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 .