Development and application of one-step ELISA for the detection of neomycin in milk

A sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin (NEO) in milk was developed. Two conjugates were synthesized as the immunogen and coating antigen. Four BALB/c female mice were immunised and the antisera were screened by indirect competitive ELISA (icELISA). The icELISA was further optimised using different coating methods, pH values, ionic strengths of assay buffer and reaction times. After optimised, the indirect competitive ELISA (icELISA) could be finished in 85min with an IC₅₀ value of 0.74±0.05 ng/mL. One-step indirect competitive ELISA (icELISA) exhibited similar sensitivity with an IC₅₀ value of 0.73±0.03 ng/mL, which was sensitive enough for NEO detection and could be finished in 55 min. No cross-reactivity of the antibody was observed with other aminoglycosides based on icELISA, indicating that the antibody is highly specific for neomycin. Milk samples were further analyzed with recovery ranging from 85 to 110% at spiked level of 0.25–10 ng/mL, and the detection limit was 0.08 ng/mL.     

Determination of zearalenone in corn based on a biotin-avidin amplified enzyme-linked immunosorbent assay

A biotin-avidin amplified enzyme-linked immunosorbent assay (BA-ELISA) method was developed to detect zearalenone (ZEN) residues in corn. The concentration of coating antigen, biotinylated monoclonal antibody specific to ZEN, avidin-horseradish peroxidase and reaction time in the system of BA-ELISA method were optimised. Under the optimum conditions, a good linear relationship between binding ratio and ZEN concentration in the range of 0.54 to 7.99 ng/mL was obtained. The regression equation was y=?21.26 ln(x)+66.89 (R²=0.9989). The limit of detection and limit of quantification were 0.35 ng/mL and 0.812 ng/mL. The IC₅₀ was calculated to be 2.071 ng/mL. In comparison with the traditional ELISA method, the sensitivity of BA-ELISA method developed has been elevated by six times. The recovery and coefficient of variation with the spiked corn samples were in the range of 86.6% to 93.7% and less than 7.8%, respectively. It promises a bright future for the sensitive and rapid detecting method of ZEN residues in corn.     

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 an immunoassay for carbendazim based on a class-selective monoclonal antibody

In the present study, a class-selective monoclonal antibody (mAb) for carbamate fungicides was obtained using two haptens. One hapten was designed as immunogen and the other one for coating. Under the optimum assay conditions of coating antigens H2-ovalbumin, in the assay buffer of sodium chloride 1.6%, no methanol, and pH 6.5, the assay system was more sensitive. The linear range was from 0.06 to 7.47 ng/mL. The cross-reactivity test was carried out with IC₅₀ values of 0.45 ng/mL for carbendazim, 0.57 ng/mL for benomyl, and 9.70 ng/mL for thiabendazole. Other related compounds thiophanate methyl, fenbendazole, mebendazole, methyl paraoxon, methyl parathion, chlorpyrifos, and chlorothalonil did not cross-react significantly in this assay. To evaluate the proposed immunoassay, real samples such as apples, tomatoes, and cucumbers were subjected to recovery test with carbendazim. The recovery and coefficient variation range from 73% to 125% and 0.36% to 11.80%, respectively. The developed method was also compared with the commercial kit for assaying carbendazim in the fruits and vegetables and the results were consistent. All results indicated that this developed enzyme-linked immunosorbent assay (ELISA) is useful for the detection of carbendazim residues in fruits and vegetables.     

Development of ELISA for melamine detection in milk powder

A sensitive ELISA method was developed based on a monoclonal antibody to detect melamine in milk powder. Two haptens were prepared using the melamine structural analogue, 2-chloro-4, 6-diamino-1, 3, 5-triazine. The obtained antibody belonged to the IgG1κ family and had high affinity for melamine (affinity constant was 3.09×10⁹). The antibody could also recognise cyromazine (cross-reactivity was 131%) and had low cross-reactivity toward the haptens. The effects of pH, ionic strength and organic solvents on ELISA performance were evaluated. The lowest IC₅₀ value (6.0±0.55 ng/mL) and the limit of detection in milk powder, 50 ng/g, were achieved under optimised conditions. Milk powder spiked at two levels of melamine (100 ng/g and 500 ng/g) was analysed with this ELISA method. Good recovery was obtained (76.2±7.3% at the 100 ng/g spiked level and 91.4±5.0% at 500 ng/g).     

Development of an immunochromatographic strip for detection of acetamiprid in cucumber and apple samples

An immunochromatographic strip was developed for detection of acetamiprid (AC) and cross-reactivity with thiacloprid (TC) in cucumber and apple samples. This detection system was developed based on a sensitive and specific monoclonal antibody produced in our laboratory. Under optimized conditions, the cut-off limits of the test strip for AC were found to be 1 and 0.25?ng/mL for TC in phosphate-buffered saline. Meanwhile, with simple preparation for detection cucumber and apple samples, the cut-off limits for AC were found to be 5 and 2.5?ng/mL for TC in cucumber samples. The immunochromatographic assay also revealed AC cut-off values of 30 and 15?ng/mL in apple samples. Each test can be evaluated within 5?min. The data indicate that the method is sensitive, rapid, specific, and has the advantages of simplicity; therefore, this immunochromatographic assay is a useful screening method for detection of AC and TC residues in cucumber and apple samples.     

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.     

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.     

Development of indirect competitive enzyme-linked immunosorbent and immunochromatographic strip assays for carbofuran detection in fruits and vegetables

Carbofuran is a highly toxic pesticide used in fruits and vegetables. In this study, we produced a specific and sensitive monoclonal antibody (mAb) which was prepared based on a hapten that was derivatized with benzofuranol against carbofuran. Following mice immunization and cell fusion, we obtained three monoclonal cell lines: 6D5, 3H2, and 3C6. The cell line 3H2 generated mAb with the highest affinity and sensitivity. The half maximal inhibitory concentration was 0.3?ng/mL, and the cross-reactivity was <1%. Based on this mAb, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and immunochromatographic test strip (ICS) assay were developed to detect carbofuran residues in cucumbers and apples. The working range of ic-ELISA was 0.1–1?ng/mL, and the cutoff value of ICS was 1?ng/mL. The analytical recovery of carbofuran in cucumber and apple samples ranged from 81% to 97%. Both methods represent rapid screening tools for carbofuran detection in fruits and vegetables.     

Development of an indirect enzyme-linked immunosorbent assay and lateral-flow test strips for pefloxacin and its analogues in chicken muscle samples

An anti-pefloxacin (PEF) monoclonal antibody (mAb), an indirect competitive enzyme-linked immunosorbent assay and lateral-flow test strip methods were developed to detect fluoroquinolone (FQ) residues in chicken muscle samples. Under optimised conditions, the anti-PEF mAb showed reasonable cross-reactivity with nine FQs with a limit of detection of 0.082?ng/mL assayed in 0.01?M phosphate buffered saline (PBS) solution. The intra- and inter-assay recoveries from spiked samples were within the range of 62.42–111.47% and 63.5–113.79%, respectively. The visual cut-off values of the lateral flow test strip in 0.01 M PBS and in food matrices were within the range of 2.5–50?ng/mL and 5–100?µg/kg, respectively. These results show that the anti-PEF mAb immunoassay and lateral flow test strip methods are suitable for simultaneous detection and routine monitoring of FQ residues in food.     

Synthesis of olaquindox metabolite,methyl-3-quinoxaline-2-carboxylic acid for development of an immunoassay

A novel synthesis method of methyl-3-quinoxaline-2-carboxylic acid (MQCA) and the preparation of its hapten were described. MQCA was synthesised involving in two steps with a high purity. For improving the sensitivity of detection, five different haptens were synthesised and corresponding immunogens and coating antigens were prepared. After comparing the sensitivity of immunoassay with different pairs of antibody and coating antigen, a specific immunoassay was obtained using an antibody raised against hapten (four-atom spacer arm) – BSA and a suitable coating antigen with a heterologous spacer arm group. The 50% inhibitory concentration of MQCA by indirect competitive enzyme-linked immunosorbent assay with optimised antibody and coating antigen was 3.84 ng/ml and the limit of detection was 0.25 ng/ml.     

Immunochemical and molecular characteristics of monoclonal antibodies against organophosphorus pesticides and effect of hapten structures on immunoassay selectivity

To produce broad-selective monoclonal antibody (Mab) for organophosphorus (OP) pesticides, heterologous enzyme-linked immunosorbent assays (ELISAs) were used for hybridoma screening. Six Mabs with different cross-reactivity to OP pesticides were produced. Based on the broad-selective Mab 5F7, homologous and heterologous indirect competitive ELISAs were developed and the influence of hapten structure on assay selectivity was investigated. Moreover, the receptor–ligand interactions between 5F7 and OP pesticides were simulated to evaluate the broad-selectivity of the Mab. Results showed that, selectivity of the Mab was determined by its molecular properties, and the assay selectivity could be modified when the assay sensitivity was improved sufficiently by heterology. With the most suitable competitor, a broad-selective heterologous ELISA was developed. The IC₅₀ values were estimated to be 7.06 ng/mL for parathion, 32.34 ng/mL for methyl parathion, 164.84 ng/mL for fenitrothion, 96.97 ng/mL for phoxim and 500.94 ng/mL for fenthion.     

An ultrasensitive immunochromatographic assay for non-pretreatment monitoring of chloramphenicol in raw milk

A non-pretreatment monitoring method based on immunochromatographic test (ICT) strips was developed to test for chloramphenicol (CAP) in raw milk. This assay was designed to measure competitive binding affinities for CAP molecules in raw milk and CAP antigen immobilized on strips with colloidal gold nanoparticles (GNPs, average diameter of 30 nm) labeled with anti-CAP monoclonal antibody (CAP mAb). Several working conditions, including the CAP solvent, different types and concentrations of CAP mAb, coating antigen, and GNP, were optimized for performance. After optimization, the detection process was carried out in 8 min with a visual limit of detection (LOD) of 0.3 ng/mL for qualitative detection and a LOD of 0.064 ng/mL for semi-quantitative detection. No cross-reactivity was detected toward CAP analogs. Based on these results, this simple and ultrasensitive assay could be thoroughly applied in the on-site testing of CAP in raw milk.     

Development of an enzyme-linked immunosorbent assay for the pyrethroid fenpropathrin

An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the detection of fenpropathrin was developed. Two haptens, FPa (α-carboxy-3-phenoxyphenyl-2,2,3,3-tetramethyl-cyclopropane-carboxylate) and FPb (α-(N-butyrical)-3-phenoxybenzyl-2,2,3,3-tetramethyl-cyclopropane-carboxylate), were synthesised and conjugated with ovalbumin (OVA) by the mixed anhydride method as coating antigens (FPa–OVA and FPb–OVA), and the hapten FPb was conjugated to bovine serum albumin (BSA) by the carbodimide method to produce an immunogen (FPb–BSA). Polyclonal antibody against fenpropathrin was raised for screening the more sensitive coating antigen. Under optimised assay conditions, the 50% inhibitory concentration (IC₅₀) was 0.34±0.090 mg/L and the limit of detection (LOD) was 0.0093±0.00065 mg/L. The cross-reactivities with other pyrethroids, such as deltamethrin, cypermethrin, fenvalerate and cyhalothrin, were all lower than 0.1%. Water samples spiked with different concentrations of fenpropathrin (0.01–1.0 mg/L) were analysed according to this method. The ic-ELISA developed could successfully be applied to residue analysis of fenpropathrin in aquatic.     

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

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

Development of an indirect competitive enzyme-linked immunosorbent assay to detect extracellular polymeric substances (EPS) secreted by the marine stromatolite-forming cyanobacteria,Schizothrix sp

An indirect competitive enzyme-linked immunosorbent assay was developed using polyclonal antibody to detect extracellular polymeric secretions (EPS) produced by the marine stromatolite-forming cyanobacteria, Schizothrix sp. The cross-reactivity of this assay with other EPSs and polymers were low (< 0.5%). This assay can detect Schizothrix sp. EPS as low as 0.5 ng/mL. Intra-assay and inter-assay comparisons showed that coefficient variations were low, ranging from 3.34 to 10.30% and from 6.30 to 12.8%, respectively, for standards between 2 and 1000 ng/mL. Also, the seawater matrix effect was negligible. Our results indicated that this assay is a useful tool for quantification of Schizothrix sp. EPS in a range of ng/mL.     

Development of an ic-ELISA and immunochromatographic strip for detection of sparfloxacin in honey

In this study, a sensitive monoclonal antibody (mAb) 3D5 against sparfloxacin (SPFX) was generated. Based on the mAb 3D5, an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method was developed for the detection of SPFX in honey. The half maximal inhibitory concentration and limit of detection for the ic-ELISA method were 0.12 and 0.02?ng/mL, respectively. The experiments showed a negligible cross-reaction with other drugs. The average recovery rates for spiked SPFX honey extracts ranged from 90% to 101%, indicating an accepted accuracy. Furthermore, a lateral-flow immunochromatographic assay strip method with a cut-off value of 2?ng/mL was developed. Therefore, both of the developed methods are suitable for future use as rapid screening methods to detect and control the content of SPFX residues in honey samples.     

表皮生长因子受体Ⅲ型突变体(EGFRvⅢ)单链抗体亲和力检测的间接ELISA的建立及优化

目的建立检测表皮生长因子受体变体Ⅲ(EGFRvⅢ)单链抗体PD0721亲和力检测的ELISA,并且对实验条件进行优化。方法利用本课题组制备的单链抗体PD0721,建立检测PD0721单链抗体亲和力的间接ELISA,并采用棋盘滴定法对实验条件中的抗体抗原浓度、二抗浓度、包被条件等方面进行优化,对本方法的灵敏度及精密度进行考察。结果使用PBS稀释抗原至1.25 mg/L于4℃包被12 h,加入120 ng/mL PD0721单链抗体以及1∶8000的酶标抗体为最佳检测结果。优化后的结果,在PD0721单链抗体浓度为15 ng/mL~480 ng/mL范围内回归模型拟合效果良好,最低检测限为7.5 ng/mL。组内差异系数为0.11%~0.99%,组间差异系数为0.68%~3.15%。结论建立了能准确、稳定检测PD0721单链抗体亲和力的间接ELISA。     

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.