人胰高血糖素樣肽1(GLP-1)酶聯免疫分析（ELISA）試劑盒使用說明書
本試劑僅供研究使用
實驗目的：本試劑盒用于測定人血清，血漿及相關液體樣本中胰高血糖素樣肽1(GLP-1)的含量。
實驗原理：
應用雙抗體夾心法測定標本中人胰高血糖素樣肽1(GLP-1)水平。用純化的人胰高血糖素樣肽1(GLP-1)抗體包被微孔板，制成固相抗體，往包被單抗的微孔中依次加入胰高血糖素樣肽1(GLP-1)，再與HRP標記的胰高血糖素樣肽1(GLP-1)抗體結合，形成抗體-抗原-酶標抗體復合物，經過*洗滌后加底物TMB顯色。TMB在HRP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胰高血糖素樣肽1(GLP-1)呈正相關。用酶標儀在450nm波長下測定吸光度（OD值），通過標準曲線計算樣品中人胰高血糖素樣肽1(GLP-1)濃度。
試劑盒組成：
試劑盒組成 | 48孔配置 | 96孔配置 | 保存 |
說明書 | 1份 | 1份 | |
封板膜 | 2片（48） | 2片（96） | |
密封袋 | 1個 | 1個 | |
酶標包被板 | 1×48 | 1×96 | 2-8℃保存 |
標準品：144pg/ml | 0.5ml×1瓶 | 0.5ml×1瓶 | 2-8℃保存 |
標準品稀釋液 | 1.5ml×1瓶 | 1.5ml×1瓶 | 2-8℃保存 |
酶標試劑 | 3 ml×1瓶 | 6 ml×1瓶 | 2-8℃保存 |
樣品稀釋液 | 3 ml×1瓶 | 6 ml×1瓶 | 2-8℃保存 |
顯色劑A液 | 3 ml×1瓶 | 6 ml×1瓶 | 2-8℃保存 |
顯色劑B液 | 3 ml×1瓶 | 6 ml×1瓶 | 2-8℃保存 |
終止液 | 3ml×1瓶 | 6ml×1瓶 | 2-8℃保存 |
濃縮洗滌液 | （20ml×20倍）×1瓶 | （20ml×30倍）×1瓶 | 2-8℃保存 |
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轉/分）。仔細收集上清，保存過程中如出現沉淀，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分鐘后，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，應該再次離心。
3. 尿液：用無菌管收集，離心20分鐘左右（2000-3000轉/分）。仔細收集上清，保存過程中如有沉淀形成，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轉/分）。仔細收集上清。檢測細胞內的成份時，用PBS（PH7.2-7.4）稀釋細胞懸液，細胞濃度達到100萬/ml左右。通過反復凍融，以使細胞破壞并放出細胞內成份。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。保存過程中如有沉淀形成，應再次離心。
5. 組織標本：切割標本后，稱取重量。加入一定量的PBS，PH7.4。用液氮迅速冷凍保存備用。標本融化后仍然保持2-8℃的溫度。加入一定量的PBS（PH7.4），用手工或勻漿器將標本勻漿充分。離心20分鐘左右（2000-3000轉/分）。仔細收集上清。分裝后一份待檢測，其余冷凍備用。
6. 標本采集后盡早進行提取，提取按相關文獻進行，提取后應盡快進行實驗。若不能馬上進行試驗，可將標本放于-20℃保存，但應避免反復凍融.
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。
實驗結果計算：
以標準物的濃度為橫坐標，OD值為縱坐標，
在坐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
準曲線的直線回歸方程式，將樣品的OD值
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。
試劑盒性能：
1.樣品線性回歸與預期濃度相關系數R值為0.92以上。
2.批內與批間應分別小于9%和13%
檢測范圍：
2pg/ml -100pg/ml
試劑盒保存：2-8℃
有效期：6個月
FOR RESEARCH USE ONLY
Human ghcagons-like pepfide 1 |
Drug Names
Generic Name：Human ghcagons-like pepfide 1 (GLP-1)ELISA Kit
Purpose
This kit allows for the determination of GLP-1 concentrations in Human serum, blood plasma, and other biological fluids.
Principle of the assay
The kit assay Human GLP-1 level in the sample，use Purified Human GLP-1antibody to coat microtiter plate wells, make solid-phase antibody, then add GLP-1 to wells, Combined GLP-1 which With HRP labeled , become antibody - antigen - enzyme-antibody complex, after washing Compley, 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 GLP-1 in the
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：144pg/ml | 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
- 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.
- 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.
- 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.
- 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 PBS（PH7.2-7.4）, 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.
- Tissue samples- After cutting samples, check the weight,add PBS（PH7.2-7.4）, Rapidly frozen with liquid nitrogen, maintain samples at 2-8℃ after melting,add PBS（PH7.4）, Homogenized by hand or Grinders, centrifugation 20-min at the speed of 2000-3000 r.p.m. remove supernatant.
- 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.
- Can’t detect the sample which contain NaN3, because NaN3 inhibits HRP active.
Important notes
- 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.
- washing buffer will Crystallization separation, it can be heated the water helps dissolve when dilute . Washing does not affect the result.
- 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 .
- 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）.
- Closure plate membrane only limits the disposable use, to avoid cross-contamination.
- The substrate evade the light preservation.
- Please according to use instruction strictly, The test result determination must take the microtiter plate reader as a standard.
- All samples, washing buffer and each kind of reject should according to infective material process.
- Do not mix reagents with those from other lots.
2pg/ml -100pg/mlAssay range
Storage and validity
1．Storage： 2-8℃.
2．validity： six months.
