A microtiter plate assay for the detection of inhibitors of the Na +, K + -ATpase

A rapid microtiter plate assay for the detection of inhibitors of the Na⁺, K⁺-ATPase has been developed. The assay is based on the measurement of inorganic phosphate released from the substrate, ATP, and has been designed to be carried out in the individual wells of a microtiter plate. Since the production of inorganic phosphate is determined colorimetrically, multiple samples can be tested simultaneously using a microtiter plate reader. This microtiter plate assay is particularly useful for screening large numbers of samples, such as microbial culture supernatants.     

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles’ colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core–shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient’s sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.     

An immunoassay for terbutryn using direct hapten linkage to a glutaraldehyde network on the polystyrene surface of standard microtiter plates

2-Aminobutylamino-4-ethylamino-6-isopropylamino-1,3,5-triazine (ABA-atrazine) has been synthesized and used as a coating hapten in an immunoassay with a monoclonal antibody against terbutryn. Coating was achieved by covalently linking ABA-atrazine to a glutaraldehyde polymer network directly bound to the polystyrene surface of a standard 96-well microtiter plate. The assay was carefully optimized. In particular, the coating hapten concentration had a strong effect on the ELISA sensitivity. By including a pre-incubation step a low test midpoint (IC50-value) of 0.130 microg L(-1) was achieved. As far as we are aware this is the most sensitive ELISA for terbutryn yet reported. The coating-hapten-format presented is proposed as generally applicable, because the glutaraldehyde-modified microtiter plate surface enables stable immobilization of a broad variety of coating haptens.     

Comparative surface plasmon resonance and enzyme-linked immunosorbent assay characterisation of a monoclonal antibody with N-acyl homoserine lactones

This study shows the detection of (N-acyl) homoserine lactones (AHLs or HSL) with monoclonal antibodies via a surface plasmon resonance (SPR)-based immunosensor in comparison to conventional microtiter plate-based enzyme-linked immunosorbent assay (ELISA). An HSL derivative, named HSL2 (Table 1), was attached to bovine serum albumin (BSA) and the conjugate (HSL2-BSA-r2) was either covalently immobilised on the SPR sensor chip surface via free amino groups or via adsorption on the ELISA polystyrene plate surface. With a newly developed rat monoclonal antibody (mAb HSL1/2 2C10), AHLs were detected sensitively in a competitive format with SPR and ELISA. Well comparable experiments between SPR and ELISA could be obtained in buffers. Moreover, the SPR sensor surface with the immobilised conjugate HSL2-BSA-r2 could be regenerated at least 340 times (regeneration cycles) without loss of activity. The measurement time per cycle was approximately 15 min. The competitive detection format for SPR and ELISA allowed the detection in the μg L⁻¹ range.     

A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol

A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 mul and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity.     

Quantitative analysis of 17beta-estradiol in river water by fluorometric enzyme immunoassay using biotinylated estradiol.

A sensitive and simple immunoassay to determine 17beta-estradiol (E2) in fresh water was developed. The method is based on a solid-phase avidin-biotin binding assay and solid phase extraction. The binding event of E2 to the antibody is detected indirectly by the competitive reaction between E2 and biotinylated estradiol (BE) as a tracer for the limited binding sites of antibodies immobilized onto the wall of a microtiter plate. Namely, E2 concentrations are determined from the strong interaction between BE and avidin conjugated with horseradish peroxidase (avidin-HRP). In order to achieve a sensitive measurement for the binding of BE to the antibody immobilized on the microtiter plate substrate, QuantaBlu fluorogenic peroxidase substrate (QFPS) was employed. The detection limit and the linear range of E2 determination were 27 pM and 27 - 7480 pM, respectively. The relative standard deviations (RSD) for the E2 assay were between 0.3 and 12.0% (n = 3). The cross-reactivities of several other estrogens in this assay system were also investigated. No serious influences from any cross-reaction caused by other estrogens tested in this experiment were observed. The determination of E2 in water samples from eight rivers and a marsh in Hokkaido was performed by the immunoassay combined with solid phase extraction. It was found that the concentration of E2 was in the range between 0.06 and 67 pM.     

A Microtiter Plate Assay for Sulfonamides

Abstract

Sulfamethoxazole, sulfisoxazole, and sulfadiazine are sulfonamides used in the treatment of several infectious diseases. Several studies have demonstrated that the amino substituent plays an important role in both the toxicity and the therapeutic effect of these drugs. In view of these findings, a rapid and convenient method of analysis would be useful for monitoring selected patients receiving these drugs. With the increasing use of microtiter plate methodology in the clinical laboratory, an assay based upon the Bratton-Marshall reaction with the amino substituent was adapted to the microtiter plate format. The results indicate that the microtiter plate assay for sulfonamides retains the sensitivity and linearity necessary for analysis of sulfonamides in biological fluids at clinically relevant concentrations. The assay is simple, rapid, convenient, and suitable for monitoring procedures where only the measurement of the active drug concentration is required.     

A flow-based enzyme-linked immunosorbent assay on a polydimethylsiloxane microchip for the rapid determination of immunoglobulin A

A flow-based enzyme-linked immunosorbent assay (ELISA) on a polydimethylsiloxane (PDMS) microchip has been developed for the rapid determination of immunoglobulin A (IgA). The analytical principle of this integrated method is the same as the conventional sandwich-type ELISA. A primary antibody (anti-IgA) was adsorbed on the surface of a PDMS microchannel, and then an antigen (IgA) and a secondary antibody (anti-IgA HRP labeled) were reacted successively. The resulting antigen-antibody complex, fixed on the surface of the microchannel, was detected using Amplex^® Red and a fluorescent imaging system. The calibration curve of the IgA standard solution was linear in the range of 0-50 ng/mL at the flow rate of 10 μL/min. This flow rate corresponds to the reaction time of 4.8 s. Compared to the conventional assay on a 96-well microtiter plate, the present assay on the microchip dramatically shortened the reaction time necessary for the enzyme-substrate reaction from 30 min to 4.8 s, i.e., to 1/375. The amounts of the reagent and sample were also reduced to 1/100 compared to the 96-well microtiter plate.     

Uncertainty analysis of the microtiter plate method for determining trypsin inhibitor activity

Protease inhibitor activity is frequently measured in legume seeds as protease inhibitors are thought to have anti-nutritional as well as anti-carcinogenic properties. Trypsin inhibitor activity (TIA) can be measured using different methods. The microtiter plate method is very convenient and routinely used; therefore, in this study, we analyzed the measurement uncertainty of the microtiter plate method to understand what affects the measurement results, as well as to compare TIA values obtained by similar and different methods. For uncertainty analysis of TIA measurement, we used the soybean variety ‘Vojvodjanka,’ which is known to have TIA greater than 80 trypsin units inhibited (TUI) per mg of seed. We followed the Guide to the Expression of Uncertainty in Measurement (GUM) for our uncertainty analysis of the microtiter plate method for TIA testing, which we present in the form of an uncertainty budget. Absorbance measurement and preparation of sample reaction mixture took the largest percent (71 %) of overall uncertainty of TIA value. The TIA of soybean variety ‘Vojvodjanka’ was (94.1 ± 8.4) TUI/mg, and this result is consistent with those obtained by other authors. The microtiter plate method is a reliable method for TIA measurement, making seed quality testing more efficient.     

Stable competitive enzyme-linked immunosorbent assay kit for rapid measurement of 11 active beta-lactams in milk, tissue, urine, and serum

Beta-lactams are used as veterinary drugs for the treatment of food-producing animals. For consumer protection, legislation is in place to set limits for their residues. An enzyme-linked immunosorbent assay (ELISA) was developed which allowed, in a single reaction, the class-specific measurement of 11 beta-lactams, with limits of detection below European maximum residue limits. Determinations were feasible in milk, tissue, urine, and serum with simple and rapid sample preparation. In this format, the specific capture antibodies were precoated on the microtiter plate and horseradish peroxidase-labeled conjugate was used to compete with free beta-lactams. The stability of the precoated microtiter plate and conjugate was at least 1 year when stored at 2 to 8 degrees C; upon reconstitution, the conjugate was stable for 6 days at 2 to 8 degrees C. The stability of lyophilized ampicillin standards was at least 6 months when stored at 2 to 8 degrees C and at least 1 year when stored at -20 degrees C. A low cross-reactivity, 3.6%, was observed with ampicillin with open beta-lactam ring relative to 100% for intact ampicillin. Generic recognition was shown by relative cross-reactivity values ranging from 22 (penicillin V) to 144% (nafcillin). Cross-reactivity for cephalosporins was <0.1%. Intra- and interassay precisions expressed as coefficient of variation were typically 2-8%. The inhibitory concentration with 50% binding for ampicillin was typically 2 ppb. Recovery for different spiked levels was >70% with all the matrixes.     

Capillary zone electrophoresis separation and collection of spermatozoa for the forensic analysis of sexual assault evidence

The processing of sexual assault kits (SAKs) relies on the genetic analysis of material extracted from swabs collected from the assault victim. A vital step in producing an identifiable DNA profile of the perpetrator is the effective separation of perpetrator (sperm) and victim (epithelial) DNA that have been isolated from the collected evidence. We report the use of capillary zone electrophoresis for the separation of intact sperm from whole and lysed epithelial cells in SAKs. The separated components are deposited into wells of a microtiter plate using a computer-controlled fraction collector, and quantitative PCR is used to verify the collection of sperm cells by targeted amplification of male DNA. We present results from simulated sexual assault samples that have been aged for up to 18 months, as well as vaginal swabs from authentic forensic kits. Components extracted from the vaginal swabs from the SAK comigrated with an aged semen sample at 6.25 ± 0.25 min. Epithelial cells migrated from 10-12 min, producing baseline resolution of the components. Sperm cells were collected in a microtiter plate for downstream analysis.     

Quantitative turbidity assay for lipolytic enzymes in microtiter plates

A clearing assay for lipolytic enzymes has been realized in 96-well microtiter plates. A thin layer containing emulsified tributyrin as turbidity-generating substrate was placed on a thicker supporting aqueous layer. Both layers were stabilized by a gel-forming agent. Enzyme addition leads to clearing of the emulsion detected with a standard microtiter plate reader as a decrease of extinction. Dependencies of the signal kinetics on the substrate and enzyme concentrations were studied. For 0.5–1 % tributyrin content the reaction rate is not substrate-limited. An initial slope of the signal kinetics is proportional to the lipase activity. A detailed characterization of the assay was performed. Lipolysis of tributyrin was confirmed by glycerol detection. Various gel-forming agents were compared and diffusion conditions in these gels were analyzed. Agar and agarose were found to be the most suitable gel-forming agents, which do not affect enzyme diffusion whereas polyacrylamide gels block lipase diffusion and therefore are not suitable for the assay. The optimized assay prepared from 1 % tributyrin emulsion in 2 % agar gel was tested with six microbial lipases and porcine pancreatic lipase. The detection limit is 20–60 ng/well which is equivalent to 30 μU/well for T. lanuginosus lipase.

Direct hapten-linked competitive inhibition enzyme-linked immunosorbent assay (CIELISA) for the detection of O-pinacolyl methylphosphonic acid

Immunoassay detection of O-pinacolyl methylphosphonic acid (PMPA) employing direct coating of N-2-aminoethyl-O-pinacolyl methylphosphonate (hapten B) on microtiter plates is reported. Coating was achieved by covalently linking hapten B to a glutaraldehyde (GA) polymer network directly bound to the polystyrene (PS) surface of a standard 96-well microtiter plate. 4-(2-(O-Pinacolylmethylphosphoryl amino)ethyl amino)-4-oxobutanoic acid (hapten A)-ovalbumin (OVA) conjugate served as the coating antigen for comparison with direct hapten B-coated plates in the CIELISA format. The developed assay employing direct hapten B coated plates demonstrated enhanced sensitivity with the IC(50) value for PMPA being 0.027 μg mL(-1). The assay could detect PMPA even at the concentration of 0.006 μg mL(-1). The mean recovery of standard PMPA (spiked in water) was found to be 83.7%.     

Aptamer sandwich assays: label-free and fluorescence investigations of heterogeneous binding events

We studied aptamer binding events in a heterogeneous format using label-free and fluorescence measurements for the purpose of developing an aptamer-based sandwich assay on a standard microtiter plate platform. The approach allowed visualization of the underlying aptamer immobilization and target binding events rather than relying on only an endpoint determination for method optimization. This allowed for a better understanding of these multi-step assays and optimal conditions specific to aptamers. α-thrombin was chosen as a prototypical analyte as two well-studied aptamers (15 and 29-mer) binding distinct epitopes are available. The Corning Epic? system, which utilizes a resonance waveguide diffraction grating in a 384-well microtiter plate format, was employed to measure relative immobilization and binding levels for various modified aptamers. Parameters investigated included the effects of aptamer orientation, label orientation, spacer length, spacer type, immobilization concentration, and binding buffer. Most notably, the 15-mer aptamer was preferable for capture over the 29-mer aptamer and aptamers with increasing poly(dT) spacer length between the biotin modification and the aptamer yielded decreased immobilization levels. This decreased immobilization resulted in increased α-thrombin binding ability for 15-mer aptamers with the poly(dT) spacer. Fluorescence measurements of fluorescein-labeled 29-mer aptamers with varying spacers were used to visualize sandwich complex formation. Using both label-free and traditional fluorescence measurements, an in-depth understanding of the overall assay was obtained, thus the inclusion of label-free measurements is recommended for future method development.     

Surface chemical approach to single-step measurement of antibody in human serum using localized surface plasmon resonance biosensor on microtiter plate system

In clinical settings, serum antibody levels serve as markers of pathology. For example, antibodies related to autoimmune diseases are among the conventional targets in laboratory tests. Simple clinical tests can improve the efficacy of laboratory practice. This study describes a single-step, wash-free technique for optically detecting antibodies in human serum through the localized surface plasmon resonance (LSPR) of gold nanoparticles. As a proof-of-concept experiment, the amount of antibiotin dissolved in human serum was measured with a LSPR-based biosensor in a wash-free manner using a conventional 96-well microtiter plate and a plate reader. For an efficient surface modification of biosensors, zwitterionic copolymer was used as a scaffold on the gold nanoparticle surface to immobilize antigen and blocking reagent. Single-step, wash-free measurement of antibiotin in human serum was successfully achieved. In addition, nonspecific responses from serum contents were significantly reduced because both the copolymer and hydrophilic antigen reagent that we employed were composed of poly(ethylene oxide) spacer. Comparative experiments of the antigen-antibody reaction in serum to that in buffered solution revealed that serum is a favorable environment for the biological reaction. In conclusion, our gold-nanoparticle-based LSPR method may provide a rapid and simple way to measure the amount of antibody in serum quantitatively in clinical practice.

Gold nanoparticles catalyzed chemiluminescence immunoassay for detection of herbicide 2,4-dichlorophenoxyacetic acid

We present a novel immunoassay format utilizing the catalytic properties of gold nanoparticles in the luminol-silver nitrate-gold nanoparticle based chemiluminescence (CL) system for the detection of widely used herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Highly sensitive anti-2,4-D antibody was produced and conjugated with gold nanoparticles of various sizes. In the present assay format, employing a competitive inhibition approach, a well-characterized hapten-protein conjugate (2,4-D-BSA) was used to coat the microtiter plates. The analyte (2,4-D) was pre-incubated with anti-2,4-D antibody labeled with gold nanoparticles and added to each well of the microtiter plate. The gold label triggered the reaction between luminol and silver nitrate generating a luminescence signal at 425 nm. Under the optimized conditions, the CL based immunoassay showed the detection limit of 2,4-D in standard water samples around 3 ng mL(-1). The CL based immunoassay format, based on gold nanoparticles as a catalyst, could be used as a fast screening methodology (<30 min) for pesticide detection.     

The radiation refinement of polystyrene surface applied in immunoanalysis

Polystyrene, material frequently used for ELISA microtiter plates, was modified by gamma irradiation in order to enhance the passive absorption binding capacity of immunoglobuline E. The dependences of this capacity increase on dose and irradiation conditions (atmosphere, presence of water) permitted the optimization this treatment of plates up to the technological scale. However the increase of passive absorption in the case of low-molecular weight synthetic peptides, such as anti-EBNA, was not sufficient and the reason for this remains to be clarified.

Radiation grafting made it possible to fix the hydrazide functional group onto the polysterene. It offers the possibility to of binding the proteins, via covalent linkage, with the carbonyl group formed by the oxidation of carbohydrates included in the protein molecule.     

A rapid sandwich immunoassay for human fetuin A using agarose-3-aminopropyltriethoxysilane modified microtiter plate

A rapid sandwich immunoassay (IA) with enhanced signal response for human fetuin A (HFA) was developed by modifying the surface of a KOH-treated polystyrene microtiter plate (MTP) with agarose and 3-aminopropyltriethoxysilane (APTES). The agarose-APTES complex binds covalently to the hydroxyl moiety of the MTP plate to serve as a binding platform for bioconjugation of EDC-activated anti-HFA antibody (Ab) via carbodiimide coupling. The one-step kinetics-based sandwich enzyme-linked immunosorbent assay (ELISA) enabled the detection of HFA in 30 min with a limit of detection (LOD) and a linear range of 0.02 ng mL⁻¹ and 1–243 ng mL⁻¹, respectively. It detected HFA spiked in diluted human whole blood and serum, and HFA in ethylenediaminetetraacetic acid (EDTA)-plasma of patients with high precision similar to that of conventional ELISA. The anti-HFA Ab-bound agarose-functionalized MTPs retained their functional activity after 6 weeks of storage in 0.1 M PBS, pH 7.4 at 4 °C.     

Self-aspirating atmospheric pressure chemical ionization source for direct sampling of analytes on surfaces and in liquid solutions

A self-aspirating heated nebulizer probe is described and demonstrated for use in the direct analysis of analytes on surfaces and in liquid samples by atmospheric pressure chemical ionization (APCI) mass spectrometry. Functionality and performance of the probe as a self-aspirating APCI source is demonstrated using reserpine and progesterone as test compounds. The utility of the probe to sample analytes directly from surfaces was demonstrated first by scanning development lanes of a reversed-phase thin-layer chromatography plate in which a three-component dye mixture, viz., Fat Red 7B, Solvent Green 3, and Solvent Blue 35, was spotted and the components were separated. Development lanes were scanned by the sampling probe operated under computer control (x, y plane) while full-scan mass spectra were recorded using a quadrupole ion trap mass spectrometer. In addition, the ability to sample the surface of pharmaceutical tablets (viz., Extra Strength Tylenol and Evista tablets) and to detect the active ingredients (acetaminophen and raloxifene, respectively) selectively was demonstrated using tandem mass spectrometry (MS/MS). Finally, the capability to sample analyte solutions from the wells of a 384-well microtiter plate and to perform quantitative analyses using MS/MS detection was illustrated with cotinine standards spiked with cotinine-d3 as an internal standard.