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PROTOCOLBetaMark x-40 ELISA ProtocolCatalog No.: SIG-38954-kitTABLE 1.Tube NumberVolume of StandardVolume of StandardDiluent (mL)Final Concentration(ng/mL)Reconstituted Standard 0 80 250,000Intermediate #1Intermediate #210mL of reconstitutedstandard10mL of intermediate#1990 2,500990 25IV.Preparation of Standard Curve (Refer to Table2)A. Label (8) 1.5mL microcentrifuge tubes as #1-8 (used enclosed tubes).B. Aliquot 480mL of the working incubation buffer (made previously in step I) to each of the standard curve tubes (#2-8)and 990mL to tube #1 (standard curve top point).C. Remove 10mL from intermediate #2 and add to 990mL working incubation buffer in tube #1 (this will be the toppoint of the standard curve, 250pg/mL).D.Mix well by inversion, do not vortex.E. Continue making 1.8 fold serial dilutions by adding 600mL of the previous dilution to 480mL of working incubationbuffer in tubes #2-7. Mix well by inversion between each dilution.TABLE 2Tube NumberVolume of Standard(mL)Volume WorkingIncubation Buffer (mL)Final Concentration(pg/mL)1 10mL intermediate #2 990 250.02 600 mL of #1 480 138.93 600 mL of #2 480 77.24 600 mL of #3 480 42.95 600 mL of #4 480 23.86 600 mL of #5 480 13.27 600 mL of #6 480 7.48 0 480 0V.Sample PreparationA. Dilute samples in working incubation buffer.B. Run samples in duplicate or triplicate.Note: The ideal dilution is 1:2. If your sample needs to be diluted more than that, dilute to 2X in PBS then dilute 1:2 in the workingincubation buffer.Intended Use: Research Use Only (RUO)Intended Storage: Store Use: between Research 2-8°C Use Only (RUO)Storage: Store between 2-8°Cwww.covance.comwww.covance.comPage 2 of 4Revision Date: 04/20/2009

PROTOCOLBetaMark x-40 ELISA ProtocolCatalog No.: SIG-38954-kitData:Human A Beta 403.0002.5002.000A620nm1.5001.0000.5000.0000 50 100 150 200 250 300Concentration (pg/mL)The graph above demonstrates the sensitivity and specificity of Covance’s Colorimetric BetaMark x-40 ELISA.Beta amyloid 40 standard curve exhibits a linear response to 7.4 pg/mL. Negligible cross-reactivity to Humanbeta amyloid 42 was detected.COVANCE RESEARCH PRODUCTS +1.800.922.2226 +1.510.594.3800www.covance.comIntended Use: Research Use Only (RUO)Intended Storage: Store Use: between Research 2-8°C Use Only (RUO)Storage: Store between 2-8°Cwww.covance.comwww.covance.comPage 4 of 4Revision Date: 04/20/2009