Determination of metolcarb residues by a biotin–streptavidin-amplified enzyme-linked immunosorbent assay in vegetables and edible fungus

An effective biotin–streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) to rapidly detect metolcarb (MTMC) residues is reported. Nonspecific adsorption was minimized by using 0.5% skimmed milk powder as a blocking buffer and 0.5% bovine serum albumin/phosphate-buffered saline (PBS) as a buffer for streptavidin–horse radish peroxidase conjugates. The established method is four times sensitive than direct competitive enzyme-linked immunosorbent assay, with the IC₅₀ of 10.0?ng?mL^?1. The samples were prepared from mustard, cucumber and mushroom with simple extraction and dilution methods, including use of PBS without concentration or cleanup steps. The samples prepared from spinach and shiitake were quantified after 2-fold methanol extraction and 20-fold dilution with 2.0% fish glutin/PBS. Good accuracy and precision were obtained with mean recoveries between 80.0% and 95.6%, and mean coefficients of variation below 12.1%. A good correlation (R^2?=?0.9931) between the BA-ELISA and high-performance liquid chromatography was observed for MTMC analysis in different samples. This method could be potentially useful for high-throughput food inspection.     

Development of a highly sensitive biotin–streptavidin enzyme-linked immunosorbent assay for detecting diethyl phthalate based on a specific polyclonal antibody

An indirect competitive biotin–streptavidin enzyme-linked immunosorbent assay (BA-ELISA) has been established for the determination of diethyl phthalate (DEP) in wine samples. A highly sensitive and specific polyclonal antibody (pAb-DEP) targeting DEP was prepared. Under optimized conditions, good linearity was achieved within a range of 0.021–9.512 μg·L^?1. The limit of detection (IC₁₀) was 0.0079 μg·L^?1 and the median inhibitory concentration (IC₅₀) was 0.443 μg·L^?1. Besides, the BA-ELISA was highly selective, with low cross-reactivity values with DEP analogs (below 5%). Finally, the concentrations of DEP in wine samples ranged from 5.93 μg·L^?1 to 59.15 μg·L^?1 by BA-ELISA. Satisfactory recoveries (89.19–112.33%) and variation coefficient values (5.81–9.43%) were successfully obtained. The consistency between the results of BA-ELISA and gas chromatography–mass spectrometry (GC–MS) was 97.48%, which further confirmed that the proposed BA-ELISA immunoassay is reliable, rapid, sensitive, and accurate for monitoring DEP in wine samples.     

Rapid detection of zearalenone and its metabolite in corn flour with the immunochromatographic test strip

Zearalenone (ZEN) is a mycotoxin produced by fungal species which are widespread in nature and commonly contaminate many cereal grains, such as wheat, maize, corn, barley, and other cereal grains. An immunochromatographic (ICA) test strip was developed for the rapid simultaneous detection of ZEN and its metabolite in corn flour samples. For the ICA test, antigen (ZEN-BSA) and goat anti-mouse IgG were, respectively, drawn on nitrocellulose membrane as control line and test line. Under the optimized condition, the cut-off limits of test strips for ZEN, α-ZAL, and β-ZAL were found to be 0.5?ng/mL in 0.01?M PBS and 50?ng/mL in corn flour samples, meanwhile α-ZOL, β-ZOL, and ZEA were found to be 1?ng/mL in 0.01?M PBS and 75?ng/mL in corn flour samples. Our data indicate that the ICA is sensitive, rapid, cost-effective, and specific on-site screening for ZEN and its metabolite.     

Residues determination of carbofuran in vegetables based on sensitive time-resolved fluorescence immunoassay

Using direct competitive time-resolved fluorescence immunoassay (TRFIA), a rapid, highly selective and sensitive method was developed for the determination of carbofuran residues in lettuce and carrot. The method was based on a direct competitive immunoassay using europium-labelled anti-carbofuran monoclonal antibody and carbofuran-ovalbumin as coated antigen. The sensitivity, estimated as the I ₅₀ value, was 34.54 ng/mL, with a detection limit (I ₁₀) of 1.94 ng/mL and a practical working range between 1.33 and 341.6 ng/mL. The average recoveries of carbofuran from lettuce and carrot were 81.26–108.0% and 113.9–124.8%, respectively. Eventually, confirmation test between TRFIA and enzyme-linked immunoassay (ELISA) was performed. It confirmed that the results given by the TRFIA method were in agreement with those of the ELISA method.     

Development of a monoclonal antibody assay and immunochromatographic test strip for the detection of amikacin residues in milk and eggs

An amikacin-sensitive monoclonal antibody (MAb) assay and immunochromatographic test strip were developed and applied for the detection of amikacin residues in bovine milk and chicken eggs. The immunoassay was specific to amikacin and showed no cross-reactivity with other aminoglycosides. The half maximum inhibitory concentration (IC₅₀) of the assay was 0.65?ng/mL and the results were obtained within 90?min. Recoveries from spiked food matrices were within the range of 73.55–84.61% for bovine milk and 73.70–105.75% for whole egg. The strip test results were obtained within 10?min and showed a visual detection limit of 5.0?ng/mL for both food matrices. These results show that the MAb immunoassay and strip test developed in this study are very specific to amikacin and sufficiently sensitive for detection and routine monitoring of amikacin residues in food.     

DEVELOPMENT OF A SENSITIVE BIOTIN–AVIDIN AMPLIFIED ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETERMINATION OF KETAMINE IN BIOLOGICAL SAMPLES

An effective biotin–avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) was developed to determine ketamine in biological samples. A conjugate of ketamine and ovalbumin (OVA) was used for immunization to produce polyclonal antibody. The conjugate of ketamine and bovine serum albumin (BSA) with polyclonal antibody was calculated to have an affinity constant (K_aff) of 3.30 × 10⁸(mol/L)^?1. The linear range of ketamine was 0.1–1000 µg/L with recoveries from 89.6% to 99.9% in spiked sample analysis. The detection limit of ketamine was 0.03 µg/L, which is more sensitive than that of the traditional ELISA. The results obtained by BA-ELISA agreed well with that of the traditional ELISA, with a correlation coefficient of 0.98.     

Development of an indirect competitive enzyme-linked immunosorbent assay and immunochromatographic assay for hydrocortisone residues in milk

An anti-hydrocortisone (HDS) monoclonal antibody, 2G8, based on a HDS succinic anhydride derivative hapten, was prepared and used to develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic assay for the detection of HDS in milk samples. The half inhibitory concentration (IC₅₀) of the antibody was 0.095?ng/mL, its limit of detection was 0.013?ng/mL, its linear range of detection was 0.026–0.356?ng/mL, and its cross-reactivity with HDS analogs was <5%. In spiked samples and a recovery test, the recovery rates ranged from 92% to 98.5%, indicating the suitability of this ic-ELISA for the analysis of HDS in milk. The immunochromatographic strip had a cutoff value of 2?ng/mL in milk and could be used for the semiquantitative analysis of HDS. When milk samples were added to the sample pad of the strip, a bright test line indicated <0.2?ng/mL HDS, a weak test line indicated 0.2–2?ng/mL HDS, and no test line indicated ≥2?ng/mL HDS. Analysis of HDS in milk samples showed that results acquired by the immunochromatographic assay agreed well with results acquired by ic-ELISA. Thus, the ic-ELISA and strip assay developed in this study rapidly and sensitively detect HDS residues in milk samples.     

BA-ELISA检测谷胱甘胱转移酶对肝癌的诊断意义

应用抗总的谷胱甘肽转移酶(EC 2.5.1.18 GSTs)的抗体,建立了生物素-亲和素-酶联免疫吸附法(BA-ELISA),并用以检测肿瘤病人血清中总GSTs浓度.本法非常敏感,其最低检出浓度可达0.05ng/ml,在0.05～6.0ng/ml的范围内,血清中总GSTs浓度和其相应之光密度值呈很好的直线关系.批内和批间的变异系数分别为8.2％和12.0％.肝癌、肝炎、消化道肿瘤、良性疾病患者血清总GSTs浓度分别为3.29±2.64,0.83±0.71,0.57±0.47和0.58±0.51ng/ml(mean±SD),而正常对照组仅为0.33±0.29ng/ml.肝癌病人血清总GSTs浓度显著高于其他疾病患者和正常人,总GSTs可望成为肝癌诊断的一个肿瘤标志。     

Development and validation of an immunochromatographic test strip for rapid detection of doxycycline residues in swine muscle and liver

A one-step lateral flow immunochromatographic technique using colloidal gold-labelled monoclonal antibody (Mab) was developed for the rapid detection of doxycycline (DOX) residues in swine tissues. For this purpose, a Mab against DOX named as 2.3/3A6 with low cross-reactivity (CR) to other tetracyclines (TCs) was produced. The sensitivity of the test strip was as low as 7 ng/mL for DOX and the half maximal inhibitory concentration (IC₅₀) was calculated to be 22±2 ng/mL by relative optical density. The test can be completed within 10 min and the detection limit was 20 ng/mL in unaided visual assessment. Recoveries in samples spiked with DOX at concentrations of 20, 40 and 80 ng/g, were demonstrated to be from 81% to 95% for muscle samples and from 81% to 92% for liver samples. The intra-assay and inter-assay coefficients of variation (CVs) ranged from 3% to 6% and from 4% to 8%, respectively. Comparing with high performance liquid chromatography (HPLC) method in sensitivity and accuracy for the detection of DOX in swine tissues, the test strip showed good agreement. Therefore, the test strip is suitable for rapid and reliable determination of DOX residues in swine tissues semi-quantitatively or qualitatively.     

人血清癌胚抗原的生物素-亲和素ELISA检测方法的建立

目的:建立检测血清癌胚抗原(CEA)的高灵敏度生物素-亲和素酶联免疫检测(BA-ELISA)方法。方法:亲和层析纯化得到CEA,免疫新西兰家兔制备多克隆抗体。将得到的抗体连接生物素和辣根过氧化物酶,在生物素化BSA包被的96微孔板上建立生物素-亲和素ELISA(BA-ELISA)。对这一体系的线性、灵敏度、特异度、稳定性、回收率等参数进行鉴定,并和常规ELISA、放射免疫检测以及化学发光法相比较检测了临床标本中的CEA浓度。结果:线性检测范围为(0.42～50)U/mL,检测结果表明,体系稳定性良好;灵敏度为0.42 U/mL,批内、批间差异均小于10.0%。该研究建立的方法与常规ELISA对比差异有统计学意义,与放射免疫检测对比差别无统计学意义。与化学发光法相比较,回归方程为:y=0.04825+0.99674x,r=0.994,差异无统计学意义。结论:建立的CEA的BA-ELISA检测方法易于操作、灵敏度高、价格低廉,适合应用于临床检测。     

Development of an immunochromatographic assay for rapid detection of clorprenaline in pig urine

A clorprenaline (CLP) monoclonal antibody, 4G1, raised against the CLP-hapten was successfully prepared for the development of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic assays for the detection of CLP in pig urine samples. The half maximal inhibitory concentration of the antibody was 0.435?ng/mL, and the limit of detection was 0.104?ng/mL. The linear range was 0.104–1.818?ng/mL, and cross-reactivity with the CLP analogue was <5%. In the spiked sample and recovery test, the recovery ranged from 81.8% to 100.5%, indicating that the ic-ELISA was suitable for CLP analysis in pig urine. The critical value of the immunochromatographic strip in pig urine was 10?ng/mL and could be used for semi-quantitative analysis of CLP. Thus, this immunochromatographic strip was suitable for quickly and sensitively detecting CLP residues in pig urine samples.     

A rabbit monoclonal antibody-based sensitive competitive indirect enzyme-linked immunoassay for rapid detection of chloramphenicol residue

A sensitive competitive indirect enzyme-linked immunoassay (ciELISA) based on a rabbit monoclonal antibody (RabMAb) against chloramphenicol (CAP) has been developed and validated in this study. After optimisation of several key physicochemical factors, such as Tween-20 percentage, pH value and ionic strength, the immunoassay showed excellent performance within the linear range of 0.18–6.37 ng mL^?1, with the 50% inhibition concentration (IC₅₀) of 1.06 ng mL^?1 and the limit of detection (LOD) of 0.1 ng mL^?1. In addition, the cross-reactivities of RabMAb towards chloramphenicol succinate, florfenicol and thiamphenicol were 2.09, 12.45 and 18.10%, respectively. Finally, the developed method was applied in spiked swine urine, milk and honey samples, with recoveries ranging from 71.03 to 109.62%. The result demonstrated that the developed immunoassay is a valuable method for screening and quantitation of CAP residues in real samples.     

An ultrasensitive chemiluminescent ELISA for determination of chloramphenicol in milk,milk powder,honey, eggs and chicken muscle

A competitive, direct, chemiluminescent enzyme-linked immunosorbent assay (CL-ELISA) for chloramphenicol (CAP) residues in milk, milk powder, honey, eggs and chicken muscle has been developed. The method gave a detection limit of 0.7 ng L^?1 and a linear range of 2.1–92.4 ng L^?1, with the IC₅₀ of 13.6 ng L^?1 under optimal conditions, dramatically better than any previously reported ELISA method for CAP detection. Spiked at levels of 5–60 ng L^?1 in different food samples, recoveries were in the range of 72.1–116.0%, with coefficient of variations of 4.2–20.2%. In a study of incurred residues, the chicken muscle samples diluted 5-, 10- and 20-fold, results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector and traditional ELISA. The developed CL-ELISA method is, therefore, suitable for rapid screening trace CAP residues in food samples.     

Development of a specific monoclonal antibody assay and a rapid testing strip for the detection of apramycin residues in food samples

A specific monoclonal antibody (MAb) against apramycin (AP) was produced and used to develop an indirect competitive enzyme-linked immunosorbent assay (idcELISA) and a rapid testing strip for the detection of AP residues in foods. MAb exhibited negligible cross-reactivity with other aminoglycosides. Under optimized conditions in 0.01 M PBS, the half maximum inhibitory concentration (IC₅₀) of MAb was 0.41?ng/ml with a limit of detection (LOD) of 0.15?ng/ml. The ELISA results were obtained within 90?min. The mean recoveries from all the spiked food samples were within the range of 79.02–105.49%, with coefficients of variation in the range of 2.21–11.4%. The strip test results obtained within 5?min had visual LODs in the range 2.5–5?µg/kg (ng/ml) for all food samples tested. Therefore, the developed strip test represents a fast and convenient detection method of AP residues in foods.     

Development of a fluorescence-linked immunoassay based on quantum dots for fenvalerate

The glutathione-coated CdTe quantum dots (QDs) were synthesized in a paper. After purification, the QDs were coupled with monoclonal antibody to fight against fenvalerate. The conjugates had the same emission wavelength as that of QDs. The excitation wavelength and emission wavelength of the conjugates were determined to obtain the highest signal-to-noise ratio. After the antibody concentration optimization, the fluorescence-linked immunoassay method was developed. The method used the QDs as the signal to quantify the fenvalerate. Compared with the enzyme-linked immunosorbent assay (ELISA), it saved more than 1 h and decreased the false-positive rate using the specified emission wavelength of QDs. The 50% inhibitory concentration (IC₅₀) of the method was 0.28 µg mL^?1. The detection limit was 25 ng mL^?1 and the linear range was 60 ng mL^?1–3.83 µg mL^?1. Via preliminary application, fenvalerate residues in spiked samples were determined. The recovery of fenvalerate in water samples ranged from 84.5% to 96.2% and that in vegetables ranged from 72.5% to 125.7%. It was a rapid detection of the fenvalerate residue in environment and vegetables.     

Preparation of an anti-dexamethasone monoclonal antibody and its use in development of a colloidal gold immunoassay

A lateral flow colloidal gold (CG) immunoassay strip has been developed for detection of dexamethasone (DEX) residues in milk samples. For this purpose, an anti-DEX monoclonal antibody (McAb), based on a DEX succinic anhydride derivative hapten, was prepared and characterized. The McAb showed a high specificity to DEX, the half inhibitory concentration of the antibody was 0.095?ng/mL, its limit of detection (LOD) was 0.017?ng/mL, and its linear range of detection was 0.034–0.265?ng/mL. The developed CG immunoassay had a visual cut-off value of 0.3?ng/mL in phosphate buffered saline (PBS) and 0.5?ng/mL in milk samples. Each test requires 10 min. Analysis of DEX in milk indicated that the results of strip assay had a strong agreement with indirect competitive enzyme-linked immunosorbent assay. Therefore, the CG immunoassay is a sensitive screening method for semi-quantitative and qualitative detection of DEX residues in milk samples.     

Antibody for the development of specific immunoassays to detect nadifloxacin in chicken muscles

A convenient and specific competitive enzyme-linked immunosorbent assay (ELISA) for nadifloxacin (NDF) was developed. The modified 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) method was employed to synthesise the artificial antigen of NDF. The indirect competitive ELISA developed to detect NDF has a concentration at 50% inhibition (IC₅₀) of 0.72 ng/mL and a limit of detection of 0.04 ng/mL. This ELISA kit could be used as a screening method to detect and control the illegal content of NDF in food products. In this study, the kit was applied to NDF detection in chicken muscle and produced a satisfactory result with recovery rates from samples spiked with NDF of 92% (10 ng/mL) and 101% (50 ng/mL).     

Development of chemiluminescent enzyme immunoassay for the determination of aflatoxin M1 in milk products

In this study, an indirect competitive chemiluminescent enzyme immunoassay (ic-CLEIA) for determining aflatoxin M₁ (AFM₁) in milk products has been developed. A luminol–hydrogen peroxide chemiluminescence system catalysed by horseradish peroxidase (HRP) was used as the signal detecting system. The effects of several factors, including concentration and pH of phosphate buffer, dilution ratio of antibody and antigen and other relevant variables on the immunoassay, were studied and optimised by single-factor experiments. The developed method presented an IC₅₀ of 0.05 ng/mL, a detection limit of 0.01 ng/mL and a linear range from 0.018 to 0.13 ng/mL. This method has been successfully applied to the evaluation of AFM₁ in milk products, the recoveries ranging from 71.9% to 109.0%. A good correlation with the commercial available ELISA kit for AFM₁ (r = 0.9978) was obtained, indicating that the ic-CLEIA method developed can be used to determine AFM₁ in real samples.     

Development of a lateral flow immunoassay for the detection of total malachite green residues in fish tissues

We developed a sensitive lateral flow immunoassay (LFI) for the detection of total malachite green (MG) residues in fish tissues. LFI was based on a colloidal gold nanoparticle-labeled monoclonal antibody against MG , which was generated by fast immunization of carboxymalachite green-bovine serum albumin. The half-maximum inhibition concentration (IC₅₀) of MG monoclonal antibody (MG mAb) was 0.057 ng/ml with a linear range of 0.020–0.162 ng/ml. Following the optimization of LFI, a qualitatively visual limit of detection of 1 ng/ml and a semi-quantitatively limit of detection of 0.418 ng/ml (IC₅₀ of the calibration curve) were obtained for total MG residues in 8 min. Cross-reactivity with most MG analogs was negligible, expect with crystal violet (CV; 78.6% cross-reactivity). LFI is a novel and sensitive assay based on MG mAb that has applications for the rapid detection of total MG and CV in fish products.     

Development of chemiluminescent enzyme immunoassay for the determination of malachite green in seafood

An indirect competitive chemiluminescent enzyme immunoassay method was developed for screening malachite green (MG). Assay conditions, including the concentration of coating, antibody dilution, incubation time, dilution buffer, ionic strength and pH, were optimised. Under the optimised conditions, coating antigen concentration was 125 ng/mL; and dilution fold of antibody was 40,000. The 50% inhibition values of 0.22 ng/mL for MG were achieved with a limit of detection of 0.01 ng/mL, the linear range was from 0.03 ng/mL to 3.27 ng/mL. The assay showed a little cross-reactivity of 3.4%, 2.7% and 1.0% with methylene blue, brilliant green and crystal violet, respectively, and negligible cross-reactivity with other analogues of MG. The developed method has been used to quantify MG in seafood samples. The recovery of MG ranged from 82.43% to 108.0% at four concentrations (0.1, 1, 3 and 5 ng/mL), and the coefficients of variation were less than 13%. A comparison results between the high-performance liquid chromatography and the developed assay showed good relativity. The results indicated that the assay was a sensitive and stable method for screening MG.