|
| Status |
Public on May 27, 2016 |
| Title |
mAbs 31E10E7 R3 peptide array |
| Sample type |
protein |
| |
|
| Source name |
purified murine monoclonal antibody
|
| Organism |
Mus musculus |
| Characteristics |
antigen: Neisserial Heparin Binding Antigen
antibody subclass: IgG2a
antibody production: hybridoma cell line
|
| Extracted molecule |
protein |
| Extraction protocol |
The murine anti-NHBA 31E10/E7 IgG2a mAb was produced and purified by conventional hybridoma production methods. The mAb was purified from culture supernatants by Protein G affinity columns (GE Healthcare). The mAb subclass was determined using a mouse mAb isotyping kit (Roche).
|
| Label |
AlexaFluor®647-labelled anti-mouse 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 |
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-mouse IgG-specific analysis. After washing with 0.1% Tween 20 in PBS buffer (PBST), arrays were incubated with an AlexaFluor®647-labelled anti-mouse 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.
|
| 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.
|
| |
|
| Submission date |
May 12, 2016 |
| Last update date |
May 12, 2017 |
| 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 |
GPL21866 |
| Series (2) |
| GSE81377 |
Epitope mapping of a monoclonal antibody directed against Neisserial Heparin Binding Antigen by peptide microarray |
| GSE81379 |
Epitope mapping of a monoclonal antibody directed against Neisserial Heparin Binding Antigen |
|
