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| Status |
Public on Mar 19, 2009 |
| Title |
Allogeneic antibodies specifically target AML antigen NuSAP1 after bone marrow transplantation |
| Organism |
Homo sapiens |
| Experiment type |
Protein profiling by protein array
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| Summary |
Identifying the targets of immune response after allogeneic hematopoietic cell transplantation (HCT) promises to provide relevant immune therapy candidate proteins. We used protein microarrays to serologically identify Nucleolar and Spindle Associated Protein 1 (NuSAP1) and Chromatin Assembly Factor 1, subunit B (p60) [CHAF1b] as targets of new antibody responses that developed after allogeneic HCT. Western blots and ELISA validated their post-HCT recognition and enabled ELISA testing of 120 other allo-HCT patients. CHAF1b specific antibodies were predominantly detected in AML patients whereas NuSAP1 specific antibodies were exclusively detected in AML patients one year post-transplant (p<0.0001). Complete genomic exon sequencing failed to identify a nonsynonymous SNP for NuSAP1 and CHAF1b between the donor and recipient cells. Expression profiles and RT-PCR showed NuSAP1 was predominately expressed in the bone marrow CD34+CD90+ hematopoietic stem cells (HSC), leukemic cell lines and B lymphoblasts as compared to other tissues or cells. Thus, NuSAP1 is recognized as an immunogenic antigen in 65% AML patients and suggests a tumor antigen role. In conclusion, clinically important tumor antigens can be identified as new antibody targets after allogeneic HCT using high density protein microarrays
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| Overall design |
Plasma Profiling using ProtoArrays: Protein microarrays were obtained (ProtoArrays™ V3, Invitrogen Corporation, Carlsbad, CA) displaying 5,056 full-length human proteins, derived from the Ultimate ORF collection (Invitrogen Corp., Carlsbad, CA) printed in duplicate with N-terminal GST epitopes expressed in Baculovirus and affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. The arrays also contained control spots consisting of buffer, empty spots, and human IgG printed in four different concentrations (IgG[1] to IgG[4]). The arrays were incubated with blocking buffer for one hr followed by application of the plasma sample diluted 1:150 for 90 min. After washing the array four times for 10 min each with PBST buffer, secondary antibody was added (goat anti-human Alexa 647, Molecular Probes, Eugene, OR) for 90 min. After washing the slides with PBST buffer, the arrays were dried and fluorescent intensity was detected using a microarray scanner (GenePix 4000B, Molecular Devices, CA). All incubations were carried out on a rotating platform at 4 ºC.
ProtoArray data acquisition and measurement: The slides were scanned at a PMT gain of 60% with a laser power of 90% and a focus point of 0 μm. Fluorescence intensity data were acquired using GenePix Pro 6.0 software (Molecular devices, Sunnyvale, CA) with the appropriate “.gal” file downloaded from the ProtoArray central portal on the Invitrogen website (http://www.invitrogen.com/protoarray) by submitting the barcode of each ProtoArray slide
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| Contributor(s) |
Wadia PP, Coram M, Armstrong RJ, Mindrinos M, Butte AJ, Miklos DB |
| Citation(s) |
20053754 |
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| Submission date |
Mar 17, 2009 |
| Last update date |
Mar 21, 2012 |
| Contact name |
Persis P Wadia |
| E-mail(s) |
pwadia@stanford.edu
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| Phone |
1-650-725-2989
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| Fax |
1-650-724-5623
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| Organization name |
Stanford University
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| Department |
Medicine
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| Lab |
Miklos Lab
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| Street address |
269 W Campus Drive, CCSR 2210
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platforms (1) |
| GPL6454 |
ProtoArray Human Protein Microarray v3.0 |
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| Samples (9)
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| Relations |
| BioProject |
PRJNA116549 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE15255.xls.gz |
8.9 Mb |
(ftp)(http) |
XLS |
| GSE15255_RAW.tar |
8.7 Mb |
(http)(custom) |
TAR (of GPR) |
| Processed data included within Sample table |
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