Rat L-Aromatic Amino Acid Decarboxylase (AADC) ELISA Kit

User manual in Chinese

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Detection principle

The kit uses a double antibody one-step sandwich enzyme-linked immunosorbent assay (ELISA). The coated microcapsules were pre-coated with rat L-aromatic amino acid decarboxylase (AADC) capture antigen, and samples, standards, and HRP-labeled detection antibodies were sequentially added, incubated and thoroughly washed. Using the substrate TMB to develop color, TMB is converted to blue under the catalysis of peroxidase and converted to the final yellow color by the action of an acid. The color depth is positively correlated with the rat L-aromatic amino acid decarboxylase (AADC) in the sample. The absorbance (OD value) was measured at 450 nm using a microplate reader to calculate the sample concentration.

Sample collection, processing and storage methods

1. Serum: Use a tube containing no pyrogen and endotoxin. Avoid any cell irritation during the procedure. After collecting the blood, centrifuge and centrifuge for 10 minutes at 3000 rpm to quickly and carefully separate the serum and red blood cells.

2. Plasma: EDTA, citrate or heparin anticoagulation. The supernatant was taken by centrifugation at 3000 rpm for 30 minutes.

3. Cell supernatant: Centrifuge at 3000 rpm for 10 minutes to remove particles and polymer.

4. Tissue homogenization: The tissue is added to the appropriate amount of physiological saline and chopped. The supernatant was taken by centrifugation at 3000 rpm for 10 minutes.

5. Storage: If the sample is not detected in time after collection, please dispense it once, freeze it at -20 Â°C, avoid repeated freezing and thawing, thaw at room temperature and ensure that the sample is fully thawed evenly.

Bring your own items

1. Microplate reader (450nm)

2. High-precision sampler and tip: 0.5-10uL, 2-20uL, 20-200uL, 200-1000uL

3. 37 Â° C incubator

Operational precautions

1. Store the kit at 2-8 Â° C and equilibrate for 20 minutes at room temperature before use. The concentrated washing liquid taken out from the refrigerator will crystallize, which is a normal phenomenon, and the water bath is heated to completely dissolve the crystals before use.

2. The slats not used in the experiment should be immediately put back into the ziplock bag and sealed (low temperature dry) for storage.

3. The pre-treated sample does not need to be diluted, and 10 Î¼L of the sample can be directly added.

4. Incubate the operation strictly in accordance with the time indicated in the instructions, the amount of liquid added, and the sequence.

5. Shake well all liquid components before use.

Kit composition

name

96-well configuration

48-hole configuration

Remarks

Microporous plate

8 holes Ã— 12

12 holes Ã— 4

Standard (100 ng/ml)

0.6mL

Dilute according to the instructions

Standard dilution

6mL

3mL

Dilute according to the instructions

Sample diluent

6mL

3mL

Dilute according to the instructions

Detection antibody-HRP

10mL

5mL

20Ã— washing buffer

25mL

15mL

Dilute according to the instructions

Substrate A

6mL

3mL

Substrate B

6mL

3mL

Stop solution

6mL

3mL

Sealing film

2 sheets

Instruction manual

1 serving

Ziplock bag

Note: Standards are diluted with standard dilutions to: 100, 50, 25, 12.5, 6.25, 0 ng/ml.

Reagent preparation

Dilution of 20Ã— Wash Buffer: Distilled water was diluted 1:20, ie 1 part of 20Ã— Wash Buffer plus 19 parts of distilled water.

Washing method

1. Manually wash the plate: Drain the liquid in the hole, fill each hole with the washing liquid, let stand for 1 min, then drain the liquid in the hole, pat dry on the absorbent paper, and wash the plate 5 times.

2. Automatic washing machine: Inject 350Î¼L of washing solution into each hole, soak for 1min, and wash the plate 5 times.

Steps

1. Remove the required slats from the foil pouch after equilibrating for 20 min at room temperature. The remaining slats are sealed back to 4 Â°C with a ziplock bag.

2. Set standard hole, sample hole and blank hole, blank hole is added; standard product hole is added with different concentration of standard product 50Î¼L;

3. The sample hole to be tested is first added with 10 Î¼L of the sample to be tested, and then 40 Î¼L of the sample diluent is added;

4. Then add 100 Î¼L of horseradish peroxidase (HRP)-labeled detection antibody to the standard well and the sample well (without blank). Seal the well with a sealing membrane, 37 Â° C water bath or incubator temperature Breed for 60min.

5. Discard the liquid, pat dry on the absorbent paper, fill each well with the washing solution, let stand for 1 min, remove the washing solution, pat dry on the absorbent paper, and repeat the washing 5 times (can also be washed with a washing machine).

6. Add 50 Î¼L of substrate A and B to all wells and incubate for 15 min at 37 Â° C in the dark.

7. Add 50 Î¼L of stop solution to all wells and measure the OD value of each well at a wavelength of 450 nm within 15 min.

Result judgment

Draw a standard curve: In the Excel worksheet, the standard concentration is used as the abscissa, and the OD value is plotted as the ordinate. The linear regression curve of the standard is drawn, and the concentration values â€‹â€‹of each sample are calculated according to the curve equation.

Kit performance

1. Accuracy: The R value of the linear regression of the standard and the expected concentration is greater than or equal to 0.9900.

2. Sensitivity: The minimum detection concentration is less than 1.0 ng/ml.

3. Specificity: Does not cross-react with other soluble structural analogs.

4. Repeatability: The coefficient of variation between the plates and the plates is less than 15%.

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

6. Validity: 6 months

7. Detection range: 3.12 ng/ml -100ng/ml

Disclaimer

1. The kit is for research use only and should not be used in clinical trials or rat experiments. Otherwise, the consequences will be borne by the experimenter and the company will not be responsible.

2. Strictly follow the instructions. Different batch numbers cannot be exchanged. The experimenter violates the instructions and the consequences are borne by the experimenter.

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