Ultra-sensitive biomarker quantification in 1µl to 5µl sample volume

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1 Ultra-sensitive biomarker quantification in 1µl to 5µl sample volume Dr. Beena Punnamoottil Sen. Project Manager Chimera Biotec GmbH

2 Microsampling I t=0 t=1 t=2 t=3 t=4 t=5 t=0 t=5 II III Assay A Assay B Assay C I II I II I II I II III Pre-Clinical PK à composite sampling vs. serial sampling from one animal Only small sample size available à limited biofluids (e.g. liquor, synov. fluid ) Non-invasive sample drawing desirable à prenatal, neonatal, pediatric One sample à multiple biomarker analysis 2

3 Biomarker support feasible? Low sample volume vs. High assay sensitivity? 2x 30µl Pipetting volume = 70µl The lower the neat sample volume, the higher the required sample dilution 3

4 Biomarker support feasible? Imperacer : Exponential signal amplification ² fine-tune sensitivity vs. sample consumption ² tolerate higher sample dilutions ² minimize neat sample volume 4

5 Is biomarker quantification possible from less than 10µl neat sample? Interleukin x 30µl Imperacer [delta Ct] pg/ml 2.3pg/ml 0.3pg/ml NC Buffer A Buffer B Buffer C Buffer D 1. Fine-tuning of sample dilution in optimal buffer 5

6 Is biomarker quantification possible from less than 10µl neat sample? Interleukin 6 Vol. 5µl 2x 1:14 dilution => 70µl 30µl Neat Sample (vol. 5µl) Diluted Sample (1:14) 27 Imperacer [delta Ct] Fine-tuning of sample dilution in optimal buffer 2. Fine-tuning of quantitative assay range 3. Assay sensitivity: 3pg/ml pg/ml NC IL-6 [pg/ml] 6

7 Is biomarker quantification possible from less than 10µl neat sample? Interleukin 6 Vol. 5µl 1:14 dilution => 70µl nominal Calc. Conc. inter-assay intra-assay [pg/ml] average CV% %RE CV% %RE Standards QCs

8 How low can we go to trade off sample consumption vs. sensitivity? Interleukin 2 Vol. 3.5µl Vol. 0.7µl 2x 30µl Dilution 1:20 Dilution 1:100 8

9 How low can we go to trade off sample consumption vs. sensitivity? Interleukin Standards 1:20 Standards 1:100 Vol. 3.5µl Vol. 0.7µl delta Ct pg/ml 1000 IL-2 [pg/ml] #1 #2 #3 #4 0.1 endogenous + 10 pg/ml pg/ml endogenous + 10 pg/ml pg/ml nominal 10 pg/ml nominal 100 pg/ml Dilution Samples 1: Samples Dilution 1:

10 How low can we go to trade off sample consumption vs. sensitivity? Interleukin Standards 1:20 Standards 1:100 Vol. 3.5µl Vol. 0.7µl delta Ct pg/ml Biomarker quantification is possible with less than 5µl neat sample volume! 10

11 Multiple target quantification in less than 5µl neat sample? Interleukin 2 Vol. 2.5µl 1:28 dilution => 70µl 2x 30µl Interleukin 6 Vol. 1µl 1:70 dilution => 70µl nominal Calc. Conc [pg/ml] Recovery [%RE] [pg/ml] IL2 IL6 IL2 IL NC

12 Multiple target quantification in less than 5µl neat sample? Interleukin 6 Vol. 1µl 1:70 dilution => 70µl Interleukin 2 Vol. 2.5µl 1:28 dilution => 70µl Ø Preliminary assay range from 3pg/ml to 729pg/ml for IL-2 and IL-6 Ø Required neat sample volume 2.5µl and 1µl respectively Ø 5µl neat sample material is sufficient for multiple target quantification 12

13 Multiple target quantification in less than 5µl neat sample? Interleukin 6 Vol. 1µl Imperacer [delta Ct] :35 dilution => 35µl 1x refrozen 2x refrozen 3x refrozen NC IL-6 [pg/ml] 1: Neat Sample (vol. 1µl) 2: Pre-diluted Sample (1:35) Ø Pre-diluted sample stability confirmed Ø Assay range: 3pg/ml pg/ml 3: Frozen Sample (Vol. 35µl) 13

14 Multiple target quantification in diseased sample material? Average IL-6 3 healthy control sera 3 psoriatic arthritis sera 3 rheumatoid arthritis sera Cytokine (IL-2 or IL-6) [pg/ml] Average IL-2 Healthy Control Psoriatic Arthritis Rheumatoid Arthritis IL-6: Vol. 1µl 1:70 dilution IL-2: Vol. 2.5µl 1:28 dilution Each target quantified under best assay conditions in parallel (Imperacer polyplex): Ø Different assay strategies: Sequential (IL-6) & Combined incubation (IL-2) Ø Imperacer polyplex assay: Sample analysis in parallel Ø Total sample: Vol. 5µl for both targets in double determination 14

15 Multiple target quantification in diseased sample material? Average IL-6 3 healthy control sera 3 psoriatic arthritis sera 3 rheumatoid arthritis sera Cytokine (IL-2 or IL-6) [pg/ml] Average IL-2 Healthy Control Psoriatic Arthritis Rheumatoid Arthritis Imperacer polyplex assays for microsamples: Ø Diseased individuals reveal significantly higher concentrations of tested cytokines Ø Individual disease status evaluation possible Ø Polyplex assays enable multiple target quantification with 5µl neat sample only 15

16 Summary & Conclusion Is biomarker quantification possible with less than 10µl neat sample? ü Yes, low level target quantification with Vol. 5µl neat sample How low can we go in regard to neat sample volume? v Ultra-sensitive Imperacer & fine tuning of dilution + assay strategy à low level target quantification with Vol. 1µl neat sample What is the clinical/diagnostic value? Ø Multiple target analysis is feasible in microsampling volume Ø Biomarker quantification in microsamples can benefit pre-clinical and clinical applications in challenging matrices and low volume samples Challenging Matrices Low Volume Samples Multiple Targets 16

17 Imperacer Ultra-Sensitive Ligand-Binding Assays and Method Development Thanks to: Michael Adler Hendrik Schröder Mark Spengler Chris<an Pieper Chimera Lab- Team Contact: Chimera Biotec GmbH Dr. Beena Punnamoottil Phone: + 49 (0) Fax: + 49 (0) Dive deeper into the proteome

18 Imperacer Ultra-Sensitive Ligand-Binding Assays and Method Development Contact: Thank you for your aden<on Chimera Biotec GmbH Dr. Beena Punnamoottil Phone: + 49 (0) Fax: + 49 (0) Dive deeper into the proteome

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22 Is biomarker quantification possible with less than 10µl neat sample? GM-CSF Vol. 5µl Imperacer assays for microsamples: Ø Best sample dilution buffer for each target-matrix composition Ø Best incubation strategy for each target-matrix composition Ø Maintenance of assay sensitivity with 5µl neat sample only 22

23 Assay Strategies Assay Strategy Combined incubation 2x 1: 2: Spiked Sample 3: Diluted Sample 1+9 / : 1+1 with Detection Conjugate (70 µl) 5: Incubate on plate 30 µl Sequential incubation 2x 2x 1: Neat sample (vol. 5 µl) 2: Diluted sample (1+13µl) 3: Freeze diluted sample 4: Thaw stored sample 5: Incubate (30 µl/well) 6: Detection conjugate 23