ELISA Protocol(biotin)

Protocol for BEK1001

(Biotin conjugated antibody + ABC System)

• Preparation of sample and reagents

1. Sample

Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20℃ for long term. Avoid multiple freeze-thaw cycles.

• Body fluids, tissue lysates and cell culture supernatants: Centrifuge to remove precipitate, analyze immediately or aliquot and store at -20℃.
• Serum: Coagulate the serum at room temperature (about 2 hours). Centrifuge at approximately 1000 × g for 15 min. Analyze the serum immediately or aliquot and store at -20℃.
• Plasma: Collect plasma with heparin as the anticoagulant. Centrifuge for 15 min at 1000 x g within 30 min of collection. Analyze immediately or aliquot and store frozen at -20°C. EDTA and citrate can not be used as anticoagulant here.

Note: 1. Coagulate blood samples completely, then, centrifuge, and avoid hemolysis and particle.

          2. NaN₃ can not be used as test sample preservative, since it is the inhibitor for HRP.

>> Sample Dilution Guideline

End user should estimate the concentration of the target protein in the test sample first, and select a proper dilution factor to make the diluted target protein concentration falls the optimal detection range of the kit. Dilute the sample with the provided diluent buffer, and several trials may be necessary in practice. The test sample must be well mixed with the diluent buffer.

• High target protein concentration (100-1000 ng/ml): Dilution: 1:100. i.e. Add 1μl of sample into 99 μl of Sample / Standard diluent buffer (Kit Component 3).  
• Medium target protein concentration (10-100 ng/ml): Dilution: 1:10. i.e. Add 10 μl of sample into 90 μl of Sample / Standard diluent buffer (Kit Component 3).
• Low target protein concentration (156-10,000 pg/ml): Dilution: 1:2. i.e. Add 50 μl of sample into 50 μl of Sample / Standard diluent buffer (Kit Component 3).
• Very low target protein concentration (≤156 pg/ml): Unnecessary to dilute, or dilute at 1:2.       

2. Wash buffer

Dilute the concentrated Wash buffer 25-fold (1:25) with distilled water (i.e. add 30ml of concentrated wash buffer into 720ml of distilled water).

3. Standard

Reconstitution of the Lyophilized human ACE standard (Kit Component 2): standard solution should be prepared no more than 2 hours prior to the experiment. Two tubes of standard are included in each kit. Use one tube for each experiment. (Note: Do not dilute the standard directly in the plate)

   a. 10,000 pg/ml of standard solution: Add 1 ml of Sample / Standard diluent buffer (Kit Component 3) into one Standard (Kit Component 2) tube, keep the tube at room temperature for 10 min and mix thoroughly.

   b. 5000 pg/ml → 156 pg/ml of standard solutions: Label 6 Eppendorf tubes with 5000 pg/ml, 2500 pg/ml, 1250 pg/ml, 625 pg/ml, 313 pg/ml, 156 pg/ml, respectively. Aliquot 0.3 ml of the Sample / Standard diluent buffer (Kit Component 3) into each tube. Add 0.3 ml of the above 10,000 pg/ml standard solution into 1st tube and mix thoroughly. Transfer 0.3 ml from 1st tube to 2nd tube and mix thoroughly. Transfer 0.3 ml from 2nd tube to 3rd tube and mix thoroughly, and so on.  

Note: The standard solutions are best used within 2 hours. The 10,000 pg/ml standard solution should be used within 12 hours. Or store at -20℃ for up to 48 hours. Avoid repeated freeze-thaw cycles.

4. Preparation of Biotin conjugated anti-human ACE antibody (Kit Component 4) working solution: prepare no more than 2 hours before the experiment.

   a. Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)

  b. Dilute the Biotin conjugated anti-human ACE antibody (Kit Component 4) with Antibody diluent buffer (Kit Component 5) at 1:100 and mix thoroughly. i.e. Add 1 μl of Biotin conjugated anti-human ACE antibody into 99 μl of Antibody diluent buffer.

5. Preparation of Avidin-Biotin-Peroxidase Complex (ABC) (Kit Component 6) working solution: prepare no more than 1 hour before the experiment.

   a. Calculate the total volume of the working solution: 0.1 ml / well × quantity of wells. (Allow 0.1-0.2 ml more than the total volume)

   b. Dilute the Avidin-Biotin-Peroxidase Complex (ABC) (Kit Component 6) with ABC diluent buffer (Kit Component 7) at 1:100 and mix thoroughly. i.e. Add 1 μl of Avidin-Biotin-Peroxidase Complex (ABC) into 99 μl of ABC diluent buffer.

• Assay procedure

Before adding to wells, equilibrate the ABC working solution and TMB substrate (Kit Component 8) for at least 30 min at room temperature (37℃). It is recommend 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 recommend to measure each standard and sample in duplicate.
2. Aliquot 0.1ml of 10,000 pg/ml, 5000 pg/ml, 2500 pg/ml, 1250 pg/ml, 625 pg/ml, 313 pg/ml, 156 pg/ml  standard solutions into the standard wells.
3. Add 0.1 ml of Sample / Standard diluent buffer (Kit Component 3) into the control (zero) well.
4. Add 0.1 ml of properly diluted sample (human serum, plasma, body fluids, tissue lysates or cell culture supernatants) into test sample wells.
5. Seal the plate with a cover and incubate at 37℃ for 90 min.
6. Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Do not wash plate!
7. Add 0.1 ml of Biotin conjugated anti-human ACE antibody work solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8. Seal the plate with a cover and incubate at 37℃ for 60 min.
9. Remove the cover, and wash plate 3 times with Wash buffer (Kit Component 10) using one of the following methods:

    Manual Washing: Discard the solution in the plate without touching the side walls. Clap the plate  on absorbent filter papers or other absorbent material. Fill each well completely with Wash buffer (Kit Component 10) and vortex mildly on ELISA shaker for 2 min, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material. Repeat this procedure two more times for a total of THREE washes.

    Automated Washing: Aspirate all wells, then wash plate THREE times with Wash buffer (Kit Component 10) (overfilling wells with the buffer). After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer be set for a soaking time of 1 min or shaking.

10. Add 0.1 ml of ABC working solution into each well, cover the plate and incubate at 37℃ for 30 min.
11. Remove the cover and wash plate 5 times with Wash buffer (Kit Component 10), and each time let the wash  buffer stay in the wells for 1-2 min. (See Step 9 for plate wash method).
12. Add 90 μl of TMB substrate (Kit Component 8) into each well, cover the plate and incubate at 37℃ in dark for 18-23 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated human ACE standard solutions), the other wells show no obvious color.
13. Add 0.1 ml of Stop solution (Kit Component 9) into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader within 30 min after adding the stop solution.

For calculation, (the relative O.D.₄₅₀) = (the O.D.₄₅₀ of each well) – (the O.D.₄₅₀ of Zero well). The standard curve can be plotted as the relative O.D.₄₅₀ of each standard solution (Y) vs. the respective concentration of the standard solution (X). The human ACE concentration of the samples can be interpolated from the standard curve.

     Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.

Typical Data & Standard Curve

Results of a typical standard run of a human ACE ELISA Kit are shown below. This standard curve was generated at our lab for demonstration purpose only. Each user should obtain their own standard curve as per experiment. (N/A=not applicable)

Incubate TMB substrate at 37℃ for 18 min.