Design of a compact disk-like microfluidic platform for enzyme-linked immunosorbent assay

This paper presents an integrated microfluidic device on a compact disk (CD) that performs an enzyme-linked immunosorbent assay (ELISA) for rat IgG from a hybridoma cell culture. Centrifugal and capillary forces were used to control the flow sequence of different solutions involved in the ELISA process. The microfluidic device was fabricated on a plastic CD. Each step of the ELISA process was carried out automatically by controlling the rotation speed of the CD. The work on analysis of rat IgG from hybridoma culture showed that the microchip-based ELISA has the same detection range as the conventional method on the 96-well microtiter plate but has advantages such as less reagent consumption and shorter assay time over the conventional method.     

Enzyme-linked immunosorbent assay for monitoring toxic dioxin congeners in milk based on a newly generated monoclonal anti-dioxin antibody

To develop an enzyme-linked immunosorbent assay (ELISA) for monitoring the toxicity due to polychlorinated dibenzo-p-dioxins and dibenzofurans contaminated in human breast milk, we have generated novel monoclonal antibodies using some haptenic derivatives linked to bovine serum albumin via the C-1 or C-2 position on the dioxin skeleton. BALB/c or A/J mice were repeatedly immunized with the immunogen, and spleen cells were fused with P3/NS1/1-Ag4-1 myeloma cells. After five fusion experiments, a hybridoma clone was established that secretes an antibody D9-36 group specifically recognizing the major toxic congeners, 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD), 1,2,3,7,8-pentachlorodibenzo-p-dioxin, and 2,3,4,7,8-pentachlorodibenzofran. An ELISA is developed on the basis of the competitive and labeled-antigen format. The toxic congeners extracted from butter or milk specimens by a novel extraction cartridge and a peroxidase-labeled dioxin analogue were sequentially reacted with a fixed amount of D9-36 in the presence of Triton X-100. The bound fraction was captured on a microtiter plate, immobilizing a second antibody, and the enzyme activity was colorimetrically determined. This ELISA afforded a practical sensitivity (measurable range, 1-100 pg/assay; detection limit, 1.0 pg/assay as 2,3,7,8-TCDD equivalent). The assay values for milk and butter samples were in reasonable accordance with the sum of the toxicity-equivalent quantity of each congener, which had been determined by a high-resolution gas chromatography/high-resolution mass spectrometry method.     

Comparison of an enzyme-linked immunosorbent assay and a gas chromatographic procedure for the determination of molinate residues

An enzyme-linked immunosorbent assay (ELISA) was compared to a gas chromatographic method for the analysis of the thiocarbamate herbicide molinate (S-ethyl hexahydroazepine-1-carbothioate). Apparent recoveries from water spiked at 1 ppb to 1 ppm levels were comparable when liquid-liquid extraction was used. Solid-phase extraction was also examined and apparent recoveries by both ELISA and gas chromatography (GC) were comparable to each other as well as to the liquid-liquid extraction method. Methanol, acetonitrile, and ethyl acetate were equally effective in eluting molinate from solid-phase columns. An excellent correlation was obtained between the ELISA and GC method for field-treated water samples extracted by using the solid phase method and either ethyl acetate or methanol as the eluting solvent. Air and soil samples from this same study correlated well when analyzed by ELISA or GC, but ELISA results for soil were generally higher than GC data and of slightly lower precision than GC. Tests with a coated plate, pipettors, and the plate reader amounted to 8.0% error, the majority of which was attributable to the coating antigen binding and to antigen-antibody reactions.     

Enzyme-linked immunosorbent assay for glycyrrhizin using anti-glycyrrhizin monoclonal antibody and an eastern blotting technique for glucuronides of glycyrrhetic acid.

Hybridomas secreting a monoclonal antibody against glycyrrhizin were produced by fusing splenocytes from a mouse immunized against a glycyrrhizin-bovine serum albumin conjugate with the hypoxanthine-aminopterin-thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. A very weak cross-reaction with glycyrrhetinic acid monoglucuronide and glycyrrhetic acid occurred. An enzyme-linked immunosorbent assay (ELISA) that had an effective measuring range of 20 -200 ng/mL of glycyrrhizin was established using this monoclonal antibody. In addition, a method named eastern blotting for the detection of glycyrrhizin was investigated. In this method, we developed a new way to separate the glycyrrhizin molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups on the protein and covalently bound to the adsorbent membrane. The monoclonal antibody bound to the aglycone part of the glycyrrhizin molecule for immunostaining. This method was validated by immunocytolocalization of glycyrrhizin in fresh Glycyrrhiza root.     

Quantitative enzyme-linked immunosorbent assay for determination of polychlorinated biphenyls in environmental soil and sediment samples

An enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of Aroclors 1242, 1248, 1254, and 1260 in soil and sediments was developed and its performance compared with that of gas chromatography (GC). The detection limits for Aroclors 1242 and 1248 in soil are 10.5 and 9 ng/g, respectively. The assay linear dynamic range is 50-1333 ng/g. Cross-reactivity of the assay with 37 structurally related potential cocontaminants in environmental soil samples was examined; none of the chlorinated anisoles, benzenes, or phenols exhibited >3% cross-reactivity, with <0.1% cross-reactivity being the norm. Soil spike recoveries of 107% and 104% were obtained for Aroclors 1242 and 1248, respectively, for a spike level of 5 mg/kg, with corresponding relative standard deviations of 14% and 17%. One hundred forty-eight environmental soil, sediment, and paper pulp samples, obtained from two EPA listed Superfund sites, were analyzed by ELISA and standard GC methods. Samples were extracted for ELISA analysis by shaking with methanol. Additional extractions of the same samples were performed either with supercritical carbon dioxide or by Soxhlet extraction with methanol. ELISA results for both the supercritical fluid and the Soxhlet extracts were in close agreement with the GC results, while the ELISA results for the methanol shake extracts were not. The data for the environmental samples demonstrated the capability of the ELISA to provide accurate results and reinforced the dependence of any detection method, including ELISA, on appropriate extraction procedures.     

Validation of accuracy of enzyme-linked immunosorbent assay in hybridoma screening and proposal of an improved screening method

The 96-well plate format of enzyme-linked immunosorbent assay (ELISA) is the de facto standard in screening hybridomas for active antibody. Despite its widespread use, there have been few or no systematic attempts to validate its accuracy and answer the fundamental question, is it finding all the positives? We report here on a comparison between ELISA and a semiautomated flow-based kinetic exclusion assay (KinExA), both used in screening the same hybridoma cell line. Our finding is that ELISA is both overreporting (false positives) and underreporting (false negatives) compared to the KinExA system. The large number of hybridoma cells (e.g., cultured in six 96-well plates) that must be checked is daunting in considering any method other than ELISA for routine screening. To overcome this, we devised a sampling strategy in which wells are combined in a specified pattern, allowing a significant reduction in the total number of measurements required.     

Synthesis of carboxyl-capped and bright YVO4:Eu,Bi nanoparticles and their applications in immunochromatographic test strip assay

Carboxyl-capped YVO₄:Eu,Bi nanoparticles with average diameter of ∼10 nm were synthesized via a copolymer of phosphono and carboxylic acid mediated hydrothermal method. Under a 350 nm ultraviolet light excitation, the YVO₄:Eu,Bi NPs exhibit sharp and bright red emission peaked at 615 nm and with highest quantum yield of ∼43%. Furthermore, the nanoparticles show good water/buffer stability and feasible bioconjugation benefiting from the carboxylic groups on their surface. Based on these kind optical and surface properties of the YVO₄:Eu,Bi nanoparticles, an immunochromatographic test strip assay for quantitative determination of human IgG was achieved. This protocol can be extended to other rare-earth nanoparticles with the purpose of developing bioprobes for desired applications.     

Energy transfer-based multiplexed assay of proteases by using gold nanoparticle and quantum dot conjugates on a surface

Rapid and sensitive assay of proteases and their inhibition in a high-throughput manner is of great significance in the diagnostic and pharmaceutical fields. We developed a multiplexed assay system of proteases and their inhibition by measuring the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) on a glass slide. In this system, while the photoluminescence (PL) of donor QDs immobilized on a surface was quenched due to the presence of AuNPs (energy acceptor) in close proximity, the protease activity caused modulation in the efficiency of the energy transfer between the acceptor and donor, thus enabling the protease assay. In comparison to the QD-dye system, the conjugate of the QD-AuNP gave rise to higher energy transfer efficiency, resulting in quantitative assay of proteases with more sensitivity. When matrix metalloproteinase, caspase, and thrombin were tested, a multiplexed assay was successfully achieved since the AuNP could be used as a common energy acceptor in conjunction with QDs having different colors. Our system is anticipated to find applications in the diagnosis of protease-related diseases and screening of potential drugs with high sensitivity in a high-throughput way.     

Development of zeptomole and attomolar detection sensitivity of biotin-peptide using a dot-blot gold nanoparticle immunoassay

An ultrasensitive, simple, and fast immunoassay for biotin-peptide detection using gold nanoparticles conjugated with antibodies has been developed. Biotin was covalently attached to a peptide and the biotin-peptide bound on a nitrocellulose membrane. Antibody-coated gold nanoparticles bound to the biotin-peptide formed red dots. With this method, 100 amol of the biotin-peptide was detected and no immunogold was bound to the membrane in the absence of biotin. The relative intensity of each dot was scored using Quantity One, a quantitative analysis software program. The linear working range of this assay was between 1 pmol and 1 micromol. The assay sensitivity was increased by silver enhancement to 100 zmol, and the linear working range was between 100 zmol and 100 fmol. This assay can be extended to detect target molecules, such as dioxin, digoxin, mercury, and so on, with matched antibodies and has potential broad applications in immunoassay.     

Development of an enzyme-linked immunosorbent assay for the determination of the linear alkylbenzene sulfonates and long-chain sulfophenyl carboxylates using antibodies generated by pseudoheterologous immunization

ELISA methods have been developed for screening contamination of water resources by linear alkyl benzene sulfonates (LAS) or the most immediate degradation products, the long chain sulfophenyl carboxylates, SPCs. The assay uses antibodies raised through pseudoheterologous immunization strategies using an equimolar mixture of two immunogens (SFA-KLH and 13C(13)-SPC-KLH) prepared by coupling N-(4-alkylphenyl)sulfonyl-3-aminopropanoic acid (SFA) and p-(1-carboxy-13-tridecyl)phenylsulfonic acid (13C(13)-SPC) to keyhole limpet hemocyanin (KLH). The immunizing haptens have been designed to address recognition versus two different epitopes of the molecule. The SFA hapten maximizes recognition of the alkyl moiety while preserving the complexity of the different alkyl chains present in the LAS technical mixture. The 13C(13)-SPC hapten addresses recognition of the common and highly antigenic phenylsulfonic group. The antisera raised using this strategy have been shown to be superior to those obtained through homologous immunization procedures using a single substance. By using an indirect ELISA format, LAS and long-chain SPCs can be detected down to 1.8 and 0.2 microg L(-1), respectively. Coefficients of variation of 6 and 12% within and between assays, respectively, demonstrate immunoassay reproducibility. The assay can be used in media with a wide range of pH and ionic strength values. Preliminary experiments performed to assess matrix effects have demonstrated the potential applicability of the method as a screening tool to assess contamination by these types of surfactants in natural water samples.     

Preparation of antibodies for the designer steroid tetrahydrogestrinone and development of an enzyme-linked immunosorbent assay for human urine analysis

A highly sensitive enzyme-linked immunosorbent assay (ELISA) has been developed for tetrahydrogestrinone (THG), the new designer anabolic steroid responsible for the well-known Balco scandal announced in year 2003. Antibodies have been raised against 18a-homo-pregna-4,9,11-trien-17beta-ol-3-carboxymethyl oxime coupled to horseshoe crab hemocyanin. The hapten has been synthesized from gestrinone by controlled reduction of the triple bond of the ethinyl group at position C-17, without affecting the double bonds of the steroidal rings, followed by reaction of the keto group at C-3 with (carboxymethoxy)amine hemihydrochloride to form the oxime bond. The antisera obtained has been used in combination with 18a-homo-pregna-4,9,11-trien-17beta-ol-20-yn-3-carboxymethyl oxime, a hapten derivative of gestrinone, coupled to bovine serum albumin to establish a competitive ELISA. Under the conditions used, THG can be detected in buffer with a limit of detection (LOD) of 0.045 +/- 0.015 microg L(-1) (N = 9). The assay is very selective since other steroids assessed are not recognized. Preliminary experiments performed with human urine samples demonstrate that the assay can be applied to the analysis of these samples after a simple sample treatment method reaching a LOD of 0.25 +/- 0.14 microg L(-1). Accuracy is very good as demonstrated by the excellent correlation obtained when analyzing blind spiked urine samples (slope 0.93, R2 = 0.992).     

Preparation and optical scattering characterization of gold nanorods and their application to a dot-immunogold assay

We describe optical monitoring of the synthesis of gold nanorods (NRs) based on seed-mediated growth in the presence of the soft surfactant template cetyltrimethyilammonium bromide. To separate NRs from spheres and surfactants we fractionated samples in the density gradient of glycerol. The optical properties of NRs were characterized by extinction and differential light-scattering spectra (at 90 degrees, 450-800 nm) and by the depolarization light-scattering ratio, I(vh)/I(vv), measured at 90 degrees with a helium-neon laser. Theoretical spectra and the I(vh)/I(vv) ratios were calculated by the T-matrix method as applied to randomly oriented NRs, which were modeled by right-circular cylinders with semispherical ends. The simulated data were fitted to experimental observations by use of particle length and width as adjustable parameters, which were close to the data yielded by transmission electron microscopy. The sensitivity of the long-wavelength resonance of NRs to the dielectric surroundings was examined both experimentally and theoretically by comparison of the extinction spectra of NRs in water and in a 25% glycerol solution. Finally, we discuss the application of NR-protein A conjugates to a dot-immunogold assay with the example of biospecific staining of human IgG molecules adsorbed onto small membrane spots.     

Conjugation behaviours of CdTe quantum dots and antibody by a novel immunochromatographic method

Three water-soluble CdTe quantum dots (QDs) (green-emitting, yellow-emitting and red-emitting) were synthesised for different refluxing time with 3-mercaptopropionic acid (MPA) as stabiliser. Then the red-emitting CdTe QDs and mouse immunoglobulin G (IgG) were taken as the representative to study the conjugation behaviour of QDs and antibody by a novel immunochromatographic method. After comparing with several methods, that is, direct conjugation, 1-ethyl-3(3-dimethylaminopropyl) carbodiimides hydrochloride (EDC)-mediated conjugation, N-hydroxysuccinimide (NHS)-mediated conjugation, EDC/NHS-mediated conjugation by immunochromatographic strips, EDC and NHS were selected together as coupling agents to conjugate QDs with antibody efficiently. Finally, the K562 leukaemia cells were incubated with the EDC/NHS-mediated conjugates to evaluate the performance in practical application, and the result from fluorescence images showed that it was successfully applied to label cells. The immunochromatographic strip was a superior method to study the conjugation of the fluorophore and antibody.     

罗丹明B酶联免疫分析方法的建立与应用

建立一种用于检测罗丹明B的间接竞争酶联免疫分析方法。该方法的IC50为1.3ng/m L,检测限为0.786ng/g。该方法检测豆瓣酱,加标浓度5 ng/g,平均回收率为83%,加标浓度10 ng/g,平均回收率为89%;检测辣椒酱,加标浓度5 ng/g,均回收率为88%,加标浓度10 ng/g,平均回收率为104%;检测辣椒油,加标浓度5 ng/g,平均回收率为94%,加标浓度10 ng/g,平均回收率为101%;检测辣椒粉,添加浓度5 ng/g,平均回收率为82%,加标浓度10 ng/g,平均回收率为90%。该方法的平均批内差为9.0%,批间差为10.5%,与其他常见染料未见交叉反应。该方法操作简便,结果准确,适用于罗丹明B的现场大量样品快速检测。     

A colorimetric gold nanoparticle sensor to interrogate biomolecular interactions in real time on a surface

This paper presents a new label-free optical method to study biomolecular interactions in real time at the surface of an optically transparent substrate. The method relies on the change in the absorbance spectrum of a self-assembled monolayer of colloidal gold on glass, as a function of biomolecular binding to the surface of the immobilized colloids. Using this approach, we demonstrate proof of principle of a label-free optical biosensor to quantify biomolecular interactions in real time on a surface in a commercially available UV-visible spectrophotometer and of a colorimetric end-point assay using an optical scanner. The spectrophotometric sensor shows concentration-dependent binding and a detection limit of 16 nM for streptavidin. The sensor is easy to fabricate, is reproducible in its performance, has minimal technological requirements, namely, the availability of an UV-visible spectrophotometer or an optical scanner, and will enable high-throughput screening of biomolecular interactions in real time in an array-based format.     

Optical and electrical characterization of a gold nanoparticle dispersion in a chiral liquid crystal matrix

We report on the effect of gold nanoparticle (Au NP) dispersion in a chiral nematic liquid crystal (LC). Polarized optical microscopy and X-ray diffraction measurements evidence the insurgence of an order change in the LC host. Moreover, a comparative analysis based on dielectric and voltammetric spectroscopies performed on pure LC and on Au NP-doped LC shows that Au NP’s presence besides affecting LC order influences its electric properties: ion conductivity results importantly reduced, and beyond a threshold value of the applied field electrophoresis phenomena are induced.     

Adenosine-dependent assembly of aptazyme-functionalized gold nanoparticles and its application as a colorimetric biosensor

Previous work has shown that DNAzyme-directed assembly of gold nanoparticles can be utilized to make effective colorimetric biosensors. However, the method is restricted to analytes that are directly involved in phosphodiester cleavage. To expand the methodology to a broader range of analytes, a colorimetric adenosine biosensor based on the aptazyme-directed assembly of gold nanoparticles is reported here. The aptazyme is based on the 8-17 DNAzyme with an adenosine aptamer motif that can modulate the DNAzyme activity through allosteric interactions depending on the presence of adenosine. In the absence of adenosine, the aptazyme is inactive and the substrate strands can serve as linkers to assemble DNA-functionalized 13-nm-diameter gold nanoparticles, resulting in a blue color. However, the presence of adenosine activates the aptazyme, which cleaves the substrate strand, disrupting the formation of nanoparticle aggregates. A red color of separated gold nanoparticles is observed. Concentrations of adenosine of up to 1 mM can be measured semiquantitatively by the degree of blue to red color changes or quantitatively by the extinction ratio at 520 and 700 nm. Under the same conditions, 5 mM guanosine, cytidine, or uridine resulted in a blue color only, indicating good selectivity of the sensor. The color difference can be clearly observed by the naked eye by spotting the resulting sensor solution onto an alumina TLC plate. Since aptamers that can target many classes of important analytes have already been selected, they can be adapted into aptazyme systems through rational design or further selection. Thus, colorimetric biosensors for many analytes of interest can be designed using the method presented here, regardless of whether the analytes are directly involved in the cleavage reaction or not.