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· 資料簡介： 人腎損傷因子 1（Kim-1）酶聯(lián)免疫分析(ELISA)

試劑盒使用說明書

本試劑僅供研究使用 目的：本試劑盒用于測定人血清、血漿、組織

勻漿及相關(guān)液體樣本中腎損傷因子 1（Kim-1）的含量。

實驗原理：

本試劑盒應(yīng)用雙抗體夾心法測定標本中人腎損傷因子 1（Kim-1）水平。用純化的人腎

損傷因子 1（Kim-1）捕獲抗體包被微孔板，制成固相抗體，往包被的微孔中依次加入人腎

損傷因子 1（Kim-1），再與 HRP 標記的檢測抗體結(jié)合，形成抗體-抗原-酶標抗體復合物，經(jīng)

過徹底洗滌后加底物 TMB 顯色。TMB 在 HRP 酶的催化下轉(zhuǎn)化成藍色，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腎損傷因子 1（Kim-1）呈正相關(guān)。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腎損傷因子 1（Kim-1）含量。

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 2 片

密封袋 1 個 1 個

酶標包被板 1×48 1×96 2-8℃保存

標準品 0.3ml×6 管 0.3ml×6 管 2-8℃保存

酶標試劑 5 ml×1 瓶 10 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 3 ml×1 瓶 6 ml×1 瓶 2-8℃保存

20×濃縮洗滌液 15ml×1 瓶 25ml×1 瓶 2-8℃保存

注：標準品濃度依次為：8、4、2、1、0.5、0 ng/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 轉(zhuǎn)/分）。仔細收集

上清，保存過程中如出現(xiàn)沉淀，應(yīng)再次離心。

2. 血漿：應(yīng)根據(jù)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(zhuǎn)/分）。仔細收集上清，保存過程中如有沉淀形成，應(yīng)該再次

離心。

3. 尿液：用無菌管收集，離心 20 分鐘左右（2000-3000 轉(zhuǎn)/分）。仔細收集上清，保存過程

中如有沉淀形成，應(yīng)再次離心。胸腹水、腦脊液參照實行。

4. 細胞培養(yǎng)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(zhuǎn)

/分）。仔細收集上清。檢測細胞內(nèi)的成份時，用 PBS（PH7.2-7.4）稀釋細胞懸液，細胞2

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(nèi)成份。離心 20 分

鐘左右（2000-3000 轉(zhuǎn)/分）。仔細收集上清。保存過程中如有沉淀形成，應(yīng)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?zhèn)?

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(zhuǎn)/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(guān)文獻進行，提取后應(yīng)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(yīng)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。

操作步驟

1. 標準品的加樣：設(shè)置標準品孔和樣本孔，標準品孔各加不同濃度的標準品 50μL；。

2. 加樣：分別設(shè)空白孔（空白對照孔不加樣品及酶標試劑，其余各步操作相同）、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，然后再加待測樣品 10μl

（樣品zui終稀釋度為 5 倍）。加樣將樣品加于酶標板孔底部，盡量不觸及孔壁，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：用封板膜封板后置 37℃溫育 60 分鐘。

5. 配液：將 20 倍濃縮洗滌液用蒸餾水 20 倍稀釋后備用。

6. 洗滌：小心揭掉封板膜，棄去液體，甩干，每孔加滿洗滌液，靜置 30 秒后棄去，如此

重復 5 次，拍干。

7. 顯色：每孔先加入顯色劑 A50μl，再加入顯色劑 B50μl，輕輕震蕩混勻，37℃避光顯

色 15 分鐘.

8. 終止：每孔加終止液 50μl，終止反應(yīng)（此時藍色立轉(zhuǎn)黃色）。

9. 測定：以空白孔調(diào)零，450nm 波長依序測量各孔的吸光度（OD 值）。 測定應(yīng)在加終止液

后 15 分鐘以內(nèi)進行。

注意事項：

1． 試劑盒從冷藏環(huán)境中取出應(yīng)在室溫平衡 15-30 分鐘后方可使用，酶標包被板開封后如未

用完，板條應(yīng)裝入密封袋中保存。

2． 濃洗滌液可能會有結(jié)晶析出，稀釋時可在水浴中加溫助溶，洗滌時不影響結(jié)果。

3． 各步加樣均應(yīng)使用加樣器，并經(jīng)常校對其準確性，以避免試驗誤差。一次加樣時間zui好

控制在 5 分鐘內(nèi)，如標本數(shù)量多，推薦使用排槍加樣。

4． 請每次測定的同時做標準曲線，zui好做復孔。如標本中待測物質(zhì)含量過高（樣本 OD 值

大于標準品孔第yi孔的 OD 值），請先用樣品稀釋液稀釋一定倍數(shù)（n 倍）后再測定，計

算時請zui后乘以總稀釋倍數(shù)（×n×5）。

5． 封板膜只限一次性使用，以避免交叉污染。

6． 底物請避光保存。

7． 嚴格按照說明書的操作進行，試驗結(jié)果判定必須以酶標儀讀數(shù)為準.

8． 所有樣品，洗滌液和各種廢棄物都應(yīng)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坐標，OD 值為縱坐標，

在坐標紙上繪出標準曲線，根據(jù)樣品的 OD

值由標準曲線查出相應(yīng)的濃度；再乘以稀釋

倍數(shù)；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(shù)，即為樣品的實際濃度。

（此圖僅供參考）

試劑盒性能：

1.樣品線性回歸與預(yù)期濃度相關(guān)系數(shù) R 值為 0.95 以上。

2.批內(nèi)變異系數(shù)與批間變異系數(shù)應(yīng)分別小于 10%和 15% 。

檢測范圍：

0.25 ng/mL - 8 ng/mL

靈敏度：

zui低檢測濃度小于 0.1 ng/mL

保存條件及有效期：

1.試劑盒保存： 2-8℃。

2．有效期： 6 個月

3Human Kidney injury molecule 1

FOR RESEARCH USE ONLY

Drug Names

Generic Name：Human Kidney injury molecule 1 (Kim-1) ELISA Kit.

Purpose

This kit allows for the determination of Kim-1 concentrations in Human

serum, plasma, tissue homogenates ａnd other biological fluids.

Principle of the assay

The kit assay Human Kim-1 level in the sample, use Purified Human Kim-1

antibody to coat microtiter plate wells, make solid-phase antibody, then add

Kim-1 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 Kim-1 in the samples is then

determined by comparing the O.D. of the samples to the standard curve.

45

Materials provided with the kit

Materials provided

with the kit

48determination

s 96 determinations Stora

ge

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:

8、4、2、1、0.5、0 ng/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 suspensionwith 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.

5. 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.

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 can’t, 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.

66.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.

7Important 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.

8Calculate

Take the standard density as the horizontal,

the OD value for the vertical ,draw the standard

curve on graph paper, Find out the corresponding

density according to the sample OD value by the

Sample curve, multiplied by the dilution multiple,

or calculate the straight line regression equation

of the standard curve with the standard density

and the OD value ,with the sample OD value in

the equation, calculate the sample density,

This chartis for reference only

multiplied by the dilution factor, the result is the

Assay range

0.25 ng/mL - 8 ng/mL

Sensitivity

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

Storage ａnd validity

1．Storage： 2-8℃.

2．validity： six months.

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