本試劑盒只能用于科學研究，不得用于醫(yī)學診斷
人（Human）葉酸（folic acid）ELISA檢測試劑盒
使用說明書
檢測原理
試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗（ELISA）。往預
先包被葉酸（folic acid）抗體的包被微孔中，依次加入標本、標準品、
HRP標記的檢測抗體，經(jīng)過溫育并*洗滌。用底物TMB顯色，TMB
在過氧化物酶的催化下轉(zhuǎn)化成藍色，并在酸的作用下轉(zhuǎn)化成zui終的
黃色。顏色的深淺和樣品中的葉酸（folic acid）呈正相關(guān)。用酶標儀
在450nm 波長下測定吸光度（OD 值），計算樣品濃度。
樣品收集、處理及保存方法
1. 血清：使用不含熱原和內(nèi)毒素的試管，操作過程中避免任何細胞
刺激，收集血液后，3000 轉(zhuǎn)離心10 分鐘將血清和紅細胞迅速小心地
分離。
2. 血漿：EDTA、檸檬酸鹽或肝素抗凝。3000 轉(zhuǎn)離心30 分鐘取上清。
3. 細胞上清液：3000 轉(zhuǎn)離心10 分鐘去除顆粒和聚合物。
4. 組織勻漿：將組織加入適量生理鹽水搗碎。3000 轉(zhuǎn)離心10 分鐘
取上清。
5. 保存：如果樣本收集后不及時檢測，請按一次用量分裝，凍存于
-20℃，避免反復凍融，在室溫下解凍并確保樣品均勻地充分解凍。
自備物品
1. 酶標儀（450nm）
2. 高精度加樣器及槍頭：0.5-10uL、2-20uL、20-200uL、200-1000uL
3. 37℃恒溫箱
操作注意事項
1. 試劑盒保存在2-8℃，使用前室溫平衡20 分鐘。從冰箱取出的
濃縮洗滌液會有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶*溶解
后再使用。
2. 實驗中不用的板條應立即放回自封袋中，密封（低溫干燥）保存。
3. 濃度為0 的S0 號標準品即可視為陰性對照或者空白；按照說明Materials supplied
Name 96 determinations 48 determinations
Microelisa stripplate 12*8strips 12*4strips
Standard 0.3ml*6tubes 0.3ml*6tubes
Sample Diluent 6.0ml 3.0ml
HRP-Conjugate reagent 10.0ml 5.0ml
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:0,1.5,3,6,12,24 ng/ml
Reagent preparation
20×wash solution:Dilute with Distilled or deionized water 1:20.
Assay procedure
1. Prepare all r e a g e n t s 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 esting sample 10μl then add Sample Diluent 40μl to testing
sample well; Blank well doesn’t add anyting.
4. Add 100μl 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
1. This standard curve is used to determine the amount in an unknown sample.
The standard curve is generated by plotting the average O.D. （450 nm）
obtained for each of the six standard concentrations on the vertical （Y） axis