sFasL Assay Kit

Prepare Wash Buffer and Assay Buffer before starting with the test procedure.

A. Wash Buffer: If crystals have formed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved. Pour entire contents (50 mL) of the Wash Buffer Concentrate into a clean 1,000 mL graduated cylinder. Bring final volume to 1,000 mL with glass-distilled or de-ionized water. Mix gently to avoid foaming. The pH of the final solution should adjust to 7.4. Transfer to a clean wash bottle and store at 4° to 25°C. Please note that the Wash Buffer is stable for 30 days.

B. Assay Buffer: Mix the contents of the bottle well. Add contents of Assay Buffer Concentrate (5.0 mL) to 95 mL distilled or de-ionized water and mix gently to avoid foaming. Store at 4°C. Please note that the Assay Buffer is stable for 30 days.

C. Preparation of Calibrator: Dissolve lyophilized Calibrator by addition of distilled water. Reconstitution volume is stated on the label of the vial. Swirl gently to ensure complete solubilization.

D. Preparation of Biotin-Conjugate: Dilute the Biotin-Conjugate 1:100 just prior to use with diluted Assay Buffer in a clean plastic tube. Mix the contents of the tube well. Please note that the Biotin-Conjugate should be used within 30 minutes after dilution.

E. Preparation of Streptavidin-HRP: Make a 1:200 dilution of the concentrated Streptavidin-HRP solution with diluted Assay Buffer as needed.

F. Addition of color-giving reagents: Blue Dye, Green Dye, Red Dye In order to help our customers to avoid any mistakes in pipetting the sFas Ligand ELISA, we now offer a new tool that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colors to each step of the ELISA procedure. This procedure is optional, does not in any way interfere with the test results, and is designed to help the customer with the performance of the test, but can also be omitted, just following the package insert.

Cell culture supernatants, human serum, EDTA, or heparinized plasma, amniotic fluid, or other body fluids are suitable for use in the assay. Remove the serum or plasma from the clot or red cells, respectively, as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples must be stored frozen at -20°C to avoid loss of bioactive sFas Ligand. If samples are to be run within 24 hours, they may be stored at 4°C. Avoid repeated freeze-thaw cycles. Prior to assay, frozen sera or plasma should be brought to room temperature slowly and mixed gently and properly diluted with Sample Diluent.

Calculate the average absorbance values for each set of duplicate calibrators and samples. Duplicates should be within 20 percent of the mean.
Create a calibration curve by plotting the mean absorbance for each calibrator concentration on the ordinate against the sFas Ligand concentration on the abscissa. Draw a best-fit curve through the points of the graph.
To determine the concentration of circulating sFas Ligand for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the calibration curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding sFas Ligand concentration.
For samples that have been diluted 1:2 according to the instructions given in this manual, the concentration has to be multiplied by the dilution factor (x2).
It is suggested that each testing facility establishes a control sample of known sFas Ligand concentration and runs this additional control with each assay. If the values obtained are not within the expected range of this control, the assay results may be invalid.