Selection of Anti-Sulfadimidine Specific ScFvs from a Hybridoma Cell by Eukaryotic Ribosome Display

BACKGROUND:Ribosome display technology has provided an alternative platform technology for the development of novel low-cost antibody based on evaluating antibiotics derived residues in food matrixes. METHODOLOGY/PRINCIPAL FINDINGS:In our current studies, the single chain variable fragments (scFvs) were selected from hybridoma cell lines against sulfadimidine (SM(2)) by using a ribosome library technology. A DNA library of scFv antibody fragments was constructed for ribosome display, and then mRNA-ribosome-antibody (MRA) complexes were produced by a rabbit reticulocyte lysate system. The synthetic sulfadimidine-ovalbumin (SM(2)-OVA) was used as an antigen to pan MRA complexes and putative scFv-encoding genes were recovered by RT-PCR in situ following each panning. After four rounds of ribosome display, the expression vector pCANTAB5E containing the selected specific scFv DNA was constructed and transformed into Escherichia coli HB2151. Three positive clones (SAS14, SAS68 and SAS71) were screened from 100 clones and had higher antibody activity and specificity to SM(2) by indirect ELISA. The three specific soluble scFvs were identified to be the same molecular weight (approximately 30 kDa) by Western-blotting analysis using anti-E tag antibodies, but they had different amino acids sequence by sequence analysis. CONCLUSIONS/SIGNIFICANCE:The selection of anti-SM(2) specific scFv by in vitro ribosome display technology will have an important significance for the development of novel immunodetection strategies for residual veterinary drugs

Plasmid-Mediated Quinolone Resistance Genes and Antibiotic Residues in Wastewater and Soil Adjacent to Swine Feedlots: Potential Transfer to Agricultural Lands

Background: Inappropriate use of antibiotics in swine feed could cause accelerated emergence of antibiotic resistance genes, and agricultural application of swine waste could spread antibiotic resistance genes to the surrounding environment. Objectives: We investigated the distribution of plasmid-mediated quinolone resistance (PMQR) genes from swine feedlots and their surrounding environment. Methods: We used a culture-independent method to identify PMQR genes and estimate their levels in wastewater from seven swine feedlot operations and corresponding wastewater-irrigated farm fields. Concentrations of (fluoro)quinolones in wastewater and soil samples were determined by ultra-performance liquid chromatography–electrospray tandem mass spectrometry. Results: The predominant PMQR genes in both the wastewater and soil samples were qnrD, qepA, and oqxB, whereas qnrS and oqxA were present only in wastewater samples. Absolute concentrations of all PMQR genes combined ranged from 1.66 × 10(7) to 4.06 × 10(8) copies/mL in wastewater and 4.06 × 10(6) to 9.52 × 10(7) copies/g in soil. Concentrations of (fluoro)quinolones ranged from 4.57 to 321 ng/mL in wastewater and below detection limit to 23.4 ng/g in soil. Significant correlations were found between the relative abundance of PMQR genes and (fluoro)quinolone concentrations (r = 0.71, p = 0.005) and the relative abundance of PMQR genes in paired wastewater and agricultural soil samples (r = 0.91, p = 0.005). Conclusions: Swine feedlot wastewater may be a source of PMQR genes that could facilitate the spread of antibiotic resistance. To our knowledge, this is the first study to examine the occurrence of PMQR genes in animal husbandry environments using a culture-independent method