AA in electrolyte solution containing 18C6H₄, in which the analyte (D or L amino acid) interacts freely. Among 14 DL-AA investigated, the enantiomers of nine (Glu, Ile, Met, PheG, Phe, Ser, Tyr, Val, and Thr) were successfully recognized in 4-15 mM 18C6H₄. Indirect photometric detection with a cationic dye, chrysoidine, was used to monitor non-chromophoric DL-AA. Among nine successfully recognized DL-AA, the D forms of Ser, Thr and Met migrated faster than the corresponding L forms. The strengths of interactions predicted from the order of migration of each enantiomer in CE were different from those in HPLC analysis. The different enantiomer recognition probably can be ascribed to the difference between CE in which the selector is not immobilized and HPLC in which the selector is immobilized by means of a spacer.     

Evaluation of the mode of binding of immunoglobulin to activated agarose.

Determining the orientation of the immobilization of proteins to solid-phase matrices is of critical importance in the development of systems that employ immobilized proteins. Among these are enzyme-linked immunoassays, immobilized enzymes and affinity chromatography matrices. To determine the orientation of immunoglobulin G (IgG) on activated agaroses, we coupled the immunoglobulin covalently to various activated matrices. The IgG was then cleaved with papain and the liberated fragments collected and analyzed using high-performance liquid chromatography. Only Fab fragments could be detected regardless of the activation method used. This implies that IgG binds to these matrices predominantly via the Fc domain. In order to develop a quantitative method of measuring the Fab and Fc fragments, we compared the binding of IgG and its papain cleavage fragments to S-Zephyr columns and Mono-S columns. Comparison between these columns showed that IgG is bound more tightly to the S-Zephyr column and, in contrast, its retention on Q-Zephyr is less than on a comparable Mono-Q column. The resolution of IgG and its fragments was better in all cases on S-Zephyr than on Mono-S under the conditions employed.     

Pseudo-homogeneous immunoextraction of epitestosterone from human urine samples based on gold-coated magnetic nanoparticles

A pseudo-homogeneous immunoextraction method based on gold-coated magnetic nanoparticles (MNPs) for the specific extraction and quantitative analysis of epitestosterone (17α-hydroxy-4-androsten-3-one, abbreviated as “ET”) from human urine samples by high-performance liquid chromatography (HPLC) has been developed. Half-IgG of anti-ET monoclonal antibodies were covalently immobilized onto (Fe₃O₄)_core-Au_shell (Fe₃O₄@Au) MNPs. An external magnetic field was applied to collect the MNPs which were then rinsed with distilled water followed by elution with absolute methanol to obtain ET as the analyte. The obtained extraction solution was analyzed by HPLC with UV detection (244 nm) within 12 min. The standard calibration curve for ET showed good linearity in the range of 20-200 ng mL⁻¹ in phosphate-buffered saline (PBS) solutions with acceptable accuracy and precision. Limit of detection for ET was 0.06 ng mL⁻¹ due to an enrichment factor of 100-fold was achieved. The results obtained by the present method for spiked human urine samples were in agreement with those from indirect competitive enzyme-linked immunoadsorbent assays (ELISAs). The antibody-conjugated Fe₃O₄@Au MNPs are novel materials for immunoaffinity extraction. Compared with the conventional technique using immunoaffinity column, the method described here for sample pretreatment was fast, highly specific, and easy to operate.     

NBD-Cl as a Post-Column Reagent for Primary and Secondary Amines after Separation by Ion-Exchange Chromatography

In this study 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is proposed as a post-column derivatization (PCD) reagent for the fluorescence detection of aliphatic primary and secondary amines after HPLC separation. Five primary (methylamine, isoamylamine, 2-phenylethylamine, putrescine, and histamine) and one secondary amine (dimethylamine) were separated isocratically on a cation-exchange column using HNO₃ (5 × 10^?3 mol L^?1) as the mobile phase. Post-column derivatization was based on two steps: 1) the derivatization of amines with NBD-Cl in alkaline medium, and 2) the acidification of the resulted mixture in order to minimize the background signal of the reagent and improve dramatically the sensitivity and determination range. The variables of the post-column reaction (concentration of NBD-Cl, buffer concentration and pH, reaction temperature, concentration of HCl, flow rates of the reagents) were thoroughly investigated. Critical chromatographic parameters such as the concentration of HNO₃, the percentage of organic solvent, and the column temperature were also examined to achieve adequate separation. An internal standard of 1,7-diaminoheptane was used. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LODs 20–100 µg L^?1 with S/N = 3), and it requires less sample preparation. The applicability of the proposed analytical scheme was demonstrated by the determination of histamine (HIS) in tuna fish tissues according to US Food and Drug Administration (FDA) guidelines.     

SPE of 5‐lipoxygenase metabolites and the effect of head‐column field‐amplified sample stacking in MEKC

The SPE of leukotrienes and eicosatetraenoic acids using anion exchange materials was compared to the classical extraction with C₁₈ columns. A silica‐based strong anion exchanger, a polymer‐based weak anion exchanger, and a polymer‐based mixed‐mode strong anion exchanger were studied. All anion exchange materials displayed a higher recovery of the analytes with values between 70 and 90% when extracting standard solutions and analyzing by HPLC. The effect was less pronounced for the analysis of the compounds in incubations of polymorphonuclear leukocytes. Using MEKC with head‐column field‐amplified sample stacking for analyte quantification, much lower values of the peak areas were observed compared to the determination of the recovery of the analytes by HPLC. Using MEKC analysis, the highest values were found for the polymer‐based weak anion exchange material, while values below 10% were found for the polymer‐based mixed mode strong anion exchanger. This could be attributed to the presence of electrolytes in the eluates that compromised the stacking efficiency. The extent of residual electrolytes depended on the SPE protocol, resulting in large differences of the amount of analyte determined by MEKC when applying head‐column field‐amplified sample stacking for online analyte concentration.     

Magnetic microparticle antibodies and their application to RIAs

Three types of magnetic microparticle antibodies were developed: 1) magnetic second antibody I (MSA-I) where the antibody molecules were directly immobilized by physical adsorption on Fe₃O₄ microparticles (magnetic nucleus, MN) 10nm±34% in diameter, 2) magnetic second antibody II (MSA-II) where the antibody molecules were immobilized by chemical coupling on the MN coated with polyacrolein, and 3) magnetic, first antibody (MFA-T₃) where the anti-T₃ antibody molecules were specifically immobilized by immunoadsorption to the second antibody molecules of the MSA-II. The optimal conditions for their preparation were elaborated. Some physical, chemical and immunological characteristics of these magnetic microparticles were described. The application of the MSA-I, MSA-II and MFA-T₃ to RIAs for evaluation of thyroid function, such as triiodothyronine (T₃), reverse T₃(rT₃), free T₃ (fT₃), thyroxine (T₄), free T₄(fT₄), thyroid-stimulating hormone (TSH), thyroglobulin (TG) and TG-antibody (TG-Ab), etc., was reported.It is a final report of Research Contract No. 6485/ RB and part of Coordinated Research Programme on Antibodies Immobilized on Magnetic Particles for Radioimmunoassay and Immunoradiometricassay of Hormones, supported by International Atomic Energy Agency.     

Capillary Electrophoresis Immunoassay for 2, 4-Dichlorophenoxyacetic Acid

ABSTRACT

A capillary electrophoresis (CE) immunoassay format for 2, 4-dichlorophenoxyacetic acid (2, 4-D) is demonstrated. A fluorescent labeled 2, 4-D analog competes with the analyte of interest for a finite number of binding sites provided by anti-2, 4-D monoclonal antibodies. CE then provides a means of separating and measuring both the free and antibody-bound fluorescent tracer using laser-induced fluorescence detection. For this assay format, the amount of free tracer is a sensitive indicator for the concentration of analyte present in the sample. A sequential injection format allows the rapid analysis of a small number of samples. The dynamic concentration range for 2, 4-D in either buffer or river water is 5 ppb to 1000 ppb.     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Site–specific immobilization of Fab’ fragments of goat antihuman IgG on quartz surfaces

This paper presents a strategy for immobilizing biomolecules onto a solid surface. We used the free thiol group directing method to immobilize Fab’ fragments to the MPTMS derived quartz substrate and results of X-ray photoelectron spectroscopic studies on the binding of MTPMS and Fab’ fragments on the quartz surfaces are reported. We also prepared a new type of immuno-labeled protein which was the rare earth element labeled antigen (human IgG) to recognize Fab’ fragment. Four characteristic peaks has been determined on the surface specifically bound human IgG labeled Tb³⁺.     

离子色谱-柱后衍生紫外检测法测定化妆品中碘酸盐与溴酸盐

建立了同时检测化妆品中溴酸盐和碘酸盐的离子色谱-柱后衍生紫外检测器定量分析方法。样品用水提取,经RP柱净化,采用IonPac AS11-HC色谱柱分离,以EG40自动淋洗液发生器生成的KOH为淋洗液梯度洗脱,溴化钾、亚硝酸钠为衍生试剂,二极管阵列检测器检测,外标法定量。结果表明:该方法在2.0～1 000 mg/kg范围内线性关系良好(r≥0.999 8);加标回收率为84%～99%;相对标准偏差小于2%。方法简单、快速、选择性强,可用于化妆品中碘酸盐和溴酸盐的测定。     

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     

HPLC Determination of Colistin in Human Urine Using Alkaline Mobile Phase Combined with Post-Column Derivatization: Validation Using Accuracy Profiles

In this study, the development, validation, and application of a new liquid chromatography post-column derivatization method for the determination of Colistin in human urine samples is demonstrated. Separation of Colistin was performed using a core–shell C₁₈ analytical column in an alkaline medium in order (i) to be compatible with the o-phthalaldehyde-based post-column derivatization reaction and (ii) to obtain better retention of the analyte. The Colistin derivative was detected spectrofluorometrically (λ_ext/λ_em = 340/460 nm) after post-column derivatization with o-phthalaldehyde and N-acetyl cysteine. The post-column derivatization parameters were optimized using the Box–Behnken experimental design, and the method was validated using the total error concept. The β-expectation tolerance intervals did not exceed the acceptance criteria of ±15%, meaning that 95% of future results would be included in the defined bias limits. The limit of detection of the method was adequate corresponding to 100 nmol·L⁻¹. The mean analytical bias (expressed as relative error) in the spiking levels was suitable, being in the range of −2.8 to +2.5% for both compounds with the percentage relative standard deviation lower than 3.4% in all cases. The proposed analytical method was satisfactorily applied to the analysis of the drug in human urine samples.