This reagent kit can only be used for scientific research and cannot be used for medical diagnosis

People（Human）Glucose-6-phosphate dehydrogenase (G6PD)

ELISA detection kit

Instruction Manual

Detection Principle

The reagent kit adopts a one-step sandwich enzyme-linked immunosorbent assay (ELISA) with double k à ng body. Add the sample, standard substance, and HRP labeled detection kit into the pre coated micropores of glucose-6-phosphate dehydrogenase (G6PD) k à ng body, incubate and wash with a ch è bottom. Using substrate TMB for color development, TMB is converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The depth of color is positively correlated with glucose-6-phosphate dehydrogenase (G6PD) in the sample. Measure the absorbance (OD value) at 450nm wavelength using an enzyme-linked immunosorbent assay (ELISA) reader and calculate the sample activity.

Sample collection, processing, and preservation methods

1. Serum: Use test tubes free of pyrogens and endotoxins, avoid any cell irritation during the operation, collect blood, centrifuge at 3000 rpm for 10 minutes, and quickly and carefully separate serum and red blood cells.

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

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

4. Tissue homogenate: Crush the tissue by adding an appropriate amount of physiological saline. Centrifuge at 3000 rpm for 10 minutes and collect the supernatant.

5. Storage: If the sample is not tested in a timely manner after collection, please divide it into batches according to a single dose, freeze it at -20 ℃, avoid repeated freezing and thawing, thaw it at room temperature, and ensure that the sample is evenly filled and thawed.

Personal belongings

1. ELISA reader (450nm)

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

3. 37 ℃ constant temperature box

Operation precautions

1. The reagent kit should be stored at 2-8 ℃ and equilibrated at room temperature for 20 minutes before use. The concentrated washing solution taken out of the refrigerator will have crystals, which is a normal phenomenon. Heating in a water bath will dissolve the crystals completely before use.

2. The Flat noodles not used in the experiment shall be immediately put back into the self sealing bag and sealed (low-temperature dry) for storage.

The S0 standard with a concentration of 0 can be considered as a negative control or blank; When operating according to the instructions, the sample has already been diluted 5 times, and the actual concentration of the sample is obtained by multiplying the final result by 5.

4. Strictly follow the time, liquid dosage, and sequence indicated in the instructions for incubation operation.

5. Shake all liquid components thoroughly before use.

Composition of reagent kit

Note: The concentrations of standard samples (S0-S5) are 0, 1.5, 3, 6, 12, and 24 U/L, respectively

Preparation of reagents

20 x dilution of washing buffer: Dilute distilled water at a ratio of 1:20, which means adding 19 parts of distilled water to 1 part of 20 x washing buffer.

Washing method

1. Manual board washing: Shake off the liquid in the holes, fill each hole with washing solution, let it stand for 1 minute, shake off the liquid in the holes, pat dry on absorbent paper, and wash the board 5 times in this way.

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

Operation Steps

1. Take out the required Flat noodles from the aluminum foil bag after 20 min of room temperature balance, and seal the remaining Flat noodles with a self sealing bag and put it back at 4 ℃.

2. Set up standard wells and sample wells, and add 50 μ L of standard samples of different concentrations to each standard well;

3. Add 10 μ L of the test sample to the sample well first, and then add 40 μ L of sample diluent; Blank holes are not added.

4. Except for blank wells, add 100 μ L of horseradish peroxidase (HRP) labeled detection reagent to each well of the standard and sample wells. Seal the reaction well with a sealing plate and incubate at 37 ℃ in a water bath or constant temperature incubator for 60 minutes.

5. Discard the liquid, pat dry on absorbent paper, fill each hole with detergent, let it stand for 1 minute, shake off the detergent, pat dry on absorbent paper, repeat washing the board 5 times (or use a board washing machine).

6. Add 50 μ L of substrate A and B to each well, and incubate at 37 ℃ in the dark for 15 minutes.

7. Add 50 μ L of termination solution to each well and measure the OD value of each well at a wavelength of 450nm within 15 minutes.

result judgment

Draw standard curve: In an Excel worksheet, use the standard concentration as the horizontal axis and the corresponding OD value as the vertical axis to draw a linear regression curve of the standard. Calculate the concentration values of each sample according to the curve equation.

Test kit performance

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

2. Sensitivity: The low detection concentration is less than 0.1 U/L.

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

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

5. Storage: Store at 2-8 ℃, away from light and moisture.

6. Validity period: 6 months

Disclaimers

1. The reagent kit is for research purposes only and should not be used for clinical or human experiments. Any consequences arising from this shall be borne by the experimenter, and our company shall not be held responsible.

2. Strictly follow the instructions for operation. If the experimenter violates the instructions, the consequences shall be borne by the experimenter.