|
| Status |
Public on Mar 07, 2018 |
| Title |
HumAb 1A12 |
| Sample type |
protein |
| |
|
| Source name |
recombinant antibody
|
| Organism |
Homo sapiens |
| Characteristics |
antigen: fHbp
antibody subclass: IgG1
cell line used to produce recombinant antibody: Expi 293
|
| Extracted molecule |
protein |
| Extraction protocol |
Optimized gene fragments corresponding to VH and VL of desired human monoclonal antibodies were inserted in a expression vector containing a human Ig gene signal peptide sequence and human IgG1, IgK or IgL costant regions under the CMV promoter. Transient production of recombinant antibodies was performed in suspension Expi293 (Thermo Fisher Scientific) according to manufactoring protocol. Recombinant antibodies were purified from supernatants with protein G beads according to manufacturer's (GE Healthcare) instructions.
|
| Label |
AlexaFluor®647-labelled anti-human secondary antibody
|
| Label protocol |
The secondary antibody used for arrays was from Jackson Immunoresearch, AlexaFluor®647-conjugated anti-Human IgG secondary antibody (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 |
Peptide 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 mAb diluted 1:200 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 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 recombinant human monoclonal antibody
|
| 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 3 replicated spots was determined, after subtraction of the background value surrounding each spot.
|
| |
|
| Submission date |
May 12, 2017 |
| Last update date |
Mar 07, 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 |
GPL23475 |
| Series (2) |
| GSE98879 |
Epitope mapping of 5 monoclonal antibodies directed against meningococcal factor H binding protein by peptide microarray |
| GSE98883 |
Epitope mapping of human antibodies directed against 4CmenB protein components |
|
