antigen: NadA antibody subclass: fab cells used to produce the antibody: expressed in E.coli
Extracted molecule
protein
Extraction protocol
H and L chain V region genes of single plasmablasts isolated from peripheral blood were amplified separately and then joined by overlap extension PCR. Products were cloned into pET22 as bicistronic expression cassettes encoding for Fab antibody fragments and sequenced. Fab clones encoded by unique germ line were expressed in Escherichia coli Rosetta2 (DE3) strain (Novagen) in Enpresso B medium (Biosilta), according to manufacturer’s instructions. Induction was carried out with 1mM IPTG for 24h at 25°C. Total cell lysis was performed in CelLytic Express (Sigma-Aldrich). Recombinant Fabs were purified by Ni2+-affinity chromatographychromatography (Ni-NTA agarose resin, Qiagen), buffer-exchanged to Tris-HCl 20mM, NaCl 300mM, sodium azide 0.05%, pH8 and stored at 4°C. Recombinant Fabs were quantified by Bradford protein assay (Sigma-Aldrich) and their purity was assessed by SDS-PAGE after coomassie staining in non-reducing conditions.
The secondary antibody used for arrays was from Jackson Immunoresearch, (872 W Baltimore Pike, West Grove, PA 19390 USA). It was conjugated to the Alexafluor dye (Alexa 647) by the company.
Hybridization protocol
Protein arrays were first blocked 60 min in Blocking Buffer (Block it, Arrayit Sunnyvale, CA 94085, USA). The array was then incubated 1h a room temperature with Fabs diluted 1:50 for anti-human IgG-specific analysis. After washing with 0.1% Tween 20 in PBS buffer (PBST), arrays were incubated with an AlexaFluor®647-labelled anti-human IgG, fab specific secondary antibody (1:800 – Jackson Immunoresearch) at RT for 1 h to detect interactions. Slides were washed again as before, then rinsed in sterile water and dried.
Scan protocol
Fluorescence signals were detected by using a Powerscanner (Tecan Trading AG, Switzerland) and the 16-bit images were generated with Powerscanner software at 10 µm per pixel resolution.
Description
purified fragment antigen binding expressed in E.coli
Data processing
Images were processed using ImaGene 9.0 software (Biodiscovery Inc, CA, USA). Elaboration and analysis of image raw fluorescence Intensity (FI) data was performed using in-house developed software and R scripts. For each sample, the mean fluorescence intensity (MFI) of replicated spots was determined, after subtraction of the background value surrounding each spot.
