Fetuin A (PTM) ELISA (DNlite-DKD)
The Fetuin A (PTM) ELISA is For Research Use Only
Size: 12×8 wells
Incubation Time: 5 Hours
Dynamic Range: 7.813 – 500 ng/mL
Sample Type: Urine
Sample Size: 180μl
Alternative Names: Unique Fetuin-A with specific post translational modification (PTM) for Diabetic Kidney Disease (DKD)
Fetuin A (PTM) ELISA (DNlite-DKD) Principle
The Fetuin A (PTM) ELISA (DNlite-DKD) (unique Fetuin-A with specific post translational modification (PTM) for Diabetic Kidney Disease (DKD)) is a competitive immunoassay. In this Fetuin A (PTM) ELISA (DNlite-DKD), calibrators or unknown urine samples are mixed with anti-unique PTM Fetuin-A monoclonal antibody (mAb), and then incubated in a microplate pre-bounded with unique PTM Fetuin-A. The monoclonal antibody recognizes unique PTM Fetuin-A in calibrators or unknown samples under competition in microplate wells. After an incubation, an Horseradish Peroxide (HRP) conjugated secondary antibody is added, followed by an incubation with 3,3’,5,5’-tetramethylbenzidine (TMB) substrate. Their relative reactivity is determined by absorbance measurement at 450 nanometers (nm) and plotted by comparison with a predetermined unique PTM Fetuin-A calibration curve.
Storage and Stability:
- Coated Microplate – Stable at -20°C in plate pouch with desiccant until expiration date
- Calibrator – Stable at -20°C until expiration date. Reconstituted Calibrators (5µg/mL) are stabled for one week -20°C and repeated freeze-thaw cycles should be avoided
- 1000X mAb anti-unique PTM Fetuin-A (E103) – Stable at -20°C until expiration date
- 4000X HRP Conjugate – Stable at -20°C until expiration date
- Diluent – Stable at 4°C until expiration date
- 10X Wash Buffer – Stable at 4°C until expiration date. Prepared 1X Wash Buffer is stable for one week at room temperature
- TMB Substrate – Stable at 4°C until expiration date
- Stop Solution – Stable at 4°C until expiration date
Reagent Preparation:
1.unique PTM Fetuin-A (E103) Calibrators
Reconstitute the E103 Calibrator with 0.2 mL distilled or deionized water, sit for 10 minutes at room temperature until completely dissolved and mix gently. The reconstituted E103 Calibrator is now at a concentration of 5 μg/mL. Dilute the 5 μg/mL E103 Calibrator with Diluent by 5-fold, then mix gently to get a 1 μg/mL E103 Calibrator. Procedures for the serial dilution to generate all calibrators for establishing E103calibration curve are shown in the instructions for use and all calibrators must be prepared and mixed well immediately prior to use.
2. 1X Wash Buffer
Recover 10X Wash Buffer to room temperature prior to use until all the salt crystals are dissolved. Calculate the required amount of 1X Wash Buffer for each Fetuin A (PTM) ELISA (DNlite-DKD). For each microplate, mix 50 mL 10X Wash Buffer with 450 mL distilled or deionized water. Homogenize but gently.
3. 1X mAb anti-unique PTM Fetuin-A (E103)
Calculate the required amount of 1X mAb anti-unique PTM Fetuin-A (E103) for each uPTM3-DKD assay, and mix 1000XmAb anti-unique PTM Fetuin-A (E103) with Diluent according to the amount required. For each microplate, mix 8 μL 1000X mAb anti-unique PTM Fetuin-A (E103) with 8 mL Diluent. Homogenize but gently.
4. 1X HRP Conjugate
Calculate the required amount of 1X HRP Conjugate for each Fetuin A (PTM) ELISA (DNlite-DKD), and mix 4000X HRP Conjugate with Diluent according to the amount required. For each microplate, mix 3 μL 4000X HRP Conjugate with 12 mL Diluent. Homogenize but gently.
