Ferreira, A. C. Silva

Ferreira, João Paulo M.

Gomes, Francisco Brás

Riedstra, Symon

English

Repositório Institucional da Universidade Católica Portuguesa

Francisco Brás Gomes1,2,*, Symon Riedstra1, A. C. Silva Ferreira1, and João Paulo M. Ferreira1Development of an immunoassay for ciprofloxacin using phage-displayed antibody fragmentsCiprofloxacin (CPFX), one of the most widely prescribed antibiotic [1], is a large spectrum synthetic drug of the fluoroquinolone’s group. Because of its impact on health and environment, the EU has set a regulatory residual limit of 30 µg kg–1 for CPFX in edible animal tissues [2]. Therefore, ultra sensitive methods of detection and quantification need to be developed. CPFX is commonly determined by chromatographic procedures, namely HPLC and GC, but these techniques are laborious and time demanding. We report the isolation of phage-displayed antibody fragments (phAbs) against CPFX from a semi-synthetic phagemid library [3], as a step for the development of an immunoassay for CPFX. 4 ROUNDS PANNINGNext round of selection2. WashingWash with 4x PBST, 4x PBS Removal of non-specific phAbs3. ElutionIncubation of bound phAbs with excess CPFX (competition)• 1st and 2nd rounds with 250 mM CPFX• 3rd round with 25 mM CPFX• 4th round with 100 µM CPFX4. Gel filtrationSeparation of CPFX from eluted phAbs, (in order to prevent bacterial death in next step)5. InfectionAmplification of selected phAbs in E. coli ER25377. Magnetic Bead-ELISACPFXMagnetic bead with CPFXCPFX specific phAbs2. Selection by Phage Display3. Magnetic Beads-ELISAAll five positive monoclonal phAbs will be tested to determine their ability to bind free CPFX (and other fluoroquinolones) by competitive elution. Selected ones will be used to produce soluble and purified VH fragments. Their affinity constants will be estimated by competitive ELISA. Also, their ability to bind similar molecules will be evaluated. One or more VH fragments will be used in immunoassays to determine and quantify CPFX on environmental samples.Five positive monoclonal antibodies-fragments against CPFX were efficiently isolated from a semi-synthetic phage antibody library by panning against immobilized CPFX on magnetic beads. PCR and RFLP analyses showed that isolated clones were VH fragments. ELISA testing demonstrated affinity towards CPFX.1 Escola Superior de Biotecnologia - Universidade Católica Portuguesa2 Departamento de Biologia - Universidade de Aveiro* Corresponding author - brunobgomes@gmail.comCPFX pH 4, 2h, TroomAmine-derivatized magnetic beadEDC1. CPFX immobilization on magnetic beadsIn order to determine the content of CPFX-binding phAbs in samples, an ELISA using CPFX-magnetic beads was developed.• Blocking of beads with soy proteins.• Incubation of phAbs solution (approx,1010 phAbs) with 50 µg beads-CPFX, 2h, RT.• Washing with PBST + PBS.• Detection of bound phAbs using Anti-M13-HRP, 2h.• Washing with PBST + PBS.• Color development with ABTS solution for 30 min. • Transfer to an ELISA plate and reading of OD at 405 nm.CPFX was efficiently immobilized onto amine-derivatized magnetic beads using EDC (figure 1). Coupling efficiency was determined by quantification of unreacted CPFX by HPLC-UV. Using a molar excess of 1.3x CPFX, all amine groups were bound to ciprofloxacin. Under these conditions 1 µg of beads suspension corresponds to about  2.5 nmol immobilized CPFX.Enrichment of positive polyclonal phAbs against CPFX was confirmed by magnetic-beads ELISA. ELISA signal increased after each round. Library 1 2 3 4Control0,081 0,112 0.118 0,199 0,249 0,652Round of selectionELISA signalE. coli ER2537Magnetic barRFLP of individual amplified VH fragments from the 4th round was performed by digestion with the BstNI restriction enzyme.It was observed that there were 5 different phAbs, while 2 were identical (clones C and D). Introduction[1] Ferech, M. et al., 2006. J. Antimicrob. Chemother. 58. 401-407[2] Zonghui, Y. et al. 2001. Food Agr. Immunol. 13. 199-204[3] Griffiths, A.D. et al., 1994. EMBO J. 13, 3245-3260Methods ResultsConclusionsFuture workpH 4, TroomTable 1 - Results of immobilization assays. Percentage of CPFX unreacted was determined by HPLC-UV.Figure 5 - PCR amplification using LMB3 and FdSeq1 primers of antibody fragment genes from individual ampicillin-resistant clones isolated from 4th round. The expected size of the PCR products was about 1000 bp (scFv). Interestingly, all are about 500 bp, suggesting display of a VH fragment (See figure 4)500 bpEight PCR positive monoclonal phAbs were tested by ELISA in order to identify CPFX binding ones.Six were positive to CPFX while 2 were negative. A control was performed with phAbs specific to one other ligand.Figure 2 - Schematic representation of selection of antibody fragments against CPFX by phage displayFigure 3 - Magnetic beads ELISA of polyclonal phAbs obtained from library to 4th round. Control was with any phAbs.Figure 4 - Schematic representation of the phagemid pHEN2 from the synthetic library [3]. 1. SelectionRatio CPFX/Beads ( mol COO-/ mol NH3+) % CPFX unreacted % NH3+ reacted 0.5 10 53 0.9 9 82 1.1 16 94 1.3 12 100 2.5 58 100  The PCR results indicate that all antibody fragments’ genes ampli f ied from individual clones obtained after the 4th round were VH genes. Magnetic barFigure 6 - Magnetic bead ELISA of monoclonal phAbs isolated from the 4th roundFigure 7 - RFLP analysis of PCR amplified antibody fragment genes6. AmplificationMagnetic barVH fragment VL fragment      A B C D E FN1 N1100 bp ruler AB C D E FFigure 1 - Reaction scheme of immobilization of CPFX onto magnetic beads by the activation of carboxylic groups on CPFX with carbodiimide (EDC) and thenthe formation of an ester linkage to amine groups on magnetic beadsIncubation of 1010 phages displayingdifferent antibody fragments [3] with50 µg magnetic beads-CPFX

Development of an immunoassay for ciprofloxacin using phage-displayed antibody fragments

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Development of an immunoassay for ciprofloxacin using phage-displayed antibody fragments

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Repositório Institucional da Universidade Católica Portuguesa