cELISA Kit for Detection of Brucella Antibody

布鲁氏菌 cELISA 抗体检测试剂盒使用说明书
Instructions of cELISA Kit for Detection of Brucella Antibody

【兽用名称】
Veterinary Names

【主要成分与含量】
Main Components and Contents

【作用与用途】    用于检测光滑型牛、羊布鲁氏菌血清抗体。 

Function and Use: It is used to detect serum antibodies of Smooth Brucella bovis and sheep.

【用法与判定】
Usage and Judgment

1      试剂配制 Reagent preparation

1.1     所有的试剂应在检验前室温下放置30分钟，使所用试剂恢复至室温。

1.1 All reagents shall be left at room temperature for 30 minutes before inspection to restore the reagents used to room temperature.

1.2     1×PBS-Tween洗涤液配制，PBS-Tween洗涤液（20×）应恢复至室温（18～25℃），如有沉淀可在37℃温浴5～10分钟，用灭菌纯化水作20倍稀释，即为pH值7.4的1×PBS-Tween洗涤液，2～8℃可保存7日。

1.2 1×PBS-Tween washing solution, PBS-Tween washing solution (20 ×) should be restored to room temperature (18 ~ 25℃), if there is precipitation, it can be bathed in 37℃ for 5 ~ 10 minutes, diluted by 20 times with sterilized purified water, i.e. 1 × PBS-Tween washing solution with pH value 7.4, which can be stored for 7 days at 2 ~ 8℃.

1.3     样品稀释液为1×PBS-Tween洗涤液。

1.3 The sample diluent is 1×PBS-Tween washing solution

1.4      对照血清，使用前30分钟使各对照血清恢复至室温，用1×PBS-Tween洗涤液将强阳性对照血清、弱阳性对照血清、阴性对照血清按所需量作10倍稀释，室温下备用。

1.4 Control serum, restore each control serum to room temperature 30 minutes before use, dilute strong positive control serum, weak positive control serum and negative control serum 10 times according to the required amount with 1 × PBS-Tween washing solution, and set aside at room temperature.

1.5     被检血清稀释，被检血清用1×PBS-Tween洗涤液作10倍稀释，室温放置备用。

1.5 Dilute the tested serum. The tested serum is diluted 10 times with 1 × PBS-Tween washing solution and placed at room temperature for standby.

1.6     HRP标记山羊抗鼠IgG    使用前30分钟使其恢复至室温备用。

1.6 HRP-labeled goat anti-mouse IgG was restored to room temperature 30 minutes before use.

1.7     单克隆抗体配制，用1×PBS-Tween洗涤液6ml溶解冷冻干燥单克隆抗体，使用前30分钟放置于室温下备用。

1.7 Formulation of monoclonal antibody, lyophilized monoclonal antibody dissolved with 6ml of 1×PBS-Tween wash solution, and set aside at room temperature 30 minutes prior to use.

1.8     底物显色液，使用前30分钟放置于室温下备用。

1.8 Substrate color developing solution, leave at room temperature for standby 30 minutes before use.

2      cELISA方法

2     cELISA method

2.1取抗原包被板（根据样品多少，可拆开分次使用），加入1×PBS-Tween洗涤液，每孔200μl，洗板4次，每次3分钟。弃去洗涤液，取10倍稀释的待检血清50μl，加入到抗原包被板1孔中，立即加入稀释的单克隆抗体溶液50μl，轻轻振匀孔中样品（勿溢出），室温放置45分钟。同时设强阳性血清对照、弱阳性血清对照、阴性血清对照和无血清空白对照（1×PBS-Tween洗涤液）各2孔。

2.1 Take the antigen coating plate (which can be disassemble and used in several times according to the number of samples), add 1×PBS-Tween washing solution, 200μl per well, and wash the plate 4 times for 3 minutes each time. Discard the washing solution, take 50μl of 10-fold diluted serum to be tested, add 50μl of diluted monoclonal antibody solution into hole 1 of antigen coating plate, immediately add 50μl of diluted monoclonal antibody solution, gently shake the sample in the hole (do not overflow), and leave at room temperature for 45 minutes. At the same time, two wells of strong positive serum control, two wells of weak positive serum control, two wells of negative serum control and two wells of serum-free blank control (1×PBS-Tween washing solution) were set up.

2.2     弃去板中液体，用1×PBS-Tween洗涤液每孔200μl，洗板4次，每次3分钟。

2.2 Discard the liquid in the plate and washing the plate 4 time for 3 minutes with 200μl of 1×PBS-Tween washing solution per well.

2.3     每孔加HRP标记山羊抗鼠IgG100μl，室温放置45分钟。

2.3 IgG100μl goat anti-mouse labeled with HRP was added to each well and kept at room temperature for 45 minutes.

2.4     用1×PBS-Tween洗涤液洗板4次，方法同上。

2.4 Plate was washed 4 time with 1×PBS-Tween washing solution, the same method as above.

2.5     每孔加底物显色液100μl，室温下避光显色10分钟。

2.5 Add 100μl substrate color developing solution to each well and develop color at room temperature for 10 minutes without light.

2.6     每孔加终止液50μl，15分钟内用酶标仪测定并记录各孔在450nm波长处的吸光度（OD）值。

2.6 Add 50μl of termination solution to each well, and measure and record the absorbance (OD) value of each well at 450nm wavelength with enzyme labeling instrument within 15 minutes.

3      结果判定

3. Result Judgment

判定标准：抑制率（PI）≥30%判为阳性，抑制率（PI）＜30%判为阴性。

Criteria: The inhibition rate (PI) ≥ 30% was judged as positive, and the inhibition rate (PI) < 30% was judged as negative.

抑制率公式：抑制率（PI）=（空白对照OD450nm―检测样品OD450nm）／空白对照OD450nm×100%。

Inhibition rate formula: inhibition rate (PI) = (blank control OD450nm-detection sample OD450nm)/blank control OD450nm × 100%.

各对照的测定值在如下范围内时，试验有效：

The test is valid when the measured values of each control are within the following ranges:

【注意事项】
Precautions

（1）试剂盒使用前各组分应室温平衡30分钟，使用后即放回2～8℃。

(1) The components of the kit should be balanced at room temperature for 30 minutes before use, and then put back to 2 ~ 8℃ after use.

（2）不同批号试剂盒的试剂组分不得混用，使用过程中，各试剂组分应避免交叉污染。

(2) Reagent components of kits with different batch numbers shall not be mixed. During use, cross contamination shall be avoided for each reagent component.

（3）底物显色液避免暴露于强光和接触氧化剂。

(3) The substrate color developing solution should avoid exposure to strong light and contact with oxidant.

（4）终止液中含有H2SO4，使用时注意不要接触到身体，衣物。

(4) The termination solution contains H2SO4, so be careful not to touch the body and clothes when using it.

（5）强阳性对照血清、弱阳性对照血清、阴性对照血清及HRP标记山羊抗鼠IgG在使用前先要低速（500～1000转）离心3～5秒，使其集中于管底。

(5) Strong positive control serum, weak positive control serum, negative control serum and HRP labeled goat anti-mouse IgG should be centrifuged at low speed (500 ~ 1000 revolutions) for 3~5 seconds before use to concentrate them at the bottom of the tube.

（6）抗原包被板拆封后应避免受潮或沾水（每次将剩余的抗原包被板用封口袋扎紧后尽快置于2～8℃）。

(6) Avoid damp or water exposure after unpacking the antigen coating plate (place the remaining antigen coating plate at 2 ~ 8℃ as soon as possible after tightening it with sealing pockets each time).

（7）待检血清样品数量较多时，应采用血清稀释板，先稀释所有待检测血清，再将稀释好的血清转移到反应板。操作过程应使各孔的反应时间一致。

(7) When the number of serum samples to be tested is large, a serum dilution plate shall be used to dilute all the serum to be tested first, and then the diluted serum shall be transferred to the reaction plate. The operation process should make the reaction time of each hole consistent.

（8）PBS-Tween洗涤液（20×）用灭菌纯化水稀释。如果发现有结晶，37℃温浴5～10分钟后再使用。

(8) PBS-Tween washing solution (20×) was diluted with sterilized purified water. If crystallization are found, that product is reused after a warm bath at 37℃ for 5 to 10 minute

（9）在操作过程中移液时，应避免气泡产生。

(9) Bubbles should be avoided during operation.

（10）严格按照操作说明书要求进行。操作过程中移液、定时和洗涤等过程必须精确。

(10) Strictly follow the requirements of the operation instructions. The process of pipetting, timing and washing must be accurate.

【规格】（1）1块板（96孔）/盒；（2）5块板（480孔）/盒

Specification: (1) 1 plate (96 holes)/box; (2) 5 plates (480 holes)/box

【贮藏与有效期】2～8℃保存，有效期为6个月。

Storage and Validity Period: Stored at 2 ~ 8℃, the validity period is 6 months.

【批准文号】
Approval No.

【生产企业】长春西诺生物科技有限公司
Manufacturer: Changchun Sino Biotechnology Co., Ltd.

地址：吉林省长春市高新产业开发区普天路58号

Address: No.58 Putian Road, High-tech Industrial Development Zone, Changchun City, Jilin Province

邮编：130012

Zip Code: 130012

电话：0431-81918209

Tel: 0431-81918209