Transcript 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.

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 cancerassociated 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
Large sample #s
Refine target list
ROC calculations
Identification of many
targets from a small number
of samples
Biomarker Screening
& Quantitative Evaluation
• Luminex Bead Arrays
• Mid-Density Multiplexed methods
Clinical Assays
• Luminex Bead Arrays
• Low-Density Multiplexed
methods
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 Antigens
Any 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 Kit
Assay Reagents, Control Bead Mix, Positive Control
combine with
TruePlex™ Antibody
Profiling Array
(pre-defined array)
and/or
Custom TruePlex™
Antibody Profiling
Array
Multiple profiling arrays can be mixed together and analyzed
as a larger multiplex
Dilute sample
Mix with beads,
incubate
Wash, add conj. &
incubate
Assay Protocol
Total time – about 2.5 hours for 96 samples
Up to 100 results per well
Quantitative results
Wash
Read in Luminex
Analyze Data
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 Arrays
Panel
Human Autoimmune I
Symbol
SNRPA
SSB
TROVE2
SNRNP70
SNRPC
Name
U1-snRNP A Protein [SNRPA 31 kD]
La/SS-B (Sjogren syndrome antigen B))
Ro/SS-A (TROVE domain family, member 2)
U1-snRNP 68 Protein (68 kDa)
U1-snRNP C Protein [SNRPC 18 kd]
Human Autoimmune II
TRIM21
PCNA
XRCC6
CENPA
AARS
Ro/SS-A (52 kDa) (Tripartite motif-containing 21)
Proliferating Cell Nuclear Antigen (PCNA)
Ku (p70/p80)
Centromere Protein A (CENP-A)
Alanyl-tRNA Synthetase (PL-12)
Human Autoimmune III
APOH
GIF
PRTN3
FTCD
CYP2D6
Apolipoprotein H
Intrinsic Factor (Gastric)
Proteinase 3
Formiminotransferase Cyclodeaminase (LC1)
Cytochrome P450 2D6 (LKM 1)
CENPB
MPO
RPLP0
Centromere Protein B, 80 kD (CENP-B)
Myeloperoxidase
Ribosomal Phosphoprotein P0
IFIH1
SNRPB
Melanoma differentiation-associated protein 5 (MDA5)
U-snRNP B/B'
Human Autoimmune IV
Autoimmune profiles in serum
35000
Median Fluorescent Intensity
30000
Lupus (SLE)
25000
Scleroderma
20000
Sjorgren's Syndrome
15000
10000
5000
0
Antigen bound to Luminex bead
SJO-5
SJO-4
SJO-3
TRIM21 (Ro/SS-A)
SJO-2
25000
SJO-1
SSB (La/SS-B)
SCL-5
30000
SCL-4
SCL-3
SCL-2
SCL-1
SLE10
SLE9
SLE8
SLE7
SLE6
SLE5
SLE4
SLE3
SLE2
SLE1
Median Fluorescent Intensity
Autoimmune sera – data from SSB and TRIM21
35000
20000
15000
10000
5000
0
500
450
400
350
300
250
200
150
100
50
0
SSB
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Sample Number
MFI
MFI
Normal (non-disease) Sera
500
450
400
350
300
250
200
150
100
50
0
SNRPA
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25
Sample Number
Dynamic Range
Control Results
30000
Median Fluorescent Intensity
25000
Positive Control
Negative Serum
20000
Lupus (SLE) Serum
15000
10000
5000
0
Antigen Coupled to Bead
Breast cancer samples tested with 20-plex tumor
antigen panel
4500
4000
3500
BC-4
BC-5
3000
2500
2000
1500
1000
500
0
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-ESO1/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 Available
Autoantibodies to tailor-made panels of
tumor-associated antigens in breast
carcinoma.
E Piura and B Piura
J Oncol, Jan 2011; 2011: 982425.
Seromic profiling of ovarian and pancreatic
cancer.
S. Gnjatic S, et al.
PNAS U S A. Mar 2010 16;107(11):5088-93.
A Distinct Repertoire of Autoantibodies in
Hepatocellular Carcinoma Identified by
Proteomic Analysis
François Le Naour, et al.
Mol. Cell. Proteomics, Mar 2002; 1: 197 203.
Multiple serological biomarkers for colorectal
cancer detection.
CC Chan, et al.
Int J Cancer, Apr 2010; 126(7): 1683-90.
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 discoveries
Improve human health
Thank you for attending !
Questions ?
Contact us:
[email protected]
[email protected]
240-620-0237 (direct)
www.origene.com/assays