1、PRODUCT INFORMATION 610 nm to 650 nm is acceptable). Blank the plate reader according to the manufacturers instructions by using the blank wells. Determine the absorbance of both the samples and the standards. Note: In case of incubation without shaking the obtained O.D. values may be lower than ind
2、icated below. Nevertheless the results are still valid. 22 BMS635 and BMS635TEN rat IL-17A 11 Calculation of Results Calculate the average absorbance values for each set of duplicate standards and samples. Duplicates should be within 20 per cent of the mean value. Create a standard curve by plotting
3、 the mean absorbance for each standard concentration on the ordinate against the rat IL-17A concentration on the abscissa. Draw a best fit curve through the points of the graph (a 5-parameter curve fit is recommended). To determine the concentration of circulating rat IL-17A for each sample, first f
4、ind the mean absorbance value on the ordinate and extend a horizontal line to the standard curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding rat IL-17A concentration. If instructions in this protocol have been followed samples have been diluted 1:
5、2 (50 l sample + 50 l Sample Diluent), the concentration read from the standard curve must be multiplied by the dilution factor (x 2). Calculation of samples with a concentration exceeding standard 1 may result in incorrect, low rat IL-17A levels. Such samples require further external predilution ac
6、cording to expected rat IL-17A values with Sample Diluent in order to precisely quantitate the actual rat IL-17A level. It is suggested that each testing facility establishes a control sample of known rat IL-17A concentration and runs this additional control with each assay. If the values obtained a
7、re not within the expected range of the control, the assay results may be invalid. A representative standard curve is shown in Figure 8. This curve cannot be used to derive test results. Each laboratory must prepare a standard curve for each group of microwell strips assayed. BMS635 and BMS635TEN ra
8、t ILFigure 8 Representative standard curve for diluted in serial 2-fold steps in Sample Diluentcurve to derive test results. A standard curve must be run for each group of microwell strips assayed.23 -17A rat IL-17A ELISA. Rat IL. Do not use this standard -17A was 24 BMS635 and BMS635TEN rat IL-17A
9、Table 2 Typical data using the rat IL-17A ELISA Measuring wavelength: 450 nm Reference wavelength: 620 nm Standard rat IL-17A Concentration (pg/ml) O.D. at 450 nm Mean O.D. at 450 nm C.V. (%) 1 100.0 2.297 2.299 0.1 2.300 2 50.0 1.037 1.018 1.8 0.999 3 25.0 0.509 0.508 0.3 0.506 4 12.5 0.344 0.313 9
10、.9 0.282 5 6.3 0.178 0.179 0.4 0.180 6 3.1 0.114 0.111 2.9 0.108 7 1.6 0.091 0.089 2.6 0.086 Blank 0 0.082 0.075 9.8 0.067 The OD values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects). Fur
11、thermore shelf life of the kit may affect enzymatic activity and thus colour intensity. Values measured are still valid. 25 BMS635 and BMS635TEN rat IL-17A 12 Limitations Since exact conditions may vary from assay to assay, a standard curve must be established for every run. Bacterial or fungal cont
12、amination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. Improper or insufficient washing at an
13、y stage of the procedure will result in either false positive or false negative results. Empty wells completely before dispensing fresh wash solution, fill with Wash Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods. 26 BMS635 and BMS635TEN r
14、at IL-17A 13 Performance Characteristics 13.1 Sensitivity The limit of detection of rat IL-17A defined as the analyte concentration resulting in an absorbance significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 1.0 pg/ml (mean of 6 independent
15、 assays). 13.2 Reproducibility 13.2.1 Intra-assay Reproducibility within the assay was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 7 serum samples containing different concentrations of rat IL-17A. 2 standard curves were run on each plate. Data below show
16、the mean rat IL-17A concentration and the coefficient of variation for each sample (see Table 3). The calculated overall intra-assay coefficient of variation was 8.5%. 27 BMS635 and BMS635TEN rat IL-17A Table 3 The mean rat IL-17A concentration and the coefficient of variation for each sample Sample
17、 Experiment Mean rat IL-17A Concentration ( pg/ml) Coefficient of Variation (%) 1 1 77.62 4.8 2 78.87 4.9 3 70.01 7.8 2 1 48.59 7.2 2 40.97 7.4 3 40.80 19.2 3 1 21.35 5.3 2 21.05 5.1 3 18.83 13.1 4 1 13.56 3.8 2 10.88 7.7 3 12.26 8.3 5 1 70.74 4.2 2 72.92 5.7 3 81.50 4.6 6 1 24.60 4.8 2 25.17 6.8 3
18、25.81 8.9 7 1 5.42 8.4 2 4.53 24.3 3 4.98 16.1 28 BMS635 and BMS635TEN rat IL-17A 13.2.2 Inter-assay Assay to assay reproducibility within one laboratory was evaluated in 3 independent experiments. Each assay was carried out with 6 replicates of 7 serum samples) containing different concentrations o
19、f rat IL-17A. 2 standard curves were run on each plate. Data below show the mean rat IL-17A concentration and the coefficient of variation calculated on 18 determinations of each sample (see Table 4). The calculated overall inter-assay coefficient of variation was 7.6%. Table 4 The mean rat IL-17A c
20、oncentration and the coefficient of variation of each sample Sample Mean rat IL-17A Concentration ( pg/ml) Coefficient of Variation (%) 1 75.50 6.4 2 43.45 10.2 3 20.41 6.7 4 12.23 11.0 5 75.05 7.6 6 25.19 2.4 7 4.98 9.0 13.3 Spike Recovery The spike recovery was evaluated by spiking 3 levels of rat
21、 IL-17A into serum, plasma (EDTA, citrate, heparin) and cell culture supernatant samples. Recoveries were determined with 4 replicates each. For recovery data see Table 5. The unspiked serum, plasma, cell culture supernatant was used as blank in these experiments. Recoveries were shown to depend on
22、the serum used. 29 BMS635 and BMS635TEN rat IL-17A Table 5 Sample matrix Spike high (%) Spike medium (%) Spike low (%) Serum 44 34 39 Plasma (EDTA) 31 20 26 Plasma (citrate) 59 37 29 Plasma (heparin) 64 45 37 Cell culture supernatant 112 92 103 13.4 Dilution Parallelism Serum, plasma cell culture su
23、pernatant samples with different levels of rat IL-17A were analysed at serial 2 fold dilutions with 4 replicates each. For recovery data see Table 6. Table 6 Sample matrix Recovery of Exp. Val. Range (%) Mean (%) Serum 85 - 122 102 Plasma (EDTA) 93 - 125 107 Plasma (citrate) 95 - 114 106 Plasma (hep
24、arin) 64 - 126 97 Cell culture supernatant 79 - 89 86 30 BMS635 and BMS635TEN rat IL-17A 13.5 Sample Stability 13.5.1 Freeze-Thaw Stability Aliquots of spiked serum samples were stored at -20C and thawed 5 times and the rat IL-17A levels determined. There was no significant loss of rat IL-17A immuno
25、reactivity detected by freezing and thawing. 13.5.2 Storage Stability Aliquots of spiked serum samples were stored at -20C, 2-8C room temperature (RT) and at 37C, and the rat IL-17A level determined after 24 h. There was no significant loss of rat IL-17A immunoreactivity detected during storage at -
26、20C, 2-8. A significant loss of rat IL-17A immunoreactivity was detected during storage at RT and at 37C after 24 h. 13.6 Specificity Crossreactivity and interference of circulating factors of the immune system were evaluated by spiking these proteins at physiologically relevant concentrations into a rat IL-17A positive sample. There was no crossreactivity detected, notably not with rat IFN-, rat TNF-, rat IL-1a, rat IL-4, rat MCP-1, rat GM-CSF. 13.7 Expected Values There were no detectable rat IL-17A levels found. Elevated rat IL-17A levels depend on the type of immunological disorder.
