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(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged. (2) ELISA Wash Concentrate must be diluted to working solution prior use. Please see REAGENTS section for details. (3) Reconstitute all assay standards and controls by adding 0.5 mL of deminerialized water to each vial. Allow the standards and controls to sit undisturbed for 10 minutes, and then mix well by inversions or gentle vortexing. Make sure that all solid is dissolved completely prior to use. These reconstituted standards and controls should be stored at 2-8 C for up to 30 days. It is not recommended to freeze the reconstituted standards and controls.
(1) Place a sufficient number of streptavidin coated microwell strips in a holder to run human NSE standards, controls and unknown samples in duplicate. (2) Test Configuration (3) Prepare NSE Tracer Antibody and Capture Antibody working solution by 1:21 fold dilution of the Tracer Antibody with the biotinylated Capture Antibody . For each strip, it is required to mix 1 mL of the Capture Antibody with 50 µL of the Tracer Antibody in a clean test tube. (4) Add 10 µL of standards, controls and patient samples into the designated microwell. (5) Add 100 µL of above mixture of Tracer Antibody and Capture Antibody solution to each of the wells. (6) Cover the plate with the plate sealer and incubate plate at room temperature, shaking at 170 rpm for 1 hour. (7) Remove plate sealer. Aspirate the contents of each well. Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used. (8) Add 100 µL of ELISA HRP Substrate into each of the wells (9) Cover the plate with one plate sealer and also with aluminum foil to avoid exposure to light. (10) Incubate plate at room temperature for 10 minutes or less. (11) Remove the aluminum foil and plate sealer. Add 100 µL of ELISA Stop Solution into each of the wells. Mix gently. (12) Read the absorbance at 450 nm within 10 minutes in a microplate reader. NOTE: to reduce the background, one can set the instrument to dual wavelength measurement at 450 nm with background wavelength correction set at 595 nm, 620 nm or 630 nm.