Multiplexed method to calibrate and quantitate fluorescence signal for allergen-specific IgE

Using a microarray platform for allergy diagnosis allows for testing of specific IgE sensitivity to a multitude of allergens, while requiring only small volumes of serum. However, variation of probe immobilization on microarrays hinders the ability to make quantitative, assertive, and statistically relevant conclusions necessary in immunodiagnostics. To address this problem, we have developed a calibrated, inexpensive, multiplexed, and rapid protein microarray method that directly correlates surface probe density to captured labeled secondary antibody in clinical samples. We have identified three major technological advantages of our calibrated fluorescence enhancement (CaFE) technique: (i) a significant increase in fluorescence emission over a broad range of fluorophores on a layered substrate optimized specifically for fluorescence; (ii) a method to perform label-free quantification of the probes in each spot while maintaining fluorescence enhancement for a particular fluorophore; and (iii) a calibrated, quantitative technique that combines fluorescence and label-free modalities to accurately measure probe density and bound target for a variety of antibody-antigen pairs. In this paper, we establish the effectiveness of the CaFE method by presenting the strong linear dependence of the amount of bound protein to the resulting fluorescence signal of secondary antibody for IgG, β-lactoglobulin, and allergen-specific IgEs to Ara h 1 (peanut major allergen) and Phl p 1 (timothy grass major allergen) in human serum.     

Magnetophoretic immunoassay of allergen-specific IgE in an enhanced magnetic field gradient

We demonstrate a novel magnetophoretic immunoassay of allergen-specific immunoglobulin E (IgE) based on the magnetophoretic deflection velocity of a microbead that is proportional to the associated magnetic nanoparticles under enhanced magnetic field gradient in a microchannel. In this detection scheme, two types of house dust mites, Dermatophagoides farinae (D. farinae) and Dermatophagoides pteronyssinus (D. pteronyssinus), were used as the model allergens. Polystyrene microbeads were conjugated with each of the mite extracts followed by incubation with serum samples. The resulting mixture was then reacted with magnetic nanoparticle-conjugated anti-human IgE for detection of allergen-specific IgE by using sandwich immuno-reactions. A ferromagnetic microstructure combined with a permanent magnet was employed to increase the magnetic field gradient ( approximately 10(4) T/m) in a microfluidic device. The magnetophoretic velocities of microbeads were measured in a microchannel under applied magnetic field, and the averaged velocity was well correlated with the concentration of allergen-specific IgE in serum. From the analysis of pooled sera obtained from 44 patients, the detection limits of the allergen-specific human IgEs for D. farinae and D. pteronyssinus were determined to be 565 (0.045 IU/mL) and 268 fM (0.021 IU/mL), respectively. These values are 1 order of magnitude lower than those by a conventional CAP system. For evaluation of reproducibility and accuracy, unknown sera were subjected to a blind test by using the developed assay system, and they were compared with the CAP system. As a result, coefficient of variance was less than 10%, and the developed method enabled a fast assay with a tiny amount of serum ( approximately 10 microL).     

High-throughput solution-based medicinal library screening against human serum albumin

High-throughput screening of combinatorial libraries has evolved from studying large diverse libraries to analyzing small, structurally similar, focused libraries. This paradigm shift has generated a need for rapid screening technologies to screen both diverse and focused libraries in a simple, efficient, and inexpensive manner. We have proactively addressed these needs by developing a high-throughput, solution-based method combining size exclusion (SEC), two-dimensional liquid chromatography (2-D LC), and mass spectrometry (MS) for determining the relative binding of drug candidates in small, focused medicinal libraries against human serum albumin (HSA). Two types of libraries were used to evaluate the performance of the system. The first consisted of five diverse ligands with a wide range of hydrophobicities and whose association constants to HSA cover 3 orders of magnitude. A beta-lactam library composed of structurally similar compounds was used to further confirm the validity of the methodology. The ability to distinguish site-specific interactions of drugs competing for individual domains of the HSA receptor is also demonstrated. Comparison of chromatographic profiles of the library components before and after incubation with the receptor using multiple reaction monitoring allowed a ranking of the ligands according to their relative binding affinities. The observed rankings correlate closely with literature values of the association constants between the respective ligands and HSA. This simple, rugged methodology can screen a wide spectrum of chemical entities from combinatorial mixtures in less than 6 min.     

A screening method for antigen-specific IgE using mast cells based on intracellular calcium signaling

A simple screening method is presented for the measurement of antigen-specific IgEs in sera in which mast cells are used. This method is based on the intracellular calcium signal in mast cells induced by cross-linking the surface high-affinity Fc receptors (FcepsilonRIs) with IgEs and multivalent antigens. When a serum containing various antigen-specific IgEs is added to the mast cell suspension, various antigen-specific IgEs are captured by FcepsilonRIs on the cell surface. However, the required antigen-specific IgE can be specifically detected after the addition of the corresponding antigen. The resulting increase in intracellular calcium concentration ([Ca2+]i), monitored by Ca2+-fluorometry, was found to be an analytical measure for the screening of IgEs. Two kinds of rodent mast cells, cell-lined RBL2H3 cells and primary cultured BMMCs, were used as a representative model system of mast cells. A DNP hapten (DNP35-HSA) and ovalbumin (OVA) were chosen for illustrative antigens, and these antigen-specific IgEs (DNP-specific IgE, OVA-specific IgE) in the corresponding rodent sera were target antibodies. It was found that [Ca2+]i increased linearly with IgE concentrations ranging from 25 to 5000 ng/mL for DNP-specific IgE and from 5 to 50 ng/mL for OVA-specific IgE. For these dynamic ranges, optimum concentrations of antigens were found to be 10 ng/mL and 1 microg/mL for DNP35-HSA and OVA, respectively. It was concluded that by monitoring the increase of [Ca2+]i in mast cells, we could determine the antigen-specific IgEs. The present immunological assay based on the Ca2+ signal transduction in mast cells offers new possibilities for efficient screening of antigen-specific IgEs and the immunogenicity of IgE in sera.     

In vitro selection of high-affinity DNA ligands for human IgE using capillary electrophoresis

Aptamers with high affinity for IgE were selected using capillary electrophoresis to demonstrate the compatibility of this technique with SELEX. The high selectivity and efficiency of CE gave rise to a very high rate of enrichment, allowing high-affinity, high-selectivity aptamers to be obtained in only four rounds of selection. Decreasing the number of rounds shortens the selection procedure from the 4-6 weeks typical of SELEX to several days. The use of "bulk" dissociation constant measurements was introduced as a method for assessing the DNA pool after each round of selection. The average dissociation constant of the sequences in the DNA pool for IgE after four rounds of selection was 29 nM. The distribution of the dissociation constants for the sequences in the pool was very narrow with a standard deviation of only 6 nM. All of the sequences assessed exhibited high specificity for human IgE when compared with human IgG or mouse IgE.     

Magnetic beads based immunoaffinity capillary electrophoresis of total serum IgE with laser-induced fluorescence detection

A magnetic beads based immunoaffinity capillary electrophoresis method for total Immunoglobulin E quantification in serum has been developed. The method combines speed, automation ability, and minimal sample consumption. Only 1 microL of serum is required while the whole immunoaffinity capillary electrophoresis method is performed in less than 50 min. The concomitant use of online immunocapture, transient isotachophoresis, and laser-induced fluorescence detection provides a sensitivity in the low picomolar range and a highly linear fluorescence response over 4 orders of magnitude (IgE concentration ranging from 2.4 to 2400 ng/mL). After validation with a reference material, the method has been successfully applied to the quantification of total IgEs in patient sera. The results compared well with classical ImmunoCap data.     

Apta-biosensors for nonlabeled real time detection of human IgE based on carbon nanotube field effect transistors

In this study, we demonstrated the aptamer-based biosensor (apta-biosensor) using CNT-FET devices for label free detection of allergy diagnosis by IgE detection. In order to detect the IgE, two kinds of receptor (monoclonal IgE antibody and anti-IgE aptamer)-modified CNT-FET devices were fabricated. The binding event of the target IgE onto receptors was detected by monitoring the gating effect caused by the charges of the target proteins. Since the CNT-FET biosensors were used in buffer solution, it was crucial to use small-size receptors like aptamers than whole antibodies so that the charged target IgE could approach the CNT surface within the Debye length distance to give a large gating effect. The results show that CNT-FET biosensors using monoclonal IgE antibody had very low sensitivity (minimum detectable level 1000 ng/mL), while those based on anti-IgE aptamer could detect 50 ng/mL. Moreover, the aptamer-modified CNT-FET herein could successfully block non-target proteins and could selectively detect the target protein in an environment similar to human serum electrolyte. Therefore, aptamer-based CNT-FET devices enable the production of label-free ultrasensitive electronic biosensors to detect clinically important biomarkers for disease diagnosis.     

Aptamer biosensors for label-free colorimetric detection of human IgE based on polydiacetylene (PDA) supramolecules

In this study, we demonstrate an aptamer-based biosensor (apta-biosensor) using PDA liposomes for label-free detection of allergy diagnosis by hIgE detection. In order to detect the target hIgE, the surface of PDA liposome were functionalized with hIgE antibody and anti-hIgE aptamer as a receptor, and the target hIgE onto the receptors was detected by the change of fluorescence signal. The hIgE antibody-modified PDA liposome biosensor had a serious problem that the immune reaction between receptor and target could not powerfully affect the change of florescence signal on PDA liposome. In order to solve this problem, the anti-hIgE aptamer which was far smaller than whole antibody was introduced as the receptor for the PDA liposome system. An aptamer-based PDA liposome biosensor was able to measure a quantity of target protein with various concentrations and at this time the detection limit was 141 ng/mL of the hIgE concentration. These results enabled diagnosis of allergy disease by an aptamer-based PDA liposome biosensor because real allergic patients showed high concentration of hIgE in serum (greater than 290 ng/mL). Therefore, we suggest that aptamer-modified PDA supramolecules as promising candidates for development of label-free colorimetric biosensors.     

Single-molecule detection of surface-hybridized human papilloma virus DNA for quantitative clinical screening

We present an improved method to quantify viral DNA in human cells at the single- molecule level. Human papilloma virus (HPV)-16 DNA was hybridized to probes that were covalently bound to a glass surface and detected with a single-molecule imaging system. In the single-probe mode, the whole genome and target DNA were fluorescently labeled before hybridization. In the dual-probe mode, a second probe was introduced that has a fluorescently labeled 1-kb DNA strand connected to the 50-nt probe sequence. With the single-probe method, the detection limit was 0.7 copy/cell, which was similar to that reported in a flow system earlier. With the dual-probe method, the linear dynamic range covers 1.44-7000 copies/cell, which is typical of early infection to near-cancer stages. Both methods were applied to cell line samples with known HPV-16 infection, and the result showed a good match with the reported viral load. DNA from cervical cells, collected with the Pap smear sampling method, was spiked with HPV-16 DNA and submitted to this assay to show compatibility with conventional sampling methods. The dual-probe method was further tested with a crudely prepared sample. The cells were heat lyzed and spun down, and the supernatant was immediately submitted to hybridization. Even with reduced hybridization efficiency caused by the interference of cellular materials, we were still able to differentiate infected cells with 600 copies/cell from healthy cells.     

Microarrays assembled in microfluidic chips fabricated from poly(methyl methacrylate) for the detection of low-abundant DNA mutations

Low-density arrays were assembled into microfluidic channels hot-embossed in poly(methyl methacrylate) (PMMA) to allow the detection of low-abundant mutations in gene fragments (K-ras) that carry point mutations with high diagnostic value for colorectal cancers. Following spotting, the chip was assembled with a cover plate and the array accessed using microfluidics in order to enhance the kinetics associated with hybridization. The array was configured with zip code sequences (24-mers) that were complementary to sequences present on the target. The hybridization targets were generated using an allele-specific ligase detection reaction (LDR), in which two primers (discriminating primer that carriers the complement base to the mutation being interrogated and a common primer) that flank the point mutation and were ligated joined together) only when the particular mutation was present in the genomic DNA. The discriminating primer contained on its 5'-end the zip code complement (directs the LDR product to the appropriate site of the array), and the common primer carried on its 3' end a fluorescent dye (near-IR dye IRD-800). The coupling chemistry (5'-amine-containing oligonucleotide tethered to PMMA surface) was optimized to maximize the loading level of the zip code oligonucleotide, improve hybridization sensitivity (detection of low-abundant mutant DNAs in high copy numbers of normal sequences), and increase the stability of the linkage chemistry to permit re-interrogation of the array. It was found that microfluidic addressing of the array reduced the hybridization time from 3 h for a conventional array to less than 1 min. In addition, the coupling chemistry allowed reuse of the array > 12 times before noticing significant loss of hybridization signal. The array was used to detect a point mutation in a K-ras oncogene at a level of 1 mutant DNA in 10,000 wild-type sequences.     

Multiwalled carbon nanotube-based bi-enzyme electrode for total cholesterol estimation in human serum

This study aims at fabricating multiwalled carbon nanotubes (MWCNTs) based enzymatic bioelectrode for total cholesterol estimation in human serum. For this purpose, a gold (Au) electrode was modified with MWCNTs uniformly dispersed in nafion (Nf) matrix. Cholesterol oxidase (ChOx) and cholesterol esterase (ChEt) were immobilised onto this Nf–MWCNTs film-modified Au electrode using layer-by-layer technique to fabricate the final bioelectrodes. The immobilisation of ChOx and ChEt onto the electrodes was demonstrated by scanning electron microscopy. The fabricated bioelectrodes were electrochemically characterised using cyclic voltammetry. The bioelectrodes offer reliable response characteristics towards cholesterol and stable electrochemical properties in terms of extended linear response range of 0.080–0.950?mM, detection limit up to 0.01?mM and optimum storage stability up to three weeks. Experimental results reveal that the fabricated bioelectrode offer optimum repeatability and reproducibility towards the cholesterol estimation and can also efficiently exclude interference by the commonly coexisting ascorbic acid, uric acid, lactic acid, glucose and urea, which is favourable for its efficient use in the highly selective analysis of total cholesterol in serum samples.     

Surface-enhanced Raman scattering based approach for quantitative determination of creatinine in human serum

A novel surface-enhanced Raman scattering (SERS) based approach for the quantitative determination of creatinine in human serum is described. Using isotopically labeled (2-13C, 2,3-15N2) creatinine as internal standard, SERS acquires the character of a ratio method that works similar to the well-established isotope dilution techniques. In conjunction with multivariate data analysis, the method was successfully applied for quantifying creatinine at clinically relevant levels and below. A partial least-squares regression model was generated from a set of 87 calibration spectra covering the full range of mole fractions of neat creatinine. The prediction performance of the model was thereafter validated with independent reference samples giving a standard deviation of less than 2%. Finally, a conditioning procedure to prepare real serum samples for SERS-based creatinine analysis was worked out and validated. Measured serum creatinine concentrations are within 3% of the values obtained from gas chromatography/isotope dilution mass spectrometry on the same serum starting material.     

Canine cancer screening via ultraviolet absorbance and fluorescence spectroscopy of serum proteins

A cost-effective optical cancer screening and monitoring technique was demonstrated in a pilot study of canine serum samples and was patented for commercialization. Compared to conventional blood chemistry analysis methods, more accurate estimations of the concentrations of albumin, globulins, and hemoglobin in serum were obtained by fitting the near UV absorbance and photoluminescence spectra of diluted serum as a linear combination of component reference spectra. Tracking these serum proteins over the course of treatment helped to monitor patient immune response to carcinoma and therapy. For cancer screening, 70% of dogs with clinical presentation of cancer displayed suppressed serum hemoglobin levels (below 20 mg/dL) in combination with atypical serum protein compositions, that is, albumin levels outside of a safe range (from 4 to 8 g/dL) and globulin levels above or below a more normal range (from 1.7 to 3.7 g/dL). Of the dogs that met these criteria, only 20% were given a false positive label by this cancer screening test.     

Development and validation of a one-step immunoassay for determination of cadmium in human serum.

A sensitive and simple one-step immunoassay was developed and validated for quantitative determination of Cd(II) in human serum. In this method, a monoclonal antibody that recognizes Cd(II)-EDTA complexes was directly immobilized onto microwell plates. The serum sample containing metallothionein(MT)-bound and non-MT-bound Cd(II) was acidified to displace the Cd(II) from MT. The sample was then treated with metal-free EDTA to convert Cd(II) to Cd(II)-EDTA complexes. A mixture of Cd(II)-EDTA complexes derived from serum samples and Cd(II)-EDTA conjugated with peroxidase enzyme was incubated in the wells to compete for binding sites of the immobilized antibody. After addition of peroxidase substrate, the bound fraction of the enzyme conjugate was measured by a microplate reader, and the signal was inversely proportional to the concentration of the Cd(II) in the sample. The assay limit of detection was 0.24 microg/L, and the effective working range at coefficient of variation of < or = 10% was 0.24-100 microg/L. Analytical recovery of spiked Cd(II), in the concentration range between 0.8 and 50 microg/L, was 97.8 +/- 4.0%. The assay was selective for Cd(II); other metal ions (Mn, Co, Cu, Zn, Mg, Hg, Ca, Ni, Fe, and Pb), tested at concentrations considerably higher than those present in human serum, did not significantly interfere with the assay. The assay results correlated well with those obtained by graphite furnace atomic absorption spectrometry (r = 0.984).     

Microfluidic capillary system for immunoaffinity separations of C-reactive protein in human serum and cerebrospinal fluid

A miniaturized system based on microfluidic capillaries is presented for point-of-care testing and clinical assessment. The approach relies on microsyringe pump-generated flow to deliver reagents and immunoaffinity chromatography to isolate the antigen from biological matrixes. Capillary sandwich immunoassays for C-reactive protein (CRP) were demonstrated in human serum and cerebrospinal fluid (CSF), which are relevant matrixes for cardiovascular disease risk and meningitis research, respectively. Capillaries packed with antibody-coated silica beads were used to capture CRP from the matrix and a second, dye-labeled antibody was introduced to form a sandwich complex. An acidic elution buffer dissociated the antibody-antigen complexes, and the labeled antibody was detected with diode laser-induced fluorescence. Four parameter logistic functions and % relative error plots were used to model and assess the data. The calibration ranges for CRP were 0.05-3.0 microg/mL in 1:10 diluted serum and 0.01-30 microg/mL in undiluted CSF. The microfluidic apparatus employed a flow rate of 2 microL/min and a sample injection volume of 250 nL. Since it was not necessary to reach antibody-antigen reaction equilibrium and the assay platform dimensions were minimal, run times were as short as 10 min.     

Development and certification of a standard reference material for vitamin D metabolites in human serum

The National Institute of Standards and Technology (NIST), in collaboration with the National Institutes of Health's Office of Dietary Supplements (NIH-ODS), has developed a Standard Reference Material (SRM) for the determination of 25-hydroxyvitamin D [25(OH)D] in serum. SRM 972 Vitamin D in Human Serum consists of four serum pools with different levels of vitamin D metabolites and has certified and reference values for 25(OH)D(2), 25(OH)D(3), and 3-epi-25(OH)D(3). Value assignment of this SRM was accomplished using a combination of three isotope-dilution mass spectrometry approaches, with measurements performed at NIST and at the Centers for Disease Control and Prevention (CDC). Chromatographic resolution of the 3-epimer of 25(OH)D(3) proved to be essential for accurate determination of the metabolites.     

Automated Formation of Lipid Membrane Microarrays for Ionic Single‐Molecule Sensing with Protein Nanopores

Efficient use of membrane protein nanopores in ionic single‐molecule sensing requires technology for the reliable formation of suspended molecular membranes densely arrayed in formats that allow high‐resolution electrical recording. Here, automated formation of bimolecular lipid layers is shown using a simple process where a poly(tetrafluoroethylene)‐coated magnetic bar is remotely actuated to perform a turning motion, thereby spreading phospholipid in organic solvent on a nonpolar surface containing a <1 mm² 4 × 4 array of apertures with embedded microelectrodes (microelectrode cavity array). Parallel and high‐resolution single‐molecule detection by single nanopores is demonstrated on the resulting bilayer arrays, which are shown to form by a classical but very rapid self‐assembly process. The technique provides a robust and scalable solution for the problem of reliable, automated formation of multiple independent lipid bilayers in a dense microarray format, while preserving the favorable electrical properties of the microelectrode cavity array.     

Application of solid-phase microextraction in the determination of diazepam binding to human serum albumin

In this paper, protein-drug interactions were studied by solid-phase microextraction (SPME) using diazepam binding to human serum albumin as a model system. Since drug compounds are normally polar and nonvolatile by nature, direct SPME is used in this work. The SPME extraction is an equilibrium process among the concentrations of the analyte partitioned onto the SPME fiber, free and bound drug in the solution. A calibration curve was first constructed by employing the amount of the analytes partitioned on the fiber versus the free analyte concentration in the solution in the absence of protein. In method I, the extraction was performed in the protein solution with known diazepam concentration. In method II, diazepam was first loaded onto the fiber by extracting in solution with known diazepam concentration. This fiber was subsequently transferred into the protein solution for desorption. The amount of the analyte left on the fiber was analyzed after the system reached equilibrium. The free drug concentration was then obtained from the calibration curve for both methods. The Scatchard plot was finally employed to obtain the number of binding sites and the equilibrium binding constants. Since only a very small amount of the protein solution is required (150 microL for each extraction), method II is very useful for circumstances where the protein amount is very limited. The direct measurement method proposed in this paper does not need a GC response factor, which significantly decreases the experimental error. The only measurement needed is the area count change (ratio) of the fiber injections before and after the protein was introduced into the solution. The difference between the direct measurement method for method I and method II is discussed. The result illustrated that the SPME direct measurement method provided both theoretical accuracy and simplicity in such applications.