Development and characterisation of an immunoaffinity column for the selective extraction of methandrostenolone from food and feed samples

Methandrostenolone (MA) is used as veterinary medicine for promoting food-producing animal growth. Accumulation of MA in a human body would cause multiple side effects. In this study, an anti-MA monoclonal antibody (mAb)-based immunoaffinity column (IAC) was prepared and its application to the selective extraction of MA from actual samples was evaluated. The employed antibody exhibited good sensitivity and specificity towards MA with an IC₅₀ value of 7.89 ng/mL and less than 1% cross-reactivity towards structurally related compounds. By coupling CNBr-activated Sepharose with the antibody, an IAC was prepared. Two percent methanol and 70% methanol were selected, respectively, as loading and eluting solution by optimisation. The maximum binding capacity of the column for MA reached 4760 ng/mL gel. The average recovery of 50, 250 and 500 ng MA standards from IACs was 88.6% with a relative standard deviation (RSD) among columns of 8.5%. After three times usage, the binding capacity and recovery still remained 93.4% and 81.1%, respectively. To further verify the effect of matrices on the extraction efficiency, the IACs were challenged with MA-fortified animal tissue and feed samples and the recovery were found to be in the range of 78.7%–95.1%.     

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.     

Improved preparation of a chitosan-based immunoaffinity column using antibody against methandrostenolone as ligand

Chitosan is obtained by N-deacetylation of chitin, which is the second most abundant natural biopolymer. In this study, an improved preparation and characterisation of a chitosan-based immunoaffinity chromatography (IAC) column were performed. The immunoaffinity adsorbent was prepared by covalently coupling monoclonal antibody (mAb) against methandrostenolone (MA) to glutaraldehyde cross-linked chitosan (chitosan CL). Scanning electron micrograph of chitosan CL indicated that the shape of the particles was spherical with a diameter range from about 300 to 500 µm. Infrared spectral analysis suggested that the immunoaffinity adsorbent was successfully prepared by coupling antibody with chitosan CL. For chromatographic extraction, 90% methanol was selected as eluant. Under optimum conditions, the maximum binding capacity of the IAC column was 3900 ng MA/mL adsorbent. The average recovery of 50, 250 and 500 ng of MA standards from IAC columns was 96.9% with a relative standard deviation among columns of 1.48%. After eight uses, the extraction recovery of the IAC column remained 82.1%. To further verify the effect of matrices on the extraction efficiency, the IAC columns were challenged with MA-fortified animal tissue and feed samples. Recoveries were 84.9–87.1%, demonstrating the effectiveness of these IAC columns for sample clean-up in MA residue determination.     

Development of an immunochromatographic strip for the rapid detection of 10 β-agonists based on an ultrasensitive monoclonal antibody

A sensitive monoclonal antibody, 3G10, derived from a mouse immunized with an immunogen of salbutamol hapten conjugated to keyhole limpet hemocyanin was developed against β-agonists, with a view to establishing a rapid gold nanoparticle immunochromatographic strip. The antibody displayed a 50% inhibitory concentration (IC₅₀) of 1.04?ng/mL for salbutamol and simultaneously detected nine other β-agonists (clenbuterol, cimatero, brombuterol, mabuterol, terbutaline, clenpenterol, carbuterol, mapenterol, pirbuterol) with uniform cross-reactivity. The immunochromatographic strip had cut-off values of 5?ng/mL for salbutamol and clenbuterol, 20?ng/mL for cimaterol, brombuterol and mabuterol, and 50?ng/mL for terbutaline, clenpenterol, carbuterol, mapenterol and pirbuterol. The collective advantages of the newly developed strip, such as high affinity, sensitivity and rapidity, support its utility in detecting β-agonists from actual biological samples, such as urine.     

ON-LINE IMMUNOAFFINITY EXTRACTION FOLLOWED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND RADIOIMMUNOASSAY FOR A NOVEL RETINOBENZOIC ACID,AM-80, IN HUMAN PLASMA

On-line coupled immunoaffinity chromatography and reversed-phase high performance liquid chromatography (on-line IAC-HPLC) with detection by radioimmunoassay (RIA) was developed for 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthyl)-carbamoyl] benzoic acid (Am-80) in human plasma. A 0.05-mL sample was directly loaded onto an immunoaffinity pre-column packed with immobilized polyclonal antibodies against Am-80. The immunoaffinity extract was then automatically introduced to reversed-phase HPLC column for separation by column switching. The immunoreactivity in the HPLC-eluted fraction was measured by competitive RIA.

The within-assay precision and accuracy of the method were 4.0–10.6% and ?6.0–+8.3%, respectively, and between-assay precision and accuracy were 6.8–11.9% and ?16.0–+9.0%, respectively. The limit of quantitation was 0.5 ng/mL in human plasma. The proposed method yields highly purified analyte in complex matrices, detects it with high sensitivity, not only by radioimmunoassay, but also by other methods, such as mass-spectrometry, and can be used for routine assay of many samples without manual sample extraction and purification.     

Anti‐idiotype and anti‐anti‐idiotype antibodies generated from polyclonal antibodies against aflatoxin b1

Polyclonal anti‐idiotype antibodies (anti‐id, pAb2) for aflatoxin were generated from mice ascites after immunization with affinity‐purified rabbit polyclonal antibody (pAbl) against aflatoxin B₁ (pAFB₁). After affinity chromatography purification, pAb2 were subjected to various analyses, and were used to generate anti‐anti‐id antibodies (pAb3) in the ascites of Balb/c mice. ELISA analyses revealed that the anti‐id antibodies bound specifically to the original pAb1, but not to other types of monoclonal antibodies or normal mouse IgG. The inhibition of binding of AFB₁ to pAb1 by pAb2 was demonstrated in both regular and biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) systems in which AFB₁‐bovine serum albumin (BSA) was coated on to the microtiter plate. The concentrations causing 50% inhibition (ID₅₀) of the binding of pAbl to AFB₁‐BSA by pAb2 were found to be 6.5 and 1.7 μg/assay respectively. Inhibition of the binding of pAb1 to the solid‐phase pAb2 by free AFB₁ (ID₅₀ = 0.63 μ/assay) was also demonstrated in the ELISA. pAb3 were found to have similar characteristics to the original pAb1. In the direct ELISA, the ID₅₀ of binding of AFB₁‐horseradish peroxidase to the solid‐phase pAb3 by AFB₁ was found to be 0.20 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of pAb3 to the solid‐phase AFB₁‐BSA by AFB₁ was found to be 1.84 ng ml^‐1 Analysis of a naturally contaminated corn sample for AFB₁ by the pAb3‐based ELISA gave a result similar to that obtained from the regular pAb1‐based ELISA (19.7 and 21.8 μg kg^‐1 respectively).     

p53蛋白的免疫亲和层析纯化

建立了ｐ５３单克隆抗体ｐＡｂ１８０１的免疫亲和层极法纯化ｐ５３蛋白，所纯化的ｐ５３蛋白经Ｗｅｓｔｅｒｎ Ｂｌｏｔ（ＥＣＬ法）检测证明：用此法从ｐ５３阳性的ＳＷ４８０细胞中分离到ｐ５３蛋白。银染显示ｐＨ２．０甘氨酸缓冲液比ｐＨ２．８的甘氨酸缓冲液洗脱效果好，这种方法的建立将为分离肿瘤细胞中引起ｐ５３蛋白功能失活和研究肿瘤发生机制提供一种有效途径。     

Development of an icELISA and immunochromatographic strip for detection of norfloxacin and its analogs in milk

We developed a monoclonal antibody (mAb) to detect norfloxacin (NOR) using a novel hapten by introducing an amino group via amido linkage into the piperazine ring of NOR. The sensitivity of an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb showed an improved IC₅₀ value from 1.18 to 0.12?ng/mL and the limit of detection from 0.37 to 0.05?ng/mL by using a heterologous coating (pazufloxacin coupled to ovalbumin). The recovery rate for NOR in milk ranged from 77.3% to 117.8%. Cross-reactivity (CR) experiments showed good CR with nine common fluoroquinolone analogs: pefloxacin, lomfloxacin, ciprofloxacin, enrofloxacin, dafloxacin, sparfloxacin, ofloxacin, marbofloxacin and flumequine. Furthermore, based on this antigen–antibody combination, an immunochromatographic strip with cut-off value of 1?ng/mL was developed. Given their good sensitivity and CR, the developed icELISA and immunochromatographic strip could be useful for detection of NOR and its analogs in milk.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of vancomycin in raw milk and animal feed

A highly sensitive monoclonal antibody (mAb) 3H4 against vancomycin (VAN) was prepared. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) were developed based on the mAb. The 50% inhibition concentration (IC₅₀) value and limit of detection (LOD) value of ic-ELISA method for vancomycin were 0.59 and 0.06?ng/mL, and for norvancomycin were 1.51 and 0.13?ng/mL under optimized conditions as pH 7.4, 0.4% (m/v) NaCl, and 5% (v/v) acetonitrile. In lateral-flow ICA, the visual limit of detection (vLOD) value and cut-off values for vancomycin were 1 and 2.5?ng/mL, and for norvancomycin were 5 and 10?ng/mL under optimized conditions as pH 8.6 with 1?mg/mL coating antigen and 1?µg/mL gold nanoparticle-labeled mAb. In raw milk and animal feed samples, recovery rates from ic-ELISA ranged from 89.2% to 121.6%. The vLOD and cut-off value were 5–10?ng/g and 100–200?μg/kg, respectively. Therefore, both methods were sensitive, rapid, and effective for the on-site detection and rapid mass screening of samples.     

Development of an ultrasensitive ic-ELISA and immunochromatographic strip assay for the simultaneous detection of florfenicol and thiamphenicol in eggs

An ultrasensitive monoclonal antibody-based gold nanoparticle immunochromatographic strip assay and indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) were developed to detect florfenicol (FF) and thiamphenicol (TAP) in egg samples. The ic-ELISA, with optimized pH, methanol content and sodium chloride content, exhibited an IC₅₀ value of 0.2?ng/mL for FF and 0.27?ng/mL for TAP, with the working range of 0.05–0.77 and 0.05–1.42?ng/mL, respectively. The optimized ic-ELISA showed negligible cross-reactivity with other phenols and broad-spectrum antibiotics. The recoveries in egg samples using the ic-ELISA ranged from 84% to 115% with a coefficient of variation of less than 5%. Based on this monoclonal antibody, a rapid and ultrasensitive immunochromatographic strip assay was developed with a cutoff value of 1?ng/mL for FF and TAP. Our results indicated that both developed methods were highly useful for screening FF and TAP in eggs.     

Comparison of an Enzyme-Linked Immunosorbent Assay with an Immunochromatographic Assay for Detection of Lincomycin in Milk and Honey

An enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic assay were constructed for the detection of lincomycin (LIN) in both milk and honey samples based on the monoclonal antibody named 5F6. The half-maximum inhibition of ELISA was 0.3?ng/mL after optimizing pH and ionic strength conditions; the limit of detection was 0.07?ng/mL. The cross-reactivity with clindamycin was 0.6%. LIN recovery in spiked milk and honey samples ranged from 84.6% to 115.6% with intra-assay coefficient variations of 1.7–25.4% and inter-assay coefficient variations of 2.7–8.9%. The detection limits were estimated as 2.1?µg/L for milk and 2.1?µg/kg for honey samples. The immunochromatographic assay revealed a LIN cut-off value of 10?ng/mL in PBS, 5?ng/mL in milk, and 120?ng/g in honey, and a visual lower detection limit of 2.5?ng/mL, 1?ng/mL and 30?ng/g in PBS, milk and honey, respectively. The immunochromatographic assay is preferred for large-scale practical application for its simpler pretreatment and satisfied sensitivity compared with ELISA assay.     

Immunoaffinity chromatography for purification of Salbutamol and Clenbuterol followed screening and confirmation by ELISA and GC-MS

Screening and confirmatory methods for the determination of Salbutamol (SAL) and Clenbuterol (CL) in swine urine were established. The polyclonal antibody against SAL was prepared, which was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) with the limit of detection of 0.5 ng/ml, and to prepare the immunoaffinity chromatography column. The immunoaffinity column could extract and purify SAL and CL simultaneously, with a binding capacity of 400 ng for SAL and 416 ng for CL. After purification, the swine urine samples were screened by ELISA for the presence of SAL and/or CL, and the positive samples were further confirmed and quantified by gas chromatography-mass spectrometry (GC-MS). The results showed that 95% of the positive samples were confirmed by GC-MS with various levels of SAL (1.1-4.6 ng/ml) and/or CL (1.9-229.1 ng/ml) residues in the incurred samples, and all the negative samples were confirmed with no SAL and/or CL residues.     

Development of ic-ELISA and an immunochromatographic strip assay for the detection of methylmercury

In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic strip assay based on a monoclonal antibody (mAb) against methylmercury (MeHg) to detect the presence of MeHg in tap water. Under optimum conditions (pH 8.0, 0.8% NaCl, and 0.1% Tween 20), the 50% half maximal inhibitory concentration (IC₅₀) and limit of detection (LOD) were 16.64 and 2.03?ng/mL, respectively. The anti-MeHg mAb was speci?c to mercury with no cross-reactivity with other metal ions. The cut-off value of the immunochromatographic strip assay was 500?ng/mL for semi-quantitative detection, and the LOD was 11.3?ng/mL for quantitative detection. The average recovery rates of the ic-ELISA and immunochromatographic strip assay were 98.13% and 107.87%, respectively, in tap water. Therefore, ic-ELISA and the immunochromatographic strip assay can be used to detect MeHg in tap water.     

Immunochromatographic strip for rapid detection of phenylethanolamine A

A monoclonal antibody (mAb) directed against phenylethanolamine A (PEA) was successfully prepared with the immunization of mice and cell fusion, and used to develop a detection method for a colloidal gold immunoassay. The 50% inhibitory concentration of mAb PEA-2G10 was 0.213?ng/mL, the limit of detection was 0.033?ng/mL, and its detection range 0.033–0.679?ng/mL. The recovery rate for PEA in swine urine was 84.5–90.8%, with good stability and accuracy. “Dry” immunoassay test strips were successfully prepared based on antibody 2G10. When the strip was tested in swine urine, the cutoff value for PEA was 5?ng/mL, with highly sensitivity. The method developed here can be used to test swine urine on-site for the rapid detection of PEA.     

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.     

Rapid and sensitive immunoassays for the detection of lomefloxacin and related drug residues in bovine milk samples

The misuse and illegal use of fluoroquinolones (FQs) in animal-based food products have drawn considerable attention in several countries. As a result, there has been an increased demand for efficient detection methods of FQs in food products. In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic strip device based on a monoclonal antibody against lomefloxacin (LFLX), a second generation FQ. Ic-ELISA had an IC₅₀ value of 0.19?ng/mL with a limit of detection of 0.04?ng/mL in 0.01?M phosphate-buffered saline (PBS). The intra- and inter-assay recovery rates of LFLX in bovine milk samples were 98.02–107.40% and 100.65–107.82%. The immunochromatographic assay of LFLX in PBS and spiked bovine milk samples had visual cutoff values at 1 and 5.0?ng/mL, respectively, with significant cross-reactivity with norfloxacin and enoxacin. The developed ic-ELISA and strip method may assist in the detection of FQs in foods.     

Anti‐idiotype and anti‐anti‐idiotype antibodies for aflatoxin from laying hens

Anti‐idiotype (anti‐id) antibodies (IgY2)for aflatoxin (AF) were obtained from the egg yolks of laying hens immunized with affinity‐purified rabbit polyclonal anti‐aflatoxin B₁ (AFB₁) carboxymethyloxime‐bovine serum albumin (BSA) antibodies (pAb1). The IgY2 were affinity purified and then subjected to various analyses. Inhibition of the binding of pAbl to the solid‐phase AFB₁‐BSA by IgY2 and the binding of pAb1 to the solid‐phase IgY2 by free AFB₁ were demonstrated in a biotin‐avidin amplified enzyme‐linked immunosorbent assay (ELISA) system. The concentration of IgY2 causing 50% inhibition (ID₅₀) of the binding of pAb1 to AFB₁‐BSA was found to be 2.45 μg/assay. The ID₅₀ concentration of the binding of pAb1 to IgY2 by free AFB₁ was found to be 0.30 μg/assay. Inhibition of the binding of AFB₁‐horseradish peroxidase (HRP) to the solid‐phase pAbl by IgY2 (ID50 = 9.65 μg/assay) was also demonstrated in the direct ELISA. Egg yolk anti‐anti‐id antibodies (IgY3) were obtained by immunizing laying hens with rabbit pAb2 against anti‐AFB₃‐hemisuccinate‐BSA monoclonal antibody. IgY3 was subjected to affinity chro‐matography purification with Sepharose gel armed with AFB₂‐carboxymethytoxime, and then subjected to various analyses. ELISA analysis revealed that IgY3 has characteristics similar to other anti‐AFB antibodies induced in various experimental animals. In the direct ELISA, the ID₅₀ of the binding of AFB₁‐HRP to solid‐phase lgY3 by AFB₁ was found to be 0.12 ng ml^‐1. In the indirect ELISA, the ID₅₀ of the binding of IgY3 to solid‐phase AFB₁‐BSA by AFB₁ was found to be 2.2 ng ml^‐1. The IgY3‐based ELISA analysis showed higher sensitivity than that of the egg yolk antibodies directly against AFB‐protein conjugates (IgY1). A good correlation was found for the data obtained from IgY3‐based and pAb1‐based ELISAs in the analysis of AFB in the fungal culture filtrates.     

Determination of marbofloxacin in plasma samples by high-performance liquid chromatography using fluorescence detection.

A simple and sensitive HPLC method has been developed for the determination of marbofloxacin (MAR) in plasma. Sample preparations were carried out by adding phosphate buffer (pH 7.4, 0.1 M), followed by extraction with trichloromethane. MAR and the internal standard, enrofloxacin (ENR), were separated on a reversed-phase column and eluted with aqueous solution-acetonitrile (80:20). The fluorescence of the column effluent was monitored at lambda(ex) = 338 and lambda(em) = 425 nm. The retention times were 2.20 and 3.30 min for MAR and ENR, respectively. The method was shown to be linear from 15 to 1500 ng/ml (r2 = 0.999). The detection limit was 15 ng/ml. Mean recovery was determined as 90% by the analysis of plasma standards containing 150, 750, and 1500 ng/ml. Inter- and intra-assay precisions were 3.3% and 2.7%, respectively.     

Development of ic-ELISA and lateral-flow immunochromatographic strip for detection of vitamin B2 in an energy drink and vitamin tablets

Vitamin B₂ (riboflavin) is a water-soluble vitamin that has important roles in human health. In this study, we developed a sensitive and specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip for the rapid detection of vitamin B₂. Following routine fusion and selection, the optimum monoclonal antibody against vitamin B₂ was obtained. The 50% inhibitory concentration and limit of detection of ic-ELISA were 8.18 and 1.80?ng/mL, respectively. The cut-off value of the lateral-flow ICA strip was 50?ng/mL. The results revealed that our developed methods are suitable for the on-site detection and mass screening of vitamin B₂ in food and pharmaceutical products.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the simultaneous detection of avermectin and ivermectin

A sensitive monoclonal antibody, 1H2, was generated in this study that simultaneously recognizes avermectin and ivermectin. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed, with 50% inhibitory concentrations of 0.491?ng/mL for avermectin and 0.770?ng/mL for ivermectin. A lateral-flow immunochromatographic assay (ICA) strip was also developed based on this monoclonal antibody for the semiquantitative and quantitative detection of avermectin and ivermectin. The semiquantitative (with the naked eye) analysis had visual limits of detection of 10?ng/mL for avermectin and 25?ng/mL for ivermectin, with cut-off values of 25 and 50?ng/mL, respectively. Using a strip scan reader, the quantitative results had calculated limits of detection of 1.3?ng/mL for avermectin and 2.9?ng/mL for ivermectin. In an analysis of raw milk samples, the recovery rates for the ic-ELISA ranged from 94% to 112% and for the ICA strip from 110% to 125%. Therefore, both methods are sensitive and effective for the on-site detection and rapid screening of samples, and are suitable for various other applications.