multiplex autoantibody profiling for autoimmune diseases and cancer
DESCRIPTION
Multiplex autoantibody profiling for autoimmune diseases and cancer. April, 2, 2014. Profile antibodies to over 10,000 human proteins using Luminex xMAP™ technology. Jim Lazar, Ph.D. VP, Assay Development. OriGene Introduction. OTI Locations Rockville (HQTs/R&D) Seattle (Blue Heron) - PowerPoint PPT PresentationTRANSCRIPT
Multiplex autoantibody profiling for autoimmune diseases and cancer
Profile antibodies to over 10,000 human proteins using Luminex xMAP™
technology
April, 2, 2014
Jim Lazar, Ph.D.VP, Assay Development
OriGene Introduction
• OTI Locations– Rockville (HQTs/R&D)– Seattle (Blue Heron)– Delaware (SDIX)– Maine (SDIX)– Foster City (BioCheck)– Beijing– Wuxi
Antibodies as Biomarkers• The immune system functions as a an early warning
system.– Malignant transformation, infections, and many
diseases can trigger an immune response• Antibodies are abundant, easily detectable and stable in
serum.
Autoantibodies to Tumor Antigens
• Tumors typically over-express multiple normal and mutant antigens
• Autoantibodies to tumor antigens can be detected much earlier than other cancer biomarkers
• Frequency of autoantibody responses to an individual tumor antigen is typically 15-20%.
• Responses to a panel of antigens can be higher than 90%
• Multiplexed autoantibody detection to a panel of antigens is necessary.
What is EarlyCDT–Lung?EarlyCDT-Lung is a simple, physician-ordered, diagnostic blood test that measures the presence of autoantibodies to a panel of seven lung cancer-associated antigens.
Autoimmune Diseases
• Autoimmune diseases affect an estimated 3% of the world population.
• Over 150 recognized autoimmune diseases.
• Each disease is characterized by autoantibodies to multiple antigens.
• Profiles may help classify disease variants and indicate organ-specific involvement.
• Autoantibody profiles may help guide the development of antigen-specific tolerizing therapy.
Biomarker Discovery• Proteomic /2D gel methods• Proteomic Mass Spec methods• High Density Protein Microarrays
Biomarker Screening & Quantitative Evaluation• Luminex Bead Arrays• Mid-Density Multiplexed methods
Clinical Assays
Identification of many targets from a small number of samples
• Luminex Bead Arrays• Low-Density Multiplexed
methods
Large sample #sRefine target listROC calculations
10,000+ Full Length Human Proteins
• Produced from TrueORF Gold™ cDNA clones– Fully sequenced– Expression validated
• Expressed in HEK293 cells– Human-expressed– Affinity purified
• Native presentation– Optimal preservation of protein
structure, – post-translational modifications
Available AntigensAny of OriGene’s >10,000 purified proteins can be custom-coupled to Luminex beads
Luminex xMAP™ technology
• Multiplex Bead Array• Efficient high-throughput
microplate format• Up to 100 targets per well• OriGene is a Luminex partner
and Certified Assay Developer
Assay Principle
Protein-coupled Luminex Beads
Sample containing human IgG
Mix with Anti-human PE Conjugate
+
+
Read Signal in Luminex
Product Configuration
TruePlex™ Human Antibody Profiling KitAssay Reagents, Control Bead Mix, Positive Control
combine with
TruePlex™ Antibody Profiling Array
(pre-defined array)
Custom TruePlex™ Antibody Profiling
Arrayand/or
Multiple profiling arrays can be mixed together and analyzed as a larger multiplex
Assay ProtocolDilute sample
Mix with beads, incubate
Wash, add conj. & incubate
WashRead in Luminex
Analyze Data
Total time – about 2.5 hours for 96 samples
Up to 100 results per well
Quantitative results
Stringency Options
• Sample Diluent can be prepared at different stringencies – High, medium or low
• High-stringency Sample Diluent will minimize non-specific signals and is recommended for initial sample testing.
• The medium- or low-stringency Sample Diluent may give better results for samples – with low autoantibody levels,
– in which the antibody affinity may be low
– for higher sample dilutions (1:500 or greater),
Available TruePlex™ Profiling ArraysPanel Symbol Name
Human Autoimmune I
SNRPA U1-snRNP A Protein [SNRPA 31 kD]SSB La/SS-B (Sjogren syndrome antigen B))
TROVE2 Ro/SS-A (TROVE domain family, member 2)
SNRNP70 U1-snRNP 68 Protein (68 kDa)SNRPC U1-snRNP C Protein [SNRPC 18 kd]
Human Autoimmune II
TRIM21 Ro/SS-A (52 kDa) (Tripartite motif-containing 21)PCNA Proliferating Cell Nuclear Antigen (PCNA)XRCC6 Ku (p70/p80)CENPA Centromere Protein A (CENP-A)AARS Alanyl-tRNA Synthetase (PL-12)
Human Autoimmune III
APOH Apolipoprotein HGIF Intrinsic Factor (Gastric)
PRTN3 Proteinase 3FTCD Formiminotransferase Cyclodeaminase (LC1)
CYP2D6 Cytochrome P450 2D6 (LKM 1)
Human Autoimmune IV
CENPB Centromere Protein B, 80 kD (CENP-B) MPO Myeloperoxidase
RPLP0 Ribosomal Phosphoprotein P0
IFIH1 Melanoma differentiation-associated protein 5 (MDA5)SNRPB U-snRNP B/B'
Autoimmune profiles in serum
TRIM
21SN
RPASS
BAPOH GIF
PCNAPRTN
3RPLP
OFT
CD
CYP2D6
TROVE2
SNRNP70
XRCC6CEN
PASN
RPCMPO
SNRPB
0
5000
10000
15000
20000
25000
30000
35000
Lupus (SLE)
Scleroderma
Sjorgren's Syndrome
Antigen bound to Luminex bead
Med
ian
Fluo
resc
ent I
nten
sity
Autoimmune sera – data from SSB and TRIM21
SLE1
SLE2
SLE3
SLE4
SLE5
SLE6
SLE7
SLE8
SLE9
SLE1
0
SCL-
1
SCL-
2
SCL-
3
SCL-
4
SCL-
5
SJO
-1
SJO
-2
SJO
-3
SJO
-4
SJO
-5
0
5000
10000
15000
20000
25000
30000
35000
SSB (La/SS-B)
TRIM21 (Ro/SS-A)
Med
ian
Fluo
resc
ent I
nten
sity
Normal (non-disease) Sera
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250
50100150200250300350400450500 SSB
Sample Number
MFI
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 250
50100150200250300350400450500 SNRPA
Sample Number
MFI
Dynamic Range
Control Results
anti-human Ig
G
Human IgG
Albumin
BSA-Tag
Donkey Ig
G
SNRP70
0
5000
10000
15000
20000
25000
30000
Positive Control
Negative Serum
Lupus (SLE) Serum
Antigen Coupled to Bead
Med
ian
Fluo
resc
ent I
nten
sity
Breast cancer samples tested with 20-plex tumor antigen panel
001_DBT
002_PDCIM
1
004_MYC
006_SF3
A1
009_PDCD6IP
010_PPIA
011_BCCIP
012_EIF4
A3
013_CSNKIE
014_NR2E3
015_ERBB2
017_PRDX2
018_BIRC5
021_IGF2
BP2
022_HSP
D1
024_MUC1
027_CDKN2A
028_HSPA14
029_CTAG1A
044_TP53
0
500
1000
1500
2000
2500
3000
3500
4000
4500
BC-4
BC-5
BC-7
BC-9
Available cancer tumor antigens known to generate autoantibodies
• Breast Cancer– ASB-9, BAT4, BDNF, Survivin, Livin, BMX, BRCA2, P16, CSNK1E, NY-ESO-1/LAGE1, CTBP1,
DBT, EIF3E, HER2, Fibulin, FKBP4, GIPC-1, HSP-90, HSPA4, HSP-27, HSP-60, IMP1 , P62, IMP2, MUC1, C-Myb, c-myc, PDCD6IP, PPIA, PRDX2, RAB5A, RAC3, Lipophilin B, SERPINH1, SF3A1, SOX2, p53, TRIM32, UBAP1
• Prostate Cancer– Caldesmon 1, Clusterin, c-myc, HER2/neu, HSP70, HSP71, IMP1, MCP1, NY-ESO-1/LAGE1,
p53, p62, p90, PARK7, PSA, RACE, SSX2, SSX4, Survivin, TARDBP, TTLL12
• Pancreatic Cancer– CTDSP1, DNAJB1,EIF4A3 (DDX48), ELAC1,GAS2, HCFC1R1,HERPUD1, MAPK9, NR2E3,
NR2E3, PDLIM1 (CLP36), PGK1,PPARG, PTPRA, RNF213 (KIAA1618), ROR2, SHOC2, SMOX, TMOD1, TMSB10, ULK4
• Antigens for other cancers and diseases available including– Ovarian cancer, melanoma, hepatocellular carcinoma, lung cancer, diabetes, COPD,
multiple sclerosis
http://www.origene.com/Luminex/Proteinarray
Extensive Bibliography AvailableAutoantibodies to tailor-made panels of tumor-associated antigens in breast carcinoma.E Piura and B PiuraJ Oncol, Jan 2011; 2011: 982425.
Multiple serological biomarkers for colorectal cancer detection.CC Chan, et al. Int J Cancer, Apr 2010; 126(7): 1683-90.
A Distinct Repertoire of Autoantibodies in Hepatocellular Carcinoma Identified by Proteomic AnalysisFrançois Le Naour, et al.Mol. Cell. Proteomics, Mar 2002; 1: 197 - 203.
Seromic profiling of ovarian and pancreatic cancer. S. Gnjatic S, et al.PNAS U S A. Mar 2010 16;107(11):5088-93.
Summary
• Autoantibodies are useful biomarkers for cancer and autoimmune diseases.
• OriGene’s TruePlex profiling kits are optimally suited for biomarker screening and validation.
• Choose from over 10,000 proteins expressed in human cells.
• The assay procedure is simple and fast.
Make great discoveriesImprove human health
Thank you for attending !
Questions ?
Contact us: [email protected]
240-620-0237 (direct)
www.origene.com/assays