Dezhao Kong

Dual Amplified Electrochemical Immunosensor for Highly Sensitive Detection of Pantoea stewartii sbusp. stewartii

Accurate and highly sensitive detection of Pantoea stewartii sbusp. stewartii-NCPPB 449 (PSS) is urgently required for international shipments due to tremendous agricultural economic losses. Herein, a dual amplified electrochemical sandwich immunosensor for PSS detection was developed, utilizing the good specificity and low cost of electrochemical immunoassay, the favorable conductivity and large specific surface area of gold nanoparticles (Au NPs), and the excellent catalytic ability of and horseradish peroxidase (HRP). A linear curve between current response and PSS concentration was established, and the limit of detection (LOD) was 7.8 × 10(3) cfu/mL, which is 20 times lower than that for conventional enzyme-linked immunosorbent assay (ELISA). This strategy is a useful approach for the highly sensitive detection of plant pathogenic bacterium.

Development of an Immunochromatographic Strip for Rapid Detection of Pantoea stewartii subsp. stewartii

A rapid, simple, sensitive, and specific immunochromatographic test strip was developed for the detection of Pantoea stewartii subsp. stewartii (Pss) in corn seed which was soaked overnight and then centrifuged for precipitate re-dissolved as samples. A pair of sensitive monoclonal antibodies for the immunochromatographic test strip was generated by mice immunization and cell fusion. Under optimized conditions, the lower detection limit of the strips for Pss was 1 × 105 cfu/mL both in 0.01 M phosphate buffer solution and corn seed samples, with no cross-reactivity with other common plant pathogens. The developed strip is useful and rapid for the detection of Pss in corn seed samples.

Development of an immunochromatographic strip for the rapid detection of Pseudomonas syringae pv. maculicola in broccoli and radish seeds

A portable immunochromatographic (IC) strip based on gold-labelled monoclonal antibodies was developed for the rapid detection of Pseudomonas syringae pv. maculicola, a plant pathogen. The IC strip detected 105 colony-forming units CFU/ml P. syringae pv. maculicola in pure culture within 10 min and had good specificity. Compared with sandwich enzyme-linked immunosorbent assay, the strip was equally sensitive; however, it is portable and time-efficient. On broccoli and radish seeds, the IC strips had a sensitivity of 5 × 105 CFU/ml and 106 CFU/ml, respectively. The results revealed that the IC strip could have potential applications for P. syringae pv. maculicola detection in plant seeds.

Sensitive and highly specific detection of Cronobacter sakazakii based on monoclonal sandwich ELISA

A sensitive and specific monoclonal antibody-based sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the Cronobacter sakazakii. A pair of monoclonal antibodies (mAbs) selected from mAbs produced by 11 murine hybridomas was selected for the sandwich ELISA procedure. Targets of two mAbs were 100 kDa and 42 kDa protein extracted from the bacteria, respectively, which were proved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. The limit of detection of this method was established as 1 × 104 cfu/mL, and the linear range from 1 × 105 to 1 × 108 cfu/mL. In the real sample test, 1 cfu/g C. sakazakii was detected in artificially contaminated powdered infant formula with 4 h enrichment.

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

ABSTRACT 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 assay strip for the detection of vancomycin in raw milk and animal feed

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

ABSTRACT 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 detection of citrinin in cereals

ABSTRACT 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.

Determination of quinoxaline antibiotics in fish feed by enzyme-linked immunosorbent assay using a monoclonal antibody

Olaquindox (OLA), mequindox (MEQ), and quincetone (QCT) are widely used synthetic antibiotics of the quinoxaline-1,4-dioxide family. However, no studies have focused on the detection of sum of OLA, MEQ, and QCT by mAb-based enzyme-linked immunosorbent assay (ELISA). In this study, a specific mAb 2F3 against OLA, MEQ, and QCT was successfully prepared. Furthermore, using the mAb 2F3, an indirect competitive ELISA (icELISA) was developed using MQCA (quinoxaline marker) coupled to OVA as the heterologous coating antigen. Under optimized assay conditions, the IC50 values were 1.03, 1.54, and 1.73 ng ml−1 for OLA, MEQ, and QCT, respectively. The recoveries ranged from 82.1% to 96.3%, and no cross-reactivity with other compounds was detected except for carbadox (0.9%). The developed icELISA was rapid and reliable for the determination of sum of OLA, MEQ, and QCT in fish feed.

Development of an immunochromatographic strip for the semi-quantitative and quantitative detection of biotin in milk and milk products

An immunochromatographic strip was developed for the semi-quantitative and quantitative detection of biotin in milk and milk products. This detection system was developed based on a sensitive and specific monoclonal antibody produced in our laboratory. The semi-quantitative results, which were visually obtained in 20 min, revealed that the visual limit of detection was 2.0 μg/100 g with a cut-off value of 8.0 μg/100 g. The quantitative results, which are obtained with a strip scan reader, revealed that the calculated limit of detection was 0.32 μg/100 g with a linear range of 0.65–68 μg/100 g. Biotin contents in milk and milk products were determined by using the developed immunochromatographic strip, and the results were validated by the microbiological assay method. Our developed immunochromatographic strip system is suitable for the on-site detection and rapid screening of biotin in food samples.