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Multiplex Human Antibody Profiling with TruePlex™ Protein Arrays

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.

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Multiplex Human Antibody Profiling with TruePlex™ Protein Arrays

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  1. Multiplex Human Antibody Profiling with TruePlex™ Protein Arrays Profile antibodies to over 8000 human proteins using Luminex xMAP technology

  2. 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

  3. 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

  4. 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.

  5. 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

  6. Assay Principle + Sample containing human IgG Protein-coupled Luminex Bead + Anti-human PE Conjugate Read Signal in Luminex

  7. 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

  8. 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.

  9. Assay Protocol Dilute serum or plasma in Sample Diluent (1:50 – 1:1000). Add Bead Mix to each well. Remove liquid from wells by vacuum. Add controls and diluted samples to the designated wells. Cover and incubate for 1 hour with shaking. Wash beads three times. Add anti-human IgG PE conjugate to each well. Incubate plate for 1 hour with shaking. Wash beads three times. Add 100 µl Wash Buffer to each well. Shake plate for 2 minutes. Read signals in Luminex instrument.

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. Available TruePlex™ Profiling Arrays

  15. Autoimmune profiles in serum

  16. Autoimmune sera – data from SSB and TRIM21

  17. Normal (non-disease) Sera

  18. Dynamic Range

  19. Control Results

  20. 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

  21. Ordering Information 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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