Multiplex autoantibody profiling for autoimmune diseases and cancer
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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)

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Multiplex autoantibody profiling for autoimmune diseases and cancer

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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)

    • 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

Identification of many targets from a small number of samples

Biomarker Screening

& Quantitative Evaluation

Large sample #s

Refine target list

ROC calculations

  • 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

  • Custom TruePlex™ Antibody Profiling Array

  • TruePlex™ Antibody Profiling Array

  • (pre-defined array)

and/or

Multiple profiling arrays can be mixed together and analyzed as a larger multiplex


Assay Protocol

Dilute sample

Mix with beads, incubate

Total time – about 2.5 hours for 96 samples

Up to 100 results per well

Quantitative results

Wash, add conj. & incubate

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


Autoimmune profiles in serum


Autoimmune sera – data from SSB and TRIM21


Normal (non-disease) Sera


Dynamic Range


Control Results


Breast cancer samples tested with 20-plex tumor antigen panel


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

  • ProstateCancer

    • 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 Available

Autoantibodies to tailor-made panels of tumor-associated antigens in breast carcinoma.E Piura and B PiuraJ 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


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