欧美国产日韩综合成人,日韩码一码二码三码区别,97精品国产综合久久久免费,亚洲精品国产第一区三区

網(wǎng)站首頁(yè)產(chǎn)品展示酶聯(lián)免疫ELISA試劑盒人的ELISA > 96T/48T人干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)ELISA
人干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)ELISA

人干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)ELISA

產(chǎn)品型號(hào): 96T/48T

所屬分類:人的ELISA

產(chǎn)品時(shí)間:2024-08-15

簡(jiǎn)要描述:人干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)ELISA價(jià)格公道、*,售后服務(wù)完整,并提供免費(fèi)代檢測(cè)服務(wù)!本試劑盒用于測(cè)定人血清,血漿及相關(guān)液體樣本中干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)的含量。

詳細(xì)說(shuō)明:

干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)ELISA試劑盒

本試劑僅供研究使用       目的:本試劑盒用于測(cè)定人血清,血漿及相關(guān)液體樣本中干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)的含量。

實(shí)驗(yàn)原理:

本試劑盒應(yīng)用雙抗體夾心法測(cè)定標(biāo)本中人干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)水平。用純化的人干擾素誘導(dǎo)T細(xì)胞趨化因子ITAC/CXCL11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干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11),再與HRP標(biāo)記的干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)抗體結(jié)合,形成抗體-抗原-酶標(biāo)抗體復(fù)合物,經(jīng)過(guò)*洗滌后加底物TMB顯色。TMBHRP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干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)呈正相關(guān)。用酶標(biāo)儀在450nm波長(zhǎng)下測(cè)定吸光度(OD值),通過(guò)標(biāo)準(zhǔn)曲線計(jì)算樣品中人干擾素誘導(dǎo)T細(xì)胞趨化因子(ITAC/CXCL11)濃度。

 

試劑盒組成

試劑盒組成

48孔配置

96孔配置

保存

說(shuō)明書(shū)

1

1

 

封板膜

2片(48

2片(96

 

密封袋

1個(gè)

1個(gè)

 

酶標(biāo)包被板

1×48

1×96

2-8保存

標(biāo)準(zhǔn)品:4500pg/ml

0.5ml×1

0.5ml×1

2-8保存

標(biāo)準(zhǔn)品稀釋液

1.5ml×1

1.5ml×1

2-8保存

酶標(biāo)試劑

3 ml×1

6 ml×1

2-8保存

樣品稀釋液

3 ml×1

6 ml×1

2-8保存

顯色劑A

3 ml×1

6 ml×1

2-8保存

顯色劑B

3 ml×1

6 ml×1

2-8保存

終止液

3ml×1

6ml×1

2-8保存

濃縮洗滌液

20ml×20倍)×1

20ml×30倍)×1

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(zhuǎn)/分)。仔細(xì)收集上清,保存過(guò)程中如出現(xiàn)沉淀,應(yīng)再次離心。

2. 血漿:應(yīng)根據(jù)標(biāo)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(xì)收集上清,保存過(guò)程中如有沉淀形成,應(yīng)該再次離心。

3. 尿液:用無(wú)菌管收集,離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細(xì)收集上清,保存過(guò)程中如有沉淀形成,應(yīng)再次離心。胸腹水、腦脊液參照實(shí)行。

4. 細(xì)胞培養(yǎng)上清:檢測(cè)分泌性的成份時(shí),用無(wú)菌管收集。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細(xì)收集上清。檢測(cè)細(xì)胞內(nèi)的成份時(shí),用PBSPH7.2-7.4)稀釋細(xì)胞懸液,細(xì)胞濃度達(dá)到100萬(wàn)/ml左右。通過(guò)反復(fù)凍融,以使細(xì)胞破壞并放出細(xì)胞內(nèi)成份。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細(xì)收集上清。保存過(guò)程中如有沉淀形成,應(yīng)再次離心。

5. 組織標(biāo)本:切割標(biāo)本后,稱取重量。加入一定量的PBSPH7.4。用液氮迅速冷凍保存?zhèn)溆谩?biāo)本融化后仍然保持2-8的溫度。加入一定量的PBSPH7.4),用手工或勻漿器將標(biāo)本勻漿充分。離心20分鐘左右(2000-3000轉(zhuǎn)/分)。仔細(xì)收集上清。分裝后一份待檢測(cè),其余冷凍備用。

6. 標(biāo)本采集后盡早進(jìn)行提取,提取按相關(guān)文獻(xiàn)進(jìn)行,提取后應(yīng)盡快進(jìn)行實(shí)驗(yàn)。若不能馬上進(jìn)行試驗(yàn),可將標(biāo)本放于-20保存,但應(yīng)避免反復(fù)凍融.

7. 不能檢測(cè)含NaN3的樣品,因NaN3抑制辣根過(guò)氧化物酶的(HRP)活性。

操作步驟

1.         標(biāo)準(zhǔn)品的稀釋與加樣:在酶標(biāo)包被板上設(shè)標(biāo)準(zhǔn)品孔10孔,在*、第二孔中分別加標(biāo)準(zhǔn)品100μl,然后在*、第二孔中加標(biāo)準(zhǔn)品稀釋液50μl,混勻;然后從*孔、第二孔中各取100μl分別加到第三孔和第四孔,再在第三、第四孔分別加標(biāo)準(zhǔn)品稀釋液50μl,混勻;然后在第三孔和第四孔中先各取50μl棄掉,再各取50μl分別加到第五、第六孔中,再在第五、第六孔中分別加標(biāo)準(zhǔn)品稀釋液50ul,混勻;混勻后從第五、第六孔中各取50μl分別加到第七、第八孔中,再在第七、第八孔中分別加標(biāo)準(zhǔn)品稀釋液50μl,混勻后從第七、第八孔中分別取50μl加到第九、第十孔中,再在第九第十孔分別加標(biāo)準(zhǔn)品稀釋液50μl,混勻后從第九第十孔中各取50μl棄掉。(稀釋后各孔加樣量都為50μl,濃度分別為3000 pg/ml,2000 pg/ml 1000 pg/ml,500 pg/ml,250 pg/ml)。

2.         加樣:分別設(shè)空白孔(空白對(duì)照孔不加樣品及酶標(biāo)試劑,其余各步操作相同)、待測(cè)樣品孔。在酶標(biāo)包被板上待測(cè)樣品孔中先加樣品稀釋液40μl,然后再加待測(cè)樣品10μl(樣品zui終稀釋度為5倍)。加樣將樣品加于酶標(biāo)板孔底部,盡量不觸及孔壁,輕輕晃動(dòng)混勻。

3.         溫育:用封板膜封板后置37溫育30分鐘。

4.         配液:將3048T20倍)倍濃縮洗滌液用蒸餾水3048T20倍)倍稀釋后備用。

5.         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(fù)5次,拍干。

6.         加酶:每孔加入酶標(biāo)試劑50μl,空白孔除外。

7.         溫育:操作同3。

8.         洗滌:操作同5。

9.         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.

10.     終止:每孔加終止液50μl,終止反應(yīng)(此時(shí)藍(lán)色立轉(zhuǎn)黃色)。

11.     測(cè)定:以空白空調(diào)零,450nm波長(zhǎng)依序測(cè)量各孔的吸光度(OD值)。 測(cè)定應(yīng)在加終止液后15分鐘以內(nèi)進(jìn)行。

注意事項(xiàng):

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(biāo)包被板開(kāi)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(huì)有結(jié)晶析出,稀釋時(shí)可在水浴中加溫助溶,洗滌時(shí)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(duì)其準(zhǔn)確性,以避免試驗(yàn)誤差。一次加樣時(shí)間控制在5分鐘內(nèi),如標(biāo)本數(shù)量多,推薦使用排槍加樣。

4.  請(qǐng)每次測(cè)定的同時(shí)做標(biāo)準(zhǔn)曲線,做復(fù)孔。如標(biāo)本中待測(cè)物質(zhì)含量過(guò)高(樣本OD值大于標(biāo)準(zhǔn)品孔*孔的OD值),請(qǐng)先用樣品稀釋液稀釋一定倍數(shù)(n倍)后再測(cè)定,計(jì)算時(shí)請(qǐng)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(qǐng)避光保存。

7.  嚴(yán)格按照說(shuō)明書(shū)的操作進(jìn)行,試驗(yàn)結(jié)果判定必須以酶標(biāo)儀讀數(shù)為準(zhǔn).

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(hào)組分不得混用。

10. 如與英文說(shuō)明書(shū)有異,以英文說(shuō)明書(shū)為準(zhǔn)。

計(jì)算:

以標(biāo)準(zhǔn)物的濃度為橫坐標(biāo),OD值為縱坐標(biāo),   

在坐標(biāo)紙上繪出標(biāo)準(zhǔn)曲線,根據(jù)樣品的OD     

值由標(biāo)準(zhǔn)曲線查出相應(yīng)的濃度;再乘以稀釋      

倍數(shù);或用標(biāo)準(zhǔn)物的濃度與OD值計(jì)算出標(biāo)      

準(zhǔn)曲線的直線回歸方程式,將樣品的OD      

代入方程式,計(jì)算出樣品濃度,再乘以稀釋      

倍數(shù),即為樣品的實(shí)際濃度。                  

 

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0.92以上。

2.批內(nèi)與批間應(yīng)分別小于9%15%

保存條件及有效期:

1.試劑盒保存:2-8。

2.有效期:6個(gè)月

 

FOR RESEARCH USE ONLY

Human ITAC/CXCL11

 

Drug Names

Generic NameHuman ITAC/CXCL11 ELISA Kit.

Purpose

This kit allows for the determination of ITAC/CXCL11 concentrations in Human serum, blood plasma, and other biological fluids.

Principle of the assay

The kit assay Human ITAC/CXCL11 level in the sample,use Purified Human ITAC/CXCL11 antibody to coat microtiter plate wells, make solid-phase antibody, then add ITAC/CXCL11 to wells, Combined ITAC/CXCL11 antibody 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 ITAC/CXCL11 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

Standard4500pg/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

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 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.Dilute and add sample to Standard: set 10 Standard wells on the ELISA plates coated, add Standard 100μl to the first and the second well, then add Standard dilution 50μl to the first and the second well, mix; take out 100μl form the first and the second well then add it to the third and the forth well separay. then add Standard dilution 50μl to the third and the forth well ,mix ; then take out 50μl from the third and the forth well discard, add 50μl to the fifth and the sixth well ,then add Standard dilution 50μl to the fifth and the sixth well, mix ; take out 50μl from the fifth and the sixth well and add to the seventh and the eighth well, then add Standard dilution 50μl to the seventh and the eighth well ,mix ; take out 50μl from the seventh and the eighth well and add to the ninth and the tenth well, add Standard dilution 50μl to the ninth and the tenth well, mix , take out 50μl from the ninth and the tenth well discard(add Sample 50μl to each well after Diluting ,(density: 3000 pg/ml,2000 pg/ml 1000 pg/ml,500 pg/ml250 pg/ml

2.add sampleSet blank wells separay (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.Incubate: After closing plate with Closure plate membrane ,incubate for 30 min at 37.

4.Configurate liquid: 30-foldor 20-fold)wash solution diluted 30-fold (or 20-fold) with distilled water and reserve.

5.washingUncover 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.

6.add enzymeAdd HRP-Conjugate reagent 50μl to each well, except  blank well.

7.incubateOperation with 3.

8.washingOperation with 5.

9.colorAdd Chromogen Solution A 50ul and Chromogen Solution B to each well, evade the light preservation for 15 min at 37

10.Stop the reactionAdd Stop Solution50μl to each well, Stop the reaction(the blue color change to yellow color).

11.assaytake 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.

 

 

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, multiplied by the dilution factor, the result is the sample actual density.

Calculate

 

 

This chart for reference only

 

 


 

 

 

 

Storage and validity

1Storage  2-8.

2validity six months.



留言框

  • 產(chǎn)品:

  • 您的單位:

  • 您的姓名:

  • 聯(lián)系電話:

  • 常用郵箱:

  • 省份:

  • 詳細(xì)地址:

  • 補(bǔ)充說(shuō)明:

  • 驗(yàn)證碼:

    請(qǐng)輸入計(jì)算結(jié)果(填寫(xiě)阿拉伯?dāng)?shù)字),如:三加四=7

電話咨詢
  • 服務(wù)熱線:
  • 400-665-0203
中文熟妇人妻又伦精品视频-久久午夜精品人妻一区二区三区-少妇被粗大猛进进出出-日韩av在线成人观看| 蜜臀精品国产亚洲av尤物-日韩人妻少妇中文字幕-赶碰97在线公开视频-亚洲欧美日韩天堂综合| 91免费视频国产自拍-亚洲av 综合一区二区人妖-青青草草青青在线播放-欧美精品免费一区二区二区| 亚洲av日韩av天堂影片精品-熟妇人妻丰满少妇中文-国产精品日本一区二区三区-国产精品熟女乱色一区二区| 久久精品国产普通话对白-丰满人妻中文字幕一区二区-国产日本精品视频在线观看-香港免费毛片在线观看| 日本亚洲一线二线三线-九月丁香婷婷啪啪色综合-狠狠综合欧美综合欧美色-亚洲丁香视频中文在线| 国产精品人成在线播放蜜臀-老司机午夜福利视频在线-亚洲激情av免费观看-国产情侣91在线观看| 国内精品一区二区三区香蕉-熟女少妇熟女高潮一区二区-亚洲乱码国产乱码精品精男男-国内人妻自拍偷拍视频一区| 主播高颜值极品尤物极品-精品少妇人妻av免费看-精品国产免费一区二区久久-成人国产av精品入口在线| 国产四虎视频在线观看-日本一区二区三区暖暖视频免费-91人妻人人澡人人添人人爽-在线日本高清日本免费| 国产免费无套精品视频-日本特色特黄aaa大片免费-日本精品免费一区二区三区-九九热精品视频在线免费| 国产自拍成人激情视频-欧美大香蕉在线视频观看-精品人妻一区二区三区麻豆91-经典三级一区二区三区| 亚洲区一区二区三区四区-精品亚洲国产成人av-国产美腿丝袜诱惑在线观看-美女抠逼视频免费网站| 一本久道视频无线视频试看-亚洲国产精品一区二区三区久久-中文字幕色偷偷人妻久久-久久精品99国产精品中| 久久亚洲国产高清av一级-免费国产精品自偷自偷免费看-日本a级特黄三级三级三级-欧美日韩一区二区中文字幕高清视频| 国产日本高清一区二区三区-久久亚洲成人精品性色-九九热99这里只有精品-亚洲愉拍自拍另类天堂| 青青草原精品在线观看-日本久久精品狼人狠狠操-欧美深夜福利视频网站-麻豆密入视频在线观看| 精品国产人成亚洲区中文久久-欧美日韩夫妻性生活视频-亚洲欧美日韩高清专区一-国产精品无套内射后插| 久久国产精品亚洲va麻豆-嫩模大尺度偷拍在线视频-免费三级在线观看自拍-天堂av在线男女av| 午夜狂情三级伦理涩之屋-亚洲国产精品美女嫩模综合在-久热在线观看免费视频-国产精品伦子一区二区三区| 福利一区福利二区刺激-亚洲精品久久麻豆蜜桃-久久av蜜臀人妻一区二区三区-国产av剧情精品播放网站| 久久精品中文字幕一区二区-日本夫妻性生活视频播放-综合久久精品亚洲天堂-日韩中文字幕不卡久久| 亚洲av成人午夜福利在线观看-日韩精品成人影院久久久-国产在线高清不卡一区-激情五月另类综合视频| 一本色道亚州综合久久精品-91麻豆国产专区在线观看-一级二级三级国产视频-熟女av天堂免费高清| 人妻精品一区二区视频免费-99热视频免费在线观看-亚洲av第一第二第三-乱码人妻精品一区二区三区| 欧美极品欧美精品欧美激情-人妻av中文字幕高清版-国产传媒麻豆天美在线观看-免费91麻豆精品国产自产自线| 日本免费久久精品视频-毛很浓密很多黑毛熟女-97这里只有精品在线-亚洲乱码国产乱码精品精| 日韩黄片av在线免费观看-久久精品国产亚洲av色哟哟-亚洲第一中文字幕少妇-91久久精品国产性色tv| 中文字幕亚洲综合精品一区-久久好视频久久这里有精品-国产在线传媒高清视频-日韩精品一区二区亚洲av失禁| 亚洲视频第一页在线观看-最新中文字幕国产精品-中文人妻熟妇人伦精品熟妇-国产福利91在线视频| 国产精品亚洲精品午夜-欧美日韩成人精品久久二区-自拍偷拍福利视频在线观看-91精品蜜桃一区二区三区| 亚洲av高清网站夜夜去-拍国产乱人伦偷精品视频-成人日韩欧美在线观看-无遮挡国产精品一级二级三级视频| 偷拍一区二区三区视频播放器-亚洲欧洲日产韩国综合-国产精品久久精品亚洲-国产乱淫av麻豆国产| 精品人妻中文字幕有码在线-亚洲欧美一区二区成人精品久久久-亚洲第一人伊狼人久久-亚洲国产欧美精品在线观看| 偷拍日韩女生厕所尿尿-水蜜桃一区二区三区四区-亚洲成人色黄网站久久-久久久国产综合午夜精品| 精品国产亚洲av蜜臀-欧美亚洲伦理在线视频-久久亚洲国产成人影院av-国产精品99蜜臀久久不卡二区| 久久亚洲国产高清av一级-免费国产精品自偷自偷免费看-日本a级特黄三级三级三级-欧美日韩一区二区中文字幕高清视频| 五月婷婷丁香综合入口-日本少妇免费中文字幕-96青草视频在线观看-中文字幕成人精品久久不卡| 国产激情在线观看视频-久久久精品国产视频在线-亚洲国产成人精品在线-亚洲乱码国产乱码精品视频| 天天躁夜夜躁狠狠85麻豆-操美女逼视频免费软件-国产精品一区二区在线观看-一区二区三区免费观看视频在线| 日韩中文有码字幕在线观看-黑人国产一区二区三区-久久国产精品久久精品-国产激情在线一区二区三区|