Cholic Acid ELISA 试剂盒

1. 96-well Protein Binding Plate : One strip well 96-well plate.
2. Anti-Cholic Acid Antibody (500X) : One 10 μL vial.
3. Secondary Antibody, HRP Conjugate : One 20 μL vial.
4. Assay Diluent : One 50 mL bottle.
5. 10X Wash Buffer : One 100 mL bottle.
6. Substrate Solution : One 12 mL amber bottle.
7. Stop Solution (Part. No. 310808): One 12 mL bottle.
8. Cholic Acid Standard : One 100 μL vial of 2.5 mM Cholic Acid in 50 mM KH2PO4, pH 7.4, 25 mM NaCl.

Box 2 (shipped on blue ice packs)

• Detection sensitivity limit of 0.4μM cholic acid
• Suitable for use with plasma, serum, urine, feces, and cell and tissue lysates
• Cholic acid standard included

Cholic Acid Conjugate Coated Plate: Dilute the proper amount of 100X Cholic Acid Conjugate 1:100 into 1X PBS. Add 100 μL of the diluted 1X Cholic Acid Conjugate to each well and incubate at 37 °C for two hours or overnight at 4 °C. Remove the coating solution and wash twice with 200 L of 1X PBS. Blot plate on paper towels to remove excess fluid. Add 200 μL of Assay Diluent to each well and block for 1 hr at room temperature. Transfer the plate to 4 °C and remove the Assay Diluent immediately before use. Note: The Cholic Acid-Conjugate coated wells are not stable and should be used within 24 hrs after coating. Only coat the number of wells to be used immediately. 1X Wash Buffer: Dilute the 10X Wash Buffer to 1X with deionized water. Stir to homogeneity. Anti-Cholic Acid Antibody and Secondary Antibody: Immediately before use dilute the Anti- Cholic Acid Antibody 1:500 and Secondary Antibody 1:1000 with Assay Diluent. Do not store diluted solutions. Preparation of Standard Curve 1. Prepare Cholic Acid standards by diluting in 1X Assay Diluent. First, dilute the stock Cholic Acid Standard 2.5 mM solution 1:10 in 1X Assay Diluent for a 250 μM solution. (e.g. add 10 μL of the stock 2.5 mM standard to 90 μL of 1X Assay Diluent). Vortex thoroughly. 2. Use this 250 μM solution to prepare a series of the remaining cholesterol standards according to Table 1 below. 250 μM Cholic Acid Assay Diluent Cholic Acid Standard Tubes Standard (μL) (μL) (M) 1 10 990 25 2 500 of Tube #1 500 12.5 3 500 of Tube #2 500 6.25 4 500 of Tube #3 500 3.13 5 500 of Tube #4 500 1.56 6 500 of Tube #5 500 0.78 7 500 of Tube #6 500 0.39 8 0 500 0 Table 1. Preparation of Cholic Acid Standards.

Serum: Avoid hemolyzed and lipemic blood samples. Collect blood in a tube with no anticoagulant. Allow the blood to clot at room temperature for 30 minutes. Centrifuge at 2500 x g for 20 minutes. Remove the yellow serum supernatant without disturbing the white buffy layer. Aliquot samples for testing and store at -80 °C. Perform dilutions in Assay Diluent as necessary. 4 Plasma: Avoid hemolyzed and lipemic blood samples. Collect blood with heparin or citrate and centrifuge at 2000 x g and 4 °C for 10 minutes. Remove the plasma layer and store on ice. Avoid disturbing the white buffy layer. Aliquot samples for testing and store at -80 °C. Perform dilutions in Assay Diluent as necessary. Cells, tissues, or feces: Homogenize 50-200 mg of the cell pellet, tissue, or feces in 0.5-2 mL of ice cold PBS using a mortar and pestle or by dounce homogenization. Incubate the homogenate at 4 °C for 20 minutes. Transfer the homogenate to a centrifuge tube and centrifuge at 12000 x g for 20 minutes. Recover the supernatant and transfer to a fresh tube. Store resuspended sample at -20 °C or colder until ready to test by ELISA. Perform dilutions in Assay Diluent as necessary.

1. Prepare and mix all reagents thoroughly before use. Each cholic acid sample including unknown and standard should be assayed in duplicate.
2. Add 50 μL of unknown sample or Cholic Acid standards to the wells of the Cholic Acid Conjugate coated plate. Incubate at room temperature for 10 minutes on an orbital shaker.
3. Add 50 μL of the diluted Anti-Cholic Acid antibody to each well, incubate at room temperature for 1 hour on an orbital shaker.
4. Wash microwell strips 3 times with 250 μL 1X Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer.
5. Add 100 μL of the diluted Secondary Antibody-HRP Enzyme Conjugate to all wells.
6. Incubate at room temperature for 1 hour on an orbital shaker.
7. Wash microwell strips 3 times according to step 4 above. Proceed immediately to the next step.
8. Warm Substrate Solution to room temperature. Add 100 L of Substrate Solution to each well, including the blank wells. Incubate at room temperature on an orbital shaker. Actual incubation time may vary from 2-30 minutes. Note: Watch plate carefully, if color changes rapidly, the reaction may need to be stopped sooner to prevent saturation.
9. Stop the enzyme reaction by adding 100 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time).
10. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length. 5