Protein Standard Detection and MRD are Confirmed Serial dilution of (native or recombinant) standard protein in diluent –Evaluate for standard protein.

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Presentation transcript:

Protein Standard Detection and MRD are Confirmed Serial dilution of (native or recombinant) standard protein in diluent –Evaluate for standard protein detection –Evaluate for parallelism between standard protein serial dilution and assay standard curve Serial dilution of plasma in diluent –Evaluate assay dilutional linearity –Validate predefined plasma dilution factor Serial dilution of (native or recombinant) standard protein in diluted plasma with predefined DF –Evaluate for parallelism between serial diluted protein spiked into diluted plasma and assay standard curve Kit Standards Protein serial dilution in diluent Protein serial dilution in plasma Plasma serial dilution in diluent Experimental design: Plate layout example:

Alpha-1-acid glycoprotein 1 (A1AG1) assay performance LOB (OD)*0.05 LLOQ (OD)*0.06 LLOQ (Conc)< 3.13 ng/mL ULOQ (Conc)200 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R A1AG1 Assay Standard Curve and key performance metrics: Kit VendorR&D Systems Kit Part #DAGP00 A1AG1 ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~33.8 Replicates/n22764 CV (%)<= 10% 4.4%2.4% A1AG1 precision data A1AG1 linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:20000 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Alpha 1-antitrypsin (A1AT) assay performance LOB (OD)*0 LLOQ (OD)*0 LLOQ (Conc)< 1.4 ng/mL ULOQ (Conc)90 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R A1AT Assay Standard Curve and key performance metrics: Kit VendorGenway Kit Part #GWB-1F2730 A1AT ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~4.6 Replicates/n22764 CV (%)<= 10% 6.3%2.6% A1AT precision data A1AT linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1: (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Alpha-1-antichymotrypsin (AACT) assay performance LOB (OD)*0.06 LLOQ (OD)*0.07 LLOQ (Conc)< 6.25 ng/mL ULOQ (Conc)400 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R AACT Assay Standard Curve and key performance metrics: Kit VendorGenway Kit Part #GWB-98E3CB AACT ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~17.5 Replicates/n22764 CV (%)<= 10% 9.8%12.3% AACT precision data AACT linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:10000 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Cathepsin D (CATD) assay performance LOB (OD)*0.06 LLOQ (OD)*0.06 LLOQ (Conc)< 156 pg/mL ULOQ (Conc)10000 pg/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R CATD Assay Standard Curve and key performance metrics: Kit VendorAbcam Kit Part #ab CATD ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (pg/mL) ~862 Replicates/n22764 CV (%)<= 10%<= 20%11.1%11.8% CATD precision data CATD linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (pg/mL)% Change from assay DF 1: % 1:200 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Carcinoembroyonic antigen (CEA) assay performance LOB (OD)*0.05 LLOQ (OD)*0.06 LLOQ (Conc)< 3 ng/mL ULOQ (Conc)120 ng/mL Standard Curve Points5 points Standard Curve Points > LOQ5 points Standard Curve R CEA Assay Standard Curve and key performance metrics: Kit VendorIBL Kit Part #RE59101A CEA ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~1.1 Replicates/n22764 CV (%)<= 10% 14.9%17.6% CEA precision data CEA linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1:1 (assay DF) % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank (CEA assay DF was 1. Assay dilutional linearity was not performed.)

Clusterin (CLUS) assay performance LOB (OD)*0.06 LLOQ (OD)*0.07 LLOQ (Conc)< 5 ng/mL ULOQ (Conc)160 ng/mL Standard Curve Points6 points Standard Curve Points > LOQ6 points Standard Curve R CLUS Assay Standard Curve and key performance metrics: Kit VendorBiovendor Kit Part #RD R CLUS ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~65.1 Replicates/n22764 CV (%)<= 10% 7.0%15.4% CLUS precision data CLUS linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:3000 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Complement C3 (CO3) assay performance LOB (OD)*2.35 LLOQ (OD)*2.03 LLOQ (Conc)< 0.47 ug/mL ULOQ (Conc)30 ug/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R CO3 Assay Standard Curve and key performance metrics: Kit VendorAssayPro Kit Part #EC CO3 ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ug/mL) ~4.0 Replicates/n22764 CV (%)<= 10% 13.9%10.5% CO3 precision data CO3 linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ug/mL)% Change from assay DF 1: % 1:250 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Complement component 9 (CO9) assay performance LOB (OD)*0.11 LLOQ (OD)*0.17 LLOQ (Conc)< ng/mL ULOQ (Conc)15 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ6 points Standard Curve R CO9 Assay Standard Curve and key performance metrics: Kit VendorAssayPro Kit Part #EC CO9 ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~2.2 Replicates/n22764 CV (%)<= 15%<= 10%8.8%9.4% CO9 precision data CO9 linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:20000 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Growth/differentiation factor 15 (GDF15) assay performance LOB (OD)*0.06 LLOQ (OD)*0.06 LLOQ (Conc)< 23.4 pg/mL ULOQ (Conc)1500 pg/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R GDF15 Assay Standard Curve and key performance metrics: Kit VendorR&D Systems Kit Part #DGD150 GDF15 ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (pg/mL) ~51.8 Replicates/n22764 CV (%)<= 10% 5.9%6.1% GDF15 precision data GDF15 linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (pg/mL)% Change from assay DF 1:447499% 1:8 (assay DF)479100% 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Macrophage migration inhibitory factor (MIF) assay performance LOB (OD)*0.07 LLOQ (OD)*0.07 LLOQ (Conc)< ng/mL ULOQ (Conc)10 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R MIF Assay Standard Curve and key performance metrics: Kit VendorR&D Systems Kit Part #DMF00B MIF ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~1.1 Replicates/n22764 CV (%)<= 10% 3.4%10.6% MIF precision data MIF linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:10 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Osteopontin (OSTP) assay performance LOB (OD)*0.06 LLOQ (OD)*0.08 LLOQ (Conc)< ng/mL ULOQ (Conc)20 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R OSTP Assay Standard Curve and key performance metrics: Kit VendorR&D Systems Kit Part #DOST00 OSTP ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~1.6 Replicates/n22764 CV (%)<= 10% 3.2% OSTP precision data OSTP linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:20 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

P-selectin glycoprotein ligand 1 (SELPL) assay performance LOB (OD)*0.05 LLOQ (OD)*0.06 LLOQ (Conc)< 1.56 U/mL ULOQ (Conc)50 U/mL Standard Curve Points6 points Standard Curve Points > LOQ6 points Standard Curve R SELPL Assay Standard Curve and key performance metrics: Kit VendorIBL Kit Part #BE59109 SELPL ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc U/mL ng/mL~10.2 U/mL Replicates/n22764 CV (%)<= 10% 4.6%8.0% SELPL precision data SELPL linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (U/mL)% Change from assay DF 1: % 1:30 (assay DF)322100% 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Serum amyloid A-1 (SAA1) assay performance LOB (OD)*0.07 LLOQ (OD)*0.09 LLOQ (Conc)< 9.4 ng/mL ULOQ (Conc)600 ng/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R SAA1 Assay Standard Curve and key performance metrics: Kit VendorLifeTech Kit Part #KHA0011C SAA1 ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (ng/mL) ~100 Replicates/n22764 CV (%)<= 20%<= 10%11.4%9.9% SAA1 precision data SAA1 linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (ng/mL)% Change from assay DF 1: % 1:240 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank

Prolyl endopeptidase FAP (SEPR) assay performance LOB (OD)*0.10 LLOQ (OD)*0.11 LLOQ (Conc)<= 62.5 pg/mL ULOQ (Conc)4000 pg/mL Standard Curve Points7 points Standard Curve Points > LOQ7 points Standard Curve R SEPR Assay Standard Curve and key performance metrics: Kit VendorR&D Systems Kit Part #DY3715 SEPR ELISA kit specifics: Intra-Assay PrecisionInter-Assay Precision SampleSTDsStandard protein serial dilutionPlasma sample 1 Conc (pg/mL) ~720 Replicates/n22764 CV (%)<= 10% 6.2%3.0% SEPR precision data SEPR linearity data Plasma dilution (n=16 for each DF)Dilution corrected concentration (pg/mL)% Change from assay DF 1: % 1:80 (assay DF) % 1: % * LOB = mean blank * sd blank, LLOQ = mean blank + 10 * sd blank