Solid-phase Fluorescence Immunoassay Research Articles

Dietary Phthalates and Low-Grade Albuminuria in US Children and Adolescents

Low-grade albuminuria is an indicator of endothelial dysfunction and is associated with an increased risk of cardiovascular disease. A graded level of exposure to bisphenol A was recently identified to be associated with increased risk of low-grade albuminuria in children and adults. Because bisphenol A and phthalates coexist as dietary contaminants, this study investigated whether exposure to phthalates is also associated with low-grade albuminuria. Data were examined from 667 children who participated in the 2009-2010 National Health and Nutrition Examination Survey and who had results for urinary phthalate metabolites and albumin excretion. Urinary albumin and creatinine concentrations were measured in a first morning specimen using a solid-phase fluorescent immunoassay and a Roche/Hitachi Modular P Chemistry Analyzer with an enzymatic method, respectively. Phthalate metabolites were analyzed in a separate spot urine sample from each participant, using high-performance liquid chromatography and tandem mass spectroscopy. For each (roughly) 3-fold increase in metabolites of di-2-ethylhexylphthalate (a high molecular weight phthalate commonly found in foods), a 0.55 mg/g increase in albumin/creatinine ratio (ACR) was identified (P=0.02), whereas a 1.30-fold odds of a higher ACR quartile was also identified for each (roughly) 3-fold increase (P=0.02). Higher ACR was not identified in relationship to metabolites of lower molecular phthalates commonly found in lotions or shampoos, suggesting specificity. Although reverse causation and unmeasured confounders represent alternative explanations, these findings, in conjunction with our earlier data on bisphenol A, indicate that a wide array of environmental toxins may adversely affect albuminuria and potentially increase the risk of cardiovascular disease. In view of the potential long-term health implications of ongoing exposure in this vulnerable subpopulation, our data support both further study and renewed regulatory efforts to limit exposure during childhood.

Application of a solid-phase fluorescence immunoassay to determine amoxicillin residues in fish tissue

The present study demonstrates an application of Parallux (a solid-phase fluorescence immunoassay) for amoxicillin analysis in fish tissue. Amoxicillin at the recommended therapeutic dose (400 mg/kg body weight) was orally administered to three groups of 25 olive flounder ( Paralichthys olivaceus ), 25 rockfish ( Sebastes schlegeli ) and 25 red sea bream ( Pagrus major ) for 7 consecutive days. Amoxicillin was detected in the muscle of fish treated by the 3rd day of the withdrawal period. The recovery rates of all spiked muscle samples were > 86% of the spiked values. The present study showed that solid-phase fluorescence immunoassay can be easily adopted in predicting amoxicillin residues in the muscle tissue of farmed fish.

Rheumatoid factors and glomerulonephritis

It is presently unknown whether rheumatoid factors have a pathogenic role in the development of various types of glomerulonephritis with immune deposits. Three isotypes of rheumatoid factors (RFs), which are autoantibodies to IgG, were measured using the solid-phase fluorescence immunoassay in sera from patients with diffuse proliferative lupus nephritis (DPLN), membranous lupus nephritis (MLN), IgA nephropathy (IgAN) and idiopathic membranous nephropathy (MN). RF activity of immunoglobulins deposited in the glomeruli from these patients was also studied by examining the binding of the FITC-conjugated human IgG and Fc portion of IgG to the glomeruli of renal biopsy specimens. IgG, IgA and IgM RFs were significantly increased in sera from patients with DPLN, and the increase was significantly lower in patients with MLN, IgAN and MN. Human IgG bound to immunoglobulin on the glomeruli only in DPLN, but not in MLN, IgAN or MN. The Fc portion of IgG was demonstrated to be involved in this reaction. It was suggested that RFs and IgG may play a major role in immune deposits on the glomeruli in DPLN and may be involved in the development of DPLN; however, this is not likely in MLN, IgAN or MN.

Monitoring of streptomycin and dihydrostreptomycin residual levels in porcine meat press juice and muscle via solid‐phase fluorescence immunoassay and confirmatory analysis by liquid chromatography after post‐column derivatization

A solid-phase fluorescence immunoassay (SPFIA) that was primarily developed for detection of antibiotic residues in milk was qualitatively applied for the pre-screening of the residues of aminoglycoside antibiotics, streptomycin and dihydrostreptomycin, in meat press juice. The confirmation of both analytes was performed using a validated method of highperformance liquid chromatography with post-column derivatization. The analytical performance was demonstrated by the analysis of pork meat samples spiked at three concentration levels, ranging from 0.25 to 2.5 ppm for each analyte. In general, the recoveries ranged from 80.4 to 81.5% and from 79.6 to 84.4% for streptomycin and dihydrostreptomycin, respectively, with relative standard deviations lower than 6%. The limits of detection were 0.1 and 0.15 ppm for streptomycin and dihydrostreptomycin, respectively, and the limits of quantification of 0.35 and 0.5 ppm are below the maximum residue limits of Codex, the European Union, and the Korean Food and Drug Administration (ranging from 0.5 to 0.6 ppm). Eight real samples collected from the Seoul area were first monitored using SPFIA, and none of them were found positive. These findings are in good accordance with those observed by HPLC analysis. To the best of our knowledge, this is the first report to monitor the aminoglycoside residues in pork meat press juice using SPFIA.

Microalbuminuria is a negative correlate for cognitive function in older adults with peripheral arterial disease: results from the U.S. National Health and Nutrition Examination Survey 1999–2002

Microalbuminuria (MA) has been increasingly identified as a marker of cardiovascular risk. Although poor cognitive function has been implicated as a sequelae of increased cardiovascular burden, little is known about the association between MA and cognitive function. Population-based cross-sectional study. National Health and Nutrition Examination Survey 1999-2002 in the USA. 2049 noninstitutionalized adults (>/=60 years) with nonmissing values in cognitive test, urinary albumin-to-creatinine ratio (UACR) and ankle-brachial blood pressure index (ABPI) was analysed. Participants with UACR >300 microg mg(-1) were excluded. The UACR, in the unit of microg mg(-1), was calculated by dividing the urinary albumin value by the urinary creatinine concentration. MA was defined as UACR between 30 and 300 microg mg(-1). Cognitive function was measured by a 2-min Digit Symbol Substitution Test (DSST). Peripheral artery disease (PAD) was defined as an ABPI <0.9 in either leg. Overall speaking, MA was inversely associated with DSST score after controlling for age, sex, race, body mass index and educational level (regression coefficient = -2.8, P = 0.002). There was an effect modification of PAD on the association between MA and the DSST score. Amongst participants with PAD, the DSST score for those with MA was lower than those without MA (beta = -6.3, P = 0.003) after multivariate adjustment. Moreover, participants with PAD in the highest quartile of UACR had significantly lower DSST score compared to those in the lowest quartile (beta = -8.7, P = 0.001). There was no association between MA and cognitive function amongst participants without PAD. We observed an additive effect of MA and PAD on DSST score. Participants with both MA and PAD had a lower mean DSST score compared to those without both conditions (beta = -6.2, P = 0.003). The presence of MA or a higher level of urinary albumin excretion was inversely associated with cognitive function in participants with PAD.

Application of a Solid-Phase Fluorescence Immunoassay to Determine Oxytetracycline and Tetracycline Residues in Tissue of Olive Flounder (&lt;i&gt;Paralichthys olivaceus&lt;/i&gt;)

Parallux, a solid-phase fluorescence immunoassay (SPFIA) developed for antibotics residue detection in milk, was applied for analysis of fish tissue. The recommended therapeutic doses of oxytetracycline (OTC, 100 g/ton water, withdrawal period 30 days) and tetracycline (TC, 150 g/ton water, withdrawal period 30 days) were treated to a group of 35 olive flounders (Paralichthys olivaceus) using dipping administration. Muscle was sampled before and after drug treatment 1st, 2nd, 3rd, 5th, 7th, and 14th day. The concentration of oxytetracycline in muscle, determined by SPFIA, was compared with that of internal standard (100 ppb as oxytetracycline). The S/C ratio of sample inhibition value to cutoff inhibition value was employed as an index to determine the muscle residue in olive flounder. To investigate the recovery rate, and standard solutions were added to muscle samples to give final concentrations in muscle of 0.1 and 0.5 microg/ml. The recovery rates of all spiked samples were >89% of the spiked value. OTC and TC were detected in muscle of fishes treated until the 3rd day of withdrawal period. The present study showed that the SPFIA can be easily adopted in predicting tissue residues for OTC and TC in farmed fishes.

Solid-phase immunoassay detection of peptides from complex matrices without a separation

A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps. Both direct and indirect immunoassay methods are demonstrated; the indirect is more sensitive and features a 1 pmol detection limit of neat dynorphin A solutions. To further increase the immunoassay sensitivity, a novel capillary blotting system with hydrophilic membranes was designed where optimized sample volumes of 167 nL were deposited for each spot. The area-reduced blotting method shows a 1000-fold improved, 1.3 fmol spot(-1) detection limit of a dynorphin A diluted in a buffered solution of 150 mg L(-1) of casein. Low-flow push-pull perfusates with volumes of 1 microL sampled from the striatum of the rat were assayed for dynorphin A by the method of standard addition. The detection limit was estimated to be 1.9 fmol in the low-flow push-pull perfusates. These data demonstrate a solid-phase near infrared immunofluorescence strategy for the study of peptides directly blotted from chemically complex biological fluid matrices.

Simultaneous Determination of Different Antibiotic Residues in Bovine and in Porcine Kidneys by Solid-Phase Fluorescence Immunoassay

Parallux, a solid-phase fluorescence immunoassay (SPFIA) developed for antibiotic residue detection in milk, was used for analysis of bovine and porcine kidney tissue. Four tetracyclines, 2 broad-spectrum cephalosporins, 3 beta-lactam antibiotics, and cephapirin were detected in one run after minimal sample preparation. This commercially available test system is designed as cartridges, each with a combination of 1-4 tests. One cartridge can be used to detect 4 analytes in the same sample, or 1 or 2 analytes in different samples. The cartridge with the combination tetracyclines-ceftiofur-penicillin-cephapirin was selected because tetracyclines, beta-lactam antibiotics as well as cephalosporins, are registered for oral or parenteral use in bovines and pigs in Europe. The test is qualitative and is recommended only for screening. Tetracycline, oxytetracycline, chlortetracycline, and doxycycline were easily detected at 300 ppb with the tetracyclines channel; ceftiofur at 1000 ppb and cefquinome at 200 ppb with the ceftiofur channel; penicillin G, ampicillin, and amoxicillin at 50 ppb with the penicillin channel; and cephapirin at 100 ppb with the cephapirin channel. These levels are equal to or lower than the corresponding maximal residue limits in kidney tissue. Cephalexin was not detected. The SPFIA test can be used as an alternative to classical inhibition tests and for post-screening inhibitor- positive kidneys, because it detects 3 specific groups of antibiotics, which enables selection of specific confirmatory methods for identification and quantification.

Part per trillion level determination of isoproturon in certified and estuarine water samples with a direct optical immunosensor

An optical immunosensor coupled to a FIA system was developed and applied to monitor isoproturon in various types of water samples. The assay is based on a solid phase fluorescence immunoassay with immobilised analyte derivative and free, fluorescence labelled antiisoproturon antibodies. Cross-reactivities (CRs) of linuron, diuron and chlortoluron were carried out to assess the influence of structurally similar compounds on the response of the biosensor. The highest CR values were obtained for diuron, 93%, whilst chlortoluron and linuron showed values of 53 and 46%, respectively, at 0.1 μg l −1 of cross-reactant. Detection limits of 0.01 and 0.14 μg l −1 were achieved after the analysis of MilliQ and river water samples, respectively. The validation of the biosensor was carried out analyzing Aquacheck certified river samples which contain different amounts of phenylurea pesticides. This River Analyser (RIANA) prototype with an optical immunosensor can be applied for monitoring of isoproturon in natural river water samples. Individual assays are completed within 15 min including total regeneration of the transducer.

Presence of aldehydic epitopes on a minor low-density lipoprotein fraction

A more negatively charged low-density lipoprotein (LDL), named minor LDL (mi-LDL), was separated by ionic exchange chromatography and further characterized. This mi-LDL contained lower amounts of polyunsaturated fatty acids, α- or γ- tocopherol, but higher amounts of lipid hydroperoxides than the major LDL fraction (ma-LDL). We show here for the first time that apoB of mi-LDL is modified by lipid peroxidation products, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). Using polyclonal antibodies, generated against 4-HNE- or MDA-LDL and apoB, the ratio of 4-HNE- or MDA-derived epitopes to apoB of mi-LDL and ma-LDL was estimated by means of a solid phase fluorescence immunoassay. The ratio of 4-HNE-derived epitopes to apoB on mi-LDL was fourfold higher, while the ratio of MDA-derived epitopes to apoB was twofold higher, compared with the ratios obtained with ma-LDL. In a competition assay with mi- and ma-LDL, only mi-LDL was an effective competitor to inhibit the immunoreaction of anti-4-HNE-LDL with 4-HNE-LDL (by 24%) and of anti-MDA-LDL with MDA-LDL (by 10%).

River Analyzer for Chlorotriazines with a Direct Optical Immunosensor

An optical immunosensor coupled to a FIA system was evaluated to monitor chlorotriazine pesticides in river water samples. The assay is based on a solid-phase fluorescence immunoassay with immobilized analyte derivative and free, fluorescence-labeled anti-atrazine or anti-simazine antibodies. The response of the immunoprobe in the presence of cross-reactants structurally similar to atrazine (simazine, deisopropylatrazine, and deethylatrazine) was studied. Limits of detection for atrazine and simazine varied between 0.06 and 0.2 μg L-1, depending if Milli-Q water or river water samples were used. Monitoring of river water samples in the Ebre area (Tarragona, Spain) during 3 months was carried out. Eighteen measurements were carried out, and only eight samples could be detected with simazine and atrazine levels ranging from 0.18 to 1 μg L-1. The results obtained with the immunosensor were validated by chromatographic techniques. The 50-mL water samples were preconcentrated by on-line solid-phase extraction (SPE) followed by liquid chromatography−atmospheric pressure chemical ionization−mass spectrometry (LC−APCI−MS). In this way, unequivocal confirmation of the environmental levels of the Ebre samples was accomplished. This RIver ANAlyzer (RIANA) with an optical immunosensor can be applied for the monitoring of chlorotriazines in river water samples with a total analysis time of 15 min. No false positive was detected.

Optical immunoprobe development for multiresidue monitoring in water

Aquifers used for drinking water production require regular monitoring for organic pollutants. Pollutant levels and pollutant patterns may change rapidly especially in surface water. Monitoring systems capable of unattended and automated operation are desirable e.g. at pumping sites. In this paper we report on a study of the application of immunoanalytical techniques for flexible and automated multiresidue testing. A solid phase fluorescence immunoassay with immobilised analyte derivate and free, fluorescence labelled antibody is used. Two optical transducers were tested: A simple `slab'-waveguide made of sheet glass and an integrated optical (IO) waveguide. Bulk fluorophore excitation was used to estimate the performance of each transducer. Both transducers allow an antibody surface coverage of less than 1‰ of a monolayer of protein to be detected. The direct and covalent immobilisation of analyte derivates at the transducer surface for a binding inhibition assay approach is compared to a competitive assay with immobilisation of analyte derivates via an auxiliary antibody conjugate. The use of this auxiliary system allows the testing of different analytes at the same transducer surface. Atrazine was selected as a model analyte for the first trials. The ELISA type assay gives a test midpoint at 2.2 μg/l and an estimated limit of detection of 0.3 μg/l. The fluoroimmunoprobe with a binding inhibition assay has a test midpoint for atrazine at about 6 μg/l. In the competitive assay with an auxiliary antibody conjugate signal levels were reduced by a factor of two and competition of free atrazine was poor. Titration with free analyte derivate (atrazine caproic acid) confirmed that this may be optimised by changing the competing derivate.

A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli O157:H7 in ground beef and apple cider.

A solid phase fluorescence-based immunoassay was developed for the detection of Escherichia coli O157:H7 using an antigen down competition format. A soft glass capillary tube served as the solid support, to which heat-killed E. coli O157:H7 were adsorbed. Polyclonal anti-E. coli O157:H7 antibody, conjugated with biotin, was used and the bound antigen-antibody complex was detected using avidin molecules labelled with Cy5, a fluorescent cyanine dye. Any E. coli O157:H7 in the sample would compete with the formation of this complex, reducing fluorescence. This assay was tested for sensitivity with spiked ground beef and apple cider samples. The minimum detectable number of cells present in the initial inoculum was calculated to be approximately 1 colony-forming unit (cfu) per 10 g of ground beef when samples were enriched in modified EC broth for 7 h at 37 degrees C. The minimum detectable number of cells for the apple cider samples was calculated to be approximately 0.5 cfu ml-1. The E. coli cells in the cider samples were captured with immunomagnetic beads, incubated for 7 h in the enrichment broth, and detected with the solid phase fluorescence immunoassay.

Development of a Solid-Phase Fluorescence Immunoassay for the Detection of Salmonella in Raw Ground Turkey

A solid-phase fluorescence sandwich immunoassay was developed to detect Salmonella spp. and tested for sensitivity on pure cultures as well as on spiked and naturally contaminated raw ground turkey. The solid support was a soft-glass capillary tube to which a polyclonal antibody against Salmonella spp. was adsorbed. The adsorbed antibody captured cellular antigens from the sample solution. The same polyclonal antibody was also biotinylated and, in combination with an avidin-Cy5 dye conjugate, served to allow detection of the immunologically captured cells. The minimum detectable number of Salmonella cells spiked into ground turkey was approximately 2.5 CFU/25 g. Fifty-one raw ground turkey samples were tested for the presence of Salmonella spp. using both this immunoassay and a modified Bacteriological Analytical Methods enrichment method. Fluorescence intensity values were normalized as a ratio of the sample fluorescence to the fluorescence of a standard. Fluorescence intensity ratios of 0.50 or greater were scored as Salmonella positive. The solid-phase fluorescence immunoassay yielded 10% false positives and no false negatives when results were compared with those from the modified enrichment method. Isolates recovered from the samples that produced false-positive results were identified as Citrobacter freundii and Enterobacter cloacae, both of which are known to cross-react with the antiserum used.

Detection of cytomegalovirus antibody in platelet concentrates by fluorescence immunoassay and latex agglutination

A passive latex agglutination (PLA) test for cytomegalovirus (CMV) antibody detection has been shown to be an acceptable method of screening both donor sera and plasma from units of red cells and platelets stored in CPDA-1. However, most plateletpheresis concentrates are collected in ACD, and CMV antibody testing of ACD-stored products has not been systematically evaluated by PLA. Sera and ACD-stored platelet concentrate bag segments from 104 donors were tested by PLA and by a solid-phase fluorescence immunoassay (FIAX) as a reference standard for CMV-IgM and CMV-IgG antibodies. Sera were stored at both 4 and 22 degrees C and were tested on Days 1 and 5 of storage; segments were tested daily for 5 days. Of 63 donor samples (61%) that tested negative for CMV-IgG by FIAX, there were two false-positive results in bag segments by PLA testing, one on Day 1 and the other on Day 2 of storage. PLA testing was consistently positive in sera and segments in the 40 donors (38%) who tested positive for CMV-IgG by FIAX. Potential false-negative PLA results occurred in five bag segments derived from one donor whose serum gave equivocal CMV-IgG results on FIAX. The sensitivity and specificity of the PLA assay were 100 percent for donor sera tested at both 4 and 22 degrees C and 91.5 and 98.4 percent, respectively, for platelet bag segment tests. Although no donors positive for CMV-IgM were identified, 15 (14.4%) had equivocal IgM anti-CMV test results.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteria as solid phase in a concentration fluorescence immunoassay analysis of antibodies to surface antigens

A modified solid-phase fluorescence immunoassay was developed using bacterial cells as the solid phase to screen antibodies produced against surface antigens from a clinical isolate of Escherichia coli, strain 1–149. The bacterial solid phase was used to analyze both polyclonal and monoclonal antibodies. The bacterial concentration fluorescence immunoassay (BCFIA) showed up to 50-fold greater sensitivity in bacterial cell detection as compared to ELISA (enzyme-linked immunosorbent assay). Moreover, BCFIA was considerably faster than ELISA with uniform reproducibility. This paper demonstrates the utility of using bacteria and their surface antigens as solid-phase matrices for antibody characterization in a FIA.

Flow cytometric detection of anti-gliadin antibodies

A very sensitive solid-phase fluorescent immunoassay to detect anti-α-gliadin IgA class antibodies is described. The solid phase consisted of polystyrene carboxylated microspheres, of 5 μm diameter, coated with α-gliadin. Serum-specific antibodies bound to the α-gliadin were measured by flow cytometry using fluorescein-conjugated anti-human IgA. 41 samples were tested and the results compared with those obtained by a standard method: an enzyme-linked immunosorbent assay (ELISA). A good correlation was found between the two techniques ( r = 0.96). The sera of untreated coeliac children showed significantly higher antibody values than the sera of children on a gluten-free diet or healthy control groups. The flow cytometric method was more sensitive when the Kolgomorov/Smirnov test was used to analyse the histograms. This method provides an alternative screening test for coeliac disease and may also be used to confirm borderline results obtained in the ELISA test.

Prevalence of Cytomegalovirus Antibodies in Blood Donors

Cytomegalovirus (CMV) obtained through blood products has proven to be a significant pathogen in newborn infants and immunocompromised hosts. Serum samples from 250 blood donors were evaluated by five commercial tests for the presence of CMV antibodies. Fifty percent of the 250 donors were positive by three or more of the tests and therefore were considered to be true positives. Solid-phase fluorescence immunoassay (SFI), indirect hemagglutination assay (IHA), immunofluorescent antibody (IFA), passive latex agglutination (LA), and enzyme immunoassay (ElA) were evaluated for sensitivity, specificity, and ease of use. SFI, ELISA, and LA tests had high sensitivity and specificity ratings. IHA and IF A had high sensitivity ratings but poor specificity rates. IHA and LA had the best functional ratings, followed in order by SFI, ELISA, and I FA. Our findings suggest that LA is the best choice among these tests for an accurate, reliable, and simple screening test for CMV antibodies.

A solid-phase fluorescent immunoassay for detecting canine or mink enteritis parvoviruses in faecal samples

Mink enteritis virus (MEV) and canine parvovirus (CPV) were detected in faecal samples from experimentally or naturally infected minks and dogs, respectively, using antibody-coated polyacrylamide beads (immunobeads, IB) as the solid phase for immunofluorescence (IF) tests. The specificity and sensitivity of the immunobead assay (IBA) were studied by comparing it with an enzyme-linked immunoassay (ELISA), a haemagglutination (HA) test and an IF test using tissue cultures. The IBA was as sensitive as ELISA, but more sensitive than the HA test and the IF test. Furthermore, the use of IB as the matrix for the immunological reactions allows FITC- or enzyme-conjugated antibodies to be used as indicators of the reactions and a simultaneous investigation of several pathogenic agents.

Quantitative solid phase fluorescence immunoassay of rheumatoid factor and C-reactive protein in active rheumatoid arthritis.

A quantitative, semiautomated, solid-phase fluorescence immunoassay (FIA) has been developed for measuring rheumatoid factor (RF) and C-reactive protein (CRP). The correlation of the FIA measurement of RF and CRP with standard measurements in rheumatoid arthritis (RA) patients is not known. To determine the correlation of FIA with standard assay methods, RF and CRP levels were measured by both methods in 151 patients with active RA. RF levels measured by FIA correlated very closely with charcoal agglutination method (r2 = 0.890, P less than 0.0001). CRP levels by FIA correlated very closely with CRP levels by nephelometric method (r2 = 0.886, P less than 0.0001) and Westergren erythrocyte sedimentation rates (ESR) (r2 = 0.356, P less than 0.0001). A weak statistical correlation of RF, CRP, and ESR with some clinical variables of RA disease activity was demonstrated. Measurement of RF and CRP by FIA is similar to standard methods and offers no specific advantages in evaluating RA patients at a single evaluation.