alpha Fetoprotein ELISA 试剂盒

1. All reagent should be brought to room temperature (18-22 °C) before us.
   2. Dilute 1 volume of Wash Buffer (50x) with 49 volumes of distilled water. For example, Dilute 15 mL of Wash Buffer (50x) into distilled water to prepare 750 mL of washing buffer (1x). Mix well before use.

Serum should be prepared from a whole blood specimen obtained by acceptable medical techniques. This kit is for use with serum samples without additives only.

1. Secure the desired number of coated wells in the holder.
   2. Dispense 20 mL of standard, specimens, and controls into appropriate wells.
   3. Dispense 100 mL of zero buffer into each well.
   4. Thoroughly mix for 10 seconds. It is very important to have complete mixing in this setup.
   5. Incubate at room temperature (18-22 °C) for 30 minutes.
   6. Remove the incubation mixture by flicking plate content into a waste container.
   7. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
   8. Strike the wells sharply onto absorbent paper or paper towels to remove all residual water droplets.
   9. Dispense 150 mL of Enzyme Conjugate Reagent into each well. Gently mix for 5 seconds.
   10. Incubate at room temperature for 30 minutes.
   11. Remove the incubation mixture by flicking plate contents into a waste container.
   12. Rinse and flick the microtiter wells 5 times with washing buffer (1X).
   13. Strike the wells sharply onto absorbent paper to remove residual water droplets.
   14. Dispense 100 mL TMB substrate into each well. Gentle mix for 5 seconds.
   15. Incubate at room temperature for
   2. minutes.
   16. Stop the reaction by adding 100 mL of stop solution to each well.
   17. Gently mix for 30 seconds to make sure that the blue color changes to yellow color completely.
   18. Read optical density at 450 nm with a microtiter reader within 15 minutes.
   
   Important Note:
   The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.

I. Accuracy: Comparison between Our Kits and Commercial Available Kits provides the following data N = 79 Correlation Coefficient = Slope =
1.038 Intercept = 0.729 Mean (Our) = 55.12 Mean (Abbott) = 5
2.24 0.985 II. Precision. 1]. Intra-Assay: Concentrations Replicates Mean S.D. % CV Level I 24 3
1.04
1.45
4.66 Level II 24 126.8
5.20
4.10 Level III 24 270.8 1
2.93
4.78 2]. Inter-Assay: Concentrations Replicates Mean S.D. % CV Level I 24 30.58
1.88
6.1 Level II 24 125.1
7.08
5.7 Level III 24 268.3 14.06
5.2 III. Linearity Two patient sera were serially diluted with 0 ng/mL standard in a linearity study. The average recovery was 10
2.2 %. AFP (ng/mL) Absorbance (450nm) 0 0.012 5 0.127 20 0.455 50 0.952 150
2.150 300
2.932 Sample Dilution Expected Observed % Recov. undiluted 27
1.20 27
1.20 2x 135.60 137.12 10
1.1 4x 67.80 69.45 10
2.4 8x 3
3.90 35.16 10
3.7 16x 16.95 17.20 10
1.5 32x
8.48
9.31 109.9 Average Recovery: 10
2.2 % IV. Recovery Various patient serum samples of known AFP levels were mixed and assayed in duplicate. The average recovery was 10
1.1 %. Conc. of AFP (ng/mL) Expected Concentration Observed Concentration % Recovery 9
2.70 94.20 10
1.6 77.63 79.31 10
2.2 2
3.24 2
3.10 99.4 156.18 159.23 10
2.0 206.06 210.10 10
2.0 148.94 150.52 10
1.1 149.73 148.97 99.50 Average Recovery: 10
1.1 %
Calculate the mean absorbance value (A450) for each set of reference standards, specimens, controls and patient samples. Constructed a standard curve by plotting the mean absorbance obtained from each reference standard against its concentration in ng/mL on graph paper, with absorbance values on the vertical or Y-axis and concentrations on the horizontal or X-axis. Use the mean absorbance values for each specimen to determine the corresponding concentration of AFP in ng/mL from the standard curve. Example of standard curve Results of typical standard run with optical density reading at 450 nm shown in the Y-axis against AFP concentrations shown in the X-axis. This standard curve is for the purpose of illustration only, and should not be used to calculate unknowns. Each user should obtain his or her own data and standard curve. Expected values and sensitivity In high-risk patients, AFP values between 100 and 350 ng/mL suggest a diagnosis of hepatocellular carcinoma, and levels over 350 ng/mL usually indicate the disease. Approximately 97 % of the healthy subjects have AFP levels less than 8.5 ng/mL. It is recommended that each laboratory establish its own normal range. The minimum detectable concentration of AFP by this assay is estimated to be 2.0 ng/mL.