01.Serum

Place collected whole blood in refrigerator at 4 ℃ overnight. Then centrifuge for 10mins at 1000-3000 rpm. Take supernatant and either test immediately or place samples at -20 ℃/-80 ℃(1-3 months) for storage.

02.Plasma

Take plasma where EDTA, sodium citrate, or heparin has been added as anticoagulant. Mix well. Centrifuge mixture for 10mins at 1000-3000 rpm. Take supernatant and test immediately or place samples at -20 ℃ /-80 ℃ (1-3 months) for storage.

03.Tissue Homogenate

Take tissue slices which have been washed in 0.01 M PBS and then a tissue protein extraction reagent has been added according to proportion of 1 g to 5-10 mL and then mixed on ice bath. After sufficient homogenization, please then centrifuge for 10mins at 5000-10000 rpm. Take supernatant for immediate testing, or place samples at -20 ℃/-80 ℃ (1-3 months) for storage.

04.Cell Culture

Centrifuge for 10 mins at 1000-3000 rpm. Take supernatant for immediate testing, or place samples at -20 ℃/-80 ℃ (1-3 months) for storage.

05.For Urine, Ascites, Cerebrospinal Fluid, etc

Centrifuge for 10mins at 1000-3000 rpm. Take supernatant for immediate testing, or place samples at -20 ℃/-80 ℃ (1-3 months) for storage.

1. Blood collection tubes should be both pyrogen- and endotoxin-free. 2. Hemolyzed or hyperlipidemic specimens are not recommended to be used. 3. Samples ultimately should appear clear and mostly transparent. All particulates should be removed through centrifugation. 4. If collected samples are not used immediately, they should be divided according to single usage quantities and stored frozen at -20/-80℃, carefully avoiding repeated freeze-thaw cycles. 5. Sample dilution optimization is often necessary for proper sample resolution within the standard curve. Pre-experiments are always recommended to be performed prior to running the bulk of the samples, in order to determine if an optimizing dilution should be made to the samples. 6. It is recommended to collect sufficient samples for running multiple tests, since sometimes some users can experience issues that may result in a loss of data. 7. Always wear protective outerwear while collecting/handling samples (e. g. wearing gloves, coat, respirator, etc. ) and be aware of all potential risks involved in handling your specimens. Specimen processing should be conducted inside a properly-maintained biological safety cabinet.

It is recommended that the user be well-informed regarding the general range of concentrations that are expected for their samples. We would suggest consulting the scholarly literature for references to similar samples' concentrations, and then to consider diluting your samples accordingly. 1. Please remove ELISA Kit from refrigerator 20 mins in advance, and begin test once it has been brought to room temperature. 2. Dilute the concentrated Washing Buffer with double distilled water (1:25). Return unused quantity back to the box. 3. Standard:Add 1. 0mL Standard Diluent to lyophilized standard vial and allow to sit for 30 mins. After the standard has completely dissolved, mix it slightly and mark with a label on the tube. 4. Procedure for standard sample dilution: Take 7 clean tubes and label them with their expected concentrations (STD2, STD3, STD4, STD5, STD6, STD7, STD8). Add 300 μL of Standard Diluent to each tube. Pipette 300 μL from the reconstituted STD1 into the tube labeled STD2 and mix well. Then pipette 300 μL from the STD2 tube into the STD3 tube and mix well. Repeat this procedure up to the STD7 tube. The Standard Diluent in the STD8 tube serves as the negative control. 5. Reconstituted standard stock solution cannot be reused. 6. Biotinylated Antibody: Remove the appropriate volume of Biotinylated Antibody solution for the quantity of wells intending to be assayed, and dilute with Antibody Diluent in a proportion of 1:100. This should be prepared 30mins in advance, and we would absolutely recommend not re-using for additional assaying. 7. Enzyme Conjugate: Remove the appropriate amount of Enzyme Conjugate solution for the quantity of wells intending to be assayed, and dilute with the Enzyme Diluent in a proportion of 1:100. This should be prepared 30mins in advance, and we would absolutely recommend not re-using for additional assaying. 8. Color Reagent: Prepare Color Reagent solution 30mins in advance by mix Color Reagent A and Color Reagent B at a volume ratio of 9:1.

Take the concentration values of standards as X- and OD readings as the Y-coordinates.Use a smooth line to connect each coordinate point of the standard values.The concentration of samples can be found by inputting the sample OD values into the line equation for the standard curve.It is recommended to employ professional curve software (e.g.curve expert 1.3) to analyze and compute the results.