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Multiplex Human Antibody Profiling with TruePlex™ Protein Arrays
Profile antibodies to over 8000 human proteins using Luminex xMAP
technology
Autoantibodies as Biomarkers
• The immune system functions as a an early warning system.– Malignant transformation, infections, and many
diseases can trigger an immune response• Immune response generates antibodies to self-
antigens (autoantibodies).• Autoantibodies can be detected much earlier and
with much higher sensitivity than other disease biomarkers.
• Antibodies are abundant and stable in serum.• Panels of autoantibodies are much more powerful
than single autoantibody
Autoantibodies to Tumor Antigens
• Tumors typically over-express multiple normal and mutant antigens
• Antibodies to these antigens can be found in the serum of most cancer patients.
• Autoantibodies to tumor antigens can be detected much earlier than other cancer biomarkers and are a promising approach for early detection.
• 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 optimal for autoantibody response profiling
Autoimmune Diseases
• Autoimmune diseases affect an estimated 3% of the world population.
• Over 150 recognized autoimmune diseases.• Autoimmune diseases are characterized by broad
autoantibody profiles.• There is significant overlap in the autoantibody profiles of
different autoimmune diseases.• Autoantibody profile can provide an 'autoantibody fingerprint‘
with potential diagnostic and prognostic utility.• Profiles may help classify disease variants and indicate
organ-specific involvement.• Autoantibody profiles may help guide the development and
measure the effectiveness of antigen-specific tolerizing therapy.
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 Bead
Sample containing human IgG
Anti-human PE Conjugate
+
+
Read Signal in Luminex
8000+ 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
Product Configuration
Multiple profiling arrays can be mixed together and analyzed as a larger multiplex.
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
Assay Protocol
1. Dilute serum or plasma in Sample Diluent (1:50 – 1:1000).
2. Add Bead Mix to each well.
3. Remove liquid from wells by vacuum.
4. Add controls and diluted samples to the designated wells.
5. Cover and incubate for 1 hour with shaking.
6. Wash beads three times.
7. Add anti-human IgG PE conjugate to each well.
8. Incubate plate for 1 hour with shaking.
9. Wash beads three times.
10.Add 100 µl Wash Buffer to each well.
11.Shake plate for 2 minutes.
12.Read signals in Luminex instrument.
Control Beads
• Anti-human IgG– Anti-human IgG antibody is coupled to the Luminex beads. This bead serves as a
positive control for the presence of human IgG in samples since human IgG in samples will be bound by this bead and subsequently detected.
• Human Serum Albumin– Recombinant human serum albumin is bound to Luminex beads. This bead serves
as a non-specific binding control. This protein does not contain the protein fusion tag.
• Tagged Protein– OriGene’s recombinant proteins all contain a small c-terminal fusion tag (myc-DDK).
This bead is coupled with tagged BSA and serves a non-specific binding control for samples. This bead will be positive with the Positive Control included in the kit.
• Donkey IgG– Donkey IgG is bound to Luminex beads. This bead serves as a non-specific binding
control for the detection antibody.
Profiling Kit Components
Positive Control (50X)
Buffer A (Low Stringency)
Buffer B (High Stringency)
Buffer C
Blocking Reagent
Control Bead Mix (50X)
Assay Buffer E
Wash Buffer (20X)
Detection Reagent (50X) (Anti-Human IgG-phycoerythrin)
Filter Plate
Plate Sealers
Positive and Negative Controls
• Positive Control– A 50X positive control is included in the kit.
It reacts with the tag sequence on the recombinant proteins and is detected with the anti-human-PE conjugate. It can be diluted in parallel with the samples.
• Negative Control– Sample Diluent is used as the negative
control. Normal sera or plasma samples can also be used as negative controls.
Stringency Options
• Reagents are supplied so that Sample Diluent can be prepared at different stringencies (low, medium and high).
• High-stringency Sample Diluent will minimize non-specific signals and is recommended for initial sample testing.
• For samples with low autoantibody levels, for samples in which the antibody affinity may be low or for higher sample dilutions (1:500 or greater), the medium- or low-stringency Sample Diluent may give better results.
Available TruePlex™ Profiling Arrays
Panel 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
TRIM21
SNRPASSB
APOH GIFPCNA
PRTN3
RPLPO
FTCD
CYP2D6
TROVE2
SNRNP70XRCC6
CENPASNRPC
MPOSNRPB
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
50
100
150
200
250
300
350
400
450
500 SSB
Sample Number
MF
I
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
50
100150
200250
300
350400
450500 SNRPA
Sample Number
MF
I
Dynamic Range
Control Results
anti-human IgG
Human IgG
Albumin
BSA-Tag
Donkey IgG
SNRP700
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
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
Ordering Information
TruePLEX™ Human Antibody Profiling Kit AP100001
TruePLEX™ Antibody Profiling Array: Human Autoimmune I
AP100002
TruePLEX™ Antibody Profiling Array: Human Autoimmune II
AP100003
TruePLEX™ Antibody Profiling Array: Human Autoimmune III
AP100004
TruePLEX™ Antibody Profiling Array: Human Autoimmune IV
AP100005
For questions or to inquire about a custom array containing any of OriGenes 8000+ recombinant human proteins, contact OriGene at assays@origene.com .
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