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User Manual
ELK05J0138, α-Ketoglutaric acid (α-KA)
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2-8°C in their pouch with the desiccant provided.
3. Mix all reagents before using.
Remove all kit reagents from refrigerator and allow them to reach room
temperature ( 20-25°C)
Materials supplied
| Name |
96 determinations |
48 determinations |
| Microelisa stripplate |
8\\*12strips |
8\\*6strips |
| Standard |
0.3ml\\*6tubes |
0.3ml\\*6tubes |
| Sample Diluent |
6.0ml |
3.0ml |
| HRP-Conjugate reagent |
6.0ml |
3.0ml |
| 20X Wash solution |
25ml |
15ml |
| Chromogen Solution A |
6.0ml |
3.0ml |
| Chromogen Solution B |
6.0ml |
3.0ml |
| Stop Solution |
6.0ml |
3.0ml |
| Closure plate membrane |
2 |
2 |
| User manual |
1 |
1 |
| Sealed bags |
1 |
1 |
| Note: Standard (S0 ￫S5) concentration was followed by:0､25､50､100､200､400 pg/mL |
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| Reagent preparation |
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| 20×wash solution:Dilute with Distilled or deionized water 1:20. |
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| Assay procedure |
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1.
Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.
2. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.
3. Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well;
4. Add 50μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C.
5. Aspirate each well and wash, repeating the process four times for a total of five washes. Wash by filling each well with Wash Solution (400μl) using a squirt bottle, manifold dispenser or autowasher.
Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting. Invert the plate and blot it against clean paper towels.
6. Add chromogen solution A 50μl and chromogen solution B 50μl to each well. Gently mix and incubate for 15 minutes at 37°C. Protect from light.
7. Add 50μl Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.
Calculation of results
9. Calculate the mean absorbance value A450 for each set of reference standards and samples.
10. Divide the average A450 value for each standard, control and test sample by the average A450 of standard0 and multiply by 100 to obtain %B/B0 for each sample.
11. Prepare a standard curve by plotting the average absorbance of each standard versus the corresponding concentrations of the standards on linear-log graph paper or the %B/B0 value for each standard versus the corresponding concentration of the standard on linear-log or logit-log graph paper. logit=ln(B/ B0)/(1-B/ B0)
12. Any values obtained for diluted samples must be further converted by applying the appropriate dilution factor in the calculations.
13. The standard density is a X, the B/ B0 is a Y, sitting to mark the paper in the logit- log up draw a standard curve. According to the B/ B0 that need to be measured the sample can from sit to mark the density value that the paper looks up the sample up.
14. The sensitivity by this assay is 1pg/mL
15. The standard curve presented here is an example of the data typically produced with this kit; however, your results will not be identical to these.
16. Standard curve match
Storage: 2-8℃. Validity:six months.
FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!