Rat GOT ELISA Kit

For the quantitative in vitro determination of Rat Aspartate aminotransferase concentrations in

 serum - plasma - celiac fluid - tissue homogenate - body fluid

FOR LABORATORY RESEARCH USE ONLY.

NOT FOR USE IN DIAGNOSTIC PROCEDURES.

This package insert must be read in its entirety before using this product.

ELISA

ENZYME LINKED IMMUNOSORBENT ASSAY

INTENDED USE AND TEST PRINCIPLE

This GOT 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 GOT in the sample, this GOT 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 GOT concentration. The concentration of GOT 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 2 hours at room temperature or overnight at 4℃ before centrifugation for 20 minutes at approximay 2000×g. Remove serum and assay immediay or aliquot and store samples at -20℃. Avoid repeated freeze-thaw cycles

Plasma - Collect plasma using heparin as an anticoagulant. Centrifuge samples for 30 minutes at 2000×g at 2-8℃ within 30 minutes of collection. Store samples at -20℃. Avoid repeated freeze-thaw cycles.

Cell culture supernates, tissue homogenate and other biological fluids - Remove particulates by centrifugation and assay immediay or aliquot and store samples at -20℃. 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.  37 ℃ incubator

2.  Standard microplate reader capable of measuring absorbance at 450 nm

3.  Precision pipettes, disposable pipette tips and Absorbent paper

4.  Distilled or deionized water

REAGENTS PROVIDED

All reagents provided are stored at 2-8°C. Refer to the expiration date on the label.

Note:

1.  Standard concentration was followed by: 480、240、120、60、30、15 U/L

2.  If samples generate values higher than the highest standard, please dilute the samples with Sample Diluent and repeat the assay.

PRECAUTIONS

1. Do not substitute reagents from one kit lot to another. Standard, conjugate and microtiter plates are matched for optimal performance. Use only the reagents supplied by manufacturer.
2. Allow kit reagents and materials to reach room temperature (20-25°C) before use. Do not use water baths to thaw samples or reagents.
3. Do not use kit components beyond their expiration date.
4. Use only deionized or distilled water to dilute reagents.
5. Do not remove microtiter plate from the storage bag until needed. Unused strips should be stored at 2-8°C in their pouch with the desiccant provided.
6. Use fresh disposable pipette tips for each transfer to avoid contamination.
7. Do not mix acid and sodium hypochlorite solutions.
8. Serum and plasma should be handled as potentially hazardous and capable of transmitting disease. Disposable gloves must be worn during the assay procedure, since no known test method can offer complete assurance that products derived from Rat blood will not transmit infectious agents. Therefore, all blood derivatives should be considered potentially infectious and good laboratory practices should be followed.
9. All samples should be disposed of in a manner that will inactivate viruses.
10. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%. The waste should be allowed to stand for a minimum of 30 minutes to inactivate the viruses before disposal.
11. Substrate Solution is easily contaminated. If bluish prior to use, do not use.
12. Substrate B contain 20% acetone, keep this reagent away from sources of heat or flame.
13. Remove all kit reagents from refrigerator and allow them to reach room temperature ( 20-25°C).

REAGENT PREPARATION AND STORAGE

Wash Solution (1X) - Dilute 1 volume of Wash solution (20X) with 19 volumes of deionized or distilled water. Wash Solution is stable for 1 month at 2-8°C.

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 50μl of Standard or Sample to the appropriate wells. Blank well doesn’t add anyting.

3.  Add 100μl of HRP-conjugate reagent to standard wells and sample wells except the blank well, cover with an adhesive strip and incubate for 60 minutes at 37°C.

4.  Wash the Microtiter Plate 4 times.

Manual Washing - Remove incubation mixture by aspirating contents of the plate into a sink or proper waste container. Using a squirt bottle, fill each well compley with Wash Solution (1X), then aspirate contents of the plate into a sink or proper waste container. Repeat this procedure for a total of four times. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears. Note: Hold the sides of the plate frame firmly when washing the plate to assure that all strips remain securely in frame.

Automated Washing - Aspirate all wells, then wash plates four times using Wash Buffer (1X). Always adjust your washer to aspirate as much liquid as possible and set fill volume at 350μL/well/wash. After final wash, invert plate, and blot dry by hitting plate onto absorbent paper or paper towels until no moisture appears.

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.

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 (X) axis versus the corresponding concentration on the horizontal (Y) 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 balnk well 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. Intra-assay CV(%) and Inter-assay CV（％）are less than 15%.
6. Assay range: 15 U/L –480 U/L.

7.  Sensitivity: The minimum detectable dose of Rat GOT is typically less than 10 U/L

8.  Cross-reactivity: This assay recognizes recombinant and natural Rat GOT. No significant cross-reactivity or interference was observed.

9.  Storage: 2-8℃ (Use frequently); six months (-20℃)。

10.  Standard curve

本試劑盒只能用于科學(xué)研究，不得用于醫(yī)學(xué)診斷

人抑制素B（INH-B）ELISA檢測(cè)試劑盒

使用說(shuō)明書(shū)

檢測(cè)原理

試劑盒采用雙抗體一步夾心法酶聯(lián)免疫吸附試驗(yàn)（ELISA）。往預(yù)先包被人抑制素B（INH-B）捕獲抗體的包被微孔中，依次加入標(biāo)本、標(biāo)準(zhǔn)品、HRP標(biāo)記的檢測(cè)抗體，經(jīng)過(guò)溫育并*洗滌。用底物TMB顯色，TMB在過(guò)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zui終的黃色。顏色的深淺和樣品中的人抑制素B（INH-B）呈正相關(guān)。用酶標(biāo)儀在450nm 波長(zhǎng)下測(cè)定吸光度（OD 值），計(jì)算樣品濃度。

樣品收集、處理及保存方法

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

2.  血漿：EDTA、檸檬酸鹽或肝素抗凝。3000轉(zhuǎn)離心30分鐘取上清。

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。3000轉(zhuǎn)離心10分鐘取上清。

5.  保存：如果樣本收集后不及時(shí)檢測(cè)，請(qǐng)按一次用量分裝，凍存于-20℃，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。

自備物品

1. 酶標(biāo)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(xiàng)

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(huì)有結(jié)晶，這屬于正?，F(xiàn)象，水浴加熱使結(jié)晶*溶解后再使用。
2.   實(shí)驗(yàn)中不用的板條應(yīng)立即放回自封袋中，密封（低溫干燥）保存。
3.   預(yù)處理后的樣本無(wú)需稀釋，直接取50μL加樣即可。
4.   嚴(yán)格按照說(shuō)明書(shū)中標(biāo)明的時(shí)間、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。

試劑盒組成

注：

1、標(biāo)準(zhǔn)品濃度依次為：96、48、24、12、6、3 pg/mL.

2、在樣本值超過(guò)標(biāo)準(zhǔn)品zui高濃度的情況下，可用樣本稀釋液適當(dāng)稀釋樣本。

試劑的準(zhǔn)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(nèi)液體，每孔加滿洗滌液，靜置1min后甩盡孔內(nèi)液體，在吸水紙上拍干，如此洗板5次。
2.   自動(dòng)洗板機(jī)：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(shè)置標(biāo)準(zhǔn)品孔、樣本孔和空白孔，空白孔什么都不加；標(biāo)準(zhǔn)品孔各加不同濃度的標(biāo)準(zhǔn)品50μL；
3.   待測(cè)樣本孔各加待測(cè)樣本50μL；
4.   隨后標(biāo)準(zhǔn)品孔和樣本孔中（空白孔不加）加入辣根過(guò)氧化物酶（HRP）標(biāo)記的檢測(cè)抗體100μL，，用封板膜封住反應(yīng)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(fù)洗板5次（也可用洗板機(jī)洗板）。
6.   所有孔加入底物A、B各50μL，37℃避光孵育15min。
7.   所有孔加入終止液50μL，15min內(nèi)，在450nm波長(zhǎng)處測(cè)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，以標(biāo)準(zhǔn)品濃度作橫坐標(biāo)，對(duì)應(yīng)OD值作縱坐標(biāo)，繪制出標(biāo)準(zhǔn)品線性回歸曲線，按曲線方程計(jì)算各樣本濃度值。

試劑盒性能

1.  準(zhǔn)確性：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：zui低檢測(cè)濃度小于1.0 pg/mL。

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

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

5.  貯藏：2-8℃，避光防潮保存。

6.  有效期：6個(gè)月

免責(zé)聲明

1.   試劑盒僅供研究使用，不得用于臨床實(shí)驗(yàn)或人體實(shí)驗(yàn)，否則所產(chǎn)生的一切后果，由實(shí)驗(yàn)者承擔(dān)，本公司概不負(fù)責(zé)。

2.   嚴(yán)格按照說(shuō)明書(shū)操作，實(shí)驗(yàn)者違反說(shuō)明書(shū)操作，后果由實(shí)驗(yàn)者承擔(dān)。