Immunoassay readout method using extrinsic Raman labels adsorbed on immunogold colloids

An immunoassay readout method based on surface-enhanced Raman scattering (SERS) is described. The method exploits the SERS-derived signal from reporter molecules that are coimmobilized with biospecific species on gold colloids. This concept is demonstrated in a dualanalyte sandwich assay, in which two different antibodies covalently bound to a solid substrate specifically capture two different antigens from an aqueous sample. The captured antigens in turn bind selectively to their corresponding detection antibodies. The detection antibodies are conjugated with gold colloids that are labeled with different Raman reporter molecules, which serve as extrinsic labels for each type of antibody. The presence of a specific antigen is established by the characteristic SERS spectrum of the reporter molecule. A near-infrared diode laser was used to excite efficiently the SERS signal while minimizing fluorescence interference. We show that, by using different labels with little spectral overlap, two different antigenic species can be detected simultaneously. The potential of this concept to function as a readout strategy for multiple analytes is briefly discussed.     

Polyclonal antibody-based noncompetitive immunoassay for small analytes developed with short peptide loops isolated from phage libraries

To date, there are a few technologies for the development of noncompetitive immunoassays for small molecules, the most common of which relies on the use of anti-immunocomplex antibodies. This approach is laborious, case specific, and relies upon monoclonal antibody technology for its implementation. We recently demonstrated that, in the case of monoclonal antibody-based immunoassays, short peptide loops isolated from phage display libraries can be used as substitutes of the anti-immunocomplex antibodies for noncompetitive immunodetection of small molecules. The aim of this work was to demonstrate that such phage ligands can be isolated even when the selector antibodies are polyclonal in nature. Using phenoxybenzoic acid (PBA), a major pyrethroid metabolite, as a model system, we isolated the CFNGKDWLYC peptide after panning a cyclic peptide library on the PBA/anti-PBA immunocomplex. The sensitivity of the noncompetitive enzyme-linked immunosorbent assay (ELISA) setup with this peptide was 5-fold (heterologous) or 400-fold (homologous) higher than that of the competitive assay setup with the same antibody. Phage anti-immunocomplex assay (PHAIA) was also easily adapted into a rapid and highly sensitive dipstick assay. The method not only provides a positive readout but also constitutes a major shortcut in the development of sensitive polyclonal-based assays, avoiding the need of synthesizing heterologous competing haptens.     

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.     

Antibody microarray detection of Escherichia coli O157:H7: Quantification, assay limitations, and capture efficiency

A sandwich fluorescent immunoassay in a microarray format was used to capture and detect E. coli O157:H7. Here, we explored quantitative aspects, limitations, and capture efficiency of the assay. When biotinylated capture antibodies were used, the signal generated was higher (over 5-fold higher with some cell concentrations) compared to biotinylated protein G-bound capture antibodies. By adjusting the concentration of reporter antibody, a linear fluorescent response was observed from approximately 3.0 x 10(6) to approximately 9.0 x 10(7) cells/mL, and this was in agreement with the number of captured bacteria as determined by fluorescence microscopy. Capture efficiency calculations revealed that, as the number of bacteria presented for capture decreased, capture efficiency increased to near 35%. Optimization experiments, with several combinations of capture and reporter antibodies, demonstrated that the amount of bacteria available for capture (10(6) versus 10(8) cells/mL) affected the optimal combination. The findings presented here indicate that antibody microarrays, when used in sandwich assay format, may be effectively used to capture and detect E. coli O157:H7.     

Significance of antibody orientation unraveled: well-oriented antibodies recorded high binding affinity

To investigate the effect of antibody orientation on its immunological activities, we developed a novel and versatile platform consisting of a well-defined phospholipid polymer surface on which staphylococcal protein A (SpA) was site-selectively immobilized. The application of a biocompatible phospholipid-based platform ensured minimal denaturation of immobilized antibodies, and the site-selective immobilization of SpA clarified the effect of antibody orientation on immunological activities. The phospholipid polymer platform was prepared on silicon substrates using the surface-initiated atom transfer radical polymerization (SI-ATRP) technique. An enzymatic reaction was performed for orientation-selective coupling of SpA molecules to the polymer brush surface. Orientation-controlled antibodies were achieved using enzymatic reactions, and these antibodies captured 1.8 ± 0.1 antigens on average, implying that at least 80% of immobilized antibodies reacted with two antigens. Theoretical multivalent binding analysis further revealed that orientation-controlled antibodies had antigen-antibody reaction equilibrium dissociation constants (K(d)) as low as 8.6 × 10(-10) mol/L, whereas randomly oriented and partially oriented antibodies showed K(d) values of 2.0 × 10(-7) and 1.2 × 10(-7) mol/L, respectively. Strict control of antibody orientation not only formed an approximately 100-fold stronger antigen-antibody complex than the controls but also sustained the native antibody K(d) (10(-10)-10(-9) mol/L). These findings support the significance of antibody orientation because controlling the orientation resulted in high reactivity and theoretical binding capacity.     

An attempt to develop surface plasmon resonance based immunosensor for Karnal bunt (Tilletia indica) diagnosis based on the experience of nano-gold based lateral flow immuno-dipstick test

Karnal bunt (KB) of wheat incited by Tilletia indica is an economically important quarantined fungal pathogen. Similarity in teliospore configuration makes it difficult to differentiate KB teliospores from the teliospores of other bunt fungi using conventional approaches. In order to determine the correct identity of KB teliospore— an infectious entity, it is essential to develop specific diagnostic probes and high quality of immunological reagents against infectious entities. The immuno-dipstick assay developed in our lab is quite sensitive to detect the antigens of even five teliospores. For on-site testing of KB, a nano-gold based lateral flow immuno-dipstick assay (LFID) was also developed in our lab using anti-teliospore antibodies. For development of LFID, colloidal gold was prepared and conjugated with anti-teliospore antibodies which were further characterized. However, species specific detection is yet to be achieved by generation of monoclonal antibodies against diagnostic antigens (34 and 66 kD teliospore's proteins). Based on the experience of nano-gold based LFID, we are proposing to develop surface plasmon resonance (SPR) based label-free detection system. The same may be employed not only for determination of the quality of immunological reagents in terms of sensitivity, specificity and precision but also development of suitable design of immunosensor for differential diagnosis of KB (T. indica). The approach involves the use of a mouse monoclonal antibody against diagnostic antigens and a suitable design of SPR sensor by the preparation of immuno-affinity layer over self assembled monolayer surfaces. The signal to noise ratio can further be enhanced using nano-gold particles. This will be first attempt for real-time monitoring of KB teliospores in wheat lots by SPR sensor and in a fully automated manner for establishing seed certification and plant quarantine standards.     

Development of highly fluorescent detection reagents for the construction of ultrasensitive immunoassays

We developed two kinds of highly fluorescent streptavidin-based conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 microL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 microg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 microL of sample volume was used, a detection limit of 0.001 microg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection.     

A general method to perform a noncompetitive immunoassay for small molecules.

A new general method to perform a noncompetitive immunoassay for low-molecular-mass analytes (less than 6000 Da) is described and checked using cortisol as a model system. The method is based on the use of a "polydentate ligand" (cortisol-poly(L-lysine) conjugate) able to block the antibody sites unoccupied by the analyte, followed by the replacement of an antibody-bound analyte by an enzyme-labeled analyte (cortisol-horseradish peroxidase), and permits the direct measurement of the analyte bound sites. The observed signal shows a near-linear correlation with the analyte concentration. The characteristics of interactions between the analyte and polydentate ligand with the specific antibody were studied to perform a preliminary evaluation of the noncompetitive immunoassay for cortisol. The noncompetitive assay was compared with a competitive immunoassay obtained under the same conditions and using the same reagents. The results of the experiments showed a lower detection limit for the noncompetitive model (0.15 ng mL-1 rather than 0.72 ng mL-1), emphasizing that the model is successful. Moreover, as the polydentate ligand is prepared from the same hapten used for the immunogen synthesis, this type of noncompetitive immunoassay appears generally applicable to all small molecules for which antibodies have been obtained.     

Immunofiltration: a methodology for preconcentration and determination of organic pollutants.

A new concentration procedure using an immunofiltration-based method is described. The approach enables quantitative determination of organic pollutants by filtering large volumes of sample through a poly(vinylidene difluoride) membrane where antibodies have been immobilized by passive adsorption. The analysis is based on a sequential competitive enzyme immunoassay. A wide range of sample volumes have been tested (0.2-5.0 mL) for each type of antibody. The improvement on the assay sensitivity and specificity achieved by means of this concentration procedure is discussed. Using this technique and the insecticide carbaryl as a model analyte, a concentration factor of at least 13 and a limit of detection of 4.75 ng/L are accomplished. The suitability of this methodology is demonstrated by the quantification of the insecticide in several types of water samples (bottled, estuarine, and physiological-saline solutions) with recoveries ranging between 102 and 111%. This method has proved to concentrate carbaryl directly, in an accurate way, for residue analysis without using organic solvents or any extraction process. Furthermore, this procedure offers the advantages of carrying out in the same system both preconcentration and quantitative determination of the analyte.     

Nanoencapsulated microcrystalline particles for superamplified biochemical assays

We report on the preparation and utilization of a novel class of particulate labels based on nanoencapsulated organic microcrystals with the potential to create highly amplified biochemical assays. Labels were constructed by encapsulating microcrystalline fluorescein diacetate (FDA; average size of 500 nm) within ultrathin polyelectrolyte layers of poly(allylamine hydrochloride) and poly(sodium 4-styrenesulfonate) via the layer-by-layer technique. Subsequently, the polyelectrolyte coating was used as an "interface" for the attachment of anti-mouse antibodies through adsorption. A high molar ratio of fluorescent molecules present in the microcrystal core to biomolecules on the particle surface was achieved. The applicability of the microcrystal-based label system was demonstrated in a model sandwich immunoassay for mouse immunoglobulin G detection. Following the immunoreaction, the FDA core was dissolved by exposure to organic solvent, leading to the release of the FDA molecules into the surrounding medium. Amplification rates of 70-2000-fold (expressed as an increase in assay sensitivity) of the microcrystal label-based assay compared with the corresponding immunoassay performed with direct fluorescently labeled antibodies are reported. Our approach provides a general and facile means to prepare a novel class of biochemical assay labeling systems. The technology has the potential to compete with enzyme-based labels as it does not require long incubation times, thus speeding up bioaffinity tests.     

Nanocrystal biolabels with releasable fluorophores for immunoassays

A novel signal amplification technology based on a new class of biofunctional fluorescent nanocrystals holds promise to improve the sensitivity and the limits of detection of immunoassays. A two-step approach without layer-by-layer techniques is described to encapsulate the fluorogenic precursor fluorescein diacetate (FDA) nanocrystals (107-nm average size) followed by conjugation of the antibody. Distearoylphosphatidylethanolamine (DSPE) modified with amino(poly(ethylene glycol)) (PEG(2000)Amine) is coated on the surface of the FDA nanocrystals to provide a interface for the antibody coupling. Anti-mouse antibodies are attached to the nanocrystalline FDA biolabels by adsorption. A high molar ratio of fluorescent molecules to biomolecules (2.8 x 10(4)) is achieved in this nanocrystal biolabel system. The analytical performance of the nanocrystal-based label system is evaluated in a model sandwich immunoassay for the detection of mouse IgG. After separation of the nonbound antibody nanocrystal labels, fluorophores are released by hydrolysis and dissolution of the nanocrystalline FDA. Due to the release of the fluorophores (fluoresceins) into a large volume of organic solvent/sodium hydroxide mixture, self-quenching is suppressed. The FDA[DSPE-PEG(2000)Amine]-modified biolabels have a highly stable colloidal suspension with minimized nonspecific interactions. The limit of detection was lowered by a factor of 5-28, and the sensitivity was 400-2700-fold higher compared with a state-of-the-art immunoassay using directly fluorescent-labeled antibodies. Our approach provides high sensitivity and low limits of detection without the need for long incubation times, making it an interesting alternative in biolabel technology.     

Fundamental building blocks for molecular biowire based forward error-correcting biosensors

This paper describes the fabrication, characterization and modeling of fundamental logic gates that can be used for designing biosensors with embedded forward error-correction (FEC). The proposed logic gates (AND and OR) are constructed by patterning antibodies at different spatial locations along the substrate of a lateral flow immunosensor assay. The logic gates operate by converting binding events between an antigen and an antibody into a measurable electrical signal using polyaniline nanowires as the transducer. In this study, B.?cereus and E.?coli have been chosen as model pathogens. The functionality of the AND and OR logic gates has been validated using conductance measurements with different pathogen concentrations. Experimental results show that the change in conductance across the gates can be modeled as a log-linear response with respect to varying pathogen concentration. Equivalent circuits models for AND and OR logic gates have been derived based on measured results.     

Competitive selection from single domain antibody libraries allows isolation of high-affinity antihapten antibodies that are not favored in the llama immune response

Single-domain antibodies (sdAbs) found in camelids lack a light chain, and their antigen-binding site sits completely in the heavy-chain variable domain (VHH). Their simplicity, thermostability, and ease in expression have made VHHs highly attractive. Although this has been successfully exploited for macromolecular antigens, their application to the detection of small molecules is still limited to a very few reports, mostly describing low-affinity VHHs. Using triclocarban (TCC) as a model hapten, we found that conventional antibodies, IgG1 fraction, reacted with free TCC with a higher relative affinity (IC(50) 51.0 ng/mL) than did the sdAbs (IgG2 and IgG3, 497 and 370 ng/mL, respectively). A VHH library was prepared, and by elution of phage with limiting concentrations of TCC and competitive selection of binders, we were able to isolate high-affinity clones, K(D) 0.98-1.37 nM (SPR), which allowed development of a competitive assay for TCC with an IC(50) = 3.5 ng/mL (11 nM). This represents a 100-fold improvement with regard to the performance of the sdAb serum fraction, and it is 100-fold better than the IC(50) attained with other antihapten VHHs reported thus far. Despite the modest overall antihapten sdAbs response in llamas, a small subpopulation of high-affinity VHHs is generated that can be isolated by careful design of the selection process.     

An MILP model and clustering heuristics for LED assembly optimisation on high-speed hybrid pick-and-place machines

This paper deals with a scheduling optimisation problem arising in printed circuit board (PCB) assembly. In one class of PCB assembly, light-emitting diodes are to be assembled into the placement locations on PCBs by a machine with multiple pick-and-place heads. The scheduling optimisation problem is to determine the assembly sequence of placement locations and the assignment of pick-and-place heads for locations so as to minimise the assembly time. We formulate it as a mixed integer linear programming model. To solve the problem efficiently, we classify the PCBs into two types. For the first type of PCBs, on which the locations are linearly arranged, a constructive heuristic is proposed based on the analysis of the best next location after a location is assembled. For the second type of PCBs, on which the locations are circularly arranged, a heuristic based on clustering strategy and path relinking method is proposed. Computational experiments show that the solutions obtained by the two heuristics make 2.32 and 6.82% improvements averagely for the PCBs with linearly and circularly arranged locations, respectively, as compared to the solutions used in real production, and they are also better than those obtained by a hybrid genetic algorithm.     

High-performance immunoaffinity chromatography and chemiluminescent detection in the automation of a parathyroid hormone sandwich immunoassay

An automated sandwich immunoassay was developed based on high-performance immunoaffinity chromatography and chemiluminescent detection, using the determination of parathyroid hormone (PTH) in plasma as a model system. In this method, injections of plasma and acridinium ester-labeled anti-(1-34 PTH) antibodies were made onto a column containing immobilized anti-(44-68 PTH) antibodies. Upon elution, PTH and its associated labeled antibody were combined with an alkaline peroxide postcolumn reagent, and the resulting light production was measured. Factors considered in optimizing this system included the column's dissociation properties, the rate of light production in the postcolumn reactor, and the use of sequential vs simultaneous injection of sample and labeled antibody. The final system developed required 6 min per plasma injection, following a 1-h incubation of sample with labeled antibody. The response was linear over 2-3 orders of magnitude and the lower limit of detection for a 66-microL plasma sample was only 16 amol, or 2.4 x 10(-13) M. Overall, this method had a precision and response similar to those of manual PTH methods but required 24-fold less time to perform. By using different immobilized and labeled antibodies, this method could easily be adapted for use with other analytes.     

Detection of antigens in biologically complex fluids with photografted whole antibodies

A highly sensitive (pM), efficient (t < 20 min) detection assay was developed by designing surfaces with grafted antibodies. Through this approach, a short half-life antigen, glucagon, was rapidly detected in a biologically complex plasma/blood environment. Tailoring of graft composition eliminates the need for time-consuming blocking steps, significantly reducing antigen-antibody incubation times, while maintaining antibody specificity and activity toward target antigen. Grafted antibodies were bound through solvated, mobile polymer chains, thereby circumventing problems associated with antibody accessibility, analyte diffusion, and steric limitations. The efficiency of this assay is provided through grafting synthesized, acrylated antibodies in the presence of PEG monoacrylate. This procedure eliminates the need for blocking steps, due to a decrease in nonspecific protein interactions. These polymerizable antibodies were tethered with a range of densities while retaining biological activity. Moreover, biological activity of acrylated antibodies was compared to that of unmodified antibodies and remained comparable. The acrylated antibodies were grafted from substrate surfaces using controlled radical photopolymerization, maintaining the advantages of classical antibody immobilization techniques while providing improved detection. Through integrating this antibody conjugation chemistry and immunoassay approach with photolithographic techniques, construction of spatial patterns on a microfluidic device was demonstrated for efficient, parallel screening of multiple antigens.     

Cross-linking of 17 beta-estradiol to monoclonal antibodies by direct UV irradiation: application to an enzyme immunometric assay

Ultraviolet irradiation was used to cross-link 17 beta-estradiol directly to monoclonal anti-17 beta-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradiation energy and wave-length. The best results were obtained at 254 (10 J/cm2, 45-min irradiation) and 312 nm (40 J/cm2, 160-min irradiation). The irradiation fully denatured both individual molecules (i.e., 17 beta-estradiol and monoclonal anti-17 beta-estradiol antibody), but 17 beta-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17 beta-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has considerable potential. We used this original approach to develop a new enzyme immunometric assay of 17 beta-estradiol based on our previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which uses chemical agents to cross-link haptens via amino groups to specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensitive (detection limit of 38 pg/mL in human serum). It correlated well with two competitive commercial immunoassays when tested on 40 human sera.     

Detection methodology based on target molecule-induced sequence-specific binding to a single-stranded oligonucleotide

We have recently developed a mix-and-read format homogeneous antigen peptide based assay for detection of the antibodies (Tian, L.; Heyduk, T. Anal. Chem. 2009, 81, 5218-5225) that employed for target detection a simple biophysical mechanism of target antibody induced annealing between two complementary oligonucleotides attached to the antigen peptide. In this work, we propose and experimentally validate an alternative variant of this assay format in which target antibody binding to antigen peptide-oligonucleotide conjugate produces a complex with high sequence-specific binding affinity to a single-stranded capture oligonucleotide. This new assay format can be used for preparing various solid-surface based assays by immobilizing the capture oligonucleotide. This assay design is not limited to antibody detection. We demonstrate that it can also be employed for detecting proteins or pathogenic bacteria using oligonucleotide-labeled antibodies as target recognition elements. Preparation of these solid-surface based assays is simplified because all interactions with the solid surfaces are mediated by well-understood oligonucleotide-oligonucleotide interactions and because of the relative ease of immobilizing oligonucleotides on various solid surfaces. These unique aspects of the assay design also allow microarray-style multiplexing that could be most useful for multiplexed antibody profiling for diagnosis and analysis of cancer, autoimmune, and infectious diseases.     

Multiple fluorescence labeling with europium chelators. Application to time-resolved fluoroimmunoassays

Multiple fluorescence labeling with conventional probes like fluorescein, to improve the detection limit of labeled reactants, is not usually successful because of fluorescence quenching. In contrast, we found that the europium chelator 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA) can be incorporated into proteins at very high molar ratios. Working with thyroglobulin as a model protein, we found that when 160 BCPDA molecules are incorporated into one thyroglobulin molecule, the fluorescence emitted by the labeled protein in the presence of excess Eu3+, is equivalent to that emitted by approximately 900 molecules of unconjugated BCPDA:Eu3+ complexes. We took advantage of the lack of any quenching effects and of the enhancement observed with the multiply labeled protein, to develop a universal reagent system consisting of (a) streptavidin covalently coupled to BCPDA labeled thyroglobulin and (b) excess Eu3+. With this approach, streptavidin is heavily labeled through thyroglobulin and retains its full biotin binding activity. We used the reagent to develop a highly sensitive time-resolved heterogeneous immunofluorometric assay of alpha-fetoprotein (AFP) in serum, using monoclonal antibodies. One antibody is immobilized in white microtitration wells (solid-phase) and the other is biotinylated. We demonstrate that this assay, using the newly developed reagent, is 25-fold more sensitive than the one using directly BCPDA labeled antibody and 5-fold more sensitive than an assay that uses BCPDA-labeled streptavidin. The detection limit of the assay with the new reagent was down to 60 amol of AFP per well. We conclude that multiple fluorescence labeling with europium chelators is an effective method of extending the sensitivity of currently used fluorescence immunoassay procedures.