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人促腎上腺皮質激素(ACTH)酶聯免疫分析(ELISA)

釋出時間₪✘: 2022-08-17  點選次數₪✘: 174次

人促腎上腺皮質激素ACTH酶聯免疫分析(ELISA)

試劑盒使用說明書

本試劑僅供研究使用       目的₪✘:本試劑盒用於測定人血清▩╃、血漿▩╃、組織勻漿及相關液體樣本中促腎上腺皮質激素(ACTH)的含量▩••◕▩。

 

實驗原理₪✘:

本試劑盒應用雙抗體夾心法測定標本人促腎上腺皮質激素(ACTH水平▩••◕▩。用純化的人促腎上腺皮質激素(ACTH)捕獲抗體包被微孔板↟↟,製成固相體↟↟,往包被的微孔中依次加入人促腎上腺皮質激素(ACTH↟↟,再與HRP標記的檢測抗體結合↟↟,形成抗體-抗原-酶標抗體複合物↟↟,經過徹底洗滌後底物TMB顯色▩••◕▩。TMBHRP酶的催化下轉化成藍色↟↟,並在酸的作用下轉化成最終的黃色▩••◕▩。顏色的深淺和樣品中的人促腎上腺皮質激素(ACTH呈正相關▩••◕▩。用酶標儀在450nm波長下測定吸光度(OD值)↟↟,透過標準曲線計算樣品人促腎上腺皮質激素(ACTH含量▩••◕▩。

 

試劑盒組成₪✘:

試劑盒組成

48孔配置

96孔配置

儲存

說明書

1

1


封板膜

2

2


密封袋

1

1


酶標包被板

1×48

1×96

2-8℃儲存

標準品

0.3ml×6

0.3ml×6

2-8℃儲存

酶標試劑

5 ml×1

10 ml×1

2-8℃儲存

樣品稀釋液

3 ml×1

6 ml×1

2-8℃儲存

顯色劑A

3 ml×1

6 ml×1

2-8℃儲存

顯色劑B

3 ml×1

6 ml×1

2-8℃儲存

終止液

3 ml×1

6 ml×1

2-8℃儲存

20×濃縮洗滌液

15ml×1

25ml×1

2-8℃儲存

注₪✘:標準品濃度依次為₪✘:80▩╃、40▩╃、20▩╃、10▩╃、5▩╃、0 pg/mL.

 

樣本處理及要求₪✘:

1. 血清₪✘:室溫血液自然凝固10-20分鐘↟↟,離心20分鐘左右(2000-3000/分)▩••◕▩。仔細收集上清↟↟,儲存過程中如出現沉澱↟↟,應再次離心▩••◕▩。

2. 血漿₪✘:應根據標本的要求選擇EDTA或檸檬酸鈉作為抗凝劑↟↟,混合10-20分鐘後↟↟,離心20分鐘左右(2000-3000/分)▩••◕▩。仔細收集上清↟↟,儲存過程中如有沉澱形成↟↟,應該再次離心▩••◕▩。

3. 尿液₪✘:用無菌管收集↟↟,離心20分鐘左右(2000-3000/分)▩••◕▩。仔細收集上清↟↟,儲存過程中如有沉澱形成↟↟,應再次離心▩••◕▩。胸腹水▩╃、腦脊液參照實行▩••◕▩。

4. 細胞培養上清₪✘:檢測分泌性的成份時↟↟,用無菌管收集▩••◕▩。離心20分鐘左右(2000-3000/分)▩••◕▩。仔細收集上清▩••◕▩。檢測細胞內的成份時↟↟,用PBSPH7.2-7.4)稀釋細胞懸液↟↟,細胞濃度達到100/ml左右▩••◕▩。透過反覆凍融↟↟,以使細胞破壞並放出細胞內成份▩••◕▩。離心20分鐘左右(2000-3000/分)▩••◕▩。仔細收集上清▩••◕▩。儲存過程中如有沉澱形成↟↟,應再次離心▩••◕▩。

5. 組織標本₪✘:切割標本後↟↟,稱取重量▩••◕▩。加入一定量的PBS↟↟,PH7.4▩••◕▩。用液氮迅速冷凍儲存備用▩••◕▩。標本融化後仍然保持2-8℃的溫度▩••◕▩。加入一定量的PBSPH7.4)↟↟,用手工或勻漿器將標本勻漿充分▩••◕▩。離心20分鐘左右(2000-3000/分)▩••◕▩。仔細收集上清▩••◕▩。分裝後一份待檢測↟↟,其餘冷凍備用▩••◕▩。

6. 標本採集後儘早進行提取↟↟,提取按相關文獻進行↟↟,提取後應儘快進行實驗▩••◕▩。若不能馬上進行試驗↟↟,可將標本放於-20℃儲存↟↟,但避免反覆凍融.

7. 不能檢測含NaN3的樣品↟↟,因NaN3抑制辣根過氧化物酶的(HRP)活性▩••◕▩。



 

操作步驟

1. 標準品的加樣₪✘:設定標準品孔和樣本孔↟↟,標準品孔各加不同濃度的標準品50μL▩••◕▩。

2. 加樣₪✘:分別設空白孔(空白對照孔不加樣品及酶標試劑↟↟,其餘各步操作相同)▩╃、待測樣品孔▩••◕▩。在酶標包被板上待測樣品孔中先加樣品稀釋液40μl↟↟,然後再加待測樣品10μl(樣品最終稀釋度為5倍)▩••◕▩。加樣將樣品加於酶標板孔底部↟↟,儘量不觸及孔壁↟↟,輕輕晃動混勻▩••◕▩。

3. 加酶₪✘:每孔加入酶標試劑100μl↟↟,空白孔除外▩••◕▩。

4. 溫育₪✘:用封板膜封板後置37℃溫育60分鐘▩••◕▩。

5. 配液₪✘:將20倍濃縮洗滌液用蒸餾水20倍稀釋後備用▩••◕▩。

6. 洗滌₪✘:小心揭掉封板膜↟↟,棄去液體↟↟,甩幹↟↟,每孔加滿洗滌液↟↟,靜置30秒後棄去↟↟,如此重複5次↟↟,拍幹▩••◕▩。

7. 顯色₪✘:每孔先加入顯色劑A50μl↟↟,再加入顯色劑B50μl↟↟,輕輕震盪混勻↟↟,37℃避光顯色15分鐘. 

8. 終止₪✘:每孔加終止50μl↟↟,終止反應(此時藍色立轉黃色)▩••◕▩。

9. 測定₪✘:以空白孔調零↟↟,450nm波長依序測量各孔的吸光度(OD值)▩••◕▩。 測定應在加終止液後15分鐘以內進行▩••◕▩。

 

注意事項₪✘:

1. 試劑盒從冷藏環境中取出應在室溫平衡15-30分鐘後方可使用↟↟,酶標包被板開封后如未用完↟↟,板條應裝入密封袋中儲存▩••◕▩。樣本在使用前也要在室溫平衡60分鐘▩••◕▩。

2. 濃洗滌液可能會有結晶析出↟↟,稀釋時可在水浴中加溫助溶↟↟,洗滌時不影響結果▩••◕▩。

3. 各步加樣均應使用加樣器↟↟,並經常校對其準確性↟↟,以避免試驗誤差▩••◕▩。一次加樣時間最好控制在5分鐘內↟↟,如標本數量多↟↟,推薦使用排槍加樣▩••◕▩。

4. 請每次測定的同時做標準曲線↟↟,最好做復孔▩••◕▩。如標本中待測物質含量過高(樣本OD值大於標準品孔第一孔的OD值)↟↟,請先用樣品稀釋液稀釋一定倍數(n倍)後再測定↟↟,計算時請最後乘以總稀釋倍數(×n×5)▩••◕▩。

5. 封板膜只限一次性使用↟↟,以避免交叉汙染▩••◕▩。

6. 底物請避光儲存▩••◕▩。

7. 嚴格按照說明書的操作進行↟↟,試驗結果判定必須以酶標儀讀數為準.

8. 所有樣品↟↟,洗滌液和各種廢棄物都應按傳染物處理▩••◕▩。

9. 本試劑不同批號組分不得混用▩••◕▩。

10. 如與英文說明書有異↟↟,以英文說明書為準▩••◕▩。

 

 

 

計算₪✘:

以標準物的濃度為橫座標↟↟,OD值為縱座標↟↟,    

在座標紙上繪出標準曲線↟↟,根據樣品的OD      

值由標準曲線查出相應的濃度;再乘以稀釋      

倍數;或用標準物的濃度與OD值計算出標      

準曲線的直線迴歸方程式↟↟,將樣品的OD      

代入方程式↟↟,計算出樣品濃度↟↟,再乘以稀釋      

倍數↟↟,即為樣品的實際濃度▩••◕▩。                  

 

                                              

 

(此圖僅供參考)

 

 

 

試劑盒效能₪✘:

1.樣品線性迴歸與預期濃度相關係數R值為0.95以上▩••◕▩。

2.批內變異係數與批間變異係數應分別小於10%15% ▩••◕▩。

 

檢測範圍₪✘:                                              

2.5 pg/mL - 80 pg/mL

 

靈敏度₪✘:                                              

檢測濃度小於0.1 pg/mL

 

儲存條件及有效期₪✘:

1.試劑盒儲存₪✘: 2-8▩••◕▩。

2.有效期₪✘: 6個月


Human adrencocorticotropic hormone

FOR RESEARCH USE ONLY

 

Drug Names

Generic Name₪✘:Human adrencocorticotropic hormone (ACTH) ELISA Kit.

Purpose

This kit allows for the determination of ACTH concentrations in Human serum, plasma, tissue homogenates and other biological fluids.

Principle of the assay

The kit assay Human ACTH level in the sample, use Purified Human ACTH antibody to coat microtiter plate wells, make solid-phase antibody, then add ACTH to the wells, Combined 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 ACTH in the samples is then determined by comparing the O.D. of the samples to the standard curve.


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

0.3ml×6 bottle

0.3ml×6 bottle

2-8℃

HRP-Conjugate reagent

5ml×1 bottle

10ml×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℃

20×Wash solution

15ml×1 bottle

25ml×1 bottle

2-8℃

Note: Standard concentration was followed by:

80▩╃、40▩╃、20▩╃、10▩╃、5▩╃、0 pg/mL.

Specimen requirements

1. serum- coagulation at room temperature 10-20 mins↟↟,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.

2. plasma-use suited EDTA or citrate plasma as an anticoagulant,mix 10-20 mins ,centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.

3. Urine-collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again. The Operation of Hydrothorax and cerebrospinal fluid Reference to it.

4. cell culture supernatant-detect secretory components, collect sue a sterile container, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant,detect the composition of cells, Dilut cell suspension with PBSPH7.2-7.4, Cell concentration reached 1 million / ml, repeated freeze-thaw cycles, damage cells and release of intracellular components, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant, If precipitation appeared, Centrifugal again.

5. Tissue samples- After cutting samples, check the weight,add PBSPH7.2-7.4, Rapidly frozen with liquid nitrogen, maintain samples at 2-8 after melting,add PBSPH7.4, Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.

6. 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 cant, specimen can be kept in -20  to preserve, Avoid repeated freeze-thaw cycles.

7. Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.

Assay procedure

1. Add standard: Set Standard wells, testing sample wells. Add standard 50μl to standard well.

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.add enzyme₪✘:Add HRP-Conjugate reagent 100μl to each well, except  blank well.

4.Incubate: After closing plate with Closure plate membrane ,incubate for 60 min at 37.

5.Configurate liquid: 20-fold wash solution diluted 20-fold with distilled water and reserve.

6.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.

7.color₪✘:Add Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37

8.Stop the reaction₪✘:Add Stop Solution 50μl to each well, Stop the reaction(the blue color change to yellow color).

9.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

 

 

 

 

 

 

 

 

 

 

Assay range

2.5 pg/mL - 80 pg/mL

 

Sensitivity

The minimum detectable dose is typically less than 0.1 pg/mL

 

Storage and validity

1Storage₪✘:  2-8.

2validity₪✘: six months.


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