Quantitative Working Range Research Articles

Application of luminescent Photobacterium Phosphoreum T3 for the detection of zearalenone and estimating the efficiency of their enzymatic degradation

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin, causes enormous economic losses in the food and feed industries. Simple, rapid, low-cost, and quantitative analysis of ZEN is particularly urgent in the fields of food safety and animal husbandry. Using the bioluminescent bacterium Photobacterium phosphoreum T3, we propose a bioluminescence inhibition assay to evaluate ZEN levels quickly. The limit of detection (LOD), limit of quantification (LOQ), and quantitative working range of this bioluminescence inhibition assay were 0.1 µg/mL, 5 µg/mL, and 5-100 µg/mL, respectively. The concentration-response curve of the bioluminescence inhibition rate and ZEN concentration was plotted within the range 5 to 100 μg/mL, as follows: y = 0.0069x2 – 0.0190x + 7.9907 (R2 = 0.9943, y is luminescence inhibition rate, x is ZEN concentration). First, we used the bioluminescence inhibition assay to detect the remaining ZEN in samples treated with purified lactonohydrolase ZHD101. The bioluminescence inhibition assay results showed a strong correlation with the HPLC analysis. Furthermore, we successfully evaluated the overall toxicity of samples treated with purified peroxidase Prx and H2O2 using the P. phosphoreum T3 bioluminescence inhibition assay. The results indicate that the degradation products of ZEN created by purified peroxidase Prx and H2O2 showed little toxicity to P. phosphoreum T3. In this study, a simple, rapid, and low-cost assay method of zearalenone by bioluminescent P. phosphoreum T3 was developed. The bioluminescence inhibition assay could be used to estimate the efficiency of enzymatic degradation of ZEN.

Determination of fentanyl contamination on United States paper currency by LC-QQQ-MS.

Previous research has evaluated the extent to which cocaine and other drugs were detectable on currency in the USA. The literature was in agreement that the majority of bills exhibited some degree of contamination. With the increase of fentanyl in the illicit drug supply, this study was designed to evaluate the extent that fentanyl, cocaine, methamphetamine and other substances were present on circulating currency in 2022. A quantitative assay using liquid chromatography-triple quadrupole mass spectrometry was developed and validated to detect six analytes: fentanyl, 4-anilino-N-phenethylpiperidine, acetylfentanyl, benzylfentanyl, cocaine and methamphetamine. One-dollar bills were collected from 13 cities across the country. Sample preparation consisted of soaking the bills in methanol followed by liquid-liquid extraction. Chromatographic separation was achieved using a C18 analytical column and gradient elution with ammonium formate in water (5 mM, pH 3) and 0.1% formic acid in acetonitrile. The quantitative working range for this assay was 0.1 μg to 1.0 μg per bill (equivalent to 1 ng/mL to 100 ng/mL of extract). Fentanyl was detected on the majority (63%) of samples, with 61% of samples having ≥0.1 μg of fentanyl and 4% of samples having ≥1.0 μg. Cocaine and methamphetamine were detected on 100% and 98% of bills, respectively, typically in amounts >1.0 μg. The remaining fentanyl-related substances were detected in 15% of samples in amounts no >0.69 μg per bill and exclusively in the presence of fentanyl. Unsurprisingly, areas of the country with higher incidence of fentanyl use yielded higher frequency of contaminated bills and higher concentrations. Human exposure to drugs on currency is unlikely to have any significant impacts toxicologically or pharmacologically; however, our research findings suggest that paper currency could serve as a useful substrate for surveillance of drug trends regionally, nationally and/or internationally.

An indirect competitive enzyme-linked immunosorbent assay for acrylamide detection based on a monoclonal antibody

ABSTRACTAcrylamide (AA) is formed spontaneously in heated foodstuffs and is a focus of concern in many people due to safety. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on a monoclonal antibody (MAb) to detect a derivative, which was generated from 4-mercaptobenzoic acid (4-MBA). As AA is a very small molecule (71.08 Da) and cannot elicit a homologous monoclonal antibody, we coupled the AA derivative (AA-4-MBA) to a carrier protein such as bovine serum albumin (BSA) and ovalbumin (OVA). The conjugates were used as the immunogen and coating antigen. A rapid and sensitive icELISA against AA-4-MBA was obtained by optimizing the experimental parameters. The MAb which had no specificity for AA or 4-MBA, but had high affinity for AA-4-MBA, had a satisfactory IC50 of 32 ng/ml and a limit of detection of 8.87 ng/ml. The quantitative working range was 8.87–112.92 ng/ml (IC20 to IC80). Cross-reactivity with other analogues was lower than 10%. These results indicated that the developed icELISA was a fast and efficient method for detecting AA in food.

Rapid enzyme-linked immunosorbent assay and immunochromatographic strip for detecting ribavirin in chicken muscles

ABSTRACTThe use of ribavirin (RBV) as an antiviral drug for livestock has been prohibited in China, the USA, and many other countries. In this study, we developed a rapid and sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and a gold nanoparticle immunochromatographic (ICA) strip test for detecting RBV in chicken muscles. Under the optimum assay conditions, where the assay employed phosphate-buffered saline at pH 7.4, no acetonitrile, and an ionic strength of 0.8%, the quantitative working range was 1.43–26.47 ng/ml with an IC50 of 6.15 ng/ml. The recovery rate for RBV in real samples ranged from 82.1% to 112.3%. The immunochromatographic test strip method had a visual cutoff value of 50 μg/kg. Given their high recovery rates and good sensitivity, the proposed ic-ELISA and ICA methods could be useful for the RBV analysis in chicken tissue samples.

A competitive microfluidic immunological clenbuterol analysis using a microELISA system

We present a novel method to analyze clenbuterol based on a competitive microfluidic immunoassay scheme with a micro-ELISA system, and obtain a limit of detection that is less than 0.1 ng ml−1 with a quantitative working range of 0.1 ng ml−1 to 27.0 ng ml−1. The approach was envisaged to be a promising method for efficient onsite clenbuterol control with good sensitivity and portability.

A highly sensitive enzyme-linked immunosorbent assay for the detection of dipropyl phthalate in plastic food contact materials

A highly sensitive and specific direct competitive enzyme-linked immunosorbent assay has been developed for the detection of dipropyl phthalate (DPrP) based on antibody-coating format. The specific polyclonal antibodies against DPrP raised in rabbits. The conjugates of the antigen with horseradish peroxidase (HRP) were used as the detectable probe. Under the optimal conditions, the limit of detection of the method was 0.01 ng/mL, and a good quantitative working range of 0.01–16 ng/mL (R 2=0.995) was obtained. The cross-reactivities of four structurally related phthalate esters were below 12% and did not interfere significantly the analysis of DPrP. The accuracy of the immunoassay has been evaluated and validated by analyzing real and spiked leachate from plastic food contact materials. The recoveries of DPrP ranged from 85.9% to 109.4%. This developed dc-ELISA was reliable, accurate and highly sensitive.

Rapid monitoring of dicyclohexyl phthalate in foods using the direct competitive ELISA

A direct competitive enzyme-linked immunosorbent assay (dc-ELISA) has been developed for the quantitative detection of the dicyclohexyl phthalate (DCHP) in some liquid food. Specific polyclonal antisera to DCHP was raised, using the hapten-bovine serum albumin conjugates as the immunogen. The conjugate of horseradish peroxidase (HRP) with antibody was used as the detectable probe to provide the direct measurement of the antigen and analyte, which was synthesised by a modified glutaraldehyde method. Under the optimised assay, the quantitative working range was from 0.1 to 100 ng mL−1 (R2=0.9989), with a limit of detection (LOD) of 0.03 ng mL−1, and a recovery of 96.6–112.4%. The specificity and accuracy of developed method were evaluated. The cross-reactivities of antibody with structurally related phthalate esters were less than 10%. Results obtained indicated that the dc-ELISA was a sensitive, low expense method to improve the routine monitoring of trace constituents in some liquid food.

Optimizing Hydrogen Sensing Behavior by Controlling the Coverage in Pd Nanoparticle Films

The response of quantum-conductance-based hydrogen sensors fabricated by controllable deposition of closely spaced Pd nanoparticle films between interdigital electrodes was investigated. Three typical response regions with different conductance–hydrogen pressure correlations were observed. The response characteristics of the devices were found to depend strongly on the nanoparticle coverage. In the low H2 pressure region, higher coverage gives higher sensitivity. In the high H2 pressure region, quantitative sensing can only be realized with low nanoparticle coverage. Optimizing the coverage allows the attainment of highly sensitive hydrogen sensors with a very wide quantitative working range, extending far beyond the hydrogen pressure region associated with the α-to-β phase transition of Pd.

Design and synthesis of immunoconjugates and development of competition inhibition enzyme-linked immunosorbent assay (CIEIA) for the detection of O-isopropyl methylphosphonofluoridate (sarin): An organophosphorous toxicant

Three haptens of the organophosphorus (OP) toxicant ‘sarin’ having different spacer arm were designed and synthesized. Haptens were conjugated with BSA (bovine serum albumin) and ovalbumin (OVA) for raising antibody and coating antigen. High antibody titer with higher specificity was obtained from 4-(4-(isopropoxy(methyl)phosphoryloxy)phenylamino)-4-oxobutanoic acid (hapten B) having reasonable long spacer arm. For the standard curve, an IC 50 (inhibitory concentration) of free antigen was found to be 0.415 μg mL −1 on the basis of indirect competitive ELISA. The study revealed that heterology in competition inhibition enzyme immunoassay (CIEIA) produced remarkable improvement in the sensitivity and specificity of the assay. Under the optimized conditions, the quantitative working range was found to be 0.19–1.56 μg mL −1 with a limit of detection (LOD) of 0.05 μg mL −1. The antibodies showed negligible cross reactivity (CR) with other OP toxicants and pesticides, which makes the assay suitable for the selective detection of sarin.

Development of determination of di-n-octyl phthalate (DOP) residue by an indirect enzyme-linked immunosorbent assay

In this research, the hapten di-n-octyl 4-aminophthalate (DOAP) was designed and synthesised successfully. It was used to couple with carrier proteins, bovine serum albumin (BSA) and ovalbumin (OVA) by diazotization reaction for immunogen (DOP–BSA) and coating antigen (DOP–OVA), respectively. Rabbits were immunised with DOP–BSA; polyclonal antiserum was raised and determined by competitive indirect enzyme-linked immunosorbent assay (ciELISA). After optimisation, a ciELISA was established. The quantitative working range for DOP was 5–75 ng/mL with the detection limit of 1.9±0.1 ng/mL and the IC50 of 19.2±1.1 ng/mL. The optimised ELISA had cross-reactivity of 22.6%, 17.6% and 21.2% with di-iso-octyl phthalate (DIOP), di-n-butyl phthalate (DBP) and di-hexyl phthalate (DHP), respectively. The result of the detection of polyvinyl chloride (PVC) samples showed that the immunoassay we developed had high-accuracy contrast with high-performance liquid chromatography electrospray ionisation tandem mass spectrometry analysis and it could be qualified to determine di-n-octyl phthalate (DOP) residue in PVC samples.

An Indirect Competitive Fluorescence Immunoassay for Determination of Dicyclohexyl Phthalate in Water Samples

A sensitive and specific indirect competitive fluorescence immunoassays (FIA) has been developed for the quantitative determination of dicyclohexyl phthalate (DCHP) using an antigen-coated plate format. The polyclonal antibodies raised against dicyclohexyl 4-amino phthalate conjugated to bovine serum albumin (BSA) by the amino diazotization linkage method. Antiserum with a sufficiently high titer was generated in rabbits and fluorescein isothiocyanate (FITC) was used as sensitive labels to construct the fluorescence immunoassay (FIA) for measurement of targeted compounds. Under optimized FIA condition, the quantitative working range was from 0.1 to 200 μg L−1 with a limit of detection of 0.05 μg L−1. Other similar phthalate compounds do not interfere significantly in the analysis using this immunoassay technique, and the cross-reactivity rates were less than 10%. Four kinds of water samples (tap water, lake water, river water and leachate) had been detected in this assay, the recovery was 91.3–107.8%. The proposed fluorescence immunoassay turned out to be a powerful tool for monitoring of dicyclohexyl phthalate in water samples at trace level.

Development of an Indirect Enzyme-Linked Immunosorbent Assay for the Organophosphorus Pesticide Paraoxon-Methyl

In the present study, the synthesis of hapten for the organophosphorus (OP) pesticide paraoxon-methyl was developed, with a spacer arm (aminocarboxylic acid) attached at the aromatic ring. It was conjugated to bovine serum albumin (BSA) for use as an immunogen and to ovalbumin (OVA) for coating antigen for ELISA testing. Rabbits were immunized with the immunogen and two polyclonal antisera were produced and screened against the coating antigen using competitive indirect enzyme-linked immunosorbent assay (ELISA). For application to textile samples, the influence of several factors such as organic solvent, ionic strength, and pH on the ELISA results were studied. Under optimized conditions, the quantitative working range was 0.012–1.158 μg/mL with a limit of detection (LOD) of 0.005 μg/mL and the IC50 was 0.115 μg/mL.There was negligible cross reactivity (CR) with other OP pesticides. The recoveries obtained by standard paraoxon-methyl addition to the different textile samples such as cotton, wool and muslin delaine were all from 86.0% to 108.0%. Therefore, the optimized ELISA may become a new convenient and economical analytical tool for monitoring paraoxon-methyl residues in textile samples.

Development of Competition Enzyme-Linked Immunosorbent Assay for Determination of Formaldehyde in Serum

Sick-building symptoms associated with indoor air volatile organic compounds, including formaldehyde in new or newly remodeled houses, have been increasingly highlighted, and are known as sick house syndrome in Japan. We developed a competition enzyme-linked immunosorbent assay for measuring formaldehyde in serum. The specificity of the antiserum generated with formaldehyde-rabbit serum albumin conjugates and the application to the detection of formaldehyde in serum were evaluated. The antiserum selectively reacted with formaldehyde conjugated to rabbit, human, rat and bovine-serum albumins, but not with unconjugated albumins. The quantitative working range of formaldehyde-human serum albumin conjugates was from 0.5 to 500 ng/well.

Quantitative Determination of Peptides Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.

Development of an ELISA for the detection of bromoxynil in water

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the nitrile herbicide bromoxynil, the polyclonal antibodies raised against 2,6-dibromo-4-cyano-phenoxyacetic acid (hapten) conjugated to bovine serum albumin (BSA) by the N-hydroxysuccinimide-activated ester method. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 77 days after the primary immunization. Antiserum A2 was applied to the residual analysis of some water samples, under optimized ELISA condition, the quantitative working range was from 10 to 500 ppb with a limit of detection of 5 ppb. Cross-reactivity to structurally similar agrochemicals and related chemicals was determined. The antiserum showed little cross-reactivity with 2,6-dibromophenol and bromoxynil octanoate ester which have a dibromophenol group as common structure, but showed no cross-reactivity with other herbicides. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween20 in the assay buffer. These four kinds of water samples were fortified with bromoxynil at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution, the mean recovery was 102.3%, and the mean coefficient of variation was 5.96%. The proposed ELISA turned out to be a powerful tool for monitoring of residual bromoxynil in water samples at trace level.

New approach to immunochemical determinations for triclopyr and 3,5,6-trichloro-2-pyridinol by using a bifunctional hapten, and evaluation of polyclonal antiserum.

The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.

Group-specific enzyme-linked immunosorbent assay for fenitrooxon and 3-methyl-4-nitrophenol in water samples based on a polyclonal antibody

Fenitrooxon [ O,O-dimethyl- O-(4-nitro- m-tolyl)phosphate] is the major metabolite of the organophosphorus insecticide fenitrothion, and 3-methyl-4-nitrophenol is its major degradation product. In the present study, we describe the development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection of these compounds in water samples based on a group-specific polyclonal antiserum generated with a “bifunctional hapten”, which has two functions: the conventional function of producing an antibody against an antigen and a unique function of promoting the production of the antibodies in rabbit. For application to water samples, the influence of several factors such as organic solvent, pH, and detergent was studied. Under optimized conditions, the quantitative working range of the fenitrooxon ELISA was 0.71–27 ng ml −1 with a limit of detection (LOD) of 0.32 ng ml −1, and the fenitrooxon concentration giving 50% reduction of the maximum signal (IC 50) was 4.2 ng ml −1. The quantitative working range of the 3-methyl-4-nitrophenol ELISA was 0.67–27 ng ml −1 with a LOD of 0.38 ng ml −1 and an IC 50 of 3.7 ng ml −1. No significant matrix effect originating from the water sample (river water, tap water, purified water, and bottled water) was shown by addition of Tween 20 to the assay buffer. Water samples spiked with each of these compounds at 1, 5, 10, and 20 ng ml −1 were directly analyzed without extraction and clean-up by the proposed ELISA. The mean recovery was 100.9%, and the mean coefficient of variation (CV) was 7.7% for the fenitrooxon ELISA and for the 3-methyl-4-nitrophenol ELISA, the mean recovery was 97.6%, and the mean CV was 7.2%. The proposed ELISA allows precise and accurate determination of these compounds in water at such low levels.

Enzyme-linked immunosorbent assay based on a polyclonal antibody for the detection of the insecticide fenitrothion. Evaluation of antiserum and application to the analysis of water samples.

For development of an indirect competitive enzyme-linked immunosorbent assay (ELISA) for the organophosphorus insecticide fenitrothion, the specificity of the antiserum R-3 generated with the bifunctional hapten, LysMNPA (2-[[[(3-methyl-4-nitrophenyl)oxy]methylcarbonyl]amino]-6-(2,4-dinitrophenyl)aminohexanoic acid) and the application to the residual analysis of some water samples were evaluated. At optimized ELISA conditions, the quantitative working range was from 1 to 39 ng/mL with a limit of detection of 0.3 ng/mL and an IC(50) value of 6 ng/mL. Cross-reactivity to structurally similar organophosphorus compounds and related chemicals was determined. The antiserum R-3 showed significant cross-reactivity with fenitrooxon and 3-methyl-4-nitrophenol, which have a 3-methyl-4-nitrophenoxy group as common structures, but showed relatively low cross-reactivity with other compounds. Each water sample (river water, tap water, purified water, and bottled water) had a matrix effect and was investigated by adding Tween 20 in the assay buffer. These four kinds of water samples were fortified with fenitrothion at several concentration levels and were directly analyzed with only dilution with an equal volume of antiserum solution. The mean recovery was 105.9%, and the mean coefficient of variation was 10.9%. The results suggested that the developed ELISA would be very suitable for a preliminary screening for fenitrothion in water samples at such low levels.

A general enzyme immunoassay for the licorice root component contained in traditional Chinese medicines.

The development and application of a new enzyme immunoassay for general assay of the Glycyrrhizae radix (GR) component in Chinese traditional medicines is described. Three commercial GR-based medicines, Tohoku kanzo (GRTK), Seihoku Kanzo (GRSEK) and Sinkyo kanzo (GRSK) were used as GR specimens. Anti-GRSK serum was elicited from rabbits immunized with GRSK fragments. The presence of common proteins as specific antigens of GR was first established by Western blot analysis of extracts of GRSK, GRSEK or GRTK using anti-GRSK. The specific antigens were applied to develop an ELISA for the assay of GR extract. Anti-GRSK was put in competition with a sample or standard GR extract and immobilized GRSK components in microtiter plate wells. The proportion of antibody binding to the solid-phase GRSK component was detected using an enzyme-labeled second antibody. The ELISA method was specific to GRSK extract and showed low sensitivity for the assay of GRTK extract. The technique of selected antibody enzyme immunoassay (SAEIA) was applied to develop a sensitive general assay method. Solid-phase GRTK extract, rather than immobilized GRSK extract, was used in the SAEIA. The SAEIA possessed the same quantitative working range of between 1 and 100 micrograms/ml for the assay of each extract of GRTK, GRSK and GRSEK. The SAEIA was successful in the detection and quantitative measurement of GR component contents in Chinese traditional medicines.