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[求助]
看一篇文章上的competition ELISA图，实在是看不懂，上课要讲，紧急求助！！
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发表在JAFC 上的 Decreased Immunoglobulin E (IgE) Binding to Cashew Allergens following Sodium Sulfite Treatment and Heating（见附件） 其中的c-ELISA图如下：  图名如下： Figure 6. Sodium sulfite-treated cashew extract lowered IgE binding. Cashew extracts (squares) or cashew extracts pretreated with DTT (diamonds) or sodium sulfite (triangles) were evaluated for IgE binding in a competitive ELISA after incubation at 65 °C (A) or 100 °C (B). Cashew extract samples were serially diluted 10-fold, and IgE binding was evaluated in microtiter plates coated with 1 μg of cashew extract. Serum samples from a pool of six cashew-allergic patients were combined, diluted 1:4, and analyzed for IgE binding to sodium sulfiteor DTT-treated cashew extracts. Cashew extract concentration is indicated on the x-axis, and relative IgE binding in IRdye-680 units is indicated on the y-axis. 其中的横坐标和我们平时做的正好相反，竞争物浓度高了反而抑制率变低了，我实在是想不清楚为什么。 同时贴上他的方法： Competetive ELISA. Microtiter plates were coated with 1 μg of raw cashew extract per well in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.6) and incubated overnight at 4 °C. Cashew extract was discarded, and the plate was blocked with PBST containing 1% BSA (w/v) at room temperature for 1 h. Plates were washed three times with PBST following blocking and all experimental incubation steps to remove unbound proteins. Treated or untreated cashew extract was centrifuged with 3 kDa spin columns to remove unreacted sodium sulfite or DTT. Samples were then buffer exchanged in 100 mM Tris, pH 8.3, by 10-fold dilution and centrifugation four times using the spin columns to further dilute any remaining sodium sulfite or DTT. Samples were resuspended in 200 μL of buffer and serially diluted 10-fold with 100 mM Tris, pH 8.3. Competition samples, to evaluate IgE binding, were made using 12.5 μL of treated cashew sample, 12.5 μL of pooled sera, and 25 μL of 100 mM Tris, pH 8.3. Samples were added to the microtiter plates and incubated at 37 °C for 1 h. Plates were washed, and 50 μL of biotinylated anti-IgE (1:1000 in PBST) was added to each well and then incubated at 37 °C for 30 min. Plates were washed with PBST, and 50 μL of streptavidin-labeled IRdye-680 (1:5000 in PBST) was added to each well. Plates were incubated at 37 °C for 30 min, washed, and then imaged with the Odyssey CLx instrument. The data in the plot are presented as percent of IgE inhibition using the following formula: IR680 value of uninhibited control − IR680 value of inhibited sample/IR680 value of uninhibited control × 100. 求大神帮忙紧急解答啊，明天下午要讲……我已经看了一晚上了，问了一圈人都没整明白~~~  图片1.png |
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2014-09-24 22:59:31, 4.62 M
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