Insulin ELISA Kit

Catalog No. ABIN1813709

Product Details Insulin ELISA Kit

Target: Insulin (INS)

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Application: ELISA

Purpose: For quantitative detection of Insulin in Human serum, plasma, body fluids, tissue lysates or cell culture supernates.
This kit was based on sandwich enzyme-linked immune-sorbent assay technology. Anti-Insulin monoclonal antibody was pre-coated onto 96-well plates. And the HRP conjugated anti-Insulin polyclonal antibody was used as detection antibodies. The standards, test samples and HRP conjugated detection antibody were added to the wells subsequently, and wash with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The density of yellow is proportional to the Insulin amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, and then the concentration of Insulin can be calculated.

Sample Type: Cell Culture Supernatant, Serum, Tissue Lysate

Analytical Method: Quantitative

Components:

1. One 96-well plate pre-coated with anti-Human Insulin antibody

2. Lyophilized insulin standards: 6 tubes (0 uIU/ml, 8 uIU/ml, 16 uIU/ml, 32 uIU/ml, 80 uIU/ml, 140 uIU/ml)

3. HRP conjugated anti-Human Insulin antibody (RTU): 6 mL

4. TMB substrate A: 7 mL

5. TMB substrate B: 7 mL

6. Stop solution: 7 mL

7. Wash buffer (25X): 30 mL

Material not included:

1. 37 ℃ incubator
2. Microplate reader (wavelength: 450 nm)
3. Precise pipette and disposable pipette tips
4. Automated plate washer
5. ELISA shaker
6. 1.5 mL of Eppendorf tubes
7. Plate cover
8. Absorbent filter papers
9. Plastic or glass container with volume of above 1 L

Application Details

Assay Time: 4.5 h

Plate: Pre-coated

Reagent Preparation:

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

Sample Preparation:

Isolate the test samples soon after collecting, then, analyze immediately (within 2 hours). Or aliquot and store at -20 °C 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 °C.
• Serum: Coagulate the serum at room temperature (about 2 hours) or at 4 °C overnight. Centrifuge at approximately 2000 × g for 20 min. Analyze the serum immediately or aliquot and store at -20 °C.

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.
3. After collecting samples, analyze immediately or aliquot and store frozen at -20 °C. Avoid repeated freeze-thaw cycles.

Assay Procedure:

Mix thoroughly when dilute the reagents, it is recommend to plot a standard curve for each test. 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.

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 50 μL of 8 µIU/mL, 16 µIU/mL, 32 µIU/mL, 80 µIU/mL, 140 µIU/mL standard solutions into the standard wells. (Note: Add user’s 50 μL of sample diluent buffer into the control well).
3. Add 50 μL of properly diluted sample (Human serum, body fluids, tissue lysates or cell culture supernatants) into test sample wells.
4. Add 50 μL of HRP conjugated anti-Human Insulin antibody into the above wells (Except the control well). Add the solution at the bottom of each well without touching the side wall.
5. Seal the plate with a cover and incubate at 37 °C for 120 min.
6. Remove the cover, discard the liquid of wells, clap the plate on the absorbent filter papers or other absorbent material. Do not wash!
7. Wash plate 3 times with Wash buffer (Kit Component 7) 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 7) buffer 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 7) (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.
8. Add 50 μL of TMB substrate A (Kit Component 4) into each well, and then, add 50 μl of TMB substrate B (Kit Component 5), vortex gently the plate on ELISA shaker for 30 seconds (Or shake gently by hand for 30 seconds), cover the plate and incubate in dark at 37 °C for 15 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 Insulin standard solutions), the other wells show no obvious color.
9. Add 50 μL of Stop solution (Kit Component 6) into each well and mix thoroughly. The color changes into yellow immediately.
10. Read the O.D. absorbance at 450 nm in a microplate reader within 30 min after adding the stop solution.

Precautions:

1. Before the experiment, centrifuge each kit component for several minutes to bring down all reagents to the bottom of tubes.
2. It is recommend to measure each standard and sample in duplicate.
3. Do NOT let the plate completely dry at any time! Since the dry condition can inactivate the biological material on the plate.
4. Do not reuse pipette tips and tubes to avoid cross contamination.
5. Do not use the expired components and the components from different batches.
6. The TMB substrate A & B (Kit Component 4 & 5) is colorless and transparent before use, if not, please contact us for replacement.

Calculation of Results:

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 Insulin concentration of the samples can be interpolated from the standard curve.
Note:
1. If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.
2. If end user hopes to calculate with unit “ng/mL”, please multiple the O.D.450 value with 0.04167.

Assay Precision: < 5 µIU/mL

Restrictions: For Research Use only