人布魯氏桿菌（Brucella）ELISA 檢測試劑盒

【資料簡介】

本試劑盒只能用于科學研究，不得用于醫學診斷

人布魯氏桿菌（Brucella）ELISA 檢測試劑盒

使用說明書

檢測原理

試劑盒采用雙抗體夾心法酶聯免疫吸附試驗（ELISA）。往預先

包被人布魯氏桿菌（Brucella）捕獲抗體的包被微孔中，依次加入

標本、標準品、HRP標記的檢測抗體，經過溫育并*洗滌。用底物

TMB顯色，TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用下

轉化成zui終的黃色。顏色的深淺和樣品中的人布魯氏桿菌

（Brucella）呈正相關。用酶標儀在450nm 波長下測定吸光度（OD

值），判斷樣品是否含有人布魯氏桿菌（Brucella）。

樣品收集、處理及保存方法

1. 血清：使用不含熱原和內毒素的試管，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 轉離心30 分鐘取上清。

3. 細胞上清液：3000 轉離心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

濃縮洗滌液會有結晶，這屬于正常現象，水浴加熱使結晶*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. 預處理后的樣本無需稀釋，直接取10μL 加樣即可。

4. 嚴格按照說明書中標明的時間、加液量及順序進行溫育操作。

5. 所有液體組分使用前充分搖勻。

試劑盒組成

名稱96 孔配置48 孔配置備注

微孔酶標板96 孔48 孔無

陰性對照0.3mL 0.3mL 無

陽性對照0.3mL 0.3mL 無

*6mL 3mL 無

檢測抗體-HRP 10mL 5mL 無

20×洗滌緩沖液25mL 15mL 按說明書進行稀釋

底物A 6mL 3mL 無

底物B 6mL 3mL 無

終止液6mL 3mL 無

封板膜2 張2 張無

說明書1 份1 份無

自封袋1 個1 個無

試劑的準備

20×洗滌緩沖液的稀釋：蒸餾水按1：20 稀釋，即1 份的20×洗滌

緩沖液加19 份的蒸餾水。

洗板方法

1. 手工洗板：甩盡孔內液體，每孔加滿洗滌液，靜置1min 后甩盡

孔內液體，在吸水紙上拍干，如此洗板5 次。

2. 自動洗板機：每孔注入洗液350μL，浸泡1min，洗板5 次。

操作步驟

1. 從室溫平衡20min 后的鋁箔袋中取出所需板條，剩余板條用自封

袋密封放回4℃。

2. 設置陰、陽性對照孔和樣本孔，陰、陽性對照孔中加入陰性對照、

陽性對照各50μL；

3. 待測樣本孔先加待測樣本10μL，再加*40μL；

4. 隨后陰、陽性對照孔和樣本孔中每孔加入辣根過氧化物酶（HRP）

標記的檢測抗原100μL，用封板膜封住反應孔，37℃水浴鍋或

恒溫箱溫育60min。

5. 棄去液體，吸水紙上拍干，每孔加滿洗滌液，靜置1min，甩去

洗滌液，吸水紙上拍干，如此重復洗板5 次（也可用洗板機洗板）。

6. 每孔加入底物A、B 各50μL，37℃避光孵育15min。

7. 每孔加入終止液50μL，15min 內，在450nm 波長處測定各孔的

OD 值。

結果判斷

1. 試驗有效性：陽性對照孔OD 值平均值≥1.00；

陰性對照孔OD 值平均值≤0.15。

2. 臨界值（Cut off）計算：臨界值=陰性對照孔平均值+0.15

3. 陰性判斷：樣品OD 值＜臨界值（Cut off），樣品為陰性

4. 陽性判斷：樣品OD 值＞臨界值（Cut off），樣品為陽性

試劑盒性能

1. 準確性：陽性對照孔OD 值平均值≥1.00；陰性對照孔OD 值平均

值≤0.15，說明試驗結果有效。

2. 特異性：不與其它可溶性結構類似物交叉反應。

3. 重復性：板內、板間變異系數均小于15%。

4. 貯藏：2-8℃，避光防潮保存。

5. 有效期：6 個月

免責聲明

1. 試劑盒僅供研究使用，不得用于臨床實驗或人體實驗，否則所

產生的一切后果，由實驗者承擔，本公司概不負責。

2. 嚴格按照說明書操作，實驗者違反說明書操作，后果由實驗者

承擔。

FOR RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Human Brucella (Brucella) ELISA Kit instruction

Intended use

This Brucella ELISA kit is intended Laboratory for Research use only and is not for

use in diagnostic or therapeutic procedures. The Stop Solution changes the color

from blue to yellow and the intensity of the color is measured at 450 nm using a

spectrophotometer. In order to measure the concentration of Brucella in the sample,

this Brucella ELISA Kit includes a set of calibration standards. The calibration

standards are assayed at the same time as the samples and allow the operator to

produce a standard curve of Optical Density versus Brucella concentration. The

concentration of Brucella in the samples is then determined by comparing the O.D.

of the samples to the standard curve.

Sample collection and storages

Serum - Use a serum separator tube and allow samples to clot for 30 minutes

before centrifugation for 10 minutes at approximay 3000×g. Remove serum and

assay immediay or aliquot and store samples at -20℃ or -80℃.Avoid repeated

freeze-thaw cycles

Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge

samples for 30 minutes at 3000×g at 2-8℃ within 30 minutes of collection. Store

samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates and other biological fluids - Remove particulates

by centrifugation and assay immediay or aliquot and store samples at -20℃or

-80℃. Avoid repeated freeze-thaw cycles.

Note: The samples shoule be centrifugated dequay and no hemolysis or

granule was allowed.

Materials required but not supplied

1. Standard microplate reader(450nm)

2. Precision pipettes and Disposable pipette tips.

3. 37 ℃ incubator

Precautions

1. Do not substitute reagents from one kit to another. Standard, conjugate and

microplates are matched for optimal performance. Use only the reagents supplied by

manufacturer.

2. Do not remove microplate from the storage bag until needed. Unused strips

should be stored at 2-8°C in their pouch with the desiccant provided.

3. Mix all reagents before using.

Remove all kit reagents from refrigerator and allow them to reach room temperature

( 20-25°C)

Materials supplied

Name 96 determinations 48 determinations

Microelisa stripplate 96 strips 48 strips

Negative control 0.3ml 0.3ml

Positive control 0.3ml 0.3ml

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

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. Separay add Positive control and Negative control 50μl to the Positive and

Negative well, Add testing sample 10μl Then add sample diluent 40μl to testing

sample well; Blank well doesn’t add anyting.

3. Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip

and incubate for 60 minutes at 37°C.

4. 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.

5. 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.

6. 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.

7. Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader

within 15 minutes.

Determine the result

1. Test validity: the average of Positive control well≥1.00; the average of

Negative control well ≤0.15.

2. Calculate Critical (CUT OFF): Critical= the average of Negative control

well + 0.15.

Negative Result: sample OD< Calculate Critical (CUT OFF) is Negative.

Positive Result: sample OD≥ Calculate Critical (CUT OFF) is Positive.

Storage and validity

Storage： 2-8℃.

validity： six months.

FOR RESEARCH USE ONLY;

NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS!

PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE

BEGINNING!