The AssayMax Human Complement Factor D (FD) ELISA kit is designed for detection of human FD in plasma, serum, saliva, milk, and cell culture samples. This assay employs a quantitative sandwich enzyme immunoassay technique that measures FD in less than 4 hours.
A polyclonal antibody specific for FD has been pre-coated onto a 96-well microplate with removable strips. Human FD in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for human FD, which is recognized by a streptavidin-peroxidase conjugate. All unbound material is then washed away and a peroxidase enzyme substrate is added.
The color development is stopped and the intensity of the color is measured.
Complement Factor D Microplate: A 96-well polystyrene microplate (12 strips of 8 wells) coated with a polyclonal antibody against human FD.
Sealing Tapes: Each kit contains 3 precut, pressure sensitive sealing tapes that can be cut to fit the format of the individual assay.
Complement Factor D Standard: Human FD in a buffered protein base (480 ng, lyophilized).
Biotinylated Complement Factor D Antibody (50x): A 50-fold concentrated biotinylated polyclonal antibody against human FD (140 µL).
EIA Diluent Concentrate (10x): A 10-fold concentrated buffered protein base (30 mL).
Wash Buffer Concentrate (20x): A 20-fold concentrated buffered surfactant (30 mL, 2 bottles).
Streptavidin-Peroxidase Conjugate (SP Conjugate): A 100-fold concentrate (80 µL).
Chromogen Substrate: A ready-to-use stabilized peroxidase chromogen substrate tetramethylbenzidine (8 mL).
Stop Solution: A 0.5 N hydrochloric acid to stop the chromogen substrate reaction (12 mL).
Material not included
Microplate reader capable of measuring absorbance at 450 nm.
Pipettes (1-20 µL, 20-200 µL, 200-1000 µL and multiple channel).
Deionized or distilled reagent grade water.
Freshly dilute all reagents and bring all reagents to room temperature before use.
EIA Diluent Concentrate (10x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the EIA Diluent 1:10 with reagent grade water. Store for up to 1 month at 2-8 ° C.
Standard Curve: Reconstitute the 480 ng of Complement Factor D Standard with 2 mL of EIA Diluent to generate a stock solution of 240 ng / mL. Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions. Prepare duplicate or triplicate standard points by serially diluting the stock solution (240 ng / mL) 1: 2 with equal volume of EIA Diluent to produce 120, 60, 30, 15, 7.5, 3.75, and 1.875 ng / mL. EIA Diluent serves as the zero standard (0 ng / mL). Any remaining solution should be frozen at -20 ° C and used within 30 days.
Biotinylated Complement Factor D Antibody (50x): Spin down the antibody briefly and dilute the desired amount of the antibody 1:50 with EIA Diluent. Any remaining solution should be frozen at -20 ° C.
Wash Buffer Concentrate (20x): If crystals have formed in the concentrate, mix gently until the crystals have completely dissolved. Dilute the Wash Buffer Concentrate 1:20 with reagent grade water.
SP Conjugate (100x): Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1: 100 with EIA Diluent. Any remaining solution should be frozen at -20 ° C.