本試劑盒只能用于科學(xué)研究又進了一步，不得用于醫(yī)學(xué)診斷

大鼠（Rat）γ氨基丁酸（GABA）ELISA檢測試劑盒

使用說明書

檢測原理

試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗（ELISA）。往預(yù)先包被γ氨基丁酸（GABA）抗體的包被微孔中要落實好，依次加入標(biāo)本機遇與挑戰、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測抗體助力各業，經(jīng)過溫育并洗滌發展基礎。用底物TMB顯色兩個角度入手，TMB在過氧化物酶的催化下轉(zhuǎn)化成藍(lán)色，并在酸的作用下轉(zhuǎn)化成最終的黃色同期。顏色的深淺和樣品中的γ氨基丁酸（GABA）呈正相關(guān)生產效率。用酶標(biāo)儀在450nm 波長下測定吸光度（OD 值），計算樣品濃度效果。

樣品收集使用、處理及保存方法

1.  血清：使用不含熱原和內(nèi)毒素的試管合規意識，操作過程中避免任何細(xì)胞刺激，收集血液后有效性，3000轉(zhuǎn)離心10分鐘將血清和紅細(xì)胞迅速小心地分離創新內容。

2.  血漿：EDTA、檸檬酸鹽或肝素抗凝廣泛關註。3000轉(zhuǎn)離心30分鐘取上清善於監督。

3.  細(xì)胞上清液：3000轉(zhuǎn)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就能壓製。3000轉(zhuǎn)離心10分鐘取上清更合理。

5.  保存：如果樣本收集后不及時檢測，請按一次用量分裝更優美，凍存于-20℃各方面，避免反復(fù)凍融，在室溫下解凍并確保樣品均勻地充分解凍成效與經驗。

自備物品

1. 酶標(biāo)儀（450nm）

2. 高精度加樣器及槍頭：0.5-10uL適應性、2-20uL、20-200uL稍有不慎、200-1000uL

3. 37℃恒溫箱

操作注意事項

1.  試劑盒保存在2-8℃方法，使用前室溫平衡20分鐘。從冰箱取出的濃縮洗滌液會有結(jié)晶規模最大，這屬于正撤€中求進，F(xiàn)象統籌，水浴加熱使結(jié)晶溶解后再使用最深厚的底氣。

2.  實驗中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保存振奮起來。

3.  濃度為0的S0號標(biāo)準(zhǔn)品即可視為陰性對照或者空白品質；按照說明書操作時樣本已經(jīng)稀釋5倍，最終結(jié)果乘以5才是樣本實際濃度深入各系統。

4.  嚴(yán)格按照說明書中標(biāo)明的時間解決問題、加液量及順序進(jìn)行溫育操作。

5.  所有液體組分使用前充分搖勻作用。

試劑盒組成

注：標(biāo)準(zhǔn)品（S0-S5）濃度依次為：0相互配合、0.5、1著力增加、2智能化、4、8μmol/L

試劑的準(zhǔn)備

 20×洗滌緩沖液的稀釋：蒸餾水按1：20稀釋處理，即1份的20×洗滌緩沖液加19份的蒸餾水建設。

洗板方法

1.  手工洗板：甩盡孔內(nèi)液體在此基礎上，每孔加滿洗滌液，靜置1min后甩盡孔內(nèi)液體前來體驗，在吸水紙上拍干自主研發，如此洗板5次。

2.  自動洗板機：每孔注入洗液350μL更加廣闊，浸泡1min損耗，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不斷發展，剩余板條用自封袋密封放回4℃積極影響。

2.  設(shè)置標(biāo)準(zhǔn)品孔和樣本孔，標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μL緊密協作；

3.  樣本孔先加待測樣本10μL越來越重要，再加樣本稀釋液40μL；空白孔不加發揮重要作用。

4.  除空白孔外近年來，標(biāo)準(zhǔn)品孔和樣本孔中每孔加入辣根過氧化物酶（HRP）標(biāo)記的檢測抗體100μL，用封板膜封住反應(yīng)孔發展目標奮鬥，37℃水浴鍋或恒溫箱溫育60min技術先進。

5.  棄去液體，吸水紙上拍干延伸，每孔加滿洗滌液認為，靜置1min，甩去洗滌液新趨勢，吸水紙上拍干反應能力，如此重復(fù)洗板5次（也可用洗板機洗板）。

6.  每孔加入底物A學習、B各50μL結構重塑，37℃避光孵育15min。

7.  每孔加入終止液50μL應用優勢，15min內(nèi)高質量發展，在450nm波長處測定各孔的OD值。

結(jié)果判斷

 繪制標(biāo)準(zhǔn)曲線：在Excel工作表中高效節能，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)影響力範圍，對應(yīng)OD值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線新創新即將到來，按曲線方程計算各樣本濃度值邁出了重要的一步。

試劑盒性能

1.  性：標(biāo)準(zhǔn)品線性回歸與預(yù)期濃度相關(guān)系數(shù)R值，大于等于0.9900。

2.  靈敏度：檢測濃度小于0.1μmol/L發展的關鍵。

3.  特異性：不與其它可溶性結(jié)構(gòu)類似物交叉反應(yīng)道路。

4.  重復(fù)性：板內(nèi)、板間變異系數(shù)均小于15%真諦所在。

5.  貯藏：2-8℃指導，避光防潮保存。

6.  有效期：6個月

7.上海軒澤康生物有限公司提供

免責(zé)聲明

1.   試劑盒僅供研究使用充分，不得用于臨床實驗或人體實驗進一步完善，否則所產(chǎn)生的一切后果，由實驗者承擔(dān)競爭力，本公司概不負(fù)責(zé)調整推進。

2.   嚴(yán)格按照說明書操作，實驗者違反說明書操作敢於挑戰，后果由實驗者承擔(dān)不斷創新。

FOR RESEARCH USE ONLY. 

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

Rat Gamma-aminobutyric acid (GABA) ELISA Kit instruction

Intended use

This GABA 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 GABA in the sample, this GABA 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 GABA concentration. The concentration of GABA 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 approximately 3000×g. Remove serum and assay immediately 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 immediately or aliquot and store samples at -20℃or -80℃. Avoid repeated freeze-thaw cycles.

Note:  The samples shoule be centrifugated dequately 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

Note: Standard (S0 → S5) concentration was followed by:0,0.5,1,2,4,8 μmol/L

Reagent preparation

20×wash solution:Dilute with Distilled or deionized water 1:20.

Assay procedure

1.  Prepare all reagents before starting assay procedure. It is recommended that all Standards and Samples be added in duplicate to the Microelisa Stripplate.

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

3.  Add Sample: Add testing sample 10μl then add Sample Diluent 40μl to testing sample well; Blank well doesn’t add anyting.

4.  Add 100μl of HRP-conjugate reagent to each well, cover with an adhesive strip and incubate for 60 minutes at 37°C. 

5.  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.

6.  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.

7.  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.

8.  Read the Optical Density (O.D.) at 450 nm using a microtiter plate reader within 15 minutes.

Calculation of results

1. This standard curve is used to determine the amount in an unknown sample. The standard curve is generated by plotting the average O.D. (450 nm) obtained for each of the six standard concentrations on the vertical (Y) axis versus the corresponding concentration on the horizontal (X) axis.

2. First, calculate the mean O.D. value for each standard and sample. All O.D. values, are subtracted by the mean value of the zero standard before result interpretation. Construct the standard curve using graph paper or statistical software.

3. To determine the amount in each sample, first locate the O.D. value on the Y-axis and extend a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the X-axis and read the corresponding concentration.

4. Any variation in operator, pipetting and washing technique, incubation time or temperature, and kit age can cause variation in result. Each user should obtain their own standard curve.

5. The sensitivity by this assay is 0.1 μmol/L

6. Standard curve

Storage：  2-8℃.

validity： six months.

FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH ENTIRE PROCEDURE BEFORE BEGINNING!