|
| Status |
Public on Aug 30, 2018 |
| Title |
5H2 Fab-anti-NHBA |
| Sample type |
protein |
| |
|
| Source name |
recombinant antibody
|
| Organism |
Homo sapiens |
| Characteristics |
description: produced by transient transfection using Expi293 (Life Technologies) cells in suspension
subclass: fab
|
| Extracted molecule |
protein |
| Extraction protocol |
Gene fragments corresponding to the heavy and light chains of the variable regions were synthesized by Geneart (Life Technologies) with Eco31I site flanking gene extremities. The 5H2 Fab was produced by transient transfection using Expi293 (Life Technologies) cells in suspension, according to the manufacturer’s protocol. Equal amounts (15 μg each/30 mL of transfection volume) of vector DNA encoding FabH and FabL chain were used to transfect Expi293 cells. The cells were incubated at 37°C with a humidified atmosphere of 8% CO2 in air on an orbital shaker rotating at 125 rpm. Cell culture supernatant was harvested after 36 and 72 h post transfection, clarified by centrifugation for 30 min at 4000 rpm and concentrated. The Fab was purified from the supernatant using a Strep Trap HP pre-packed column with StrepTactin Sepharose High Performance medium (GE Healthcare), followed by gel filtration chromatography on a Superdex 200 16/60 column (GE Healthcare). After purification, a cleavable C-terminal Strep tag II on the heavy chain was removed with TEV (tobacco etch virus) protease. Purity of the Fab was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after coomassie staining and its amount was quantified by absorbance at 280 nm.
|
| Label |
AlexaFluor®647-labelled anti-human IgG, fab specific secondary antibody
|
| Label protocol |
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 array was first blocked 60 min in Blocking Buffer (Block it, Arrayit Sunnyvale, CA 94085, USA). The array was then incubated 1h a room temperature with huFab 5H2 diluted 1:2000 for anti-human IgG-specific analysis. After washing with 0.1% Tween 20 in PBS buffer (PBST), array was 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. Slide was 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
n/a
|
| Data processing |
Image was 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 8 replicated spots was determined, after subtraction of the background value surrounding each spot.
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| |
|
| Submission date |
Apr 05, 2018 |
| Last update date |
Aug 30, 2018 |
| Contact name |
Erika Bartolini |
| Organization name |
GSK Vaccines
|
| Department |
AIMB
|
| Lab |
Molecular Biology
|
| Street address |
Via Fiorentina 1
|
| City |
Siena |
| State/province |
Italy |
| ZIP/Postal code |
53100 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24853 |
| Series (1) |
| GSE112752 |
Protein microarray epitope mapping of monoclonal antibody 5H2 raised by human immunization with the 4CMenB vaccine formulation containing the fusion antigen NHBA-NMB1030. |
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