How to improve the sensitivity of the multiplex detection of cancer biomarkers?multiplex detection of cancer biomarkers?

Zhugen Yanga, Yann Chevolota, Yasemin Ataman-Önalb, Geneviève Choquet-Kastylevskyb, Claude Lambertc, Eliane Souteyranda and

Emmanuelle LaurenceauaEmmanuelle Laurenceau

a Université de Lyon Institut des Nanotechnologies de Lyon UMR-CNRS 5270Université de Lyon, Institut des Nanotechnologies de Lyon , UMR CNRS 5270, Ecole Centrale de Lyon (France)

b bioMérieux, Département bioMarqueurs, Marcy l’Etoile (France)c Laboratoire d’Immunologie clinique IFR143, CHU Saint-Etienne Hôpital Nord (France)

118èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Context of the study

Colorectal cancer is the third cancer worldwide with a survival rate ~ 56%

Efficient decrease of mortality with mass-screening (FOBT)good specificity = 98%good specificity 98%low sensitivity ~ 50-60%

A tib d b d iFOBT iti it b t ifi itAntibody-based iFOBT: sensitivity but specificity

Urgent need to develop blood-based assay for cancerUrgent need to develop blood-based assay for cancer biomarkers detection

increase sensitivity and keep high specificityimprove patient’s compliancereduce mortality

228èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

reduce mortality

(Mazurczyk R et al. Sens Actuators B chem, 2008; 128(2): 552-559)

Customized antibody microarray on 3D-microstructured Chip

(Mazurczyk R et al. Sens Actuators B chem, 2008; 128(2): 552 559)

40 microwellsVolume = 250 nL

parallel screening of 40 biological samples or experimental

Volume = 250 nL

p g g p pconditions

tiny sample requirement

Detection antibody (concentration, label)

C t tib d

Target cancer biomarker (protein, peptide, carbohydrate)

Detection antibody (concentration, label)

( tti b ff b t ti )

Solid support (glass slide)Surface chemistry

Capture antibody

(fonctional groups, surface energy, composition)

(spotting buffer, probe concentration)

33

Solid support (glass slide)

8èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Previous results5 Cancer biomarkers: CEA, p53, Hsp60, PDI, DefA6

(Yang Z. et al. Sens Actuators B chem, 2012; 175: 22-28)

, p , p , ,6 surface chemistries: -COOH, -NHS / CMD, MAMVE,

-NH2 (APDMES) / Chitosan3 spotting buffers: Acetate (pH 4.5), PBS (pH 7.4), Carbonate (pH 9.6)

Optimization of capture antibody immobilization

Spotting buffer:Carbonate (pH9 6) [ t Ab] 10 M

448èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Carbonate (pH9.6)Acetate (pH4.5)

[capture Ab] = 10 µM

Previous results (Yang Z. et al. Sens Actuators B chem, 2012; 175: 22-28)

Optimization of biological recognitionOptimization of biological recognition

LODLOD

Cut off=Mean+3SDCut off Mean 3SD

Selected surfaces:NHS

Tumor antigens Optimal surfaces LOD Dynamic range

CEA MAMVE/CMD 10 pM 4.7 log/4.0 log

HSP60 NHS/Chitosan/MAMVE 10 pM 4 7 log/4 0 log /4 7 log

Tumor antigens Optimal surfaces LOD Dynamic range

CEA MAMVE/CMD 10 pM 4.7 log/4.0 log

HSP60 NHS/Chitosan/MAMVE 10 pM 4 7 log/4 0 log /4 7 log MAMVEChitosan

HSP60 NHS/Chitosan/MAMVE 10 pM 4.7 log/4.0 log /4.7 log

PDI NHS 10 pM 4.7 log

DEFA6 Chitosan 10 pM 4.7 log

HSP60 NHS/Chitosan/MAMVE 10 pM 4.7 log/4.0 log /4.7 log

PDI NHS 10 pM 4.7 log

DEFA6 Chitosan 10 pM 4.7 log

558èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

CA19-9 NHS/CMD 10 U/mL 3.0 log/3.0 logCA19-9 NHS/CMD 10 U/mL 3.0 log/3.0 log

Spotting design and conditions

B1 A t t + 0 05% PVA

IgG-Cy3 in B2

B1=Acetate + 0.05% PVA IgG-Cy3 in B1 Capture Ab in B1

Capture Ab in B2B2=Carbonate + 0.05% PVA

A ti H 60

Anti-CEA21

3

Detection AbDetection AbAnti-Hsp60

Anti-PDI4567

Capture Absera

Capture Absera

Anti-DefA6

Anti-p53

78

109

Blank = buffer (B1, B2)Positive control = purified cancer biomarker (10 nM)Positive control = purified cancer biomarker (10 nM)2 healthy donors’ sera = N1, N2 (1/250)4 CRC sera = CRC1, CRC2, CRC3, CRC4 (1/250)

668èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Results : Proof of Concept• buffer: white bar / 2 normal sera (red pink bars)buffer: white bar / 2 normal sera (red, pink bars)• 4 colorectal cancer sera (blue, orange, green, yellow bars)• purified antigen: black bar

Detection of cancer biomarker significantly depends on surfacesignificantly depends on surface chemistry

778èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Conclusion

CEA Hsp60 PDI DefA6 p53CRC1 NHS

CRC2 Chitosan Chitosan NHS NHS

CRC3 Chitosan NHSMAMVE

NHSMAMVE

CRC4 NHS Chitosan NHSPositive CRC sera 1/4 2/4 2/4 3/4 3/4

Detection of a single cancer biomarker display low sensitivityDetection of a single cancer biomarker display low sensitivity

Multiplex detection of cancer biomarkers in optimal conditions (surface chemistry, [probe], spotting buffer) significantly increase the sensitivity

888èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Perspectives

Future work will focus on:

enlarge patients’ cohorte to validate the number of cancerenlarge patients’ cohorte to validate the number of cancer biomarkers to be detected

integration of optimal chemistries on the same support to improve the accuracy of a cost-effective test

compare antibody microarrays to Vidas

determine reproducibility and linearity of the multiplex detection

evaluate LOD, LOB, LOQ

998èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013

Financial support:

Acknowledgements

Financial support:

PhD fellowship

BQRBQR

C’NANO Rhône-Alpes

Technical support:

NANOLYON Plate-forme

Prof. T. Delair (Laboratoire des Matériaux Polymères et Biomatériaux)

Zhugen Yang (PhD student) Dr. Claude LambertDr. Yann ChevolotDr. Eliane SouteyrandyDr. Thomas Géhin

Dr Yasemin Ataman-ÖnalDr Yasemin Ataman ÖnalDr Geneviève Choquet-Kastylevsky

10108èmes Journées Scientifiques du CLARA – 21 & 22 mars 2013