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PubMed1992

Disease-associated anti-bovine serum albumin antibodies in type 1 (insulin-dependent) diabetes mellitus are detected by particle concentration fluoroimmunoassay, and not by enzyme linked immunoassay.

We recently developed a particle concentration fluoroimmunoassay for the measurement of serum antibodies to bovine serum albumin in patients with Type 1 (insulin-dependent) diabetes mellitus. We observed elevated IgG-anti-bovine serum albumin antibodies in 100% of newly-diagnosed diabetic children and in 2.5% of matched control children. Here we compare the fluoroimmunoassay and the more commonly available enzyme linked immunoassay technique, exchanging coded serum samples from 40 newly-diagnosed diabetic children and 179 control children between two laboratories. Particle concentration fluoroimmunoassay detected elevated IgG-anti-bovine serum albumin antibodies in all diabetic children, enzyme immunoassay in 25% (p less than 0.0001). Fluoroimmunoassay detected elevated levels in 2.2% and enzyme immunoassay in 10% of control children (p less than 0.002). Elevated IgA-anti-bovine serum albumin antibodies in patients were slightly more often detected by fluoroimmunoassay than by enzyme immunoassay, while in control children enzyme immunoassays detected elevated levels three times more often (p less than 0.01). Values measured in either assay showed overall no correlation in either patient (IgG:rs = 0.28; IgA:rs = 0.11) or control sera (IgG:rs = 0.02; IgA:rs = -0.05). Fluoroimmunoassay for IgG was 100% disease-sensitive (enzyme immunoassay: 25%, p less than 0.0001) and more disease-specific (IgG; p less than 0.02). Our findings demonstrate that these assay techniques detected distinct subsets of anti-bovine serum albumin antibodies with little (IgG) or some (IgA) overlap. In fluoroimmunoassay procedures, antigen:antibody binding occurs within 1-2 min while hours are allowed in an enzyme immunoassay.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1989

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.

Antibodies, Monoclonal↗

PubMed1991

Evaluation of a new human immunodeficiency virus (HIV) antibody detection method based on time-resolved fluoroimmunoassay.

We have developed a time-resolved fluoroimmunoassay for the detection of anti-HIV antibody using purified virus, recombinant gp120 or a synthetic peptide derived from gp41. The results of time-resolved fluoroimmunoassay correlated well with those obtained by enzyme immunoassay and with the results of immunoblotting. This time-resolved fluoroimmunoassay has sufficient sensitivity and specificity for clinical use.

Fluoroimmunoassay↗

PubMed2006

Magnetically-assisted transport evanescent field fluoroimmunoassay.

The immunoassay, based on specific recognition of an antigen by its antibody, has garnered widespread use in clinical analysis as well as application in such areas as food industry and environmental monitoring. Fluoroimmunoassays (FIAs) are especially attractive due to the inherent sensitivity of fluorescence spectroscopy and the availability of a wide range of commercial antibodies and fluorescent labels. In current form, however, FIAs can be cumbersome, multistep procedures and often lack versatility when there is interest in measuring many different target antigens. This report is proof of a concept paper introducing a new FIA approach, Magnetically-Assisted Transport Evanescent Field Fluoroimmunoassays (MATEFFs), which seeks to preserve the advantages of current approaches to FIAs while attempting to address some of the drawbacks. MATEFFs utilize magnetic microspheres as solid supports for the fluoroimmunoassay with direct detection of bound analyte within the sample mixture effected by selectively driving the functionalized beads to a prism surface using an external magnet. An evanescent wave is generated by total internal reflection of a laser beam at the optical interface between the prism and sample and serves to excite the fluorescent species magnetically delivered into the localized field. This technique eliminates wash steps without compromising sensitivity, all the while minimizing interference from fluorescing species present in the sample matrix. Preliminary optimization studies assessing the impact of background interfering agents, incident angle, magnetic field direction, laser power density via focusing, and bead concentration on MATEFFs performance characteristics are discussed herein along with a detailed description of the experimental platform. Utilizing a model sandwich assay system with biotinylated anti-IgG as the capture antibody, rabbit IgG as the antigen, and anti-IgG-R-phycoerythrin as the reporter antibody, we demonstrate a linear dynamic range of 3 orders of magnitude, physiologically relevant detection limits of low nanograms per milliliter, and RSD values of less than 5%.

Fluoroimmunoassay↗

PubMed1993

New neonatal thyrotropin enzyme immunoassay with fluorimetric detection: comparison with time-resolved fluoroimmunoassay.

This short communication compares a novel fluorimetric microplate enzyme immunoassay (FEIA) with a commercial time-resolved fluoroimmunoassay for the determination of thyrotropin in dried blood spots. The evaluation was performed using a retrospective study design with newborn blood samples from three screening centres. Non-parametric Spearman rank correlation analysis revealed highly significant positive correlation between methods: rs = 0.465, p < 0.0001 (Hannover), rs = 0.659, p < 0.0001 (Minsk), rs = 0.755, p < 0.0001 (Helsinki). Wilcoxon signed rank test performed for paired FEIA and time-resolved fluoroimmunoassay showed that the results obtained by both tests represented the same distribution (p < 0.0001). The new method, using fluorimetric detection, can be performed with the instrumentation commonly used for the screening of congenital hypothyroidism and phenylketonuria. Results are obtained within three to four hours after arrival of the sample in the laboratory. Preliminary evaluation indicates the method to be a suitable alternative to time-resolved fluoroimmunoassay for neonatal thyroid function screening.

Congenital Hypothyroidism↗

PubMed1986

Time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine.

We describe time-resolved fluoroimmunoassay of 5-methyl-2'-deoxycytidine (5MedCyd). The assay is based on the use of a highly specific antiserum raised in rabbits against BSA-conjugated 5-methylcytidine (5MeCyd). The tracer in the solid-phase time-resolved fluoroimmunoassay (TR-FIA) was antigen-selected anti-5MedCyd labeled with Europium. Thyroglobulin-linked 5MeCyd served as the solid-phase antigen. The measuring range for the fluoroimmunoassay was from less than 1 to 5000 pmol per assay of 5MedCyd. A good correlation between the results obtained with the TR-FIA and HPLC was demonstrated when the methods were applied to the measurement of methylation in human leukemic cells and other DNA samples. TR-FIA has several advantages over the more laborious techniques available so far: (i) high sensitivity, (ii) large assay ranges, (iii) rapidity and large number of simultaneous assays, (iv) simplicity, and (v) low cost provided that the laboratory has equipment for time-resolved fluorometry.

Animals↗

PubMed1988

Development of fluoroimmunoassays for the specific detection of morphine in urine.

Two fluoroimmunoassays for the specific detection of morphine in urine are described based on the use of ovine antibodies and fluorescein-labelled normorphine. The first, a polarisation fluoroimmunoassay, is performed by adding 10 microliter of urine to 1.5 ml of a single-reagent, comprising premixed antiserum and tracer, incubation for a few minutes at ambient temperature and measurement of fluorescence polarisation. The assay gives results which compare well with those by thin-layer chromatography, EMIT d.a.u., and the Boehringer opiate drug test. Although adequate for routine screening for drug abuse, the technique is not as sensitive as some radioimmunoassays. Therefore, a second fluoroimmunoassay was developed based on the use of the same antibodies covalently coupled to magnetisable particles to facilitate both the separation of the bound and free fractions and the removal of non-specific interfering substances. Thus, larger sample volumes could be employed and greater sensitivity achieved.

Chromatography, Thin Layer↗

PubMed1985

Dual-label fluoroimmunoassay for simultaneous determination of primidone and phenobarbital.

A dual-label magnetizable solid-phase fluoroimmunoassay for direct determination of serum levels of primidone and its main metabolite, phenobarbital, in a single tube was developed and optimized. Fluorescein isothiocyanate was used to label phenobarbital and a rhodamine 101 derivative (XRITC) to label primidone; these could be independently quantitated with no fluorimetric "cross-talk." The dual-label assay employs mixed immunochemical reagents but is otherwise similar in performance to conventional, single-drug, magnetizable solid-phase fluoroimmunoassays. The ability to measure simultaneously two related analytes, such as a drug and its metabolite, represents a useful extension of immunoassay. This was illustrated in the present work by assay of sera from patients on primidone therapy. Dual-label assay results correlated well with those by a conventional enzymoimmunoassay (for primidone) and by a polarization fluoroimmunoassay (for phenobarbital).

Fluorescent Antibody Technique↗

PubMed1987

Development of fluoroimmunoassays for flecainide.

Antibodies raised in sheep against a flecainide:protein conjugate and fluorescein-labeled drug were used to develop simple fluoroimmunoassays for the measurement of flecainide acetate in serum or plasma. A rapid, nonseparation assay, based on fluorescence polarization was optimized for therapeutic drug monitoring. A separation fluoroimmunoassay, using antibodies covalently linked to magnetizable particles to avoid the need for centrifugation, was also optimized and validated for monitoring flecainide therapy. It is applicable to lipemic, hemolyzed or icteric samples unsuitable for the nonseparation approach and for laboratories with access only to a simple fluorimeter. Finally, the separation fluoroimmunoassay was modified slightly to improve markedly sensitivity for use in pharmacokinetic studies.

Flecainide↗

PubMed1979

Application of fluoroimmunoassay to cerebrospinal fluid immunoglobulin G and albumin.

Special solid-phase fluoroimmunoasssay protocols were used to measure the amount of immunoglobulin G (IgG) and albumin in 1,511 samples of cerebrospinal fluid. The fluoroimmunoassay is an inhibition test conducted on the surface of a plastic probe, whose fluorescence is inversely related to the concentration of spinal fluid IgG or albumin. A microcomputer interfaced with the fluorometer calculates the sample IgG or albumin from a calibration curve based on standard cerebrospinal fluid values. The test and interpretation take less than 90 min. Correlation coefficients for over 100 cerebrospinal fluid samples tested by both fluoroimmunoassay and radial immunodiffusion were: IgG, 0.90 (slope, 1.04); albumin, 0.95 (slope, 0.99). The within-run precision (coefficient of variation) was: IgG, 4.4%; albumin, 6.3%. Run-to-run precision on a midrange sample was: IgG, 7.7%, albumin, 11.7%. These findings establish the simplicity, speed, and precision of the modified fluoroimmunoassay system for specific cerebrospinal fluid proteins.

Adolescent↗

PubMed1992

A new highly sensitive immunoassay for cytokines by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA).

Non-isotopic immunoassays for human tumor necrosis factor alpha (TNF alpha) and human interleukin-6 (IL-6) were established by employing the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) system based on the time-resolved fluoroimmunoassay technique with europium-labeled antibody. Compared to enzyme-linked immunosorbent assays and bioassays, the sensitivity and range of measurement were significantly increased by applying the DELFIA systems to TNF alpha and IL-6. TNF alpha was measurable from 100 fg/ml to 10 ng/ml with the TNF alpha-DELFIA and IL-6 was measurable from 100 fg/ml to 1 ng/ml with the IL-6-DELFIA.

Antibodies, Monoclonal↗

PubMed1994

Syntheses of europium-labeled digoxin derivatives and their use in time-resolved fluoroimmunoassay.

A simple and efficient method of derivatizing and labeling the terminal sugar ring of digoxin (and other steroid glycosides) is described. The terminal sugar ring was oxidized by periodate to dialdehyde, followed by a reaction with the aminooxy group of heterobifunctional spacer reagent. Usually, reductive amination is needed to stabilize the Schiff base-type compounds produced in reactions between amino and aldehyde groups. Here the oxime compounds produced are stable wherefore the reductive amination step can be avoided and the reaction gives high yields without significant side reactions. After characterization the digoxin dioxime derivatives formed were labeled with europium chelates. These labeled digoxin derivatives bearing one or two europium-containing chelates, coupled via different spacers, were purified and tested in a competitive time-resolved fluoroimmunoassay. Several digoxin-specific monoclonal antibodies were also tested to determine the most suitable antibody-tracer combination, which was then employed to develop a simple competitive time-resolved fluoroimmunoassay for digoxin.

Aldehydes↗

PubMed1988

Screening of antibodies to varicella-zoster virus in leukemic patients: comparison of commercial methods of enzyme-immunoassay and fluoroimmunoassay.

Commercial methods of enzyme immunoassay and fluoroimmunoassay for the detection of varicella-zoster antibodies were compared using serum samples from individuals in a program of bone marrow transplantation. The correlation was 93.4%. Enzyme immunoassay showed to be more sensitive. Both methods are applicable to the detection of varicella-zoster antibodies. However, when fluoroimmunoassay is used, we recommend the confirmation of negative results by a sensitive enzyme immunoassay.

Antibodies, Viral↗

PubMed2002

Improved fluoroimmunoassays using the dye Alexa Fluor 647 with the RAPTOR, a fiber optic biosensor.

The performance of the fluorescent dye Alexa Fluor 647 (AF647) was explored as an alternative to Cy5 for immunoassays on the RAPTOR, a fiber optic biosensor. The RAPTOR performs sandwich fluoroimmunoassays on the surface of small polystyrene optical waveguides for analyte detection. Fluorescence and immunoassay data were examined at various dye-to-protein (D/P) ratios for both Cy5 and Alexa Fluor 647. Primarily, due to the self-quenching characteristics of Cy5, Alexa Fluor 647 is substantially more effective in fluoroimmunoassays, yielding over twice the signal for any given analyte concentration. Alexa Fluor 647 can be attached to antibodies at higher ratios, D/P=6, before self-quenching begins to limit the dye's effectiveness. Furthermore, while Alexa Fluor 647 becomes quenched at high dye-to-protein ratios, D/P=9, the net fluorescence yield reaches a maximum, as opposed to Cy5-labeled proteins, which become nearly nonfluorescent at high labeling ratios, D/P> or =6. The limitations of Cy5 were elucidated with an immunoassay for ricin, while the advantages of Alexa Fluor 647 were demonstrated in both direct binding assays as well as in a sandwich immunoassay for staphylococcal enterotoxin B.

Antibodies↗

PubMed1999

Heterogeneous fluoroimmunoassays using fluorescein as label with measurement of the fluorescence signal directly onto the solid-phase.

A heterogeneous fluoroimmunoassay approach with measurement of the fluorescence signal directly onto the solid support is described. Fluorescein was employed as fluorescent label whereas plastic microtitration wells were used as solid carriers. The influence of the type of the plastic wells on the proposed approach was investigated. Different types of commercially available plastic microtitration wells (white-opaque, black, and transparent) were tested. The solid supports were judged on the grounds of the maximum fluorescence signal, the precision, and the sensitivity they provided in a model non-competitive assay for mouse IgG. Among the solid supports tested, the white-opaque wells provided the most promising results with respect to the parameters in question. This support was also used for the development of a competitive fluoroimmunoassay for the determination of total thyroxin in human serum samples in order to assess the validity of the proposed approach under real immunoassay conditions. The assay developed had appropriate analytical characteristics for the determination of thyroxin and the serum sample values obtained were well correlated with those determined by a commercial solid-phase radioimmunoassay.

Animals↗

PubMed2002

Time-resolved fluoroimmunoassay of plasma and urine O-desmethylangolensin.

We present a method for the determination of the phytoestrogen metabolite O-desmethylangolensin (O-DMA) in plasma (serum) and in urine. O-DMA is a metabolite of daidzein, which occurs in soybeans. It has been suggested that isoflavones may afford protection against breast and prostate cancer and therefore, also the metabolites are of interest. The method is based on time-resolved fluoroimmunoassay (TR-FIA) using a europium chelate as a label. After the synthesis of 4"-O-carboxymethyl-O-DMA, this compound is coupled to bovine serum albumin, and then used as antigen in immunization of rabbits. The tracers with the europium chelate are synthesized using the same 4"-O-derivative of the alpha-methyldeoxybenzoin. After enzymatic hydrolysis and ether extraction the immunoassay is carried out by time resolved fluoroimmunoassay (TR-FIA). Cross-reactivity was tested with angolensin, dihydrogenistein, dihydrodaidzein, equol, 6'-OH-angolensin, trans-4-OH-equol, 6'-OH-O-DMA, cis-4-OH-equol and 5-OH-equol. The antiserum cross-reacted only with angolensin. This cross-reactivity seems not to influence the results, which were highly specific. Plasma samples are hydrolyzed and extracted. Urine samples are analyzed directly after hydrolysis without extraction. The correlation coefficient between the plasma TR-FIA results and the GC-MS results was high; r value was 0.985. The correlation coefficient between the urine TR-FIA results and the GC-MS results was high over the entire range of concentrations (0-1500 nmol/l); r value was 0.976, but lower in the low concentration range (0-100 nmol/l), i.e. value was 0.631. The intra-assay coefficients of variation (CVs) for plasma O-DMA concentrations and for urine O-DMA concentrations at three different concentrations varied 2.8-7.7 and 3.0-6.0%, respectively and the inter-assay CVs varied 3.8-8.9 and 4.4-6.6%, respectively. The working range of the plasma and urine O-DMA assays was 0.5-512 nmol/l.

Animals↗

PubMed2003

Time-resolved fluoroimmunoassay for equol in plasma and urine.

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4'-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4'-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography-mass spectrometry (GC-MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC-MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC-MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5-6.5 and 3.4-6.9, respectively. The inter-assay CV% varies between 5.4-9.7 and 7.4-7.7, respectively. The working ranges of the plasma and urine assay are 1.27-512 and 1.9-512nmol/l, respectively.

Animals↗

PubMed1997

A fluoroimmunoassay based on immunoliposomes containing genetically engineered lipid-tagged antibody.

Immunoliposomes were prepared by using biosynthetically lipid-tagged anti-2-phenyloxazolone single-chain antibody. Carboxyfluorescein as a fluorescent marker was encapsulated in the immunoliposomes. Some conditions for fluoroimmunoassay using the immunoliposomes were optimized by binding assays with hapten-coated microtiter wells. A competitive fluoroimmunoassay for the caproic acid conjugate of 2-phenyloxazolone as a model antigen was performed with the immunoliposomes. In the optimized assay conditions, antigen could be determined in the concentration range from 10(-7) to 10(-9) M.

Binding, Competitive↗