标题大豆凝集素（SBA）酶联免疫分析（ELISA）

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大豆凝集素（SBA）酶联免疫分析（ELISA）
试剂盒使用说明书
本试剂仅供研究使用目的：本试剂盒用于测定组织，细胞及其它相
关样本中大豆凝集素（SBA）含量。
实验原理：
本试剂盒应用双抗体夹心法测定标本中大豆凝集素（SBA）水平。用纯化的大豆凝集素
（SBA）抗体包被微孔板，制成固相抗体，往包被单抗的微孔中依次加入大豆凝集素（SBA），
再与HRP 标记的大豆凝集素（SBA）抗体结合，形成抗体-抗原-酶标抗体复合物，经过彻
底洗涤后加底物TMB 显色。TMB 在HRP 酶的催化下转化成蓝色，并在酸的作用下转化成
最终的黄色。颜色的深浅和样品中的大豆凝集素（SBA）呈正相关。用酶标仪在450nm 波
长下测定吸光度（OD 值），通过标准曲线计算样品中大豆凝集素（SBA）浓度。
试剂盒组成：
试剂盒组成48 孔配置96 孔配置保存
说明书1 份1 份
封板膜2 片（48） 2 片（96）
密封袋1 个1 个
酶标包被板1×48 1×96 2-8℃保存
标准品：900ng/L 0.5ml×1 瓶0.5ml×1 瓶2-8℃保存
标准品稀释液1.5ml×1 瓶1.5ml×1 瓶2-8℃保存
酶标试剂3 ml×1 瓶6 ml×1 瓶2-8℃保存
样品稀释液3 ml×1 瓶6 ml×1 瓶2-8℃保存
显色剂A 液3 ml×1 瓶6 ml×1 瓶2-8℃保存
显色剂B 液3 ml×1 瓶6 ml×1 瓶2-8℃保存
终止液3ml×1 瓶6ml×1 瓶2-8℃保存
浓缩洗涤液（20ml×20 倍）×1 瓶（20ml×30 倍）×1 瓶2-8℃保存
标本要求：
1．标本采集后尽早进行提取，提取按相关文献进行，提取后应尽快进行实验。若不能
马上进行试验，可将标本放于-20℃保存，但应避免反复冻融
2．不能检测含NaN3 的样品，因NaN3 抑制辣根过氧化物酶的（HRP）活性。
操作步骤：
1. 标准品的稀释与加样：在酶标包被板上设标准品孔10 孔，在第一、第二孔中分别加标
准品100μl，然后在第一、第二孔中加标准品稀释液50μl，混匀；然后从第一孔、第二
孔中各取100μl 分别加到第三孔和第四孔，再在第三、第四孔分别加标准品稀释液50μl，
混匀；然后在第三孔和第四孔中先各取50μl 弃掉，再各取50μl 分别加到第五、第六孔
中，再在第五、第六孔中分别加标准品稀释液50ul，混匀；混匀后从第五、第六孔中各
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取50μl 分别加到第七、第八孔中，再在第七、第八孔中分别加标准品稀释液50μl，混
匀后从第七、第八孔中分别取50μl 加到第九、第十孔中，再在第九第十孔分别加标准
品稀释液50μl，混匀后从第九第十孔中各取50μl 弃掉。（稀释后各孔加样量都为50μl，
浓度分别为600ng/L，400ng/L ，200ng/L，100ng/L， 50ng/L）。
2. 加样：分别设空白孔（空白对照孔不加样品及酶标试剂，其余各步操作相同）、待测样
品孔。在酶标包被板上待测样品孔中先加样品稀释液40μl，然后再加待测样品10μl（样
品最终稀释度为5 倍）。加样将样品加于酶标板孔底部，尽量不触及孔壁，轻轻晃动混
匀。
3. 温育：用封板膜封板后置37℃温育30 分钟。
4. 配液：将30（48T 的20 倍）倍浓缩洗涤液用蒸馏水30（48T 的20 倍）倍稀释后备用。
5. 洗涤：小心揭掉封板膜，弃去液体，甩干，每孔加满洗涤液，静置30 秒后弃去，如此
重复5 次，拍干。
6. 加酶：每孔加入酶标试剂50μl，空白孔除外。
7. 温育：操作同3。
8. 洗涤：操作同5。
9. 显色：每孔先加入显色剂A50μl，再加入显色剂B50μl，轻轻震荡混匀，37℃避光显色
15 分钟.
10. 终止：每孔加终止液50μl，终止反应（此时蓝色立转黄色）。
11. 测定：以空白空调零，450nm 波长依序测量各孔的吸光度（OD 值）。测定应在加终止
液后15 分钟以内进行。
注意事项：
1． 试剂盒从冷藏环境中取出应在室温平衡15-30 分钟后方可使用，酶标包被板开封后如未
用完，板条应装入密封袋中保存。
2． 浓洗涤液可能会有结晶析出，稀释时可在水浴中加温助溶，洗涤时不影响结果。
3． 各步加样均应使用加样器，并经常校对其准确性，以避免试验误差。一次加样时间最好
控制在5 分钟内，如标本数量多，推荐使用排枪加样。
4． 请每次测定的同时做标准曲线，最好做复孔。如标本中待测物质含量过高（样本OD 值
大于标准品孔第一孔的OD 值），请先用样品稀释液稀释一定倍数（n 倍）后再测定，计
算时请最后乘以总稀释倍数（×n×5）。
5． 封板膜只限一次性使用，以避免交叉污染。
6． 底物请避光保存。
7． 严格按照说明书的操作进行，试验结果判定必须以酶标仪读数为准.
8． 所有样品，洗涤液和各种废弃物都应按传染物处理。
9． 本试剂不同批号组分不得混用。
10. 如与英文说明书有异，以英文说明书为准。
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计算：
以标准物的浓度为横坐标，OD 值为纵坐标，
在坐标纸上绘出标准曲线，根据样品的OD
值由标准曲线查出相应的浓度；再乘以稀释
倍数；或用标准物的浓度与OD 值计算出标
准曲线的直线回归方程式，将样品的OD 值
代入方程式，计算出样品浓度，再乘以稀释
倍数，即为样品的实际浓度。
（此图仅供参考）
试剂盒性能：
1.样品线性回归与预期浓度相关系数R 值为0.92 以上。
2.批内与批见应分别小于9%和15%
检测范围：
30ng/L -700ng/L
保存条件及有效期：
1.试剂盒保存：；2-8℃。
2．有效期：6 个月
FOR RESEARCH USE ONLY
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soybean agglutinin
Drug Names
Generic Name：soybean agglutinin（ SBA） ELISA Kit.
Purpose
This kit allows for the determination of SBA concentrations in tissue ,cell
and other samples.
Principle of the assay
The kit assay SBA level in the sample，use Purified SBA antibody to coat
microtiter plate wells, make solid-phase antibody, then add SBA to wells,
Combined SBA antibody which With HRP labeled,become antibody - antigen -
enzyme-antibody complex, after washing Completely, Add TMB substrate
solution,TMB substrate becomes blue color At HRP enzyme-catalyzed,
reaction is terminated by the addition of a sulphuric acid solution ａnd the color
change is measured spectrophotometrically at a wavelength of 450 nm. The
concentration of SBA in the samples is then determined by comparing the O.D.
of the samples to the standard curve.
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Materials provided with the kit
Materials provided
with the kit
48determinations 96 determinations Storage
User manual 1 1
Closure plate
membrane 2 2
Sealed bags 1 1
Microelisa stripplate 1 1 2-8℃
Standard：900ng/L 0.5ml×1 bottle 0.5ml×1 bottle 2-8℃
Standard diluent 1.5ml×1 bottle 1.5ml×1 bottle 2-8℃
HRP-Conjugate
reagent 3ml×1 bottle 6ml×1 bottle 2-8℃
Sample diluent 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen
Solution A 3ml×1 bottle 6ml×1 bottle 2-8℃
Chromogen
Solution B 3ml×1 bottle 6ml×1 bottle 2-8℃
Stop Solution 3ml×1 bottle 6ml×1 bottle 2-8℃
wash solution （20ml×20 fold）
×1bottle
（20ml×30 fold）
×1bottle 2-8℃
Specimen requirements
1. extract as soon as possible after Specimen collection,and according to the
relevant literature, ａnd should be experiment as soon as possible after the
extraction. If it can’t, specimen can be kept in -20 ℃ to preserve, Avoid
repeated freeze-thaw cycles.
2. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP
active.
Assay procedure
1.Dilute ａnd add sample to Standard: set 10 Standard wells on the ELISA
plates coated, add Standard 100μl to the first ａnd the second well, then add
Standard dilution 50μl to the first ａnd the second well, mix; take out 100μl
form the first ａnd the second well then add it to the third ａnd the forth well
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separately. then add Standard dilution 50μl to the third ａnd the forth
well ,mix ; then take out 50μl from the third ａnd the forth well discard, add
50μl to the fifth ａnd the sixth well ,then add Standard dilution 50μl to the fifth
and the sixth well, mix ; take out 50μl from the fifth ａnd the sixth well ａnd add
to the seventh ａnd the eighth well, then add Standard dilution 50μl to the
seventh ａnd the eighth well ,mix ; take out 50μl from the seventh ａnd the
eighth well ａnd add to the ninth ａnd the tenth well, add Standard dilution 50μl
to the ninth ａnd the tenth well, mix , take out 50μl from the ninth ａnd the tenth
well discard（add Sample 50μl to each well after Diluting ,（density: 600ng/L ，
400ng/L ，200ng/L，100ng/L， 50ng/L）
2.add sample：Set blank wells separately （blank comparison wells don’t add
sample ａnd HRP-Conjugate reagent, other each step operation is same）.
testing sample well. add Sample dilution 40μl to testing sample well, then add
testing sample 10μl （sample final dilution is 5-fold）, add sample to wells ,
don’t touch the well wall as far as possible, ａnd Gently mix.
3.Incubate: After closing plate with Closure plate membrane ,incubate for 30
min at 37℃.
4.Configurate liquid: 30-fold （or 20-fold）wash solution diluted 30-fold （or 20-fold）
with distilled water ａnd reserve.
5.washing：Uncover Closure plate membrane, discard Liquid, dry by swing,
add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by
pat.
6.add enzyme：Add HRP-Conjugate reagent 50μl to each well, except blank
well.
7.incubate：Operation with 3.
8.washing：Operation with 5.
9.color：Add Chromogen Solution A 50ul ａnd Chromogen Solution B to each
well, evade the light preservation for 15 min at 37℃
10.Stop the reaction：Add Stop Solution50μl to each well, Stop the reaction（the
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blue color change to yellow color）.
11.assay：take blank well as zero , Read absorbance at 450nm after Adding
Stop Solution ａnd within 15min.
Important notes
1. The kit takes out from the refrigeration environment should be balanced
15-30 minutes in the room temperature, ELISA plates coated if has not use
up after opened, the plate should be stored in Sealed bag.
2. washing buffer will Crystallization separation, it can be heated the water
helps dissolve when dilute . Washing does not affect the result.
3. add Sample with sampler Each step, And proofread its accuracy frequently,
avoids the experimental error. add sample within 5 mins, if the number of
sample is much , recommend to use Volley .
4. if the testing material content is excessively higher （The sample OD is
bigger than the first standard well ）,please dilute Sample （n-fold）, Please
diluente ａnd multiplied by the dilution factor.（×n×5）.
5. Closure plate membrane only limits the disposable use, to avoid
cross-contamination.
6. The substrate evade the light preservation.
7. Please according to use instruction strictly, The test result determination
must take the microtiter plate reader as a standard.
8. All samples, washing buffer ａnd each kind of reject should according to
infective material process.
9. Do not mix reagents with those from other lots.
Calculate
Take the standard density as the horizontal,
the OD value for the vertical ,draw the standard
curve on graph paper, Find out the corresponding
density according to the sample OD value by the
Sample curve, multiplied by the dilution multiple,
or calculate the straight line regression equation
of the standard curve with the standard density
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Assay range
30ng/L -700ng/L
Storage ａnd validity
1．Storage： 2-8℃.
2．validity： six months.