Shusheng Tang

Rapid enzyme-linked immunosorbent assay and colloidal gold immunoassay for kanamycin and tobramycin in Swine tissues.

A monoclonal antibody (Mab) was produced by using the kanamycin-glutaraldehyde-bovine serum albumin (Kan-GDA-BSA) conjugate as the immunogen. The anti-Kan Mab exhibited high cross-reactivity with tobramycin (Tob) and slight or negligible cross-reactivity with other aminoglycosides. The specificity and cross-reactivity of this Mab are discussed regarding the three-dimensional, computer-generated molecular models of the aminoglycosides. Using this Mab, a rapid enzyme-linked immunosorbent assay (ELISA) and a colloidal gold-based strip test for Kan and Tob were developed. The rapid ELISA showed a 50% inhibition value (IC 50) of 0.83 ng/mL for Kan and 0.89 ng/mL for Tob with the analysis time less than 40 min, and the recoveries from spiked swine tissues at levels of 25-200 microg/kg ranged from 52% to 96% for Kan and 61% to 86% for Tob. In contrast, the strip test for Kan or Tob had a visual detection limit of 5 ng/mL in PBS, 50 microg/kg in meat or liver, and 100 microg/kg in kidney, and the results can be judged within 5-10 min. Observed positive samples judged by the strip test can be further quantitated by ELISA, hence the two assays can complement each other for rapid detection of residual Kan and Tob in swine tissues. Moreover, physical-chemical factors that affected the ELISA and strip test performance were also investigated. The effect of pH and antibody amount for gold-antibody conjugation on the strip test sensitivity was determined followed by a theoretical explanation of the effects.

Development of an immunochromatographic strip test for rapid detection of melamine in raw milk, milk products and animal feed.

A simple, rapid and sensitive immunogold chromatographic strip test based on a monoclonal antibody was developed for the detection of melamine (MEL) residues in raw milk, milk products and animal feed. The limit of detection was estimated to be 0.05 μg/mL in raw milk, since the detection test line on the strip test completely disappeared at this concentration. The limit of detection was 2 μg/mL (or 2 μg/g) for milk drinks, yogurt, condensed milk, cheese, and animal feed and 1 μg/g for milk powder. Sample pretreatment was simple and rapid, and the results can be obtained within 3-10 min. A parallel analysis of MEL in 52 blind raw milk samples conducted by gas chromatography-mass spectrometry showed comparable results to those obtained from the strip test. The results demonstrate that the developed method is suitable for the onsite determination of MEL residues in a large number of samples.

Cyclin A is essential for the p53-modulated inhibition from benzo(a)pyrene toxicity in A549 cells

Benzo(a)pyrene (B(a)P) is an environment carcinogen that can enhance cell proliferation by disturbing the signal transduction pathways in cell cycle regulation. The p53 tumor suppressor as a cell cycle check-point determinant plays a critical role in cell proliferation. However, the mechanism of p53 that accounts for the remarkable toxicity of B(a)P remains elusive. Here we reported that exposure of B(a)P to A549 cells caused G1 to S and G2/M phase transition along with increased expression of p53, cyclin D1, Cdk2, Cdk4, p21 and decreased expression of cyclin E, but no change in cyclin A and p27 expression. Up-regulation of p53 expression via transfection caused G1 phase arrest with decreased expression of cyclin A, E, Cdk2 and Cdk4, and increased expression of p21, when the expression of cyclin D1 and p27 were not significant changed. While B(a)P exposure to A549 cells following p53 transfection, up-regulation of p53 significantly attenuated the B(a)P-induced enhancement of cell proliferation and cell arrest, with increased expression of cyclin D1, Cdk2 and Cdk4, and with declined expression of cyclin A, cyclin E and p21, and p27 were not significant changed. Compared to the untreated cells, cylin A expression reduced in p53-transfected cells and in the B(a)P-treated cells following p53 transfection, but showed no change in the only B(a)P-treated cells. These results indicated that cyclin A is regulated by p53, not by B(a)P, and it is essential in the p53-modulated inhibition from benzo(a)pyrene toxicity in A549 cells, cyclin E and p21 also as downstream genes of p53 involved it, which is p27-independent.

Residue depletion of gentamicin in swine tissues after intramuscular administration.

A sensitive and robust high-performance liquid chromatographic method with fluorescence detection (HPLC-FLD) was developed for the determination of gentamicin (GEN) residues in swine tissues. The limit of quantification (LOQ) of the method was 50 ng/g for muscle and 100 ng/g for liver and kidney. Mean recoveries at all fortification levels ranged from 82.34 to 93.20% with coefficient of variation (CV) below 5.39%. Residue depletion study of GEN in swine was performed after intramuscular injections twice daily at a dose of 4 mg/kg of bw with 12 h intervals for 5 consecutive days. The concentrations of GEN were determined in injection site, muscle, liver, and kidney by the HPLC-FLD method. The highest GEN concentration was measured in kidney, indicating that kidney was the primary target tissue for GEN residue. GEN concentrations in all examined tissues were below the maximum residue limit (MRL) recommended by the European Union (EU) at 50 days post-treatment.

Development of a Monoclonal Antibody-Based ELISA for the Detection of Sulfaquinoxaline in Chicken Tissues and Serum

Abstract A monoclonal antibody (Mab) was produced by using sulfaquinoxaline-human serum albumin (SQX-HSA) conjugate as immunogen. The anti-SQX Mab exhibited negligible cross reactivity with other commonly used sulfonamides. Using this Mab, a competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed to detect SQX in chicken tissues and serum. The ciELISA showed a 50% inhibition (IC50) value of 2.60 ng/mL. The recoveries of SQX from spiked chicken muscle, liver, and serum at levels of 5–50 µg/kg were 82.6–96.5%, 75.3–94.5%, and 69.7–89.3%, respectively. The coefficient variations (CVs) were 6.22–7.17%, 4.9–8.9%, and 1.20–10.15%, respectively. Detection limits were 1.29 µg/kg in muscle, 1.32 µg/kg in liver, and 2.44 µg/kg in serum.

Monoclonal Antibody-Based Immunoassay for the Detection of Maduramicin in Chicken Tissues

Abstract This paper reports on the preparation of a monoclonal antibody (Mab) against maduramicin (MD) and the development of a simple and sensitive ELISA for MD in chicken tissues. MD was conjugated to bovine serum albumin for immunogens and ovalbumin for coating antigens by mixed anhydride (MA) and active ester (AE) methods. Hybridoma cells were generated using spleen cells from a mouse immunized with MD-BSA conjugate. After screening with ELISA, the Mab with high anti-interference ability and high affinity was selected, and it exhibited negligible cross-reactivity with other usual-used polyether antibiotics. After optimization, the developed ELISA showed an IC50 value of 2.12 ± 0.46 ng/ml (n = 20). Chicken muscle and liver samples were extracted with methanol-8% NaCl solution (4:1) and then directly diluted with PBS-10% methanol for analysis. The recoveries of MD from spiked chicken tissues at levels of 40–480 µg/kg ranged from 81.3% to 91.3% with variation coefficients of 5.2–12.1%, and the detection limits were 6.5 µg/kg in muscle and 9.2 µg/kg in liver, respectively (n = 20).

Analysis of Quinocetone and its Four Metabolites in Swine Liver by Ultra-performance Liquid Chromatography Quadrupole Time-of-flight Mass Spectrometry

A new method using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/ Q-TOF-MS) was developed for determination of quinocetone (QCT) and its major metabolites, 1–desoxyquinocetone (1– DQCT), 4–desoxyquinocetone (4–DQCT), 1,4–bisdesoxyquinocetone (DQCT) and methyl–3–quinoxaline–2–carboxylic acid (MQCA) in swine liver. Tissue samples were extracted by a two-step method. Firstly, all analytes except MQCA were extracted with ethyl acetate. Then a hydrochloric acid solution was added in the remained sediment for hydrolysis of combined MQCA. The MQCA in the acid extract was transferred to ethyl acetate by liquid-liquid extraction (LLE) approach. All the ethyl acetate extract were combined, evaporated, resolved in a phosphate buffer, and cleaned up using a Oasis MAX cartridges. The chromatographic separation of all the analytes was achieved in less than 5 min using UPLC. The identification and confirmation by accurate mass measurement and their different fragmentation pathways were performed on ESI-Q-TOF-MS (MS/MS). The quantitation was carried out in TOF mode using narrow window extracted ion chromatogram of each compound. In the range of 1-100 µg·kg–1, the calibration curve equations of MQCA, QCT, 4-DQCT, 1-DQCT and DQCT were y=0.7878x+10.49, y=4.656x+42.244, y=5.5825x+24.919, y=8.491x+21.195 and y=10.733x+160.72, respectively. And the relative coefficients of all calibration curves were higher than 0.98. The limit of detection and the limit of quantification were 1.4–4.8 µg·kg –1 and 4.6–15.9 µg·kg–1, respectively. The established method was validated for determination of incurred swine liver samples in an actual residue study.