Development of a non-competitive immunoassay for cortisol and its application to the analysis of saliva

A general method of performing non-competitive immunoassays for a low-molecular-mass analyte was developed and applied to cortisol determination in saliva samples. The method is based on the use of a “blocking reagent”, which is able to bind to antibody sites not occupied by the analyte, and in a stronger way than the analyte itself. When an enzyme-labelled analyte is added it substitutes the analyte in the antibody complex, but not the blocking reagent. The measured signal is linearly correlated to the concentration of the complex and, consequently, to the analyte concentration. The 3σ limit of detection (LOD, 0.2 nmol l⁻¹) obtained by the above method was 10 times lower than that obtained by the corresponding ELISA. As non-competitive immunoassays reported for small molecules up to now have been no more than just approaches, the suitability of the proposed assay for cortisol quantification in a real matrix was investigated. Human saliva was chosen as a matrix because of the need for very sensitive techniques to determine salivary cortisol content. The matrix effect was offset by performing the calibration experiments in acidic conditions (pH=5.6) and adding 0.1% of bovine serum albumin (BSA) to the buffer. In these conditions, the LOD was 1.4 nmol l⁻¹, which was adequate to measure normal levels of cortisol. Spiked samples were analysed and gave recoveries ranging from about 80 to 120%. Therefore, five subject samples, collected over 18 h showed salivary cortisol concentrations compatible with the circadian variation of reported normal values.     

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed.Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL⁻¹, the calibration curve was linear from 5.0 to 40 ng mL⁻¹ (R² = 0.952) with an IC₅₀ value of 18.2 ng mL⁻¹. In the extracts of 20 Chinese traditional drugs, the detection capability (CCβ) of vardenafil was 0.08 mg g⁻¹, the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue.The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.     

Development and validation of a highly sensitive ELISA for the determination of pharmaceutical indomethacin in water samples

The development and validation of a highly sensitive and specific indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of pharmaceutical indomethacin in water samples was presented. The immunogen and coating antigen were prepared by covalently linking indomethacin to bovine serum albumin and ovalbumin by anhydride ester method. Two rabbits were immunized by standard immunization processes and the superior antibody was characterized in terms of sensitivity, specificity, precision, accuracy and stability. Under optimal experimental conditions, the standard curve was constructed in the concentration range of 0.01-10 ng/mL. For 10 consecutive standard curves run in 2 weeks, IC₅₀ value (the concentration of analyte producing 50% of inhibition) were found within 0.10-0.25 ng/mL, and the detection limit (DL) at a signal-to-noise ratio of 3 (S/N = 3) was about 0.01 ng/mL. The antiserum recognized acemetacin, a precursor of indomethacin with 92.3% cross-reactivity, while the cross-reactivity values of antiserum with other tested compounds were very low. From the spiking experiments, the recoveries were found within 98-123%. The ELISA was applied for the determination of indomethacin in different water samples and the results were confirmed by conventional HPLC. The correlation coefficient of 0.988 was obtained, demonstrating a good correlation of ELISA with HPLC.     

Development of a portable biosensor for screening neurotoxic agents in water samples

A high sensitive portable biosensor system capable of determining the presence of neurotoxic agents in water has been developed. The system consists of (i) a screen-printed electrode with acetylcholinesterase (AChE) immobilized on it, (ii) a self-developed portable potentiostat with an analog to digital (A/D) converter and a serial interface for transferring data to a portable PC and (iii) an own designed software, developed with Lab-Windows CVI, used to record and process the measurements. The system has been developed to perform high precision amperometrical measurements with low drifts, low noise and a good reproducibility. In the configuration depicted, the percentage of AChE inhibition is proportional to the content of neurotoxic agents in a sample. This type of measurement is performed by the steady-state method from the first steady current (by a phosphate buffer solution) and the second steady current (by an enzymatic reaction produced by the addition of acetylthiocholine chloride to the solution). Validation was performed by analyzing spiked water samples containing pesticides. The design is specially suited for screening purposes, does not need sample preconcentration, is totally autonomous and suitable for the field detection of neurotoxic agents in water.     

酶联免疫吸附分析法测定水产品及水中孔雀石绿和无色孔雀石绿

本文报道一种同时测定水产品及水样中孔雀石绿(MG)和无色孔雀石绿(LMG)的间接竞争酶联免疫吸附分析法。对无色孔雀石绿分子进行修饰,使其与载体蛋白交联,得到免疫原和包被抗原,经过多次免疫动物制得抗无色孔雀石绿的多克隆抗体。在优化的实验条件下,IC50值(标准曲线中吸光度抑制至最大吸光度值的50%时所对应的待测物浓度)为0.9~2.6μg/L,检出限为0.02~0.10μg/L,无色孔雀石绿在水样及水产品中的回收率为76.2~95.0%,与孔雀石绿的交叉反应率为95.25%。真实样品测定中,两种食用鱼养殖水样及一个鱼样中未检出孔雀石绿和无色孔雀石绿,但在观赏鱼养殖水样及另一鱼样中检出孔雀石绿和无色孔雀石,浓度分别为1.84μg/L和1.38μg/L。     

Preparation of a multi-hapten antigen and broad specificity polyclonal antibodies for a multiple pesticide immunoassay

A multi-determinant artificial antigen was prepared by haptens of four pesticides (chlorpyrifos, triazophos, carbofuran and parathion methyl) conjugating to the carrier protein BSA in turn. Male New Zealand white rabbits were immunized with this multi-determinant immunogen to produce the polyclonal antibodies (PAbs), which can recognize the four pesticides. The PAbs displayed high level for each relative hapten-OVA conjugate, with the favorable titers of 4.49 × 10⁴, 8.98 × 10⁴, 2.24 × 10⁴ and 1.86 × 10⁴, for CHBu-OVA, THHe-OVA, BFNB-OVA and MP5-OVA, respectively. Characterization studies of the PcAbs showed that it has high affinity and specificity to the four relative pesticides. An indirect competitive ELISA was developed for multi-residue determination. The I₅₀ value for the four pesticides was 0.290, 0.065, 0.582 and 2.824 μg mL⁻¹, with the detection limit (I₁₀) of 0.022, 0.005, 0.015 and 0.115 μg mL⁻¹ for carbofuran, triazophos, chlorpyrifos and parathion methyl, respectively. The linear rang was 0.016-2.000, 0.005-0.500, 0.010-2.000 and 0.063-5.000 μg mL⁻¹, respectively, for carbofuran, triazophos, chlorpyrifos and parathion methyl. Results indicated that, this study provided a new strategy to develop immunoassays through artificial antigen design for pesticides multi-residue determination.     

Synthesis of three haptens for the class-specific immunoassay of O,O-dimethyl organophosphorus pesticides and effect of hapten heterology on immunoassay sensitivity

A general and broad class-specific enzyme-linked immunosorbent assay was developed for the O,O-dimethyl organophosphorus pesticides, including malathion, dimethoate, phenthoate, phosmet, methidathion, fenitrothion, methyl parathion and fenthion. Three haptens with different spacer-arms were synthesized. The haptens were conjugated to bovine serum albumin (BSA) for immunogens and to ovalbumin (OVA) for coating antigens. Rabbits were immunized with the immunogens and six polyclonal antisera were produced and screened against each of the coating antigens using competitive indirect enzyme-linked immunosorbent assay for selecting the proper antiserum. The effect of hapten heterology on immunoassay sensitivity was also studied. The antibody-antigen combination with the most selectivity for malathion was further optimized and tested for tolerance to co-solvent, pH and ionic strength changes. The IC₅₀ values, under optimum conditions, were estimated to be 30.1 μg L⁻¹for malathion, 28.9 μg L⁻¹ for dimethoate, 88.3 μg L⁻¹ for phenthoate, 159.7 μg L⁻¹ for phosmet, 191.7 μg L⁻¹ for methidathion, 324.0 μg L⁻¹ for fenitrothion, 483.9 μg L⁻¹ for methyl parathion, and 788.9 μg L⁻¹ for fenthion. Recoveries of malathion, dimethoate, phenthoate, phosmet and methidathion from fortified Chinese cabbage samples ranged between 77.1% and 104.7%. This assay can be used in monitoring studies for the multi-residue determination of O,O-dimethyl organophosphorus pesticides.     

Dispersive liquid-phase microextraction using ionic liquid as extractant for the enrichment and determination of DDT and its metabolites in environmental water samples

Ionic liquids are a kind of environmentally friendly solvents which have drawn great attention in many fields. The potential of ionic liquid as dispersive liquid-phase microextraction (DLPME) solvent for the enrichment of typical persistent organic pollutants, dichlorodiphenyltrichloroethane (DDT), and its metabolites including 1,1-dichloro-2,2-bis-(4′-chlorophenyl)ethane and 1,1-dichloro-2,2-bis-(4′-chlorophenyl)ethylene has been investigated. Parameters that may influence the extraction efficiency, such as the type and volume of ionic liquid, the type and volume of disperser solvent, extraction time, and sample pH, were investigated and optimized in detail. The experimental results showed the excellent linear relationship between peak area and the concentration of DDT and its metabolites over the range of 1–50 μg L⁻¹, and the precisions (RSDs) were 5.27–6.73% under the optimal conditions. The limits of detection could reach 0.33–0.63 μg L⁻¹. Satisfied results were achieved when the proposed method was applied to determine the target compounds in real-world water samples with spiked recoveries over the range 94.4–115.3%. All these facts indicated that ionic liquid DLPME coupled to HPLC was an environmentally friendly alternative for the rapid analysis of DDT and its metabolites at trace level in environmental water samples.     

Development of a heterogeneous chemiluminescent flow immunoassay for DDT and related compounds

A heterogeneous chemiluminescent (CL) flow immunoassay for DDT was optimized comparing different types of immunoaffinity supports: beads, nylon coils and membranes (membranes HyBondN+). In order to characterize solid immunoaffinity supports two basic immunoassay formats were performed, using (1) enzyme-labeled secondary and (2) enzyme-labeled specific monoclonal antibodies (MAbs). In both formats, hapten DDT5 conjugated to ovalbumin immobilized on solid supports according to the appropriate immobilization procedure, enzyme label (horseradish peroxidase, HRP) and luminescent detection (luminol/H₂O₂/p-iodophenol) were used. The lowest limit of detection (LOD), 1 nM p,p-DDT, was obtained with a membrane-based flow immunoassay with HRP-labeled specific antibody. Beads and packed tubing were discarded as appropriate supports because of the difficulties encountered for reproducible packing and the occurrence of light scatterring (beads), which seriously compromised the performance and reproducibility of the flow immunoassay.     

Development of a microparticle-based on-site immunoassay for the detection of atrazine in soil and water samples

Atrazine is widely used as a herbicide in agriculture and has been identified as a major groundwater contaminant in the US. Because of the possible hazard associated with its usage, there is a need for an efficient and economic screening method for on-site field testing of atrazine and other s-triazine herbicides in soil and water. We have developed a rapid, on-site test for the detection of atrazine based on the principle of microparticle agglutination inhibition immunoassay. The test detects 50 microg kg(-1) (0.050 ppm) atrazine in soil samples with direct extraction and 1.0 microg L(-1) atrazine in water samples when coupled with solid phase extraction.     

Development of a dipstick immunoassay for quantitative determination of 2,4-dichlorophenoxyacetic acid in water, fruit and urine samples

A sensitive dipstick assay for 2,4-dichlorophenoxyacetic acid (2,4-D) detection was developed. The assay was based on the competitive reaction of 2,4-D and enzyme tracer with monoclonal antibodies immobilised on an Ultrabind^? membrane. The binding of enzyme tracer on the test strip was determined by a simple, portable reflectometer as remission at 657 nm. Using this technique, 2,4-D could be detected in a concentration range of 0.5 μg/L to 100 μg/L. The center point of the 2,4-D test was found at a concentration of 6 μg/L. Cross-reactivity with 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) as determined by this dipstick assay was 2.5% and 3% by standard ELISA technique using microtiter plates. The assay was applied in the detection of 2,4-D in real water samples, and sensitivity was comparable to spiked water samples. If combined with an effective extraction procedure that results in recovery rates of 90%, the dipstick assay can be used to monitor human exposure to 2,4-D from contamination in water, from oranges and in testing urine samples. Received: 2 September 1998 / Revised: 29 January 1999 / Accepted: 31 January 1999     

Simple and surprisingly effective one-step extraction-cleanup by soxflo for DDT and its metabolites from environmental samples

A pilot study found that DDT breakdown at the GC inlet was extensive in extracts from some--but not all--samples with high organic carbon contents. However, DDT losses could be prevented with a one-step extraction-cleanup in the Soxflo instrument with dichloromethane and charcoal. This dry-column procedure took 1 h at room temperature. It was tested on spiked soil and peat samples and validated with certified soil and sediment reference materials. Spike recoveries from freshly spiked samples ranged from 79 to 111% at 20-4000 microg/kg concentrations. Recoveries from the real-world CRMs were 99.7-100.2% of DDT, 89.7-90.4% of DDD and 89.6-107.9% of DDE. It was concluded that charcoal cleanups should be used routinely during surveys for environmental DDX pollution in order to mitigate against unpredictable matrix-enhanced breakdown in the GC.     

Biomimetic synthesis of all-inclusive organic-inorganic nanospheres for enhanced electrochemical immunoassay

A friendly biomimetic process was adopted for the mild preparation of “all-inclusive” organic-inorganic nanospheres, which effectively integrate biorecognition function and signal amplification function. The resulted Ca₃(PO₄)₂-Ab₂-BSA nanospheres were employed as signal labels for enhancing detection of nuclear matrix protein 22 (NMP 22). The fabricated electrochemical immunosensor exhibited a linear range (0.08–77.00 U/mL) and an ultralow limit of detection (0.01 U/mL) towards NMP 22, which can be taken as a promising tool for clinical diagnosis of bladder cancer.     

A highly sensitive and selective immunoassay for the detection of tetrabromobisphenol A in soil and sediment

Tetrabromobisphenol A is the most widely used brominated flame retardant. A sensitive and selective enzyme-linked immunosorbent assay (ELISA) for the detection of tetrabromobisphenol A was developed. The limit of detection and the inhibition half-maximum concentration of tetrabromobisphenol A in phosphate buffered saline with 10% methanol were 0.05 and 0.87 ng mL⁻¹, respectively. Cross-reactivity values of the ELISA with a set of important brominated flame retardants including tetrabromobisphenol A-bis(2,3-dibromopropylether), 2,2′,6,6′-tetrabromobisphenol A diallyl ether, hexabromocyclododecane, 1,2-bis(pentabromodiphenyl) ethane, 1,2-bis(2,4,6 tribromophenoxy) ethane, bis(2-ethylhexyl)-3,4,5,6-tetrabromophthalate, 2-ethylhexyl-2,3,4,5-tetrabromobenzoate, and polybrominated diphenyl ethers were <0.05%. Concentrations of tetrabromobisphenol A determined by ELISA in the soils from farmlands, the soils from an e-waste recycling site, and the sediments of a canal were in the range of non-detectable–5.6 ng g⁻¹, 26–104 ng g⁻¹ and 0.3–22 ng g⁻¹ dw, respectively, indicating the ubiquitous pollution of tetrabromobisphenol A. The results of this assay for 16 real world samples agreed well with those of the liquid chromatography–tandem mass spectrometry method, indicating this ELISA is suitable for screening of tetrabromobisphenol A in environmental matrices.     

Rapid monitoring and sensitive determination of DDT and its metabolites in water sample using solid-phase extraction followed by ion mobility spectrometry

Dichlorodiphenyl trichloroethane (DDT) as an organochlorine compound has been globally used as a pesticide for controlling soil-dwelling insects and treating diseases such as malaria and typhus. The degradation products of DDT and its metabolites have also negative effects on the environment. The present study has investigated the determination of DDT and its metabolites in water sample using ion mobility spectrometry (IMS) as a rapid and sensitive detection technique. For this purpose, DDT and its metabolites were extracted using reverse phase solid-phase extraction (SPE) from water samples. The samples were then recovered by eluting with methanol and finally, quantified using the corona discharge IMS technique. Injection and oven temperatures and the effect of dopant were optimized as experimental parameters influencing both detection and determination efficiencies. Degradation of DDT in IMS drift tube was studied and reduced mobility values of DDT and its metabolites were calculated. The developed method was validated using water sample to obtain good results for the determination of DDT at low levels (1 ng ml^?1) while spiked recoveries were obtained to be between 95.0–96.7%. The proposed method based on IMS proved to be a simple, inexpensive, rapid and sensitive procedure for the fast monitoring and determination of DDT and its main metabolites in water sample.     

Colorimetric analysis of water and sand samples performed on a mobile phone

Analysis of water and sand samples was done by reflectance measurements using a mobile phone. The phone's screen served as light source and front view camera as detector. Reflected intensities for white, red, green and blue colors were used to do principal component analysis for classification of several compounds and their concentrations in water. Analyses of colored solutions and colorimetric reactions based on widely available chemicals were performed. Classification of iron(III), chromium(VI) and sodium salt of humic acid was observed using reflected intensities from blue and green light for concentrations 2-10 mg/l. Addition of complex forming sodium salt of ethylenediaminetetraacidic acid enabled the discrimination of Cu(II) ions in the 2-10 mg/l concentration range based on reflection of red light. An alternate method using test strips for copper solutions with the phone as reader also demonstrated a detection limit of 2 mg/l. Analysis of As(III) from 25 to 400 μg/l based on reflection of red light was performed utilizing the bleaching reaction of tincture of iodine containing starch. Enhanced sensitivity to low concentrations of arsenic was obtained by including reflected intensities from white light in the analysis. Model colored sand samples representing discoloration caused by the presence of arsenic in groundwater were analyzed as a complementary method for arsenic detection.     

A competitive immunoassay with a surrogate calibrator curve for aflatoxin M1 in milk

A green enzyme-linked immunosorbent assay (ELISA) to measure aflatoxin M₁ (AFM₁) in milk was developed and validated with a surrogate calibrator curve. Polyclonal anti-idiotype (anti-Id) antibody, used as an AFM₁ surrogate, was generated by immunizing rabbits with F(ab′)₂ fragments from the anti-AFM₁ monoclonal antibody (mAb). The rabbits exhibited high specificity to the anti-AFM₁ mAb, and no cross-reactivity to either of the other anti-aflatoxin mAbs or the isotype matched mAb was observed. After optimizing the physicochemical factors (pH and ionic strength) that influence assay performance, a quantitative conversion formula was developed between AFM₁ and the anti-Id antibody (y = 31.91x − 8.47, r = 0.9997). The assay was applied to analyze AFM₁ in spiked milk samples. The IC₅₀ value of the surrogate calibrator curve was 2.4 μg mL⁻¹, and the inter-assay and intra-assay variations were less than 10.8%; recovery ranged from 85.2 to 110.9%. A reference high-performance liquid chromatography method was used to validate the developed method, and a good correlation was obtained (y = 0.81x + 9.82, r = 0.9922).     

Investigation of the feasibility of TiO(2) nanotubes for the enrichment of DDT and its metabolites at trace levels in environmental water samples

TiO(2) nanotubes, novel nanomaterials synthesized from hydrothermal treatment, were investigated for being used as a new solid-phase extraction adsorbent with o,p'-DDT, [1,1,1-trichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl)ethane, p,p'-DDT, [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] and its principle metabolites p,p'-DDD, [1,1-dichloro-2,2-bis(4-chlorophenyl)ethane] and p,p'-DDE [1,1-(2,2-dichloro-ethanylidene)-bis(4-chlorobenzene)] as the target analytes. Several factors such as eluant and its volume, the sample pH, sample volume and the flow rate of samples, were optimized. The effect of humic acid, which is often present in natural water system, was also investigated. Under the optimal conditions, lower detection limits of 0.0031, 0.0037, 0.0053 and 0.0025 ng mL(-1) for p,p'-DDD, p,p'-DDT, o,p'-DDT and p,p'-DDE, respectively, were obtained. The proposed method was successfully applied to the analysis of the target compounds in several environmental water samples. Good recoveries over the range of 81.2-115% were obtained. These results indicated that titanium nanotubes had enormous potential in environmental field as a novel SPE adsorbent material.     

Screening and determination of melamine residues in tissue and body fluid samples

Melamine is a chemical product that was sporadically mixed into animal feeds to boost protein content. Excessive melamine in animal feed can induce renal failure and even death in animals. The residue of melamine in edible animal products also threatens human health. Currently, there is no real-time and high throughput method to detect residual melamine in animal tissues. Successful development of such methods is very important for fast and on-site screening of melamine residue in animal tissues to eliminate the potential threat to human health. Here we demonstrate the detection of residual melamine from swine and chicken tissues and body fluids using indirect competitive enzyme-linked immunosorbent assay (ELISA) method. A detection sensitivity of 0.5 μg mL⁻¹ and a limit of detection of 0.05 μg mL⁻¹ were achieved with this method. A gas chromatography-mass spectrometry (GC-MS) method was also developed to act as a confirmatory and quantitative procedure for the ELISA results. The limits of quantitation (LOQ) of were 0.01 μg g⁻¹ and 0.005 μg mL⁻¹ for tissues and body fluids, respectively. The two methods showed good agreement (r² > 0.992). The method developed was performed on samples of tissues from chickens fed with melamine-spiked feed.     

A comprehensive immunoassay for the detection of microcystins in waters based on polyclonal antibodies

Microcystins (MCs) are a group of closely related toxic cyclic heptapeptides produced by common cyanobacteria (blue-green algae), and microcystin-leucine-arginine (MC-LR) is among the most frequent and most toxic microcystin congeners. In this study, a free amino group was introduced to MC-LR at its seventh amino acid residue with 2-mercaptoethylamine, and the product aminoethyl-MC-LR was coupled to bovine serum albumin (BSA) and horseradish peroxidise (HRP) by glutaraldehyde to be complete antigen (MC-LR-BSA) and labelled hapten (MC-LR-HRP), respectively. Polyclonal antibodies against MC-LR were generated by immunization with MC-LR-BSA. A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) was established to detect the MCs in waters, which showed a good cross-reactivity with MC-LR, MC-RR, MC-YR, MC-LF, MC-LW and nodularin, and have a detection limit for MC-LR 0.12 μg L⁻¹, the 50% inhibition concentration (IC₅₀) for MC-LR was 0.63 ± 0.06 μg L⁻¹ and the quantitative detection range was from 0.17 to 2.32 μg L⁻¹, the analysis result of water samples showed good recovery and reliability. So the comprehensive and reliable dc-ELISA will well potentially suit for sensitive analysis for total MCs in drinking as well as resource water samples.