Human Asprosin ELISA Kit，abbexa，abx257694

Pre-coated 96-Well Microplate

Standard

Standard Diluent Buffer

Wash Buffer

Detection Reagent A

Detection Reagent B

Diluent A

Diluent B

TMB Substrate

Stop Solution

Plate Sealer

37°C incubator

Multi and single channel pipettes and sterile pipette tips

Squirt bottle or automated microplate washer

1.5 ml tubes

Distilled water

Absorbent filter papers

100 ml and 1 liter graduated cylinders

Microplate reader (wavelength: 450 nm)

ELISA Shaker

Serum: Samples should be collected into a serum separator tube. Coagulate the serum by leaving the tube undisturbed in a vertical position overnight at 4°C or at room temperature for up to 60 minutes. Centrifuge at approximately 1000 × g for 20 min. Analyze the serum immediately or aliquot and store at -20°C or -80°C.

Plasma: Collect plasma using heparin or EDTA as an anticoagulant. Centrifuge for 15 minutes at 1000 × g within 30 minutes of collection. Assay immediately or aliquot and store at -20°C or -80°C. Avoid hemolysis and high cholesterol samples.

Tissue homogenates: The preparation of tissue homogenates will vary depending upon tissue type - this is just an example. Rinse tissues with ice-cold PBS to remove the excess of blood. Weigh before homogenization. Finely mince tissues and homogenize with a tissue homogenizer on ice in PBS and sonicate the cell suspension. Centrifuge the homogenates at 5000 × g for 5 min and collect the supernatant. Assay immediately or aliquot and store at -20°C.

1) Standard: Prepare the standard with the recommended volume of Standard Diluent Buffer, to make the standard solution. Then use the Standard Diluent buffer to carry out serial dilutions of the standard solution, as instructed in the Protocol.

2) Wash Buffer: Dilute the concentrated Wash Buffer with distilled water, as instructed in the Protocol.

3) Detection Reagent Preparation: Calculate the total volume of working solution required. Dilute Detection Reagent A and Detection Reagent B with Diluent A and Diluent B, respectively, at 1:100.

1) Set standard, test samples and control wells.

2) Aliquot 100 μl of diluted standard into the standard wells.

3) Aliquot 100 μl of Standard Diluent buffer into control (zero) well.

4) Aliquot 100 μl of diluted samples into the sample wells. Incubate for 1 hr at 37 °C.

5) Aliquot 100 μl of Detection Reagent A to each well. Incubate for 1 hr at 37 °C.

6) Wash 3 times.

7) Aliquot 100 μl of Detection Reagent B to each well. Incubate for 30 mins at 37 °C.

8) Wash 5 times.

9) Aliquot 90 μl of TMB Substrate to each well. Incubate for 10-20 mins at 37 °C.

10) Aliquot 50 μl of Stop Solution.

11) Measure the OD at 450 nm.

Equilibrate the kit components and samples to room temperature (18 - 25 °C) before use. It is recommended to plot a standard curve for each test.

1. Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample at least in duplicate.

2. Add 100 µL of each standard, control and sample into the appropriate wells. Seal the plate with a cover and incubate for 1 h at 37°C.

3. Remove the cover and discard the liquid.

4. Add 100 μl of the detection Reagent A working solution to each well. Seal the plate with a cover and incubate for 1 h at 37°C.

5. Remove the cover and discard the solution. Wash the plate 3 times with 1X Wash Buffer.

6. Add 100 µL of Detection Reagent B working solution into each well, seal and incubate at 37°C for 30 min.

7. Discard the solution and wash the plate 5 times with wash buffer as explained in previous step.

8. Aliquot 90 μl of TMB Substrate into each well. Seal the plate with a cover and incubate at 37°C for 10-20 min. Avoid exposure to light. The incubation time is for reference use only, the optimal time should be determined by end user. Do not exceed 30 min.

9. Add 50 µL of Stop Solution to each well. Read at 450 nm immediately.