Matrix-assisted laser desorption/ionization mass spectrometry of DNA using photocleavable biotin

Oligonucleotides containing a photocleavable biotin (5'-PC-biotin) were analyzed by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) with wavelengths in the ultraviolet (UV) and infrared (IR) from solution and after capture on streptavidin-coated agarose or magnetic beads. The analysis was used to monitor the release of the oligonucleotides as a result of photochemical cleavage of the biotinylated linker. Near-UV pulses (UV-MALDI) led to predominant release of the photocleaved product. In contrast, only the uncleaved analyte was detected using IR pulses (IR-MALDI). Results from MALDI analysis are also presented for DNA containing a photocleavable 5'-amino group which can be covalently linked to a variety of activated surfaces and marker molecules. In a demonstration of this approach, a 5'-PC-biotinylated 49 nt RNA oligonucleotide was enzymatically synthesized using a PC-biotin-r(AG) dinucleotide primer, captured on streptavidin coated magnetic beads and analyzed by UV-MALDI. Potential applications of photocleavable linkers combined with MALDI for the analysis of nucleic acids are discussed.     

Preparation of single-stranded DNA from PCR products with streptavidin magnetic beads

The preparation of single-stranded DNA from double-stranded PCR products is an essential step in the identification of aptamers by Systematic Evolution of Ligands by EXponential enrichment (SELEX). The most widely used method for producing single-stranded DNA is alkaline denaturation of biotinylated PCR products attached to streptavidin-coated magnetic beads. Recently, it has been suggested that this method may be unsuitable due to the release of interfering amounts of streptavidin and biotinylated DNA. In this article, the alkaline method is compared with a thermal method that is known to release significant amounts of streptavidin and biotinylated DNA. Results show that trace amounts of streptavidin and biotinylated DNA are released in the alkaline method, but this can be curtailed by preconditioning the beads in aqueous sodium hydroxide. The main product in the alkaline method is single-stranded DNA, which is produced in high yield.     

A self-assembled quantum dot probe for detecting beta-lactamase activity

This communication describes a quantum dot probe that can be activated by a reporter enzyme, beta-lactamase. Our design is based on the principle of fluorescence resonance energy transfer (FRET). A biotinylated beta-lactamase substrate was labeled with a carbocyanine dye, Cy5, and immobilized on the surface of quantum dots through the binding of biotin to streptavidin pre-coated on the quantum dots. In assembling this nanoprobe, we have found that both the distance between substrates and the quantum dot surface, and the density of substrates are important for its function. The fluorescence emission from quantum dots can be efficiently quenched (up to 95%) by Cy5 due to FRET. Our final quantum dot probe, assembled with QD605 and 1:1 mixture of biotin and a Cy5-labeled lactam, can be activated by 32microg/mL of beta-lactamase with 4-fold increase in the fluorescence emission.     

Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N'-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.     

Protein engineering of streptavidin for in vivo assembly of streptavidin beads

Escherichia coli was engineered to intracellularly manufacture streptavidin beads. Variants of streptavidin (monomeric, core and mature full length streptavidin) were C-terminally fused to PhaC, the polyester granule forming enzyme of Cupriavidus necator. All streptavidin fusion proteins mediated formation of the respective granules in E. coli and were overproduced at the granule surface. The monomeric streptavidin showed biotin binding (0.7 ng biotin/microg bead protein) only when fused as single-chain dimer. Core streptavidin and the corresponding single-chain dimer mediated a biotin binding of about 3.9 and 1.5 ng biotin/mug bead protein, respectively. However, biotin binding of about 61 ng biotin/mug bead protein with an equilibrium dissociation constant (KD) of about 4 x 10(-8)M was obtained when mature full length streptavidin was used. Beads displaying mature full length streptavidin were characterized in detail using ELISA, competitive ELISA and FACS. Immobilisation of biotinylated enzymes or antibodies to the beads as well as the purification of biotinylated DNA was used to demonstrate the applicability of these novel streptavidin beads. This study proposes a novel method for the cheap and efficient one-step production of versatile streptavidin beads by using engineered E. coli as cell factory.     

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidin-immobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70 degrees C for 3 min, cooled slowly to 25 degrees C, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58 degrees C, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80 degrees C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs.     

Magnetic immunoseparation PCR assay (MIPA) for detection of hepatitis a virus (HAV) in American oyster (Crassostrea virginica)

In order to detect the low numbers of hepatitis A viral (HAV) particles which may potentially be present in food and cause a serious illness, an original procedure which combines immunomagnetic separation and PCR is described. The use of streptavidin magnetic beads coated with biotinylated human anti-HAV IgG allows virus capture and the removal of the RT-PCR inhibitory compounds which usually are present in shellfish extracts. Following immunomagnetic capture, the separated HAV were lysed, the beads discarded, and the supernatant containing the viral RNA subjected to the RT-PCR protocol. Levels of HAV ranging from 10 to 10⁵ pfu were successfully detected in artificially contaminated samples of shucked American oyster ( Crassostrea virginica ).     

Near real-time biosensor-based detection of 2,4-dinitrophenol

A fluorescent biosensor assay has been developed for near real-time detection of 2,4-dinitrophenol (DNP). The assay was based on fluorescent detection principles that allow for the analysis of antibody/antigen interactions in solution using the KinExA immunoassay instrument. Our KinExA consisted of a capillary flow observation cell containing a microporous screen that maintains a compact capture antigen-coated bead bed. The bead bed was comprised of polymethylmethacrylate (PMMA) beads coated with dinitrophenol-human serum albumin (DNP-HSA) conjugate. Phosphate buffered saline (PBS) solutions, containing various concentrations of free DNP, were incubated for 30 min with mouse anti-DNP monoclonal antibody to equilibrium. Solutions containing the DNP-monoclonal antibody complex and possible excess free antibodies were then passed over DNP-HSA labeled beads. The free monoclonal anti-DNP antibody, if available, was then bound to the DNP-HSA fixed on the beads. The system was then flushed with excess PBS to remove unbound reactants in the bead bed. The beads were then subjected to brief contact with PBS solutions containing goat anti-mouse fluorescein isothiocyanate (FITC)-labeled secondary antibody, once again, followed by a short PBS flush. The fluorescence was recorded during the addition of the FITC labeled secondary antibody to the bead bed through the final PBS flushing with the KinExA. The amount of DNP detected could then be determined from the fluorescent slopes that were generated or by the remaining fluorescence that was retained on the beads after final PBS flushing of the system. This assay has been able to detect a minimum of 5 ng/ml of DNP in solution and can be adapted for other analytes of interest simply by changing the capture antigen and antibody pairs.     

Quantitation of polymerase chain reaction products by hybridization-based assays with fluorescent, colorimetric, or chemiluminescent detection.

In this report two nonradioactive assays for quantitative analysis of polymerase chain reaction (PCR) products are presented. In the first assay, magnetic beads coated with streptavidin were used to capture biotinylated PCR fragments. After hybridization with a hapten-labeled probe, these beads were analyzed either by flow cytometry (method A) or by immunoenzymatic reactions (method B). Using a dilution series of purified PCR products, we consistently found a lower detection limit of 1.5 fmol for method A than the 0.15-fmol limit for method B. In the second assay we used the peroxidase-based enhanced chemiluminescence system in combination with a cooled charge-coupled device camera to quantify PCR fragments that were spotted on membranes. A linear logarithmic response was observed between the amount of light produced within a certain time interval and the number of DNA molecules. With an exposure time of 5 min, a detection limit of 0.15 fmol was found. Longer exposure times did not result in a higher sensitivity. We conclude that the assays are of sufficient sensitivity for application in quantitative PCR strategies. The nonradioactive technology facilitates implementation of these assays in routine settings.     

Magnetic bead-based solid phase for selective extraction of genomic DNA

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.     

Microarray of recombinant antibodies using a streptavidin sensor surface self-assembled onto a gold layer

We have developed a sensitive method for the detection of recombinant antibody-antigen interactions in a microarray format. The biochip sensor platform used in this study is based on an oriented streptavidin monolayer that provides a biological interface with well-defined surface architecture that dramatically reduces nonspecific binding interactions. All the antibody or antigen probes were biotinylated and coupled onto streptavidin-coated biochip surfaces (1 microL total volume). The detection limits for the immobilized probes on the microarray surface were 0.5 microgram/mL (200 fmol/spot) for the peptide antigen and 0.1 microgram/mL (3 fmol/spot) for the recombinant antibodies. Optimal concentrations for the detection of the Cy5-labeled protein target were in the range of 20 micrograms/mL. Protein microchips were used to measure antibody-antigen kinetics, to find optimal temperature conditions, and to establish the shelf life of recombinant antibodies immobilized on the streptavidin surface. For recombinant antibody fragments with a kDa of 10-100 nM, we have established an easy and direct immunoassay. In addition, we developed an indirect method for antibody detection with no need for expensive and time-consuming antibody purifications and modifications. Such a method was shown to be useful for large-scale screening of recombinant antibody fragments directly after their functional expression in bacteria. Our data demonstrate that recombinant antibody fragments are suitable components in the construction of antibody chips.     

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.     

Fabrication and characterization of 3D hydrogel microarrays to measure antigenicity and antibody functionality for biosensor applications

We report the fabrication, characterization and evaluation of three-dimensional (3D) hydrogel thin films used to measure protein binding (antigenicity) and antibody functionality in a microarray format. Protein antigenicity was evaluated using the protein toxin, staphylococcal enterotoxin B (SEB), as a model on highly crosslinked hydrogel thin films of polyacrylamide and on two-dimensional (2D) glass surfaces. Covalent crosslinking conditions were optimized and quantified. Interrogation of the modified 3D hydrogel was measured both by direct coupling of a Cy5-labeled SEB molecule and Cy5-anti-SEB antibody binding to immobilized unlabeled SEB. Antibody functionality experiments were conducted using three chemically modified surfaces (highly crosslinked polyacrylamide hydrogels, commercially available hydrogels and 2D glass surfaces). Cy3-labeled anti-mouse IgG (capture antibody) was microarrayed onto the hydrogel surfaces and interrogated with the corresponding Cy5-labeled mouse IgG (antigen). Five different concentrations of Cy5-labeled mouse IgG were applied to each microarrayed surface and the fluorescence quantified by scanning laser confocal microscopy. Experimental results showed fluorescence intensities 3-10-fold higher for the 3D films compared to analogous 2D surfaces with attomole level sensitivity measured in direct capture immunoassays. However, 2D surfaces reported equal or greater sensitivity on a per-molecule basis. Reported also are the immobilization efficiencies, inter-and intra-slide variability and detection limits.     

Lack of immune stimulation by immobilized CpG-oligodeoxynucleotide.

Selected phosphorothioate oligodeoxynucleotides containing CpG (CpG-ODN) activate immune responses, including B-cell proliferation and cytokine production. The mechanism by which cells detect CpG-motifs is not known. There are conflicting reports in the literature concerning the ability of CpG-ODN linked to solid supports to stimulate immunity. We prepared a fluorescent, biotinylated CpG-ODN, a reagent that will support the growth of 7TD1 cells, a murine B-cell hybridoma line that requires CpG-ODN or interleukin-6 (IL-6) for survival. Stimulation of 7TD1 cell growth was not reduced by complexing biotinylated CpG-ODN to streptavidin, but cell growth was not supported by CpG-ODN coupled to streptavidin-coated latex, magnetic, gold, or agarose beads. A fluorescent CpG-ODN was also covalently attached to cyanogen bromide-activated Sepharose beads via a 5'-amine group. These derivatized Sepharose beads did support 7TD1 cell growth, but incubation of the beads with 7TD1 cells resulted in the appearance of fluorescence within the cells, suggesting that growth stimulation may be due to CpG-ODN leached from the beads. Our results are consistent with the need for CpG-ODN to be internalized into cells to be immunostimulatory.     

Visualising fouling of a chromatographic matrix using confocal scanning laser microscopy

Confocal scanning laser microscopy (CSLM) was used to visualise the spatial location of foulants during the fouling of Q Sepharose FF matrix in finite batch experiments and for examining the subsequent effectiveness of clean-in-place (CIP) treatments in cleaning the heavily fouled beads. Beads were severely fouled with partially clarified E. coli homogenate by contacting the beads with the foulant for contact times of 5 min, 1 or 12 h. The use of two different fluorescent dyes, PicoGreen and Cy5.5, for labelling genomic PicoGreen-labelled dsDNA and protein respectively, allowed the direct observation of the chromatographic beads. The extent of fouling was assessed by measuring the subsequent adsorption of Cy5.5-labelled BSA to the beads. Control studies established that the labelling of BSA did not affect significantly the protein properties. In the control case of contacting the unfouled matrix with Cy5.5-labelled BSA, protein was able to penetrate the entire matrix volume. After fouling, Cy5.5-labelled BSA was unable to penetrate the bead but only to bind near the bead surface where it slowly displaced PicoGreen-conjugated dsDNA, which bound only at the exterior of the beads. Labelled host cell proteins bound throughout the bead interior but considerably less at the core; suggesting that other species might have occupied that space. The gross levels of fouling achieved drastically reduced the binding capacity and maximum Cy5.5-labelled BSA uptake rate. The capacity of the resin was reduced by 2.5-fold when incubated with foulant for up to 1 h. However, when the resin was fouled for a prolonged time of 12 h a further sixfold decrease in capacity was seen. The uptake rate of Cy5.5-labelled BSA decreased with increased fouling time of the resin. Incubating the fouled beads in 1 M NaCl dissociated PicoGreen-labelled dsDNA from the bead exterior within 15 min of incubation but proved ineffective in removing all the foulant protein. Cy5.5-labelled BSA was still unable to bind beyond the outer region of the beads. A harsher CIP treatment of 1 M NaCl dissolved in 1 M NaOH was also ineffective in removing all the foulant protein but did remove PicoGreen-conjugated dsDNA within 15 min of incubation. Cy5.5-labelled BSA was able to bind throughout the bead interior after this more aggressive CIP treatment but at a lower capacity than in the case of fresh beads. The competitive adsorption of BacLight Red-labelled whole cells or cell debris and PicoGreen-conjugated dsDNA was also visualised using CSLM.     

Effects of fluorophore-to-silver distance on the emission of cyanine-dye-labeled oligonucleotides

We examined the fluorescence spectral properties of Cy3- and Cy5-labeled oligonucleotides at various distances from the surface of silver island films. The distance to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA) and avidin, followed by binding of a biotinylated oligonucleotide. The maximum enhancement of fluorescence near a factor of 12 was observed for the first BSA-avidin layer, with the enhancement decreasing to 2-fold for six layers. The minimum lifetimes were observed for the first BSA-avidin layer, and were about 25-fold shorter than on quartz slides without silver, with the lifetimes being about 2-fold shorter for six BSA-avidin layers. These results suggest that maximum fluorescence enhancements occur about 90A from the silver surface, a distance readily obtained by one or two layers of proteins.     

Controlled release of DNA/polyamine complex by photoirradiation of a solid phase presenting o-nitrobenzyl ether tethered spermine or polyethyleneimine

Various gene transfer and automated/monitorized analytical applications require the controlled release of nucleic acid. A solid phase with spermine or polyethyleneimines (PEI, 600 MW) tethered by o-nitrobenzyl linkages was synthesized with polyethylene oxide beads (ArgoGel-NH(2)). The photolysis of test compound O-2-nitrophenethyl O,O-diethyl phosphate or solid phase with o-nitrobenzyl group as synthetic linker was completely degradable with photoirradiation at 365 nm for 10-18 min at 3.5 mW/cm(2). DNA binding with polyamine of the solid phase and releasing of DNA/polyamine were monitored by UV measurement and gel electrophoresis. The potential exists to employ a DNA-loaded solid phase for spatially, temporally, or dose-controlled release of DNA, at extracellular or intracellular sites.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

Multicomponent mesofluidic system for the detection of veterinary drug residues based on competitive immunoassay

An automated multicomponent mesofluidic system (MCMS) based on biorecognitions carried out on meso-scale glass beads in polydimethylsiloxane (PDMS) channels was developed. The constructed MCMS consisted of five modules: a bead introduction module, a bioreaction module, a solution handling module, a liquid driving module, and a signal collection module. The integration of these modules enables the assay to be automated and reduces it to a one-step protocol. The MCMS has successfully been applied toward the detection of veterinary drug residues in animal-derived foods. The drug antigen-coated beads (?250 μm) were arrayed in the PDMS channels (?300 μm). The competitive immunoassay was then carried out on the surface of the glass beads. After washing, the Cy3-labeled secondary antibody was introduced to probe the antigen-antibody complex anchored to the beads. The fluorescence intensity of each bead was measured and used to determine the residual drug concentration. The MCMS is highly sensitive, with its detection limits ranging from 0.02 (salbutamol) to 3.5 μg/L (sulfamethazine), and has a short assay time of 45 min or less. The experimental results demonstrate that the MCMS proves to be an economic, efficient, and sensitive platform for multicomponent detection of compound residues for contamination in foods or the environment.     

A generic particle-based nonradioactive homogeneous multiplex method for high-throughput screening using microvolume fluorimetry.

We have developed a novel fluorescence-based homogeneous binding assay for high-throughput screening of chemical compounds. In this assay, a Cy5- or Cy5.5-labeled ligand binds to receptor immobilized on a particle, either a bead or a cell. The resulting localized signal can be detected by a modified microvolume fluorimeter (MVF). When a molecule which competes with the labeled ligand is present, the localized fluorescence on cells or beads is reduced. Image processing software enumerates events and analyzes fluorescence intensity. We describe MVF assays for the IL-1 and IL-5 receptors. Using synthetic peptides with a range of affinities for the IL-1 receptor, we obtained IC(50) data consistent with those determined by radioligand binding assays. Because the image processing software can discriminate among events with different diameters, we were able to develop a multiplex assay, in which the IL-1R and IL-5R assays were carried out in the same well with each receptor immobilized on a different size of bead. IC(50) values generated in the multiplex assay for ligands specific to each receptor were comparable to those determined independently. Finally, similar IC(50) values were obtained in a 16-microl volume in an 864-well plate. This homogeneous, nonradioactive, miniaturizable, and multiplex-capable assay holds much promise for screening of combinatorial libraries and compound collections.