Production of single-chain Fv antibodies against melamine from a rabbit phage display library for the development of a melamine immunoassay

A rabbit phage display library of single chain Fv (scFv) antibodies was constructed to screen specific scFv antibodies against melamine. Rabbits were immunized with melamine immunogens prepared via glutaraldehyde method. Two recombinant scFv antibodies with high specificity for melamine, termed A9 and B4, were isolated via library panning. SDS-PAGE analysis showed that the soluble A9-scFv and B4-scFv antibodies were expressed successfully and the scFvs showed good sensitivity with an IC₅₀ of 12.64 and 10.32 μM to melamine, respectively. The cross-reactivity of 2 scFv antibodies with cyanuric acid and chloromycetin were below 1%. The high similarity of 2 scFv nucleotide sequences indicated that they may originate from 1 B cell clone that had undergone somatic diversification. As a consequence, the conjugation synthesis was successful and the selected scFvs could be used for sensitive detection of melamine in foods and feeds by immunoassay.     

Development of an Enzyme-Linked Immunosorbent Assay for the Detection of Difenoconazole Residues in Fruits and Vegetables

An indirect, competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of difenoconazole was developed. Two haptens were designed and successfully synthesized. Hapten 1 had a particular moiety of difenoconazole, while hapten 2 had its full structure. The polyclonal antibodies against hapten-protein conjugates were prepared by immunizing rabbits. After optimization of the conditions, the detection limit (IC₁₅) and sensitivity (IC₅₀) were 4.58 and 29.10 μg L^?1, respectively. The cross-reactivities of the antibody with 11 triazole fungicides were all less than 0.1%, which showed that the antibody had excellent specificity. The recoveries of difenoconazole from the spiked samples ranged from 89.70 to 102.31% with good accuracy. The matrix effect was easily removed using a simple, rapid, and efficient extraction method on fruits and vegetables. The detection limit was all 229 μg kg^?1 in fruits and vegetables. To validate the ic-ELISA, samples were spiked with difenoconazole at three different concentrations and simultaneously analyzed using high-performance liquid chromatography (HPLC). The results showed a good correlation between the ic-ELISA and HPLC data (R ² = 0.9970). As a result, the developed immunosorbent assay is suitable for the quantitative determination of difenoconazole in fruits and vegetables.     

Preparation of Artificial Antigen and Development of Indirect Competitive ELISA Based on Chicken IgY for the Detection of Acid Orange II in Food Samples

To evaluate the feasibility of specific egg yolk antibodies (IgY) technology for the detection of hazardous chemical residues in foodstuffs, IgY were generated to detect acid orange II (AO2) residues. Bovine serum albumin (BSA) was made to bind with AO2 by succinic anhydride (SA), and the conjugate was used to immunize the laying chickens. PEG-6000 precipitation was used to extract IgY antibodies. The titer of anti-AO2 IgY attained the peak (1:12,800) after fifth booster immunization. Checkerboard titration showed that 1:640 dilution of anti-AO2 IgY could approximately give an optical density (OD) 1.0 at 5 μg/mL AO2-OVA coating concentration. The anti-AO2 IgY based indirect competitive ELISA (ic-ELISA) showed that the IC₅₀ value of anti-AO2 IgY was 10.72 μg/mL and regression curve equation was y?=?24.41?×?+75.11 (R ²?=?0.946). The ic-ELISA showed less cross-reactivity in range between 0.09 and 21.07 %. Recoveries from dried bean curd, sausage, and chilli powder samples were from 78.80 to 152.90 %, with relative standard deviation lower than 19.13 %. The study suggests that generated anti-AO2 IgY antibodies could be used in routine screening analysis of AO2 residues in food samples.     

Development of an Indirect Competitive ELISA for Analysis of Alternariol in Bread and Bran Samples

Alternariol (AOH) is one of the major mycotoxins produced by various species of Alternaria fungi. Natural occurrences of AOH have been reported in various foods, including fruits; processed fruit products such as apple juice, tomato products; wheat and other grains; oilseeds and products thereof, such as sunflower seeds, oilseed rape meal, and flax seed/linseed; and pecans. In this study, AOH-specific polyclonal antibodies were generated and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for monitoring AOH in bread and bran samples. The assay was very sensitive with a limit of detection (LOD) of 2.4?±?0.6 ng/g and a half maximal inhibitory concentration (IC₅₀) of 15.2?±?2.6 ng/g in bread and a LOD of 8.4?±?1.2 ng/g and IC₅₀ of 52.8?±?10.8 ng/g in bran extract. The assay was very specific to AOH and showed no cross-reactivity to alternariol monomethyl ether, altertoxin, altenuene, tentoxin, or tenuazonic acid. The effect of organic solvents on the assay was tested. The ELISA system tolerated methanol and acetonitrile as co-solvents at up to 5% content without significant loss of IC₅₀ value. Recoveries in all cases were greater than 75%, and the results using this method were comparable to those obtained from mass spectrometry methods. We conclude that this method is suitable for rapid detection of AOH in bread and bran samples, without expensive analytical equipment or time-consuming sample preparation.     

A Highly Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay (ic-ELISA) by Antigen Coating for Diethyl Phthalate Analysis in Foods

As we have known, with the plasticizer disturbance in 2011 in Taiwan, long-term exposure to diethyl phthalate (DEP), one of the widely used phthalate esters, can lead to serious health problems. Therefore, a highly sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) by antigen-coated plate format for DEP in foods was proposed in this paper. The polyclonal antibodies were raised against diethyl 4-aminophthalate (4-DEAP) conjugated to bovine serum albumin by the amino diazotization linkage method. Coating antigen was prepared with 4-DEAP conjugated to ovalbumin using the same procedure. Under the optimal experimental conditions, the ic-ELISA has a linear working range of 0.005–18.6 ng/mL (R ²?=?0.9921), with a limit of detection of 0.0049 ng/mL. Low cross-reactivity (<9 %) to structurally related phthalates was observed. The method was successfully applied to the determination of DEP in fruit juice, milky tea, pure milk, and sour milk, without purification or preconcentration. Satisfactory recoveries were obtained ranging from 91.1 to 109.3 %. The results suggested that the developed ic-ELISA is a simple, sensitive, and specific method for the rapid monitoring of DEP in food samples.     

Fluorescence Polarization Immunoassay for Rapid, Accurate and Sensitive Determination of Ochratoxin A in Wheat

A sensitive and accurate fluorescence polarization (FP) immunoassay has been developed for the determination of ochratoxin A (OTA) in naturally contaminated wheat samples. A fluorescein-labeled OTA tracer was synthesized, and its binding response with three monoclonal antibodies was tested. The most sensitive competitive FP immunoassay showed an IC₅₀ value of 0.48 ng/mL with a negligible cross-reactivity for ochratoxin B (1.7 %) and no cross-reactivity with other mycotoxins commonly occurring in wheat. The wheat sample was extracted with acetonitrile/water (60:40, v/v) and purified by a rapid solid-phase extraction procedure using an aminopropyl column prior to the FP immunoassay. The overall time of analysis was less than 20 min. The average recovery from spiked wheat samples (3 to 10 μg/kg) was 87 %, with relative standard deviations generally lower than 6 %. Limits of detection and quantification were 0.8 and 2.0 μg/kg, respectively. The trueness of the method was assessed by using two reference materials for OTA showing good accuracy and precision. A good correlation (r?=?0.995) was observed between OTA contamination of 19 naturally contaminated wheat samples analyzed by both FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up used as reference method. These results show that the developed FP method is suitable for high-throughput screening, as well as for reliable quantitative determination of OTA in wheat at level far below the EU regulatory limits.     

Rapid Screening of Quinoxaline Antimicrobial Growth Promoters and Their Metabolites in Swine Liver by Indirect Competitive Enzyme-Linked Immunosorbent Assay

Quinoxaline feed additives are antimicrobial growth promoters (AGPs); use three of them is permitted, and two of them are illegally used. It results in residue of quinoxaline AGPs and their metabolites in edible animal tissues, which are potentially harmful to human health. In order to effectively monitor the multiple residues of them in swine liver, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed based on polyclone antibody preparation. Protein conjugates were synthesized and immune to New Zealand white rabbit according to designed schemes. The effective antiserum with 50 % inhibiting concentration (IC₅₀) value of 1.34 μg?L^?1 was obtained. A new synthesized quinoxaline with similar chemical structure to olaquindox but longer spacer arm was used as coating antigen. Cross-reactivity of other four quinoxaline AGPs and their eight metabolites was tested; seven of them have cross-reactivity over 10 %, with IC₅₀ of 0.10–2.50 μg?L^?1. At the spike level of 1 to 100 μg?kg^?1 in swine liver, the recoveries of all compounds ranged from 80.14 % to 96.90 %, with the inter-day variation coefficient (CV) of 5.67–13.82 % and the intra-assay CV of 6.22–14.19 %. The limit of detection ranged from 0.03?±?0.002 to 0.79?±?0.05 μg?L^?1. Positive samples were determined by the ic-ELISA method and successfully confirmed by liquid chromatography-tandem mass spectrometry. The proposed ELISA is feasible for screening quinoxaline AGPs and their metabolites in swine liver.     

A Sensitive Immunoaffinity Column-Linked Indirect Competitive ELISA for Ochratoxin A in Cereal and Oil Products Based on a New Monoclonal Antibody

A new sensitive monoclonal antibody (mAb) 1H2 against ochratoxin A (OTA) was reported herein. This mAb belonged to the immunoglobulin G1 (k chain) isotype. In the optimized indirect competitive enzyme-linked immunosorbent assay (icELISA), 1H2 showed a 50 % inhibition concentration (IC₅₀) value of 0.058 ng/mL and a detection limit (IC₁₀) of 0.001 ng/mL. The cross-reactivity of 1H2 with ochratoxin B, aflatoxins, deoxynivalenol, zearalenone, T-2 toxin, or fumonisins was below 0.3 %. Based on this mAb, an immunoaffinity column (IAC)-linked icELISA was developed for OTA detection in the cereal and oil products. The working range of the assay for solid sample was 0.36–16 μg/kg. The recoveries from spiked samples of IAC-linked icELISA ranged from 83 to 101 %. These recoveries were much higher than those of icELISA (21–78 %) and in good agreement with those obtained by using the standard high-performance liquid chromatography method (87–110 %). The results indicated that the mAb 1H2 had the values for studies of OTA in the crude agricultural products.     

糖皮质激素广谱特异性单克隆抗体的制备及其ic-ELISA方法的建立

将氢化可的松半抗原HYD通过活化脂法与钥孔戚血蓝蛋白（keyhole limpet hemocyanin,KLH）偶联获得氢化可的松完整抗原KLH-HYD,并对小鼠肌肉注射此抗原,使小鼠免疫;通过杂交瘤技术获得1 株能够稳定分泌氢化可的松的单克隆抗体细胞株HYD-C1,并通过腹水制备大量抗体。将氢化可的松半抗原HYD和地塞米松半抗原DEX通过活化脂法分别与卵清蛋白（ovalbumin,OVA）偶联作为包被原,建立糖皮质激素的间接竞争酶联免疫吸附检测（indirect competitive-enzyme linked immunosorbent assay,ic-ELISA）方法。结果：在同源包被情况下氢化可的松灵敏度为1.63 ng/mL,异源包被情况下灵敏度为0.24 ng/mL;在交叉抑制实验中,异源策略中的各糖皮质激素灵敏度均高于同源策略中的结果,异源策略条件下很好地覆盖了本研究中所检测的糖皮质激素,使该抗体具备良好的广谱特异性。在异源策略添加回收实验中平均添加回收率在77.5%～111.3%之间,落在合理范围内,满足检测需求。结论：成功制备出氢化可的松单克隆抗体,建立了同源和异源策略糖皮质激素检测方法,结果发现异源策略中糖皮质激素的灵敏度高于同源策略,并使该方法具有更好的广谱特异性。     

Development of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay for the Analysis of Diclazuril in Chicken Tissues

A highly sensitive and specific monoclonal antibody (Mab) against diclazuril was produced. The hapten with diclazuril coupled to diazotised 4-aminobenzoic acid was synthesized and conjugated to bovine serum albumin by the active ester method to form an immunogen for antibody generation. A novel diclazuril carboxymethyloxime derivative used in an ovalbumin conjugate was applied as a heterologous coating antigen and was expected to improve the immunoassay sensitivity. A sensitive and simple indirect competitive enzyme-linked immunosorbent assay (icELISA) based on the Mab for the determination of diclazuril was developed. Under the optimized conditions, the icELISA for diclazuril showed a half maximum inhibition concentration (IC₅₀) value of 1.8 ng/mL, with limit of detection of 0.24 ng/mL and negligible cross-reactivities with other coccidiostat compounds including toltrazuril, robenidine, nicarbazin, halofuginone, amprolium, monensin, and maduramycin. The icELISA was successfully applied to diclazuril residue analysis in spiked chicken tissues. The average recoveries, intra-assay, and inter-assay coefficients of variation were in a range from 77.6 to 103.7 %, 3.7 to 13.0 %, and 5.6 to 18.3 %, respectively.     

Development and validation of an indirect competitive enzyme-linked immunosorbent assay for monitoring quinoxaline-2-carboxylic acid in the edible tissues of animals

The feed drug additive carbadox is a suspected carcinogen and mutagen. To monitor effectively residues of carbadox in the edible tissues of food-producing animals, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) to detect quinoxaline-2-carboxylic acid, the marker residue of carbadox, was developed. Several haptens were synthesised and conjugated to the carrier protein. Nine female New Zealand white rabbits were immunised with the immunising conjugates to produce polyclonal antibodies according to the designed schemes of immunisation. The highly specific antibody that was very sensitive to N-butylquinoxaline-2-carboxamide with an IC₅₀ value of 7.75?µg?l^?1 was selected for the development of an ic-ELISA. The standard curves based on the N-butylquinoxaline-2-carboxamide matrix calibration ranged from 0.2 to 51.2?µg?l^?1. The decision limit and detection capability of the ic-ELISA were 0.60 and 0.83?µg?kg^?1 for liver and 0.68 and 0.79?µg?kg^?1 for muscle of swine, respectively. The recoveries were 57–108% with coefficients of variation of less than 20% when the quinoxaline-2-carboxylic acid was spiked into liver and muscle with the concentrations of 1.0–20.0?µg?kg^?1. Excellent correlations between the results of the ic-ELISA and an HPLC method (r?=?0.9956???0.9969) were observed for incurred tissues. These results suggest that the ic-ELISA is a sensitive, accurate and low-cost method that would be a useful tool for screening residues of carbadox in the edible tissues of food-producing animals.     

Selective Immunodetection of Difloxacin in Animal Muscles and Sera: Role of Hapten Orientation

The influence of sarafloxacin (SAR) hapten orientation in immunogen on antibody specificity was examined. The spatial orientation of SAR linked through its carboxyl group to the carrier resulted to SAR-selective response (Anal Methods 2016, 8:5843–5850). To provide opposite orientation of SAR in immunogen, it was linked by its secondary amine to (1) succinic anhydride-modified BSA using carbodiimide-mediated conjugation and to (2) unmodified BSA using formaldehyde condensation method. Two versions of indirect competitive enzyme-linked immunosorbent assays (ELISA) based on generated antibodies were developed. A panel of homo- and heterologous conjugates was examined as potential coating antigens. No cross-reactions were registered in ELISA-1 and ELISA-2 except for fluorophenyl-containing fluoroquinolones difloxacin (DIF), SAR, and tosufloxacin as 138, 100 and 25% and 175, 100 and 8%, respectively. Among the recognized analytes, only DIF is authorized in meat products, so the developed tests were selective for DIF determination in bovine and porcine muscles and sera samples. The values of half-maximal inhibition concentration (IC₅₀) for these assay versions were estimated to be 1.2 and 0.25 ng/ml. DIF detection limits (IC₁₀) were, respectively, 0.15 and 0.015 ng/ml that allowed measuring of MRL and ten times lower level of DIF in the animal muscles. The developed ELISAs were also suitable for DIF determination in animal sera in the concentration range 1000–1 ng/ml.     

Preparation of a Broadly Specific Monoclonal Antibody-Based Indirect Competitive ELISA for the Detection of Benzodiazepines in Edible Animal Tissues and Feed

Incorrect use of benzodiazepines could result in serious health problems. To monitor the illegal use of benzodiazepine compounds in animals, a group-specific monoclonal antibody (mAb) was prepared in this study. The obtained 3D7 mAb, which is an IgG1 isotype mAb, displayed an IC₅₀ value of 8.9 ng mL^?1 for diazepam and exhibited cross-reaction for diazepam (100 %), nitrazepam (49 %), nordiazepam (140 %), temazepam (32 %), oxazepam (17 %), estazolam (7.5 %), and alprazolam (2.4 %). Based on this mAb, for the first time in this study, an optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) protocol that did not require complicated sample preparation and clean-up was developed. The detection limits of this ic-ELISA for benzodiazepines ranged from 1.2 to 3.3 μg kg^?1 in muscle matrix and from 25.2 to 55.4 μg kg^?1 in feed matrix. The recoveries ranged from 70.9 to 111.3 % with coefficients of variation below 15.0 %. Good correlations (r?>?0.9494) between the results of the ic-ELISA and high-performance liquid chromatography were also observed. This simple method reduced the time required for sample preparation, ensured greater throughput, and met the requirements for benzodiazepine residue analyses. In conclusion, the proposed method is a sensitive and rapid multi-residue technique that offers a cost-effective alternative to current published procedures without any concession in the ability to detect benzodiazepine sedative misuse.     

Effects of protein hydrolysate from chicken feather meal on tyrosinase activity and melanin formation in B16F10 murine melanoma cells

Tyrosinase is a copper-containing enzyme that controls mammalian melanogenesis. Tyrosinase inhibitors are important for their potential application in cosmetic products. Chicken feather meal is a rich source of amino acids, which have been linked with tyrosinase inhibition activity. This study investigated the tyrosinase inhibitory properties of protein hydrolysates prepared from chicken feather meal. Protein hydrolysates prepared by pepsin-pancreatin with MW <3 kDa exhibited strong tyrosinase inhibition activity for both monophenolase (IC₅₀ 5.780 ± 0.188 µg/mL) and diphenolase activities (IC₅₀ 0.040 ± 0.024 µg/mL) in a cell-free mushroom tyrosinase system. These samples were uncompetitive inhibitors with Ki values of 18.149 and 27.189 µg/mL in monophenolase and diphenolase activities, respectively. A cell culture model showed that this hydrolysate had the strongest inhibition on the viability of B16F10 cells (IC₅₀ 1.124 ± 0.288 µg/mL) and 0.210 µg/mL of the sample exhibited inhibition of tyrosinase activity by 50.493% and melanin synthesis by 14.680% compared to the control.     

Development and Validation of a Sensitive Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Screening of Florfenicol and Thiamphenicol in Edible Animal Tissue and Feed

To monitor the illegal use of florfenicol (FF) and thiamphenicol (TAP) in edible animal tissue and feed, a sensitive monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) has been developed with simple sample preparation and cleanup. The obtained monoclonal antibody (5F4) that has isotype IgG1 showed an IC₅₀ value of 0.21 μg L^?1 for FF and 0.35 μg L^?1 for TAP, respectively. It did not exhibit measurable cross-reactivity with other antibiotics. The limits of detection (LODs) for FF and TAP in a muscle matrix ranged from 0.07 to 0.14 μg kg^?1 and in a feed matrix ranged from 2.9 to 5.2 μg kg^?1. The recoveries were 72.8 to 113.4 % with a coefficient of variation of less than 15 %. Good correlation between the ELISA and HPLC-MS/MS results in the tissues tested demonstrated the reliability of our ic-ELISA. This ELISA is a useful tool for screening FF and TAP in edible animal tissue and feed.     

间接竞争酶联免疫法测定鱼体中的亚甲基蓝

目的 本实验旨在建立快速检测鱼体中亚甲基蓝的间接竞争酶联免疫分析方法。方法 以BSA载体蛋白,以天青C(Azure C)为半抗原,利用戊二醛法合成免疫原Azure C-BSA,免疫新西兰大白兔,制备多克隆抗体。通过对包被浓度、抗体浓度和二抗浓度等一系列实验条件的优化,建立检测鱼体中亚甲基蓝的间接竞争酶联免疫吸附方法(ic-ELISA)。结果 获得的多克隆抗体特异性强、灵敏度高,在5 -500 ng/mL范围内,线性良好,线性回归方程为y=0.3658x-0.1867 (R²=0.98),IC50为75.4 ng/mL,检测限为8.3 ng/mL,样品添加回收率为77.2%-79.3%,RSD为5.3 %-8.1 %。结论 该方法灵敏度高、特异性强,可用于鱼体中亚甲基蓝的快速检测。     

表面等离子共振传感检测Cry2A蛋白

目的：建立利用表面等离子共振（surface plasmon resonance,SPR）传感器检测转基因植物中苏云金芽孢杆菌Cry2A蛋白的方法。方法：利用SPR检测技术,根据生物分子间的相互作用,在金片表面修饰Cry2A蛋白的特异性单克隆抗体,对不同质量浓度梯度的Cry2A蛋白进行检测研究。结果：该方法可有效地检测到Cry2A蛋白,检测灵敏度可达10 ng/mL,与同为抗虫基因编码的Cry1Ac和Cry2Ab蛋白未出现交叉反应。结论：利用SPR方法检测Cry2A蛋白操作简单,省时且灵敏度高、特异性强,可用于对Cry2A蛋白的定性检测。     

Two-Site Antibody Immunoanalytical Detection of Food Allergens by Surface Plasmon Resonance

Bovine protein β-lactoglobulin (βLG) and peanut protein Ara h1 are considered good targets for detecting milk and peanut allergen, respectively. We used surface plasmon resonance (SPR) to detect βLG and Ara h1 by immobilizing the affinity-purified monoclonal antibodies on the biosensor chip. Proteins that bound to the antibody surface were detected by a shift in resonance angle. Adding polyclonal antibodies in the sandwich assay enhanced the sensitivity. βLG and Ara h1 were detected at 5.54 and 0.77 ng/mL, respectively, by SPR, and the results demonstrated good linear relation with relatively low concentrations of protein. The limit of detection with SPR was comparable to that with sandwich enzyme-linked immunosorbent assay (S-ELISA) with the same polyclonal and monoclonal antibodies. Use of the SPR biosensor is a simple, fast, and reliable way to detect βLG and Ara h1.     

Development of a sensitive monoclonal-based enzyme-linked immunosorbent assay for monitoring T-2 toxin in food and feed

The consumption of food or feed contaminated with high levels of T-2 toxin may cause adverse health effects in humans and other animals. In this study, to monitor T-2 toxin rapidly in food and feed, a sensitive and specific monoclonal antibody (mAb) against T-2 toxin was generated and a simple and rapid indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) developed. T-2 toxin was first converted to T-2-hemisuccinate (T-2HS) and T-2-hemiglutarate (T-2HG), which were then conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) to prepare an immunogen and coating antigen, respectively. After the inoculation of female Balb/c mice and cell fusions, one cell line, 4D8, with the IgG1 isotype was obtained. The 4D8 antibody exhibited the ability specifically to recognise T-2 toxin with IC₅₀ 1.46 µg l^?1. Based on this 4D8 mAb, an optimised ic-ELISA protocol was developed using only methanol–water (7:3, v/v) in feed and cereal samples and ethyl acetate in muscle samples. The limits of detection of T-2 toxin in various sample matrices varied from 0.07 to 15.8 µg kg^?1; the recoveries ranged from 50.3% to 113.6%; and the CVs were less than 19.0%. These results suggest that the prepared mAb and the developed ic-ELISA method will be a useful tool for detecting T-2 toxin in foods and feeds.     

Development of a highly sensitive and specific monoclonal antibody-based enzyme-linked immunosorbent assay for determination of doxycycline in chicken muscle,liver and egg

A modified indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed using a highly sensitive and specific monoclonal antibody (McAb) to determine doxycycline (DC) residues in chicken tissues and egg. The McAb against DC was produced by hybridoma technique and a modified ic-ELISA was characterised in terms of sensitivity, specificity, precision and accuracy. At optimal experimental conditions, the standard curve was constructed at concentrations ranged from 0.01 to 100 ng/ml. The IC₅₀ value was 1.32 ± 0.18 ng/ml. The limit of detection was 0.14 ± 0.02 ng/g. The recoveries of DC from spiked chicken liver, muscle, and egg at levels of 50–600 ng/g were 84.6–85.5%, 88.2–89.1%, and 84.4–89.3%, respectively. The coefficient variations (CVs) were 5.1–9.3%, 3.7–11.3%, and 4.7–9.8%, respectively. Linear regression analysis showed good correlation, with r² values 0.9909 for chicken liver and 0.9916 for chicken muscle.