Polarization Fluoroimmunoassay Research Articles

The degree of cartilage degradation assessed by serum biomarker levels changes after arthroscopic knee synovectomy in rheumatoid arthritis patients.

In order to test the validity of the selected surgical technique as a way to manage persistent synovitis of the knee joint, as well as to slow down the cartilage and bone destruction, we studied the dynamics of biomarkers of inflammatory conditions, and bone and cartilage destruction after total arthroscopic synovectomy (TAS) of the knee joint. The sampling comprised 124 RA patients (158 knees) who had undergone the TAS procedure between 2003 and 2015. Before surgery the rheumatoid factor (IgM), C-reactive protein (CRP), erythrocyte sedimentation rate test was completed for all patients. Blood serum samples were collected (prior to surgery, and three, six and 12months after surgery) and frozen at -70°С. The content of CRP, Matrix metalloproteinase-3 (MMP-3), Cartilage Oligomeric Matrix Protein, as well as cross-linked Ctelopeptides of types I and II were measured in blood serum by means of polarization fluoroimmunoassay with the use of a standard set of reagents. The average duration of the disease in the studied group was 8.7±6.6years. Concentration of the inflammation markers showed that only MMP-3 displayed statistical significance. The obtained results can be used as basis in assessing the efficiency and effectiveness of this method of treatment of persistent knee joint synovitis associated with the RA.

Liquid Chromatography-Tandem Mass Spectrometry Method for Tdm of Vancomycin and Comparison with Results of Polarization Fluoroimmunoassay

Liquid Chromatography-Tandem Mass Spectrometry Method for Tdm of Vancomycin and Comparison with Results of Polarization Fluoroimmunoassay

Liquid Chromatography-Tandem Mass Spectrometry Method for Determination of Gentamicin Concentrations and Comparison with Two Immunoassay Methods (Chemiluminiscent Assay a Polarization Fluoroimmunoassay)

Gentamicin belongs to aminoglycoside antibiotics. Therapeutic drug monitoring (TDM) of gentamicin is recommended for all patients to assure both sufficient and safe concentrations in blood. Gentamicin is composed from a mixture of three drugs (C1, C1A and C2A+C2) with the basic share as follows: C1 25-50%, C1A 10-35% and C2A+C2 25-55%. Gentamicin concentrations are preferably measured using immunoanalytical assays. A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method with simple sample extraction and a relatively short time of gentamicin analysis was developed and validated. 50 µL of serum was precipitated using acetonitrile and formic acid. A RP BEH C18, 1.7 µm, 2.1 x 50 mm column maintained at 30oC and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode. The results were compared with analyses of polarization fluoroimmunoassay (FPIA) method (Abbott AxSYM) and a chemiluminiscent assay (Advia Centauer, Siemens). Passing-Bablok regression analysis and Bland-Altman analysis were used to compare gentamicin concentrations. Calibration curve was linear in the range of 0.1-50 mg/L. Recovery was 91.6-102.0% with a coefficient of variations of 1.4-8.4% for single types of gentamicin. The sum of gentamicin concentrations correlated significantly with both immunoanylatical assays. Presented LC-MS/MS method is fast and precise with simple sample extraction and can be applied for routine TDM of gentamicin.

New liquid chromatography-tandem mass spectrometry method for routine TDM of vancomycin in patients with both normal and impaired renal functions and comparison with results of polarization fluoroimmunoassay in light of varying creatinine concentrations

A new LC-MS/MS method with simple sample extraction and a relatively short period of vancomycin analysis for routine therapeutic drug monitoring was developed and validated. 50μL serum was precipitated using 20μL 33% trichloroacetic acid and 0.5mol/L NH4OH was added to increase pH before analysis. A RP BEH C18, 1.7μm, 2.1×50mm column maintained at 30°C and tobramycin as internal standard were used. Mass detection was performed in positive electrospray mode.The results obtained with LC-MS/MS method were correlated with an FPIA assay (Abbott AxSYM) using mouse monoclonal antibody. Subjects were divided into three groups according to creatinine levels (53.5±19.1, 150.2±48.4, 471.7±124.7μmol/L) and Passing-Bablok regression analysis and Bland-Altman analysis were used to compare vancomycin concentrations. The results of subjects with both normal and higher creatinine levels correlated very well and the linear regression model equations were near ideal (LC-MSVAN=0.947×AbbottVAN+0.192 and LC-MSVAN=0.973×AbbottVAN−0.411 respectively). Dialyzed patients with the highest creatinine levels showed about 14% greater vancomycin concentration with the FPIA assay (LC-MSVAN=0.866×AbbottVAN+2.127). This overestimation probably due to the presence of the metabolite CDP ought not to be of clinical relevance owing to the wide range of recommended vancomycin concentration.

Enzyme-linked immunosorbent assay for determination of cyclosporin a in whole blood

A test-system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of cyclosporin A (CSA) in human whole blood has been developed. The detection limit of the method was 25 ng/ml, the linearity of the method in the concentration range 60-1400 ng/ml varied from 94 to 105%, the variation coefficient did not exceed 8%. The novel method exhibited good correlation with radio-immunoassay and polarization fluoroimmunoassay methods; the linear regression coefficients were 0.965 and 0.983, respectively. The developed test system is stable for at least 9 months when stored at 4 degrees C and can be used in clinical practice.

Immunochemical methods for the determination of mycotoxins

The state-of-the-art immunochemical methods for the determination of mycotoxins are considered. Both instrumental (enzyme-linked immunosorbent assay, polarization fluoroimmunoassay, and sensor devices) and noninstrumental methods are presented. The principles of particular methods are considered, and the examples of the use of these methods for the determination of mycotoxins from various groups in food products and animal feeds are given; the main lines of development are discussed.

Hapten Design and Development of Polarization Fluoroimmunoassay for Nonylphenol

Polyclonal antibodies to nonylphenol (NP), raised against two different haptenic derivatives were characterized by a rapid, homogeneous polarization fluoroimmunoassay (PFIA). The first hapten, ω-(4-hydroxyphenyl)nonanoic acid (NP9), was designed to mimic the linear NP isomer and contains hydroxyphenyl and linear alkyl chain moieties of the NP molecule. The second hapten, 4-aminophenol (4AP), contains the hydroxyphenyl moiety of NP molecule alone and thus potentially mimics various phenolic compounds with different side chain structures. A number of fluorescent labeled antigens (tracers) with various structures of the spacer arm between the antigen and the fluorescent dye was synthesized and used to optimize the competitor structure for NP-specific PFIA. The most sensitive assay with limit of detection (LOD) and IC50 values of 8 and 53 mg L–1, respectively, was obtained when anti-NP9 antibody and NP9-labeled antigen were used. Anti-NP9 resulted in more specific assay, where the cross-reactivity toward the relative phenolic compounds did not exceed 5%. Anti-4AP displayed substantial recognition of several bis-substituted phenols, including 2-amino-4-chlorophenol and 2,4-dinitrophenol.

Preliminary Screening Method for Dioxin Contamination Using Polarization Fluoroimmunoassay for Chlorinated Phenoxyacid Pesticides

Polarization fluoroimmunoassays (PFIA) were developed for the chlorinated pesticides 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In order to optimize the PFIA procedures, a number of fluorescein-labeled 2,4-D and 2,4,5-T derivatives were synthesized and the influence of their structures on PFIA characteristics was studied. Also, several antisera were tested in developing the PFIA for 2,4,5-T. The assays were adapted for use with the Abbott TDx Analyzer and could be run in automatic mode by the adaptation of existing software and protocols. Dynamic ranges for 2,4-D and 2,4,5-T were 0.2-200 ng mL–1 and 30-10 000 ng mL–1, respectively. Total time for the automated assay of 20 samples was about 22 min. PFIA provides a suitable means for screening of a large number of samples. The rapid determination of 2,4,5-T, which is one of the precursors of polychlorinated dibenzo-p-dioxins, one of the most toxic groups of pollutants, may potentially be used to provide preliminary evidence of dioxin contamination.

Development of a Polarization Fluoroimmunoassay for the Herbicide Metsulfuron-Methyl

A polarization fluoroimmunoassay (PFIA) based on previously raised rabbit polyclonal antibodies was developed for detection and quantification of the sulfonylurea herbicide metsulfuron-methyl (MSM) in surface water samples. A 'restricted' hapten, consisting of only phenylsulfonamide moiety of MSM molecule was used to synthesize a fluorescein-labelled derivative of MSM (tracer). The limited assay cross-reactivity was observed towards most of sulfonylurea herbicides, with exception of primisulfuron-methyl and chlorimuron-ethyl. The optimized MSM PFIA had a dynamic range from 30 to 1000 w g r -1 with an IC 50 value of 123 w g r -1 and a lower detection limit of 5 w g r -1 . Intra- and inter-assay precision over the of MSM calibration curve dynamic range were 0.9 and 7.1%, respectively. Influence of several organic solvents on MSM PFIA performance was investigated, and the assay was found to tolerate up to 10% v/v of methanol, DMSO and acetone. Application of MSM PFIA technique to the analysis of surface water samples taken from lakes and rivers in Bavaria, and spiked with MSM at concentration of 50 w g r -1 , gave mean MSM recoveries of 108.6-110.8%.

Experimental study and mathematical modeling of the interaction between antibodies and antigens on the surface of liposomes

Unilamellar liposomes with incorporated hapten–phospholipid conjugates were proposed as models of polyvalent antigens with migrating determinants for quantitative analysis of their interaction with antibodies. The monovalent pesticide atrazine was used as a model antigen. For its incorporation into the lipid bilayer, the atrazine carboxylated derivative was conjugated with dimyristoylphosphatidylethanolamine (DMPE). Unilamellar liposomes were prepared with dimyristoylphosphatidylcholine/atrazine–DMPE at molar ratios of 90:10, 95:5, 98:2, 99:1 and 99.5:0.5. Their interaction with the peroxidase-labeled anti-atrazine antibodies was studied by enzyme immunoassay and polarization fluoroimmunoassay techniques. It was shown that the increase in hapten content in the liposomes from 0.5 to 10 mol% led to an increase in the equilibrium constants of the interaction with antibodies from 0.093×10 8 to 0.303×10 8 M −1. The association rate constants varied from 1.45×10 5 to 15.5×10 5 M −1 s −1 depending on the antigen content in liposomes and experimental conditions. The measured constants were applied for a mathematical model describing multi-step interaction between antibodies and polyvalent liposomal antigens. The model adequately describes the quantitative regularities of the influence of antigen content and the affinity of immunochemical interaction on the quantity and the dynamics of the immune complexes forming.

DEVELOPMENT OF A POLARIZATION FLUOROIMMUNOASSAY FOR LINEAR ALKYLBENZENESULFONATES (LAS)

ABSTRACT A polarization fluoroimmunoassay (PFIA) based on rabbit polyclonal antibodies was developed and optimized for the detection and quantification of linear alkylbenzene sulfonates (LAS). Hapten mimics with different lengths of the alkyl radical and positions of the phenyl ring were synthesized to prepare fluorescent conjugates (tracers) for PFIA. Structures of target compounds, LAS derivatives and tracers were confirmed by nuclear magnetic resonance and mass spectrometry. Fluorescent tracers in combination with antibodies raised against different LAS immunogens were used for the development of class-selective PFIAs based on different structural combinations of the aliphatic chain moiety. The most sensitive assay was found for the homologous antibody-tracer combination based on the 3-(p-sulfophenyl)butyric acid hapten mimic. The PFIA has a limit of detection of 0.5 mg/L, calibration curve midpoint of 9 mg/L and a dynamic range of 3 to 85 mg/L. Cross-reactivity studies demonstrated the assay is specific towards the sulfophenyl moiety of anionic surfactants while the recognition of aliphatic alkylsulfonates was not significant (cross-reactivity<5%). Total assay time for 10 samples was 7 min, thus allowing fast and reliable detection of LAS in the low mg/L range.

Express detection of pentachlorophenol as dioxins precursor in natural water.

A rapid detection method for the pesticide pentachlorophenol (PCP) — polarization fluoroimmunoassay (PFIA) — in the dynamic range of 10–9,000 ppb was developed. PCP may form polychlorinated dibenzo-p-dioxins, making environmental monitoring of this compound an issue of great importance. In order to optimize the PFIA procedure, a number of fluorescein-labeled PCP derivatives and similar compounds (tracers) were synthesized, and the influence of their structure on PFIA characteristics was studied. Also, two antisera were tested in developing PFIA for PCP. The developed method is highly specific for PCP and can be used for its determination in water samples at a level down to 10 ppb. Total time of the assay for 10 samples is about 7 min. The assay provides a useful and a highly practical screening tool for the processing of large numbers of samples and for the preliminary estimation of potential dioxins contamination in water resources.

Production of Antibodies and Development of Specific Polarization Fluoroimmunoassay for Acetochlor

Specific polyclonal antibodies towards acetochlor (2-chloro- N -(ethoxymethyl)- N -(2-ethyl-6-methylphenyl)acetamide) were obtained from rabbits immunized against a 3-mercaptopropionic acid derivative of acetochlor, covalently attached to bovine serum albumin. A polarization fluoroimmuoassay (PFIA) based on these antibodies was developed and optimized to detect acetochlor in water samples. The optimized PFIA had a detection limit of 9 µg/L, linear working range from 50 to 5500 µg/L and within-assay coefficient of variation less than 4%. Cross-reactivity studies demonstrated that these antibodies are capable of specific detection of acetochlor amongst structurally related chloroacetanilide herbicides. Assay cross-reactivity values were: alachlor 0%, metolachlor 2.4%, propachlor 0%, butachlor 0.2% and dimethachlor 0.5%. Five organic solvents commonly used in sample extraction were evaluated for their effect on acetochlor PFIA performance, and methanol and ethanol were found to be compatible with the assay up to 10% v/v.

Antibody production and development of a polarization fluoroimmunoassay for the herbicide triclopyr

This paper describes a homogeneous and competitive fluoroimmunoassay of polarization for the detection and quantification of the herbicide [(3,5,6-trichloro-2-pyridinyl)oxy]acetic acid (triclopyr). Antibodies against triclopyr were produced by immunizing a rabbit with triclopyr–bovine serum albumin (BSA) conjugates. The fluoresceinamine labeled triclopyr were obtained, characterized and isolated. Two standard curves were obtained, one with a linear dynamic range (LDR) between 0.86 and 100 μg ml −1 of triclopyr and the other for lower concentrations (0.0007–100 μg ml −1). The minimum detectable concentration (MDC) was 0.7 ng ml −1 and the relative standard deviation (R.S.D.) was 1.96% (average value of three concentrations of the curve for n=8). The assay of specificity of the antiserum was developed using other molecules structurally very similar or usually found in formulations. The assay was very specific for triclopyr with respect to the other molecules tested. The technique provides good sensitivity, specificity, precision and MDC. This method was applied to triclopyr previously added to tap water and the recoveries obtained were between 97.9 and 130%.

Difficulties in the assay of liposomal amikacin (MiKasome) in serum.

Antibiotic-free human serum was spiked with known concentrations of liposomal amikacin and assayed on the Abbott TDx System, using polarization fluoroimmuno assay (PFIA) kits from Abbott Laboratories, Oxis and Sigma. Although all three kits gave a linear response, the Abbott and Oxis kits showed very low recovery (<21%) with only the Sigma kit giving near 100% recovery. Heating samples at 56 degrees C for 30 min improved recovery with the Abbott and Oxis kits (75-80% of target value), but decreased recovery with the Sigma kit (85% of target value). The loss of amikacin from liposomal amikacin, as measured using the Sigma kit, was related to both temperature and duration of heating, reaching a maximal loss of 21% after 1 h at 60 degrees C.

Antigen–Antibody Interactions in the Reverse Micellar System: Quenching of the Fluorescence of Fluorescein-Labeled Atrazine by Antibodies against Atrazine

This work presents a new method for performing homogeneous fluoroimmunoassay in apolar organic media, quenching fluoroimmunoassay (QFIA). This method is based on utilization of the reverse micellar system of Aerosol OT (AOT) inn-octane as a medium for the analysis of compounds with low water solubility. It is shown using the system for determination of a hydrophobic pesticide atrazine as an example. The conjugate of atrazine with fluorescein (FA) serves as a label for fluorescence detection of antigen–antibody interaction in the reverse micellar system. The fluorescence quantum yield of this compound drastically depends on the microenvironment of the label in the reverse micelle system. Specifically, the binding of this conjugate with the antibodies solubilized in the reverse micelles results in fluorescence quenching. We found that quenching efficiency depends on the properties of the reverse micellar system (surfactant concentration, hydration degreew0,w0= [water]/[surfactant], etc.). The optimal conditions for quenching of FA fluorescence by antibodies in reverse micelles of AOT inn-octane are low surfactant concentration and hydration degree, allowing one to get large reversed micelles (w0= 15–20) capable of retaining solubilized antibodies. Addition of free atrazine results in displacement of the conjugate and restoration of its fluorescence. The sensitivity of the analysis to atrazine is only 10 times less than that of the commonly used method of homogeneous immunoassay, polarization fluoroimmunoassay, in aqueous solution using the same antibodies and conjugate. The advantage of QFIA in reverse micelles is that the analyte can be added when dissolved in nonpolar organic solvent.

Localization of the epitope in methamphetamine and its antibody use for the detection of methamphetamine and benzphetamine by polarization fluoroimmunoassay.

An antibody was prepared, using a four carbon-bridged methamphetamine molecule as an immunogen in order to develop a polarization fluoroimmunoassay for urine screening of methamphetamine and benzphetamine. Also, its binding characteristics were investigated to locate epitope sites of methamphetamine. The study showed that the antibody was highly capable of eliciting a polarization fluoroimmunoassay response. However, the detection limit was much greater for benzphetamine (0.05 ppm) than for methamphetamine (0.2 ppm) and weakly antibody binding was found with methamphetamine. This difference in sensitivity may reflect the similarity of benzphetamine to the immunogen used to produce the antibody. Both benzphetamine and the immunogen have a tertiary amine attached to a carbon bridges whereas methamphetamine has only a secondary amine and amphetamine has a primary amine group. The difference of cross-reactivity data between phenylethylamine drugs and beta-hydroxyl phenylethylamine drugs indicates that the beta-carbon position have a major influence on the antibody interaction. Thus, the substitution of hydroxyl group on beta-carbon resulted in virtually no antibody affinity, even if a tertiary amine or secondary amine group was present in the molecule. This suggests that the beta-carbon chain plays a primary role as the epitope site with cooperative binding site of tertiary amine or secondary amine in alpha-carbon position. A hydroxyl group at the beta-carbon position plays an important inhibitory role to the antibody binding.

Polarization fluoroimmunoassays of progesterone

A quick reliable homogenous polarization fluoroimmunoassay (PFIA) of progesterone was developed. The assay is carried out with Abbott TDx (USA) polarization fluorometer and it takes 5-7 min to analyze 10 samples by this method. The range of progesterone concentrations determined is 1 to 1000 ng/ml. Fluorescein labeled progesterone-3-carbo- xymethyloxime which was used as tracer (labeled antigen) during analysis has been synthesized and purified. Two types of PFIA were developed: one making use of rabbit antiserum to progesterone-3-carboxymethyloxime conjugated with bovine serum albumin (BSA) or keyhole limpet hemocianin (KLH), in the other antiserum to progesterone-11-hemisuccinate-BSA (or KLH) is used. Different combinations of the tracer and antibodies were used. The sensitivity of heterologous PFIA (with antibodies to immunogen heteroioguos to tracer by structure) was higher than that of homologous PFIA. The test is sufficienctly sensitive and specific. The method is particularly valuable for determination of progesterone in model solutions (using buffer standards).

Development of Polarization Fluoroimmunoassay for the Detection of s-Triazine Herbicides

Abstract A rapid, non-isotopic polarization fluoroimmunoassay (PFIA) for the monitoring of the simazine (striazine herbicide) level in water was developed. Polyclonal antiserum was raised in rabbits by immunization with simazine – Keyhole Limpet Haemocyanin conjugate. Sensitivity of the PFIA with the use of heterologous tracer with the shortest bridge between antigen and fluorescein proved to be the highest. All analytical criteria for PFIA were satisfied. The detection limit of simazine (3 ng/ml in 50 μl of sample) was comparable to that for liquid or gas chromatography method. The detection limit of ELISA using the same antiserum and conjugate derivative of atrazine with horseradish peroxidase was 0.1 ng/mL of simazine. The cross-reactivity for PFIA with widely used s-triazine herbicides: atrazine, propazine, terbuthylazine was 100%, 32% and 20%, respectively. The cross-reactivity for PFIA with some metabolites of s-triazines and other herbicides was negligible.

Detection of phencyclidine in urine using a polarization fluoroimmunoassay.

A polarization fluoroimmunoassay was developed for the detection of phencyclidine in urine and the reagents were adapted for use on the Abbott TDx analyser. The assay was used to look for evidence of phencyclidine abuse, over a 6 month period, amongst patients attending drug abuse clinics in the East End of London. Although over 2000 patients' samples tested negative, the assay successfully detected phencyclidine in an external quality control scheme.