Detection of Salmonella enteritidis in incubated pools of egg contents by fluorescence polarization and lateral flow immunodiffusion.

Efficient detection of Salmonella enteritidis inside eggs is critical for confirming that individual commercial laying flocks present a risk to public health. In most standard bacteriological culturing protocols, an initial incubation step is necessary to allow the typically very small population of S. enteritidis cells in pools of egg contents to multiply to more easily detectable levels. In the present study, two rapid methods were evaluated as alternatives to plating on selective media for detecting S. enteritidis in incubated egg pools. By using either fluorescence polarization or lateral flow immunodiffusion assays, S. enteritidis could be consistently detected in egg pools at 10(8) cfu/mL (and in most pools at 10(7) cfu/mL). Although the rapid assays were significantly less sensitive than culturing, they both were consistently able to detect contamination when pools of 10 eggs were inoculated with approximately 10 cfu of S. enteritidis and incubated for 72 h at 25 degrees C.

Detection of experimental Salmonella enteritidis and S. typhimurium infections in laying hens by fluorescence polarization assay for egg yolk antibodies.

Identifying infected laying flocks is a critical component in efforts to prevent eggborne transmission of Salmonella enteritidis to humans. In the present study, egg yolk samples from experimentally infected chickens were tested for specific antibodies with a very rapid fluorescence polarization assay using tracers prepared from the O-polysaccharides of S. enteritidis and S. typhimurium and a conventional ELISA using an S. enteritidis flagellin antigen. In two trials, groups of specific-pathogen-free laying hens were infected orally with 106 or 10(8) cfu of S. enteritidis (phage type 13a) or with 10(8) cfu of S. typhimurium. Eggs were collected during five weekly postinoculation intervals. Both fluorescence polarization and ELISA detected the majority of hens infected with S. enteritidis at either dose level, although they also frequently cross-reacted with samples from hens infected with S. typhimurium. Fluorescence polarization with an S. typhimurium tracer was likewise able to consistently detect S. typhimurium infection but also tended to cross-react with samples from hens infected with S. enteritidis. Fluorescence polarization appears to offer a simple and rapid alternative to conventional serological methodology, although concerns about specificity may limit the usefulness of antibody testing data.

Fluorescence polarization as a tool for the determination of deoxynivalenol in wheat.

The mould Fusarium graminearum is found worldwide as a pathogen of cereal grains, in particular of wheat and maize, and it produces a mycotoxin known as deoxynivalenol (DON or vomitoxin). Each year, the presence of this compound and related trichothecenes causes substantial losses to agricultural productivity. Rapid methods for the measurement of the toxin in grains are required to monitor and divert effectively contaminated grain from the food supply. A fluorescence polarization (FP) immunoassay using a previously described monoclonal antibody for DON was developed. The assay was based on the competition of unlabeled DON from a sample with a fluorescently tagged DON, DON-fluorescein (DON-FL), for a DON-specific monoclonal antibody in solution. The FP of the tagged DON was increased upon binding with the antibody. In the presence of free toxin, less of the DON-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of wheat with the DON-FL and antibody. The sensitivity of the assay was strongly dependent upon the time between mixing of the sample with the tracer and measurement of the fluorescence polarization, with midpoints for the competition curves ranging from 0.03 microg ml(-1) with a 15-s incubation to >1 microg ml(-1) with a 12-min incubation. Samples of wheat naturally contaminated with DON were evaluated by FP and by an HPLC-UV method, with a good correlation (r2 = 0.97). Although the FP method tended to overestimate DON slightly in the wheat samples, by approxiamtely 20%, the assay was easy to use and very useful for the screening of wheat.

Fluorescence polarization as a means for determination of fumonisins in maize.

Fumonisins, mycotoxins produced by certain species of Fusaria, are commonly found worldwide as contaminants in maize. This paper reports the development of a rapid, portable fluorescence polarization-based assay for fumonisins in maize. The assay was based on the competition of unlabeled fumonisin, from a sample, with a fluorescently tagged fumonisin (FB(1)-FL) for a fumonisin-specific monoclonal antibody in solution. The fluorescence polarization (FP) of the tagged fumonisin was increased upon binding with the antibody. In the presence of free toxin, less of the FB(1)-FL was bound and the polarization signal was decreased. The assays were very simple to perform, requiring only mixing of an aqueous extract of maize with the tagged fumonisin and antibody, and required <2 min per sample, excluding extraction time. Two permutations of the assay were tested, one with each sample matrix serving as its own blank, and the other with all of the samples compared relative to a PBS blank with normalization of the data similar to an ELISA. The limit of detection, defined as the toxin content associated with a fluorescence polarization signal 5 standard deviations from that of a fumonisin-free control, was 0.5 microg of FB(1)/g in spiked maize. Recoveries from spiked maize over the range of 0.5-20 ppm averaged 94.3 +/- 13.8%. Forty-eight samples of field-contaminated maize were tested by the FP and an established HPLC method, with a good correlation between the two (r(2) = 0.85-0.88). For these samples, the two variations of the FP assay also compared well to one another (r(2) = 0.97), suggesting the assay principle is very robust. The results, combined with the speed and ease of use for the assay, suggest that this technology has substantial potential as a screening tool for mycotoxins in foods.

Development of a fluorescence polarization-based diagnostic assay for equine infectious anemia virus.

The control of equine infectious anemia virus (EIAV) infections of horses has been over the past 20 years based primarily on the identification and elimination of seropositive horses, predominantly by a standardized agar gel immunodiffusion (AGID) assay in centralized reference laboratories. This screening for EIAV-seropositive horses has been to date hindered by the lack of a rapid diagnostic format that can be easily employed in the field. We describe here the development of a rapid solution-phase assay for the presence of serum antibodies to EIAV based on fluorescence polarization (FP) (patent pending). Peptides derived from antigenic regions of EIAV core and envelope proteins were initially screened for their utility as probes in an FP assay to select the best peptide antigen candidates. The FP assay was optimized to detect the presence of EIAV-specific antibodies by a change in the FP of a fluorescein-labeled immunoreactive peptide diagnostic antigen. The most sensitive and specific peptide probe was a peptide corresponding to the immunodominant region of the EIAV transmembrane protein, gp45. This probe was tested for its reactivity in the optimized FP assay with 151 AGID-positive horse sera and 106 AGID-negative serum samples. The results of these studies demonstrated that the FP assay reactivity correlated with reported AGID results in 106 of 106 negative serum samples (100% specificity) and in 135 of 151 positive serum samples (89.4% sensitivity). The FP assay was also found to have a very low background reactivity and to readily detect antibodies produced early in infection (

Fluorescence polarization: an analytical tool for immunoassay and drug discovery.

Fluorescence polarization (FP) is an intrinsically powerful technique for the rapid and homogeneous analysis of molecular interactions in biological/chemical systems. The technique has been successfully used to diagnose various viral and infectious diseases in humans and animals, to monitor therapeutic drug levels and substances of abuse in body fluids and to determine food born pathogens, grain mycotoxins and pesticides. It has also been used in monitoring enzyme catalyzed hydrolysis, protein-protein interactions, DNA diagnostics and high throughput screening during the course of drug discovery. Work by various groups, including our own, have demonstrated that the technique can replace a substantial number of solid phase assays. FP, defined by the equation P = [IV - IH] / [IV + IH] (where V and H are the vertical and horizontal components of the intensity I of emitted light respectively when exited by vertically plane polarized light), is independent of the intensity of the light and the concentration of the fluorophore. Hence it is functional in colored and cloudy solutions. The FP of a fluorophore is proportional to its rotational relaxation time, which in turn depends upon its molecular volume (or molecular weight) at constant temperature and solution viscosity. When a fluorophore-labeled ligand binds to a larger molecule, equilibrium is established rapidly and the FP increases. This property has been successfully exploited in many fields as described in this review.

BODIPY-alpha-casein, a pH-independent protein substrate for protease assays using fluorescence polarization.

BODIPY-alpha-casein is a new fluorescent protein substrate designed for fluorescence polarization studies to measure proteolytic activity at any pH over the range from pH 2 to 11. Kinetic protease assays in real-time were performed in 1 to 5 min using an FPM-1 fluorescence polarization instrument. A purified enzyme or bacterial culture was mixed with the BODIPY-alpha-casein in a buffer of an appropriate pH and the decrease in fluorescence polarization was automatically recorded at 0.5-min intervals. The initial decrease in fluorescence polarization with time was dependent on protease concentration. In 3-min assays at 37 degrees C, the sensitivity of detection was 8 mU for pepsin at pH 2.0, 1 mU for papain at pH 6.0, 0.6 mU for proteinase K at pH 7.4, and 2 mU for Streptomyces griseus alkaline protease at pH 11. Only 1-10 microliters of a growing culture was necessary to assay the protease activity of Porphyromonas gingivalis or Treponema denticola, oral bacteria that possess certain proteases on their surfaces. These assays have clinical applications, since certain pathogens use proteolytic activity as a virulence mechanism and differ from their nonpathogenic counterparts in this characteristic. Fluorescence polarization assays are simple, rapid, and reproducible.

Modification of the Mycobacterium bovis extracellular protein MPB70 with fluorescein for rapid detection of specific serum antibodies by fluorescence polarization.

The principle of fluorescence polarization described by Perrin (F. Perrin, J. Phys. Radium 7:390-401, 1926) was applied to the development of a novel assay that used fluorescein-labeled Mycobacterium bovis secretory protein MPB70 for rapid detection of anti-MPB70 antibodies in selected sera from three M. bovis-infected species (elk, Ilama, and bison). Labeling of purified MPB70 with fluorescein isothiocyanate resulted in the incorporation of 0.96 +/- 0.08 (mean +/- standard deviation; n = 3) fluorescein group per MPB70 molecule. The labeled protein fluoresced strongly with an emission maximum at 518 nm when excited with light of a wavelength near 493 nm, and its immunoreactivity with anti-MPB70 monoclonal antibody 4C3/17 was not altered by modification with fluorescein. The fluorescence polarization assay protocol was optimized for analysis of serum samples by incorporating into the assay buffer 0.05% lithium dodecyl sulfate, which prevents the occurrence of some nonspecific interactions. Sera from M. bovis-infected animals, selected on the basis of exhibiting the presence of anti-MPB70 antibodies, as detected by enzyme-linked immunosorbent assay (ELISA), reacted with fluorescein-labeled MPB70, resulting in an increase in polarization of up to 330 milli-polarization units, in contrast to the values for noninfected sera (167 to 178 mP), which were close to that obtained in the absence of specific antibodies (164.7 +/- 3.3 mP; n = 6). These results demonstrated the feasibility of using fluorescein-labeled MPB70 to detect anti-MPB70 antibodies by fluorescence polarization and suggested that the assay described here can be an alternative to ELISA or other antibody assay systems. The advantages of this original methodology and its general applicability to the diagnosis of infectious diseases are discussed.

Monoclonal antibodies to HIV-1 p24 core protein include pairs which exhibit synergistic binding.

Gamma and kappa chain cDNAs from four mouse monoclonal antibodies (mAbs) which bind three different sites on the core antigen (p24) of HIV-1 have been cloned and their V-region sequences determined. These mAbs are part of a larger group of seven anti-p24 mAbs analyzed in simultaneous competition assays with HIV-1 lysate as antigen and in protein blotting experiments using 10 carboxy-terminal truncations of a p24 fusion protein. One mAb, BB128, recognizes the p24 loop sequence EAAEWDRVHP and enhances the binding of two other mAbs (BI1777 and BI1279) when tested pairwise in simultaneous competition assays. The two monoclonals enhanced by BB128 recognize different antigenic sites on p24, with BI1777 binding to carboxy-terminal sequences and BI1279 to amino-terminal residues. In the pairwise assays, mAb BI1279 also acts as enhancing antibody for BI1777, as does mAb BB328, which recognizes residues in the central region of p24. Since aggregated p24 monomers form the HIV-1 capsid, p24 is a multivalent antigen in HIV-1 lysate. It seems likely, therefore, that synergistic binding of mAb pairs to p24 is effected by bivalent binding of the enhancing mAb stabilizing a conformation favorable for bivalent binding of the enhanced mAb.

Particle concentration fluorescence immunoassay: a new immunoassay technique for quantification of human immunoglobulins in serum.

A new fluorescence immunoassay technique, particle concentration fluorescence immunoassay (PCFIA), has been developed for quantifying the human immunoglobulins (IgA, IgM, and IgG). In these "two-site sandwich assays," the capture antibody is immobilized on small polystyrene spheres and the tracer is fluorescein-labeled antibody. Polystyrene particles less than 1 microm in diameter make up the solid phase, to which goat anti-human antibody for each respective assay is attached. Serum specimens are diluted (5000-fold for IgA or IgM, 20 000-fold for IgG) placed on the 96-well Pandex assay plate; and mixed with the solid phase and tracer (fluorescein-labeled goat anti-human IgA, IgM, or IgG), which are added automatically by the Pandex Screen Machine. This instrument incubates the reaction mixture for 17 min at ambient temperature, separates the bound and free label by filtration, washes the solid phase, and determines the total particle-bound fluorescence by front-surface fluorimetry or epifluorescence, calculates results, and generates detailed reports. Ninety-six specimens may be analyzed in 29 min or 960 specimens in 136 min. Results by PCFIA for IgA, IgM, and IgG in serum correlated well with those by rate nephelometry.

Particle concentration fluorescence immunoassay (PCFIA): a new, rapid immunoassay technique with high sensitivity.

A new solid-phase fluorescence immunoassay technique is described and is exemplified by the detection of murine monoclonal antibodies to human IgG in hybridoma culture supernatants and the detection of murine IgG. The assay is performed in a specially designed 96-well plate. For antibody detection, antigen bound to submicron polystyrene particles is bound to its specific antibody, which is in turn reacted with fluorescein-labeled affinity-purified goat anti-mouse IgG. The reaction is complete in 10 min at ambient temperature. The solid phase is separated from the reaction mixture by filtration, washed and the total particle-bound fluorescence is determined by front-surface fluorimetry. The sensitivity of the technique for antibody detection is equivalent to enzyme-linked immunoabsorbent assay and 2-4 ng/ml for murine IgG detection. It is readily amenable to automation.

Fluorescence polarization immunoassay IV. Determination of phenytoin and phenobarbital in human serum and plasma.

Fluorescence polarization immunoassays for phenytoin and phenobarbital in human serum or plasma are described and shown to be clinically useful. No sample pretreatment, extraction, or phase separation is required. A determination can be made with less than 20 microL of sample in 15 min, with incubation at ambient temperature. Abnormal concentrations of protein, lipid, hemoglobin, or bilirubin do not interfere. In addition, drug metabolites and commonly co-administered drugs do not affect the assay results at clinically significant concentrations. Analytical recoveries of each of the two anticonvulsant drugs from serum averaged 101%. Between-assay CVs were less than 6.5%, with sensitivities of 0.5 mg/L. Comparison of this method with "high-performance" liquid chromatography, homogeneous enzyme immunoassay, and radioimmunoassay for phenytoin determinations yielded correlations of 0.99, 0.98, and 0.99, respectively. Similar comparison studies for phenobarbital yielded correlation coefficients of 0.99, 0.98, and 0.99, respectively.

Fluorescence polarization immunoassay for the determination of therapeutic drug levels in human plasma.

Fluorescence polarization immunoassay (FPIA) is a technique which has been known for a number of years, but despite its many advantages, its has not seen clinical utility. The main reason for this is the lack of simple, rapid, high performance instrumentation suitable for use in a clinical laboratory. This problem has been addressed in this laboratory and an instrument has been developed which meets these needs. The solutions of the two other problems associated with the technique (i.e. non-specific binding of tracer to serum proteins and the intrinsic fluorescence of serum) have also been accomplished. Assays for a variety of therapeutic drugs including gentamicin, theophylline, phenytoin, and phenobarbital have been developed and shown to correlate well with a number of reference method. The assays have been totally automated with a concomitant increase in speed and ease of use and a significant improvement in performance over the manual system.

Fluorescence polarization immunoassay. iii. an automated system for therapeutic drug determination.

A fully automated system for performing fluorescence polarization immunoassay has been developed. Reagents for each assay are contained in coded reagent packs, and no reagent reconstitution is required. A common buffer is used for all assays, minimizing changeover and set-up times for each assay. A single sample may be assayed in 5 min, or 20 samples in 10 min. A single-tube blank subtraction for each sample results in highly precise polarization values and obviates sample interferences. We have used this method for assays of gentamicin, theophylline, phenytoin, and phenobarbital. CVs are 1-4%, and the results correlate well with those by other methods. Because of the instrument design and the stability of the reagents, daily calibration is not required; samples may therefore be run immediately upon receipt or batched as desired.

Fluorescence polarization immunoassay. I. Monitoring aminoglycoside antibiotics in serum and plasma.

Fluorescence polarization immunoassays of the aminoglycoside antibiotics gentamicin, tobramycin, and amikacin in plasma and serum are described and shown to be clinically useful. The aminoglycoside tracers were prepared by reacting the parent compounds with 5-[(4,6-dichlorotriazin-2-yl)-amino] fluorescein. Antisera specific for the compounds were raised in rabbits by conventional procedures. Tracer, sample, and diluted antiserum are combined and, after a 15-min incubation at ambient temperature, the polarization of the fluorescence of the tracer is determined in a specially designed fluorometer. The assays are designed to give accurate trough (i.e., minimum during therapy) values and to be free of matrix effects. Severely icteric samples may interfere, but this can be overcome by blank subtraction. The performance of the assays with clinical specimens compared favorably with that of some commercially available assays.

Tryptophanyl and carboxylic acid residues in the active centre of glucoamylase I from Aspergillus niger.

The pH-dependence of the photo-oxidation of L-tryptophan, in the presence of Rose Bengal and Methylene Blue, has been investigated. True, initial rate constants were determined in order to circumvent errors due to secondary processes. Photo-oxidation of glycoamylase I from A. niger in the presence of Methylene Blue or Rose Bengal resulted in a pH-dependent loss of enzymic activity, which was analogous to the destruction of free L-tryptophan during photo-oxidation. The loss of enzymic activity was closely associated with the destruction of tryptophan residues in the enzyme. Significant protection of both enzymic activity and tryptophanyl residues in the enzyme molecule was achieved by performing the photo-oxidation in the presence of maltose, which is a substrate for the enzyme. The tryptophanyl residues of glucoamylase I, which had been inactivated by reaction of its carboxylic acid residues with glycine methyl ester in the presence of a water-soluble carbodi-imide, were also substantially protected by maltose. It is concluded that the active centre of glucoamylase I is a cleft lined with tryptophanyl residues that participate in the binding of the substrate. One or more carboxylic acid residues are involved in bond cleavage.

Binding studies on a mouse-myeloma immunoglobulin A having specificity for beta-D-(1 yields 6)-linked D-galactopyranosyl residues.

The free energies of binding between immunoglobulin A J539 (Fab') and methyl 6-O-acetyl beta-D-galactopyranoside (1) and 6-O-beta-D-galactopyranosyl-1, 2:3, 4-di-O-isopropylidene-alpha-D-galactopyranose (2) have been measured. The values found suggest that bulky substitution on O'-6 or O-1, O-2, O-3, and O-4 in the hapten 6-O-beta-D-galactopyranosyl-D-galactose (3) does not interfere with effective binding of that ligand and the immunoglobulin. This conclusion supports the postulations that (a) the ligand 3 binds only on one side of the molecule, and (b) the combining site of the immunoglobulin J539 appears to be located on an exposed surface area.

Six BALB-c IgA myeloma proteins that bind beta-(1-6)-D-galactan. Partial amino acid sequences and idiotypes.

Six IgA myeloma proteins of BALB/c origin which bind antigens containing beta-(1 --> 6)-D-galactan side chains have been isolated by affinity chromatography on galactoside-BSA-Sepharose columns. Partial amino acid sequences of of the light chains to residue Cys23 and the heavy chains to reside 30 were determined on the automated sequencer. No differences were found among the six V(K) sequences. Among some 50 partial V(K) sequences that have thus far been determined these six chains are the only ones thus far identified in this subgroup; at least 25 V(K) subgroups in the mouse have been identified so far. The heavy chain partial sequences were also very closely related but two differences were found. One protein differed from the other five by having isoleucine instead of leucine at position 5, a second protein differed from the others by having an unidentified amino acid at position 19. Using the highly sensitive inhibition of hemagglutination method it was found that each of the proteins possessed a unique inidividual antigenic determinant.