Development of indirect competitive ELISA and lateral-flow immunochromatographic assay strip for the detection of sterigmatocystin in cereal products

Sensitive and specific anti-sterigmatocystin (STG) monoclonal antibody (mAb) 4G10 was obtained by immunization and cell fusion. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method and lateral-flow immunochromatographic assay (ICA) strip method were developed for the detection of STG in cereal products based on this mAb. The 50% inhibition concentration and limit of detection for the ic-ELISA method were 0.092 and 0.015?ng/mL, respectively. The visual limit of detection (vLOD) and cut-off value for the lateral-flow ICA strip method were 0.1 and 0.5?ng/mL, respectively. From the analysis of different cereal samples (wheat, maize and rice), the recovery rates ranged from 78.3% to 122.0% for the ic-ELISA method. For the lateral-flow ICA strip, the vLODs were 3, 1.2 and 3?ng/g, and the cut-off values were 12, 6 and 6?ng/g for wheat, maize and rice, respectively. Therefore, both of the developed methods are suitable for the on-site detection and rapid screening of numerous samples.     

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 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.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of folic acid in energy drinks and milk samples

Folic acid (FA) is an important vitamin for human growth and development, especially for pregnant women. A sensitive, rapid, and accurate FA detection method is required to assess the nutritional quality and safety of foods. A monoclonal antibody against FA was prepared for the development of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and lateral-flow immunochromatographic assay (ICA) strip. The 50% inhibitory concentration and limit of detection of ic-ELISA were 0.12 and 0.018?ng/ml, respectively. The visual limit of detection and cut-off values of the lateral-flow ICA strip were 0.5 and 2.5?ng/ml, respectively. Using the ICA strip, FA recovery rates were 89–98% from energy drinks and 73–87% for milk samples and were in good agreement with those obtained from the conventional microbiological assay method. Our developed methods are sensitive, convenient, effective, and suitable for on-site detection and rapid mass screening of food samples.     

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.     

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.     

Rapid detection of tenuazonic acid in cereal and fruit juice using a lateral-flow immunochromatographic assay strip

A lateral-flow immunochromatographic assay (ICA) strip was developed for determination of tenuazonic acid (TeA) using both semi-quantitative and quantitative methods. The ICA strip was based on produced anti-TeA monoclonal antibody, and could be used for TeA analysis in cereal and fruit juice, with visual limits of detection of 1600?ng/g and 400?ng/mL, and cut-off values of 6400?ng/g and 3200?ng/mL, respectively. The calculated limits of detection values were 126.4?ng/g and 65.3?ng/mL for wheat and apple juice samples, respectively. The recovery rates ranged from 90% to 129%. In summary, the developed strip was effective for TeA detection and would be suitable for on-site detection and rapid screening of samples.     

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.     

Development of ic-ELISA and lateral-flow immunochromatographic assay strip for the detection of citrinin in cereals

A sensitive and specific anti-citrinin (anti-CIT) monoclonal antibody 1F2 was obtained following immunization and cell fusion. An indirect competitive enzyme-linked immunosorbent assay was developed with a 50% inhibitory concentration of 0.761?ng/mL and a limit of detection of 0.089?ng/mL. The recovery rates for CIT-spiked cereals (maize, wheat, and rice) ranged from 112% to 123%. A lateral-flow immunochromatographic assay was developed for both semi-quantitative and quantitative detection. With CIT-spiked cereals, the visual limit of detection was 8?ng/g and the cut-off value was 40?ng/g (semi-quantitative analysis with naked-eye detection). Using a strip scan reader, the calculated limit of detection was 1.28–1.8?ng/g for different CIT-spiked cereals. The recovery rates ranged from 110% to 127%. Therefore, both methods were effective for CIT detection and suitable for on-site detection and rapid screening of samples.     

Development of sensitive and fast immunoassays for amantadine detection

A sensitive indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) method and a gold nanoparticle immunochromatographic strip were developed to detect amantadine (AM) in foods. A novel AM hapten was prepared, and a sensitive monoclonal antibody against AM was developed. The optimum ic-ELISA conditions included a pH of 7.4 and an ionic strength of 1.6% with no organic solvents. The half-inhibition concentration (IC₅₀) value of ic-ELISA was 1.92?ng/ml, with a limit of detection of 0.62?ng/ml. The immunochromatographic test strip method had a visual cut-off value at 5?μg/kg. Chicken samples were spiked with three concentrations of AM (1, 2, and 5?µg/kg) and analysed by ic-ELISA. Good recoveries were obtained (92.3% at 1?µg/kg AM, 116.1% at 2?µg/kg AM, and 91.8% at 5?µg/kg AM).     

Development of an immunochromatographic test strip and ic-ELISA for tetrabromobisphenol: a detection in lake water and rice pudding samples

A sensitive and selective indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic test strip were developed for the detection of tetrabromobisphenol A (TBBPA), an important brominated flame retardant. A monoclonal antibody (mAb) against TBBPA was produced and the half inhibition concentration (IC50) was 0.44?ng/ml, with the linear range 0.15–1.32?ng/ml. TBBPA recovery by ic-ELISA was 70.7–108.8% from rice pudding samples and 110.2–119.8% from lake water samples. A sensitive and rapid immunochromatographic test strip was developed using this specific mAb, with a good cut-off value of 25 and 50?ng/ml in lake water and rice pudding samples, respectively. The results of this study revealed that the ic-ELISA and immunochromatographic test strip represent useful methods of TBBPA detection in lake water and rice pudding 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 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 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.     

Rapid and ultrasensitive detection of 3-amino-2-oxazolidinone in catfish muscle with indirect competitive enzyme-linked immunosorbent and immunochromatographic assays

In this study, we developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and an immunochromatographic assay to detect the furazolidone metabolite, 3-amino-2-oxazolidinone (AOZ). The detection of AOZ was based on the AOZ derivative 3-([2-nitrophenyl]methyleneamino]-2-oxazolidinone (2-NPAOZ). 3-([(3-Carboxyphenyl)methylene]-amino)-2-oxazolidinone (2-CPAOZ) was used as the immunizing hapten and a conjugate of AOZ and glyoxylic acid (AOZ-A) was used as the coating hapten. The monoclonal anti-2-NPAOZ antibody, 5G12, was generated with a half-inhibitory concentration (IC₅₀) of 0.2?ng/mL and a line of 0.06–0.66?ng/mL. Its cross reactivity with other analogues was less than 8%. Spiked catfish samples (0.1, 0.2, and 0.5?μg/kg) were analyzed with the proposed system. The ic-ELISA showed a recovery range of 86.2–118.5% and the intra-assay and inter-assay coefficients of variation ranged from 4.3% to 9.4%. Under the optimum assay conditions, the immunochromatographic assay had a visual cut-off value of 0.5?μg/kg in catfish samples. Both methods can be used to detect AOZ in catfish samples.     

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.     

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.     

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.     

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.     

Rapid detection of tulathromycin in pure milk and honey with an immunochromatographic test strip

In this paper, we describe the development of a rapid, simple, specific, and sensitive immunochromatographic strip test based on an antibody–antigen reaction for detection of tulathromycin (TULA) in samples of 0.01 M PBS pH 7.4, pure milk and honey. The monoclonal antibodies specifically recognized the corresponding antigens produced in our laboratory. Under optimized conditions, the cut-off limits of the test strips for TULA in 0.01 M PBS pH 7.4 was found to be 2.5?ng/mL. Meanwhile, with simple preparation for detection in pure milk and honey samples, the cut-off limits for TULA were found to be 5 and 10?ng/mL, respectively. Each test can be evaluated in 5?min. In summary, this lateral-flow device provides a sensitive, effective, specific, and rapid method for detection of TULA residues in food matrix.