Fish (FTG) yolk plasmin (VTG) ELISA test kit instructions - Database & Sql Blog Articles

Detection Principle

The kit utilizes a double-antibody one-step sandwich ELISA method. This technique allows for the accurate detection of Vitreinogen (VTG) by using a coated antibody, followed by sequential addition of the test sample, standard, and HRP-labeled detection antibody. After washing, TMB is used as a substrate to develop color. The enzyme peroxidase catalyzes the conversion of TMB to blue, which then turns yellow when an acid is added. The intensity of the color is directly proportional to the concentration of VTG in the sample.

Sample Collection, Processing, and Storage

1. Serum: Collect blood using a pyrogen-free and endotoxin-free tube. Centrifuge at 3000 rpm for 10 minutes to separate serum from red blood cells.

2. Plasma: Anticoagulate with EDTA, citrate, or heparin. Centrifuge at 3000 rpm for 30 minutes to collect the supernatant.

3. Cell Supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymers.

4. Tissue Homogenate: Mince the tissue in physiological saline and centrifuge at 3000 rpm for 10 minutes to obtain the supernatant.

5. Storage: If not tested immediately, store samples at -20°C. Avoid repeated freezing and thawing. Thaw at room temperature before use.

Items to Bring

1. Microplate reader (450 nm)

2. High-precision pipettes and tips (0.5–10 μL, 2–20 μL, 20–200 μL, 200–1000 μL)

3. 37°C incubator

Operational Precautions

1. Store the kit at 2–8°C and allow it to equilibrate at room temperature for 20 minutes before use. If the washing buffer crystallizes after removal from the fridge, heat it gently in a water bath before use.

2. Return unused strips to the ziplock bag and store them at low temperature and dry conditions.

3. S0 can be used as a negative control or blank. If the sample was diluted 5 times, multiply the result by 5 to get the actual concentration.

4. Follow the instructions precisely for incubation time, volume, and sequence.

5. Shake all reagents well before use.

Kit Composition

Name	96-well configuration	48-well configuration	Remarks
Microporous ELISA plate	12 holes × 8	12 holes × 4	Not included
Standard	0.3mL*6 tubes	0.3mL*6 tubes	Not included
Sample diluent	6 mL	3 mL	Not included
Detection antibody-HRP	10 mL	5 mL	Not included
20× Washing Buffer	25 mL	15 mL	Dilute as instructed
Substrate A	6 mL	3 mL	Not included
Substrate B	6 mL	3 mL	Not included
Stop solution	6 mL	3 mL	Not included
Sealing film	2 sheets	2 sheets	Not included
Instruction manual	1 copy	1 copy	Not included
Ziplock bag	1	1	Not included

Note: Standard concentrations (S0–S5): 0, 30, 60, 120, 240, 480 ng/mL

Reagent Preparation

Dilute 20× Wash Buffer 1:20 with distilled water (1 part 20× buffer + 19 parts water).

Washing Method

1. Manual: Drain each well, add washing solution, let stand for 1 minute, drain, pat dry, and repeat 5 times.

2. Automatic: Inject 350 μL per well, soak for 1 minute, wash 5 times.

Procedure

1. Remove plates from the pouch after 20-minute equilibration at room temperature. Seal unused plates back into the ziplock bag and store at 4°C.

2. Set up standard and sample wells. Add 50 μL of standards to standard wells.

3. For sample wells, add 10 μL of sample, then 40 μL of sample diluent. Do not add anything to blank wells.

4. Add 100 μL of HRP-labeled detection antibody to each well. Seal and incubate at 37°C for 60 minutes.

5. Discard liquid, pat dry, wash 5 times with washing solution.

6. Add 50 μL of substrates A and B to each well. Incubate in the dark at 37°C for 15 minutes.

7. Add 50 μL of stop solution to each well. Measure OD at 450 nm within 15 minutes.

Result Interpretation

Create a standard curve in Excel, plotting standard concentration on the x-axis and OD values on the y-axis. Use the linear regression equation to calculate sample concentrations.

Kit Performance

1. Accuracy: R value ≥ 0.9900

2. Sensitivity: < 1.0 ng/mL

3. Specificity: No cross-reactivity with other analogs

4. Repeatability: CV < 15%

5. Storage: 2–8°C, protected from light and moisture

6. Shelf Life: 6 months

Disclaimer

1. This kit is for research purposes only. Not suitable for clinical trials or human experiments. The user assumes full responsibility for any consequences.

2. Follow instructions strictly. Any deviation may lead to incorrect results, and the user will bear all responsibility.