Detecting proton flux across chromatophores driven by F0F1-ATPase using N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt

Application of immunoassay to biosensors for use in the point-of-care setting ideally requires immunoassay without separation steps and with small volumes of both sample and reagents. The suitability of cloned enzyme donor immunoassay (CEDIA), one of a few homogeneous immunoassays available, was investigated for application to biosensors. This method is based on the bacterial enzyme beta-galactosidase, which has been genetically engineered by others into two inactive fragments, enzyme donor (ED) and enzyme acceptor (EA). Association of the ED and EA fragments in the assay results in formation of active enzyme, which acts on substrate to generate a detectable signal. Sensitivity of commercially available CEDIA kits were compared, with respect to the sample and reagent volumes, using three different signal generation processes. The CEDIA kit for valproic acid and three substrates, a colorimetric (chlorophenol red-beta-D-galactopyranoside), a chemiluminescent (Lumi-Gal 530), and a bioluminescent (Beta-Glo Assay System), were employed in the study. Our results indicate that the high sensitivity of the bioluminogenic substrate, D-luciferin-O-beta-galactopyranoside, with short assay time and small volumes of sample and reagents required for the assay, simple handling, and relatively low expense, make this substrate, together with CEDIA, suitable for application to biosensors intended for drug and metabolite monitoring in the point-of-care setting.     

An ImmunoChip prototype for simultaneous detection of antiepileptic drugs using an enhanced one-step homogeneous immunoassay

The development and characterization of a one-step homogeneous immunoassay-based multiwell ImmunoChip is reported for the simultaneous detection and quantitation of antiepileptic drugs (AEDs). The assay platform uses a cloned enzyme donor immunoassay (CEDIA) and a Beta-Glo assay system for generation of bioluminescent signal. Results of the one-step CEDIA for three AEDs (carbamazepine, phenytoin, and valproic acid), in the presence of serum, correlate well with the values determined by fluorescence polarization immunoassay. CEDIA intra- and interassay coefficients of variation are less than 10%. A microfabrication process, xurography, was used to produce the multiwell ImmunoChip. Assay reagents were dispensed and lyophilized in a three-layer pattern. The multiwell ImmunoChip prototype was used to detect and quantify AEDs in serum samples containing all three drugs. Luminescent signals generated from each well were recorded with a charge-coupled device (CCD) camera. The assays performed on an ImmunoChip were fast (5 min), requiring only small volumes of both the reagents (<1 microl/well) and the serum sample. The ImmunoChip assay platform described in this article may be well suited for therapeutic monitoring of drugs and metabolites at the point-of-care setting.     

Development of high-throughput spermidine synthase activity assay using homogeneous time-resolved fluorescence

Spermidine synthase (SPDS) catalyzes transfer of the propylamine group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine to yield methylthioadenosine (MTA) and spermidine. SPDS plays a regulatory role in cell proliferation and differentiation. This article describes the development of a high-throughput SPDS activity assay using homogeneous time-resolved fluorescence (HTRF) based on energy transfer from europium cryptate as a donor to crosslinked allophycocyanin (XL665) as an acceptor. First a highly specific anti-MTA monoclonal antibody, MTA-7H8, was generated, and then a competitive immunoassay for MTA determination was developed using europium cryptate-labeled MTA-7H8 and XL665-labeled MTA. In our homogeneous immunoassay, the percentage molar cross-reactivity of dcSAM with MTA-7H8 was 0.01% and the detection limit of MTA was 2.6 pmol/well. Our HTRF assay uses only one assay plate in which both enzyme reaction and MTA determination can be done successively. Therefore, our method can enable automatic screening of SPDS inhibitors from large numbers of samples.     

An enzyme immunoassay for secretin

A sensitive and specific enzyme immunoassay for secretin was developed with the use of enzyme-labeled antigens. Synthetic porcine secretin and its carboxy-terminal fragments (residues 11-27 and 18-27) were conjugated with beta-D-galactosidase for use in the immunoassay, and the assay method with the latter fragment (residues 18-27) linked to beta-D-galactosidase was found to be the most sensitive. The minimum amount of secretin detectable by this method was 1-2.5 pg/assay. Serum levels of secretin after intravenous injection of the peptide in rats were determined by both the enzyme immunoassay and a commercial radioimmunoassay kit. The correlation coefficient between the levels measured by the two methods was 0.984. The enzyme immunoassay could detect immunoreactive secretin levels in normal human sera, giving a value of 16.9 +/- 2.2 pg/ml (mean +/- SE of six human subjects).     

A rapid diffusion immunoassay in a T-sensor

We have developed a rapid diffusion immunoassay that allows measurement of small molecules down to subnanomolar concentrations in <1 min. This competitive assay is based on measuring the distribution of a labeled probe molecule after it diffuses for a short time from one region into another region containing antigen-specific antibodies. The assay was demonstrated in the T-sensor, a simple microfluidic device that places two fluid streams in contact and allows interdiffusion of their components. The model analyte was phenytoin, a typical small drug molecule. Clinically relevant levels were measured in blood diluted from 10- to 400-fold in buffer containing the labeled antigen. Removal of cells from blood samples was not necessary. This assay compared favorably with fluorescence polarization immunoassay (FPIA) measurements. Numerical simulations agree well with experimental results and provide insight for predicting assay performance and limitations. The assay is homogeneous, requires <1 microl of reagents and sample, and is applicable to a wide range of analytes.     

Development of homogeneous immunoassays based on protein fragment complementation

We demonstrate a functional in vitro proof-of-principle homogeneous assay capable of detecting small (<1 kDa) to large (150 kDa) analytes using TEM-1 β-lactamase protein fragment complementation. In the assays reported here, complementary components are added together in the presence of analyte and substrate resulting in colorimetric detection within 10-min. We demonstrate the use of functional mutations leading to either increased enzymatic activity, reduced fragment self-association or increased inhibitor resistance upon analyte driven fragment complementation. Kinetic characterization of the resulting reconstituted enzyme illustrates the importance of balancing increased enzyme activity with fragment self-association, producing diagnostically relevant signal-to-noise ratios. Complementation can be utilized as a homogeneous immunoassay platform for the potential detection of a range of analytes including, antibodies, antigens and biomarkers.     

Chemiluminescence-based biosensor for fumonisins quantitative detection in maize samples

A compact portable chemiluminescent biosensor for simple, rapid, and ultrasensitive on-site quantification of fumonisins (fumonisin B1+fumonisin B2) in maize has been developed. The biosensor integrates a competitive lateral flow immunoassay based on enzyme-catalyzed chemiluminescence detection and a highly sensitive portable charge-coupled device (CCD) camera, employed in a contact imaging configuration. The use of chemiluminescence detection allowed accurate and objective analyte quantification, rather than qualitative or semi-quantitative information usually obtained employing conventional lateral flow immunoassays based on colloidal gold labeling. A limit of detection of 2.5 μgL(-1) for fumonisins was achieved, with an analytical working range of 2.5-500 μgL(-1) (corresponding to 25-5000 μgkg(-1) in maize flour samples, according to the extraction procedure). Total assay time was 25 min, including sample preparation. A simple and convenient extraction procedure, performed by suspending the sample in a buffered solution and rapidly heating to eliminate endogenous peroxidase enzyme activity was employed for maize flour samples analysis, obtaining recoveries in the range 90-115%, when compared with LC-MS/MS analysis. The chemiluminescence immunochromatography-based biosensor is a rapid, low cost portable test suitable for point-of-use applications.     

A novel chemiluminescent substrate for detecting lactamase

Beta-lactamase is a well established reporter for monitoring cellular events while chemiluminescence is the preferred read-out mode in high throughput screens. Here, we report the first chemiluminescent assay for beta-lactamase using beta-galactosidase based enzyme fragment complementation technology. The enzyme fragment complementation technology employs a large protein fragment called the enzyme acceptor and a small peptidic fragment called an enzyme donor. These fragments are inactive separately but recombine rapidly in solution to yield active beta-galactosidase detected by chemiluminescence or fluorescence. A cyclic enzyme donor comprising a substituted cephalosporin moiety is used as the lactamase substrate. The cyclic substrate does not complement with enzyme acceptor to yield active beta-galactosidase, but upon cleavage with lactamase yields the linear enzyme donor which complements readily with enzyme acceptor. This methodology has been exploited in a simple, sensitive, homogeneous cell based reporter gene assay to monitor G-protein coupled receptor activation in a microtitre plate with a chemiluminescent read out.     

Rapid determination of bovine lactoferrin in dairy products by an automated quantitative agglutination assay based on latex beads coated with F(ab′)2 fragments

This study evaluated an automated immunoassay for bovine lactoferrin (LF) in dairy products based on latex beads coated with F(ab')2 fragments. Methods: F(ab')2 fragments were obtained by pepsin digestion of rabbit anti-bovine LF (IgG fraction) and polystyrene latex beads were coated with the F(ab')2 fragments. We used the beads to develop a rapid and homogeneous light scatter immunoassay employing an autoanalyzer (the Automated Latex assay). The Automated Latex assay was easy to perform and could rapidly determine bovine lactoferrin in lactoferrin-supplemented products. It was sensitive enough for testing products and showed good precision.     

Irreversible enzyme-shuttle immunoassay

The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.     

Enzyme modulator mediated immunoassay (EMMIA)

1. Enzyme modulator mediated immunoassay (EMMIA) is a separation-free (homogeneous) enzyme amplified immunoassay. 2. The assay is based on the ability of an analyte labeled enzyme modulator to modify the activity of an indicator enzyme and on ability of an anti-analyte antibody to abrogate the modifying action of the analyte-labeled enzyme modulator upon its binding to anti-analyte antibody. 3. The principle of EMMIA is elaborated in detail. The stages in the development of an EMMIA are described. 4. Criteria in selecting enzymes and modulators suitable for EMMIA are discussed. 5. Examples of EMMIA's using different kinds of modulators for different classes of analytes are presented.     

A homogeneous immunoassay for cyclic nucleotides based on chemiluminescence energy transfer.

A chemiluminescent derivative of cyclic AMP, aminobutylethylisoluminol succinyl cyclic AMP (ABEI-scAMP), was synthesized in order to develop a homogeneous immunoassay based on non-radiative energy transfer. ABEI-scAMP was chemiluminescent (5.1 X 10(18) luminescent counts X mol-1 at pH 13), pure (greater than 95%) stable and immunologically active. A conventional immunoassay was established using ABEI-scAMP and a solid-phase anti-(cyclic AMP) immunoglobulin G which could detect cyclic AMP at least down to 25fmol. A homogeneous immunoassay for cyclic AMP was established by measuring the shift in wavelength from 460 to 525nm which occurred when ABEI-scAMP was bound to fluorescein-labelled anti-(cyclic AMP) immunoglobulin G. The assay was at least as sensitive as the conventional radioimmunoassay using cyclic [3H]AMP and could measure cyclic AMP over the range 1-1000nM. The homogeneous chemiluminescent energy transfer assay was also able to quantify the association and dissociation of antibody-antigen complexes. Chemiluminescence energy transfer occurred between fluorescein-labelled antibodies and several other ABEI-labelled antigens (Mr values 314-150000) including progesterone, cyclic GMP, complement component C9 and immunoglobulin G. The results provide a homogeneous immunoassay capable of measuring free cyclic AMP under conditions likely to exist inside cells.     

Highly sensitive solid-phase enzyme immunoassay for terminal deoxynucleotidyl transferase

A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)₂ antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.     

Enzyme inhibitor screening using a homogeneous proximity-based immunoassay for estradiol

The authors have previously reported a homogeneous time-resolved fluorescence proximity immunoassay for estradiol. The assay was based on luminescence resonance energy transfer between a long lifetime fluorescent europium(III) chelate-dyed nanoparticle donor and a short lifetime, near-infrared fluorescent acceptor. The energy transfer prolonged the lifetime of the sensitized acceptor emission, and the fluorescence of the acceptor was measured using a time-resolved detection. The developed immunoassay was employed to screen inhibitors for enzyme 17beta-hydroxysteroid dehydrogenase type 1. The enzyme overexpressed in MCF-7 cells catalyzed a reversible conversion of estroneto17beta-estradiol. The inhibition efficiency of the tested molecule was obtained by comparing the final concentration of converted estradiol after 60 min of conversion reaction in a sample and in a conversion control not containing an inhibitor. The Zbeta factor calculated using the E2 concentrations of the homogeneous assay was 0.64, demonstrating a relatively good performance of the assay. The results from the homogeneous assay were comparable with the results obtained using radioactively labeled estrone as a substrate and high-performance liquid chromatography (HPLC) separation of estrone and converted estradiol after the enzyme reaction. Thus, this homogeneous assay can simplify the primary screening of potential new drug molecules by replacing a tedious radiometric HPLC method.     

Use of high-capacity surface with oriented recombinant antibody fragments in a 5-min immunoassay for thyroid-stimulating hormone

High-capacity surfaces can enhance analyte-binding kinetics and be beneficial for rapid immunoassays. Site-specifically immobilized, oriented recombinant single-chain Fv (scFv) and Fab antibody fragments were compared with a conventional, nonoriented monoclonal antibody (Mab) to capture antigen from serum to solid surface in a one-step, two-site thyroid-stimulating hormone (TSH) immunoassay with a 5-min incubation time. The assay used a ready-to-use dry reagent-based concept and time-resolved fluorescent measurement. TSH binding capacities were 3.0-fold (Fab) and at least 4.1-fold (scFv) higher when recombinant antibodies were used instead of Mab. Recombinant antibody fragments also produced faster kinetics (5 vs. 45-min saturation level) than Mab: 21-25% (Mab) versus 72-83% (scFv and Fab). Analytical sensitivities of the 5-min assay were 0.09 mIU/L TSH (Fab), 0.16 mIU/L TSH (scFv), and 0.26 mIU/L TSH (Mab). Between-run variabilities were 4.2-7.9% (Fab), 4.6-17.7% (scFv), and 5.5-7.2% (Mab). The assays correlated well with the AutoDELFIA hTSH (human TSH) Ultra assay (r = 0.99, n = 109). Fab was good in all aspects of immunoassay—capacity, kinetics, sensitivity, and analytical performance. As a homogeneous, stable, and small-sized binding molecule with optimized surface-coating properties as well as reduced risk for interference by heterophilic antibodies, Fab fragment is a promising and realistic immunoreagent for the future.     

A coupled assay for UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc).

UDP-GlcNAc:Gal beta 1-3GalNAc-R beta 1,6-N-acetylglucosaminyltransferase (GlcNAc to GalNAc) (i.e., core 2 GlcNAc-T) is a developmentally regulated enzyme of the O-linked oligosaccharide biosynthesis pathway. We have developed a coupled-enzyme assay for core 2 GlcNAc-T that is approximately 100 times more sensitive than the standard assay using UDP-[3H]GlcNAc as a sugar donor. Core 2 GlcNAc-T reactions were performed using unlabeled UDP-GlcNAc donor and Gal beta 1-3GalNAc alpha-paranitrophenyl (pNp) as acceptor. The product, Gal beta 1-3(GlcNAc beta 1-6)GalNAc alpha-pNp was then further reacted with purified bovine beta 1-4Gal-T and UDP-[3H]Gal to produce Gal beta 1-3([3H]Gal beta 1-4GlcNAc beta 1-6) GalNAc alpha-pNp, which was separated on an Ultrahydrogel HPLC column. Approximately 10% of the available GlcNAc-terminating acceptor was substituted in the Gal-T reaction, allowing 1 pmol of product to be readily detected. The increased sensitivity of the coupled assay should facilitate studies of core 2 GlcNAc-T activity where material is limiting or specific activity is low.     

GDH biosensor based off-line capillary immunoassay for alkylphenols and their ethoxylates

The application of a quinoprotein glucose dehydrogenase modified thick-film sensor as label detector in a capillary immunoassay (CIA) for xenoestrogens is presented. The detection of the alkylphenols and their ethoxylates is based on the competition between the analyte and tracer molecules for the binding sites of anti-alkylphenol ethoxylate antibodies. This assay is performed off-line in small disposable PVC capillaries coated with immobilized antibodies. This format allows the combination of the assay with a small portable device potentially useful for on-site environmental monitoring. Beside high amplification the utilization of beta-galactosidase as enzyme label allows the direct combination with a GDH biosensor at optimal pH conditions. The bioelectrocatalytic properties of this biosensor offer an additional amplification and thus allow a very sensitive quantification of 4-aminophenol, generated by the beta-galactosidase. Detection limits of the analytes in the microg/l range were obtained, while other phenolics and surfactants showed no or very little cross reactivity.     

Aequorin-based homogeneous cortisol immunoassay for analysis of saliva samples

Homogeneous assays are attractive because they are performed in only one phase, namely, the liquid phase, and thus, they do not require separation of phases as their heterogeneous counterparts do. As opposed to heterogeneous assays, the signal generation in a homogeneous assay is a direct result of analyte binding, which allows the multiple washing and incubation steps required in an indirect heterogeneous assay format to be eliminated. Moreover, homogeneous assays are usually fast and amenable to miniaturization and automation. In this article, we describe the development of a homogeneous assay for the hormone cortisol using the bioluminescent photoprotein aequorin as a reporter molecule. A cortisol derivative was chemically conjugated to the lysine residues of a genetically modified aequorin in order to prepare an aequorin-cortisol conjugate capable of binding anticortisol antibodies. The binding of anticortisol antibodies to the aequorin-cortisol conjugate resulted in a linear response reflected in the emission of bioluminescence by aequorin. A competitive binding assay was developed by simultaneously incubating the aequorin-cortisol conjugate, the anticortisol antibodies, and the sample containing free cortisol. Dose-response curves were generated relating the intensity of the bioluminescence signal with the concentration of free cortisol in the sample. The optimized homogeneous immunoassay produced a detection limit of 1 x 10 (-10) M of free cortisol, with a linear dynamic range spanning from 1 x 10 (-5) to 1 x 10 (-9) M. Both serum and salivary levels of cortisol fall well within this assay's linear range (3.0 x 10 (-7) M to 7.5 x 10 (-7) M and 1.0 x 10 (-8) M to 2.5 x 10 (-8) M, respectively), thereby making this assay attractive for the analysis of this hormone in biological samples. To that end, it was demonstrated that the assay can be reliably used to measure the concentration of free cortisol in saliva without significant pretreatment of the sample.     

Rapid and separation-free sandwich immunosensing based on accumulation of microbeads by negative-dielectrophoresis

We report here a rapid and separation-free immunoassay using a dielectrophoresis (DEP) device consisting of an interdigitated microarray (IDA) electrode and a polydimethylsiloxane (PDMS) substrate. On applying an AC voltage to the IDA in a negative-DEP (n-DEP) frequency region, goat anti-mouse immunoglobulin (anti-mouse IgG)-immobilized microbeads moved to the surface of the PDMS substrate placed above the IDA. The microbeads accumulated at designated areas of the PDMS surface that had been precoated with anti-mouse IgG. When the fluorescence microbeads bearing anti-mouse IgG were suspended in an analyte (mouse IgG) solution, the microbeads trapped the analyte to form immunocomplexes on microbeads. The microbeads reacted with mouse IgG accumulated and were captured at the designated areas of the PDMS surface via an antibody-antigen-antibody (sandwich) reaction. The captured microbeads were detected selectively by fluorescence measurements at the focused designated areas, regardless of the presence of uncaptured microbeads suspended in solution. Thus, the separation and washing-out steps usually required for conventional immunoassay are eliminated in the presented procedure. Since the formation of the sandwich structures was accelerated significantly by n-DEP, a period as short as 30s was sufficient to detect the immunoreaction at the surface. The fluorescence intensity of the captured microbeads at the designated areas increased with analyte concentration in the range 0.01-10ng/mL. The present procedure therefore yields a rapid, sensitive, and separation-free immunoassay in a simple device.     

Extending the detection limit of solid-phase electrochemical enzyme immunoassay to the attomole level

Electrochemical enzyme immunoassay methodology has been developed to take advantage of the selectivity of antibody reactions, the amplification feature of an enzyme-based assay, and the ease with which small amounts of the enzyme-generated product can be detected electrochemically. A heterogeneous sandwich enzyme immunoassay was used in this work as the model assay. In this type of assay, the antigen is sandwiched between the enzyme conjugate and a primary antibody that is adsorbed to the solid phase. Alkaline phosphatase is a suitable enzyme for electrochemical assays since it catalyzes the conversion of electroinactive phenyl phosphate to electroactive phenol. The product, phenol, is then quantitated by liquid chromatography with electrochemical detection in a thin-layer flow cell with a carbon paste electrode at 0.895 V vs Ag/AgCl. The current produced by the oxidation of phenol is directly proportional to the analyte (antigen) concentration. The problem associated with these types of solid-phase immunoassays is that the adsorption of the primary antibody is desired while the adsorption of other assay proteins is not. The detection limits are generally defined by the ability to control this nonspecific adsorption. The detection limit of a previous electrochemical assay for rabbit IgG was 100 pg/ml and was limited by a large background current observed in the absence of antigen. In the present study, each step of the assay was examined in order to determine the sources of this background current, and it was found that the major contribution was from the nonspecific adsorption of the enzyme conjugate. Using combinations of Tween 20 and bovine serum albumin as blocking agents, the level of nonspecific adsorption was reduced by 96%.(ABSTRACT TRUNCATED AT 250 WORDS)