Quantitation ofE. coli protein impurities in recombinant human interferon-γ

A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.     

Flow analysis methods for the direct ultra-violet spectrophotometric measurement of nitrate and total nitrogen in freshwaters

Allergy to tree nuts represents an acute health problem. Sensitized people can be inadvertently exposed to hidden allergens resulting from cross-contamination of foods. For this reason, reliable and highly sensitive analytical methods are needed to be developed for control and labeling of food ingredients and products. In the present paper we have proposed a new allergen specific sandwich-ELISA for hazelnut operated in optical and electrochemical modes. The ELISA was based on chicken egg yolk antibodies raised against a major hazelnut allergen, Cor a 9. The developed ELISA has a limit of detection in phosphate buffer of 4 ng mL(-1). No significant cross-reactivity with peanut, wheat or other food ingredients has been detected. Extracts of blank control cookies did not show any false positive response and the limit of detection in cookies was estimated to be 0.1 μg of hazelnut protein per g of food (0.1 ppm). The ELISA protocol was successfully adapted to operate in electrochemical mode and it was applied for the detection of hazelnut traces in cookies.     

Synthesis of haptens of organophosphorus pesticides and development of immunoassays for fenitrothion

A simple synthetic method for haptens of organophosphorus (OP) pesticides with a spacer arm (aminocarboxylic acid) attached at the pesticide thiophosphate group was developed. While the previous synthetic approach for this type of haptens requires seven steps, the present method involves only two steps. Using this method, five haptens of fenitrothion were synthesized and two of them were conjugated to proteins to be used as immunogens for production of polyclonal antibodies. Using the antibodies and a coating antigen, an indirect competitive enzyme-linked immunosorbent assay (ELISA) for fenitrothion was developed, which showed an IC₅₀ of 3.7 ng/mL with a detection limit of 0.5 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 5.6 ng/mL with a detection limit of 0.4 ng/mL. The antibodies in both assays showed negligible cross-reactivity with other OP pesticides except with the insecticide parathion-methyl only in the direct ELISA. Recoveries of fenitrothion from fortified lettuce and rice samples ranged 84-116 and 100-121%, respectively.     

Immunochemical-based method for detection of hazelnut proteins in processed foods

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.     

Determination of egg proteins in snack food and noodles

Egg is one of the 5 major allergenic foods that are responsible for more than 3/4 of food allergies in children. Food-allergic responses can be controlled by avoidance of the offending foods. The applicability of a commercial enzyme-linked immunosorbent assay (ELISA) kit for the detection of egg in food products such as cookies, crackers, pretzels, salad dressings, and raw and cooked noodles was evaluated. A preliminary evaluation of an antibody-based biosensor was also performed. A National Institute of Standards and Technology (NIST) whole dried egg powder reference material, SRM 8415, was used as a standard. A homogeneous and stable aqueous egg suspension was prepared for the evaluation of the performance of the Veratox for Egg Allergen Test (Neogen Corp., Lansing, MI). This test does not detect egg yolk proteins. Each gram of the aqueous dried egg suspension contained 643 microg whole dried egg, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. When cookies, crackers, salad dressings, noodles, and ice cream were spiked at a level of 24 mg/kg SRM 8415, recoveries for whole egg averaged about 28%. All foods containing egg as indicated on the ingredient label were found positive by the Veratox test. No false positives occurred in samples that did not contain eggs. Similar results were obtained using the Naval Research Laboratory (NRL) array biosensor, an evanescent wave fluoroimmunosensor. Results for cooked noodles showed that they contained <1% of the egg found in uncooked noodles. A comparison of extracts from cooked and uncooked noodles by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in protein profiles. The boiling of the noodles could have reduced the immunoreactivity of the egg proteins to the antibodies used in the kit or rendered the egg proteins nonextractable.     

Development of an Enzyme-Linked Immunosorbent Assay (ELISA) for the Herbicide Propanil

Although the extensively used postemergence herbicide propanil itself is of low acute toxicity in mammals, it raises environmental concerns due to its effect on aquatic organisms and other adverse impacts. Therefore, in order to obtain a rapid analytical method for this pesticide, an indirect enzyme-linked immunosorbent assay (ELISA) has been developed. Antibodies obtained against a conjugate of 3,4-dichloroaniline coupled to succinylated proteins were tested in hapten-homologous and heterologous indirect ELISA formats using various N -(dichlorophenyl)-succinamic acid derivatives conjugated to carrier proteins as coating antigens. Titers in ELISA were found to be significantly affected by the type and quantity of coating antigen. One of the optimized systems using N -(2,4-dichlorophenyl)-succinamic acid and N -(3,5-dichlorophenyl)-succinamic acid conjugated to ovalbumin allowed serum dilution of 1 : 10,000 and IC 50 values of 2.2 and 2.7 ng/mL for propanil, respectively. The limit of detection (LOD) of the immunoassays is 0.2 ng/mL. Other optimized ELISA systems based on different dichloroaniline-based coating antigens also offered similar sensitivities. The ELISA systems appeared to tolerate methanol and ethanol upto 5% concentration. For confirmatory purposes, the ELISA protocol was compared with a highly sensitive gas chromatographic method coupled with mass spectrometric detection (GC-MS). Spiked propanil content was detected both by ELISA and GC-MS in methanolic rice extract. Detection sensitivities of the two analytical systems appeared to closely correlate with each other in the range of 10-90 ng/mL (0.02-0.18 µg/g), indicating the utility of the immunoanalytical method in detecting propanil content in rice, the main commodity propanil is being applied on.     

A new LC/APCI-MS method for the determination of cholesterol oxidation products in food

Cholesterol oxidation products (COPs) can be formed in the body or in animal foods from cholesterol during food processing. A new method for the extraction and quantification of cholesterol, 7-ketocholesterol, cholestane-3beta-5alpha-6beta-triol, 25-hydroxycholesterol, 5,6alpha-epoxycholesterol, and 7beta-hydroxycholesterol by means of reversed-phase LC/atmospheric pressure chemical ionization mass spectrometry is presented. A baseline separation of all COPs was achieved, allowing a separate quantification also for isobaric compounds. The limits of detection were 15-30 ng/mL, quantification was performed from 100 ng/mL to 10 microg/mL with RSD < 2%. The method was applied successfully to the determination of cholesterol and COPs in processed foods such as pork, beef, chicken, and egg.     

Detection of soy proteins in processed foods: literature overview and new experimental work

Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 microg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 microg/mL, corresponding to 0.4 microg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.     

高效液相色谱法与酶联免疫试剂盒测定海水中痕量氯霉素

详细报道了海水样品中氯霉素的高效液相色谱检测方法及酶联免疫试剂盒检测方法。应用高效液相色谱法测定海水中氯霉素,方法的线性范围为5～50ng,检出限为0．064ng,回收率为96．2％-118．4％,相对标准偏差小于8．9％。酶联免疫试剂盒测定海水中的氯霉素背景值偏高,在加标浓度为10ng／mL时回收率为101．0％,相对标准偏差为8．32％。     

Determination of proteins at nanogram levels by their quenching effect on the chemiluminscence reaction between luminol and hydrogen peroxide with manganese-tetrasulfonatophthalocyanine as a new catalyst

The manganese-tetrasulfonatophthalocyanine (MnTSPc) catalyzed luminol-hydrogen peroxide chemiluminescence (CL) systems can be quenched in the presence of proteins. A highly sensitive CL quenching method has been developed for the determination of proteins. Under optimum conditions, the linear ranges of the calibration curves were 0.1-20 microg/mL for human serum albumin (HSA), 0.2-20 microg/mL for human gamma-IgG, and 0.5-50 microg/mL for the bovine serum albumin (BSA) with the corresponding detection limits were 1.9 ng/mL, 2.7 ng/mL, and 3.4 ng/mL. The method has been applied to the analysis of total proteins in human serum samples and the results were in good agreement with clinical data provided.     

Comparison of enzyme-linked immunosorbent assay with liquid chromatography-tandem mass spectrometry for the determination of diethylstilbesterol residues in chicken and liver tissues

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative detection of the diethylstilbesterol (DES). Polyclonal rabbit antisera, raised against protein conjugate diethylstilbesterol-mono-caroxyl-propyl-ethyl-bovine-serum-albumin (DES-MCPE-BSA), were utilized in immobilized antibody-based and competitive immunoassays. Assay conditions, including concentrations of antisera and horseradish peroxidase, (HRP)-DES, were optimized. The effects of incubation time, surfactant concentration, ionic strength and pH of the medium were also investigated. The typical calibration curve gave an average IC(50) value of 2.4 ng/mL, calibration range from 0.2 to 30.5 ng/mL and a detection limit of 0.07 ng/mL. The specificity of the assay was tested against DES structurally related compounds, and the assay proved highly selective for DES. Assay performance was validated using spiked chicken meat and liver tissue samples. Moreover, it was compared with liquid chromatography-tandem mass spectrometry. The ion pair for quantification of DES was m/z 267.4/251.4, and the linear equation of DES was y = 0.1033x + 0.0126 (r = 0.9960). The two analytical methods can be applied to monitor DES and other steroid residues in foods.     

Determination of 4- n -nonylphenol in water by enzyme immunoassay

A range of monoclonal antibody-based competitive immunoassays in the format of microtitre plate ELISA and dipstick tests for quantitative and semi-quantitative detection of 4- n -nonylphenol in water was developed. A simple visual dipstick test was based on changing of spot colour from green to brown in the presence of 4- n -nonylphenol at concentrations within the range 10-100 ng mL(-1). Two different detection systems were used for quantitative immunoassay. Application of enhanced chemiluminescence (ECL) resulted in an increase of the sensitivity of ELISA when compared to conventional colorimetric detection. Thus a detection limit of 0.06 ng mL(-1 )of 4- n -nonylphenol was achieved with IC(50) 2.0 ng mL(-1). The tests developed were applied to natural and spiked water samples.     

Comparsion of an immunochromatographic strip with ELISA for simultaneous detection of thiamphenicol,florfenicol and chloramphenicol in food samples

Rapid and sensitive indirect competitive enzyme‐linked immunosorbent assays (ic‐ELISA) and gold nanoparticle immunochromatographic strip tests were developed to detect thiamphenicol (TAP), florfenicol (FF) and chloramphenicol (CAP) in milk and honey samples. The generic monoclonal antibody for TAP, FF and CAP was prepared based on a hapten [D ‐threo‐1‐(4‐aminophenyl)‐2‐ dichloroacetylamino‐1,3‐propanediol], and the haptenwas linked to a carrier protein using the diazotization method. After the optimization of several parameters (coating, pH, sodium chloride content and methanol content), the ic‐ELISA was established. The quantitative working range for TAP was 0.11–1.36 ng/mL, with an IC₅₀ of 0.39 ng/mL. The optimized ELISA showed cross‐reactivity to CAP (300%) and FF (15.6%), with IC₅₀ values of 0.13 and 2.5 ng/mL, respectively. The analytical recovery of TAP, FF and CAP in milk and honey samples in the ic‐ELISA ranged from 81.2 to 112.9%. Based on this monoclonal antibody, a rapid and sensitive immunochromatographic test strip was also developed. This strip had a detection limit of 1 ng/mL for TAP, FF and CAP in milk and honey samples. Moreover, the test was completed within 10 min. Our results showed that the proposed ic‐ELISA and immunochromatographic test strip method are highly useful screening tools for TAP, FF and CAP detection in milk and honey samples. Copyright © 2015 John Wiley & Sons, Ltd.     

Determination of proteins at nanogram levels by their quenching effect on large particle scattering of colloidal silver chloride

A novel quantitative method for the determination of proteins in aqueous solutions has been based on the quenching of the resonance scattering light of colloidal silver chloride in the presence of proteins. The detection limits for eight kinds of proteins (BSA, HSA, egg albumin, human gamma-IgG,alpha-chymotrypsin, E. Coli. alpsase, myoglobin, alpha-casein) were at about 8 ng/mL; the linear ranges of the calibration curves were 10-400 ng/mL under optimal conditions,except for human gamma-IgG (20-400 ng/mL), myoglobin (10-300 ng/mL), and alpha-casein (10-300 ng/mL). Three wavelengths (398 nm, 475 nm, 499 nm) were all suitable for the determination and any acidity from pH 3.0 to pH 9.0 could be chosen. A few non-protein substances at high concentration levels interfered with this method, but this problem could simply be overcome by diluting the samples before the assay. Mechanism studies showed that the quenching effect of proteins on the scattering light of colloidal silver chloride was mainly due to the coagulation of AgCl particles retarded by protein. The method was employed for the determination of total protein in human serum with satisfactory results.     

A monoclonal antibody-based ELISA for multiresidue determination of avermectins in milk

Due to the widespread use and potential toxicity of avermectins (AVMs), multi-residue monitoring of AVMs in edible tissues, especially in milk, has become increasingly important. With the aim of developing a broad-selective immunoassay for AVMs, a broad-specific monoclonal antibody (Mab) was raised. Based on this Mab, a homologous indirect enzyme-linked immunosorbent assay (ELISA) for the rapid detection of AVMs in milk was developed. Under the optimized conditions, the IC?? values in assay buffer were estimated to be 3.05 ng/mL for abamectin, 13.10 ng/mL for ivermectin, 38.96 ng/mL for eprinomectin, 61.00 ng/mL for doramectin, 14.38 ng/mL for emamectin benzoate. Detection capability (CCβ) of the ELISA was less than 5 ng/mL and 2 ng/mL in milk samples prepared by simple dilution and solvent extraction, respectively. The optimized ELISA was used to quantify AVMs in milk samples spiked at different amounts. The mean recovery and coefficient of variation (CV) were 95.90% and 15.42%, respectively. The Mab-based ELISA achieved a great improvement in AVMs detection. Results proved this broad-selective ELISA would be useful for the multi-residue determination of AVMs in milk without purification process.     

基于表面等离子体共振技术用鸡蛋黄抗体 IgY测定人血清中转铁蛋白

采用表面等离子体共振(SPR)方法, 用鸡蛋黄抗体(IgY)取代传统免疫检测中哺乳动物抗体IgG作为识别分子偶联于CM5传感芯片上, 对人血清中的转铁蛋白进行了检测. 考察了IgY在传感芯片上的偶联条件及芯片的再生条件. 结果表明, 在pH=4.0, IgY浓度为100 μg/mL, 流速为5 μL/min的最佳偶联条件下, SPR响应信号和转铁蛋白浓度在50~500 ng/mL范围内具有良好的线性关系, 检出限为39.56 ng/mL, 对人血清样品检测的日间变异系数<8%, 日内变异系数<5%, 平均回收率为86.22% ~94.51%.     

Monoclonal antibody-based enzyme-linked immunosorbent assays for the detection of the organophosphorus insecticide diazinon

Four haptens of the organophosphorus (OP) insecticide diazinon were synthesized to develop enzyme-linked immunosorbent assays (ELISAs) for this pesticide. One of them was conjugated to KLH to be used as the immunogen for production of monoclonal antibodies. By using the antibodies and a coating antigen, an indirect competitive ELISA was developed, which showed an IC₅₀ of 4.0 ng/mL with a detection limit of 0.7 ng/mL. A direct competitive ELISA using an enzyme tracer was also developed, which showed an IC₅₀ of 6.0 ng/mL with a detection limit of 0.9 ng/mL. The antibodies in both assays showed negligible cross-reactivity with metabolites of diazinon and other OP pesticides. Recovery of diazinon from fortified lettuce and rice samples was satisfactory except at the fortified concentration of 100 ppb.     

Study of Immunoassay Methods for Recombinant Human Erythropoietin （rhEPO） Using Competitive ELISA

Two different immunoassay methods,competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) and amplificative competitive indirect ELISA(ACI-ELISA) using biotin-avidin complex system were studied to detect rhEPO.The linear ranges were 50-20000ng/mL and 10-50000ng/mL for CI-ELISA and ACI-ELISA,respectively.The low detection limits of CI-ELISA and ACI-ELISA were 62.8ng/mL and 8.5ng/mL,respectively.     

Application of a new anti-zearalenone monoclonal antibody in different immunoassay formats

Monoclonal antibodies against zearalenone (ZEA) were raised in mice according to the hybridoma technology and applied in different immunochemical techniques. More specifically, three formats based on the competitive direct enzyme immunoassay principle were developed: an enzyme-linked immunosorbent assay (ELISA), a flow-through gel-based immunoassay column and a flow-through membrane-based immunoassay. In ELISA, the 50% inhibitory concentration (IC50) was 0.8 ng/mL, and the limit of detection for ZEA standard solutions was 0.1 ng/mL. The antibodies showed a high ZEA (100%) and α-zearalenol (α-ZOL) (69%) recognition, while cross-reactivities with α-zearalanol, zearalanone, β-zearalenol and β- zearalanol were 42%, 22%, <1% and <1%, respectively. For standard solutions, a cut-off level at 10 ng/mL could be established for the gel- and membrane-based enzyme immunoassays. Assay time of both non-instrumental tests was 25 min for 10 samples. By including a simple sample extraction procedure, the methods were applied to wheat with IC50s in ELISA of 80 and 120 μg/kg (dilution up to 5% and 15% (v/v) of wheat matrix, respectively). The cut-off level of the gel- and membrane-based immunoassays was established at 100 μg/kg. Potentials and limitations of the developed methods were compared. The possible application for multi-mycotoxin analysis of the ELISA method based on a single monoclonal antibody was investigated. Therefore, principal component analysis and partial least squares regression data modelling were used to separate the immunoassay responses of two cross-reactants (ZEA and α-ZOL).      

Determination of thiabendazole in fruit juices by a new monoclonal enzyme immunoassay

A competitive, indirect enzyme-linked immunosorbent assay (ELISA) for thiabendazole has been developed and applied to the analysis of fruit juices spiked with this fungicide. The immunoassay is based on a new monoclonal antibody derived from a hapten functionalized at the nitrogen atom in the 1-position of the thiabendazole structure. To our knowledge, such a structure has not been previously used to obtain antibodies to thiabendazole. The I50 value and the detection limit of the ELISA for standards were 0.2 and 0.05 ng/mL, respectively. Fruit juices were analyzed by diluting samples in assay buffer, without extraction or cleanup. Samples were not even centrifuged or filtered to remove fruit pulp. Under these conditions, the immunoassay was able to accurately determine thiabendazole down to 1 ng/mL in orange and grapefruit juices, down to 5 ng/mL in banana juice, and down to 20 ng/mL in apple and pear juices. Sensitivity differences of the ELISA were caused by the minimum dilution required by each juice to minimize matrix effects: 1/10 for orange and grapefruit juices, 1/50 for banana juice, and 1/100 for apple and pear juices. In an attempt to further increase the sensitivity of the immunoassay for matrixes showing the strongest interferences, apple and pear juices spiked with thiabendazole at low levels (1-20 ng/mL) were extracted with ethyl acetate before analysis. This simple procedure entailed a significant reduction of matrix effects, which in fact allowed us to determine accurately as low as 5 ng/mL thiabendazole in apple and pear juices. Irrespective of whether samples were analyzed by the direct dilution method or after extraction, the simplicity, sensitivity, and sample throughput of this monoclonal immunoassay makes it a very convenient method for the routine monitoring of thiabendazole residues in fruit juices.