Canine Type II Collagen (COl II) ELISA Kit

The canine type II collagen (Col II) ELISA kit operates on the principle of a double-antibody one-step sandwich ELISA. The process begins by coating microwells with a specific capture antibody against canine Col II. Afterward, samples, standards, and HRP-conjugated detection antibodies are sequentially added, incubated, and thoroughly washed to remove unbound components. A TMB substrate is then introduced, which changes color under the action of peroxidase—first turning blue and then yellow when an acid is added. The intensity of the final yellow color correlates directly with the concentration of Col II in the sample. The optical density (OD) is measured at 450 nm using a microplate reader, allowing for precise quantification of Col II levels. **Sample Collection and Preparation:** 1. **Serum:** Collect blood using endotoxin-free tubes. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells. 2. **Plasma:** Use anticoagulants such as EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes to obtain plasma. 3. **Cell Supernatant:** Centrifuge at 3000 rpm for 10 minutes to remove debris and cellular components. 4. **Tissue Homogenate:** Homogenize tissue in physiological saline, then centrifuge at 3000 rpm for 10 minutes to collect supernatant. 5. **Storage:** If not tested immediately, aliquot and store at -20°C. Avoid repeated freeze-thaw cycles. Thaw at room temperature before use. **Required Equipment:** - Microplate reader (450 nm) - Precision pipettes (0.5–10 µL, 2–20 µL, 20–200 µL, 200–1000 µL) - Incubator set at 37°C **Precautions:** - Store the kit at 2–8°C. Allow it to equilibrate at room temperature for 20 minutes before use. - Dilute the washing buffer as instructed (1:20 with distilled water). - Seal unused strips in a ziplock bag and store them properly. - Do not dilute pre-treated samples; add 10 µL directly. - Follow incubation times and procedures strictly. - Shake all reagents before use. **Kit Components:** | Component | 96-well | 48-well | Notes | |----------|---------|---------|-------| | Microwells | 12×8 strips | 12×4 strips | No standards included | | Standard Dilutions | 0.5 mL | 0.5 mL | 500 pg/ml | | Standard Diluent | 6 mL | 3 mL | As per instructions | | Sample Diluent | 6 mL | 3 mL | As per instructions | | Detection Antibody-HRP | 6 mL | 3 mL | No standard included | | 20× Wash Buffer | 20 mL | 20 mL | Dilute as per instructions | | Substrate A | 6 mL | 3 mL | No standard included | | Substrate B | 6 mL | 3 mL | No standard included | | Stop Solution | 6 mL | 3 mL | No standard included | | Seal Film | 2 sheets | 2 sheets | No instructions | | Ziplock Bags | 1 piece | 1 piece | No notes | **Reagent Preparation:** - Dilute the 20× wash buffer with distilled water in a 1:20 ratio. - Manual washing: Drain each well, fill with wash solution, let sit for 1 minute, drain, and repeat 5 times. - Automatic washing: Add 350 µL of wash solution per well, soak for 1 minute, and repeat 5 times. **Procedure:** 1. Remove the required number of wells from the foil pouch after 20 minutes at room temperature. Store the rest in a sealed ziplock bag at 4°C. 2. Set up standard, sample, and blank wells. Add 50 µL of standards at different concentrations. 3. Add 10 µL of sample and 40 µL of diluent to each sample well. 4. Add 50 µL of HRP-labeled detection antibody to each well. Seal with a membrane and incubate at 37°C for 60 minutes. 5. Discard liquid, wash 5 times with wash buffer. 6. Add 50 µL of TMB A and B, incubate in the dark for 15 minutes. 7. Add 50 µL of stop solution and measure OD at 450 nm within 15 minutes. **Result Analysis:** Plot standard curve in Excel using standard concentrations on the x-axis and corresponding OD values on the y-axis. Calculate sample concentrations using the regression equation. **Kit Performance:** - Accuracy: R ≥ 0.99 - Sensitivity: <1.0 pg/mL - Specificity: No cross-reactivity with other analogs - Repeatability: CV <15% - Storage: 2–8°C, protected from light and moisture - Shelf Life: 6 months - Detection Range: 15.6 pg/mL – 500 pg/mL **Disclaimer:** This kit is for research purposes only. Not intended for clinical trials or dog experiments. The company assumes no responsibility for misuse. Always follow the instructions carefully. Do not mix different batch numbers. Any deviation may lead to inaccurate results.

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