Manual of human N-acetyl-β-D-glucosaminidase (NAG) ELISA kit
Human N-acetyl-β-D-glucosaminidase (NAG) ELISA kit is for research use only
Detection range: 0.533 mIU / ml-40 mIU / ml
Minimum detection limit: 0.03 mIU / ml
Intended application: ELISA method for quantitative determination of NAG content in human serum, plasma and other related biological fluids.
Explanation:
1. The newly opened enzyme-linked plate may contain a little water-like substance in the well. This is a normal phenomenon and will not have any impact on the experimental results.
2. When the concentrated washing liquid is stored at low temperature, salt will precipitate out. When diluted, it can be heated and dissolved in a water bath.
Experimental principle: The content of NAG was detected by competitive enzyme-linked immunoassay. First coat a microplate with a single antibody to prepare a solid-phase antibody, then add the specimen or standard to be tested and NAG labeled with horseradish peroxidase to form a coating antibody -NAG (HRP) Complex. After color development, the absorbance value (OD value) is measured in a microplate reader, the concentration-absorbance curve is fitted by computer or drawing, and the NAG content in the sample to be tested is calculated inversely.
Composition and preparation of human N-acetyl-β-D-glucosaminidase (NAG) ELISA kit:
1. Assay plate: One piece (96 wells).
2. Standard: 5 × 0.5 ml / bottle.
Standard S1 S2 S3 S 4 S 5
Concentration (mIU / ml) 0.533 1.067 2.67 10.67 40
3. Enzyme conjugate (HRP-conjugate): 1 × 6ml / bottle.
4. Developer A (Substrate A): 1 × 7ml / bottle.
5. Developer B (Substrate B): 1 × 7ml / bottle.
6. Concentrated washing buffer (Wash Buffer): 1 × 15ml / bottle, each bottle is diluted 20 times with distilled water.
7. Stop solution (Stop Solution): 1 × 7ml / bottle
Reagents and equipment needed but not provided:
1. Standard specification microplate reader
2. High-speed centrifuge
3. Electric constant temperature incubator
4. Clean test tubes and Eppendof tubes
5. Series adjustable pipettes and tips, when testing more samples at one time, it is best to use multi-channel pipettes
6 Distilled water, volumetric flask, etc.
Collection and preservation of specimens:
1. Plasma: EDTA or heparin can be used as an anticoagulant. Centrifuge the sample at 2-8 ° C 1000 xg for 15 minutes within 30 minutes after collection, or store the specimen at -20 ° C or -80 ° C, but avoid repeated freezing melt.
2. Serum: Whole blood specimens should be left at room temperature for 2 hours or overnight at 4 ° C and centrifuged at 1000 xg for 20 minutes. The supernatant can be taken for detection, or the specimens should be stored at -20 ° C or -80 ° C, but avoid repeated Freeze and thaw.
Note: The above specimens should be stored at 4 ℃ for less than 1 week, -20 ℃ or -80 ℃ should be sealed and stored, -20 ℃ should not exceed 1 month, -80 ℃ should not exceed 2 months; specimen hemolysis will affect the final The test results, so hemolytic specimens should not be tested.
Steps:
1. Equilibrate various reagents to room temperature [18-25 ° C] for half an hour, take concentrated washing solution, dilute 1:20 with distilled water according to the number of batches tested, mix well and set aside.
2. Take out the enzyme labeling plate, set a blank control well without adding any liquid; set two holes in each standard point in turn, add 50ul of the corresponding standard to each well; add 50ul of the sample to be tested directly to each remaining well
3. Add 50ul of enzyme conjugate to each well (except the blank control well), mix well, attach a self-adhesive seal, and incubate at 37 ° C for 1 hour.
4. Manually wash the plate and discard the liquid in the hole. Fill the holes with the washing solution, let it dry for 10 seconds, and then pat dry after repeating three times; wash the plate with a plate washer, choose three washing procedures, and pat dry after washing.
5. Add 50μl of Developer A solution and 50μl of Developer B solution to each well. After shaking and mixing, develop color at 37 ° C in the dark for 15 minutes. Add 50μl of stopper solution to each well.
6. Read with a microplate reader, take a wavelength of 450nm, first adjust the zero point with a blank well, and then measure the OD value of each well.
data processing:
1. Computer: Using the linear fitting function, the logarithm (Log (concentration)) of the concentration of the standard products S1-S5 should be taken as X, and the logarithm (Log ( OD value-NSB)) as Y, linear fitting. Then calculate the serum concentration to be measured from the fitted line.
2. Manual work diagram: using double logarithmic graph paper, with the standard concentration as the horizontal axis, and the corresponding 0D value as the vertical axis, draw a smooth curve or straight line, and find the corresponding concentration on the curve according to the serum OD value to be measured value.
Precautions:
1. All the bottled reagents and the required pre-coated strips in the kit taken from the refrigerated environment should be equilibrated at room temperature (18-25 ° C) for 30 minutes, and the rest should be sealed in time and put back 2- Store at 8 ° C in the dark for future use.
2. Reagents should be shaken well before use.
3. The result judgment must be completed within 10 minutes after the termination of the reaction.
4. Reagents of different batches cannot be mixed.
5. When adding samples, care should be taken to avoid contamination and contamination between the reagents and samples used.
6. During operation, use one tip for each reagent in the kit, one tip for each standard, and one tip for each sample. It is best to use the tip once.
Specificity: This kit can detect natural or recombinant NAG at the same time, and there is basically no cross reaction with other related proteins.
Validity: 6 months (stored at 2-8 ° C, protected from light)
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