膠體金方法的優(yōu)勢(shì)以及靈敏度比對(duì)數(shù)據(jù)

1、膠體金方法的優(yōu)勢(shì)以及靈敏度比對(duì)數(shù)據(jù)*公司新推出的胱抑素C測(cè)定試劑盒，采用膠體金包被抗體，膠體金顆粒在546nm有最大吸收，當(dāng)膠體金顆粒和樣本中的CysC抗原發(fā)生凝集時(shí)，在546nm的吸光度下降，吸光度值決定于CysC的濃度。此 方法避免了抗體在反應(yīng)系統(tǒng)中初始吸光度較低造成靈敏度低的缺陷。采用546nm可見光區(qū)檢測(cè)膠體金顆 粒吸光度的改變，使得測(cè)試結(jié)果特異性更強(qiáng)，而且吸光度變化更為穩(wěn)定。1.1概述免疫膠體金技術(shù)（Immunogold Technique）是以膠體金作為示蹤標(biāo)志物應(yīng)用于抗原-抗體反應(yīng)的一種免疫標(biāo)記技術(shù)。膠體金（colloidal gold）是由氯金酸（HAuCl4）在還原劑（如白磷

2、、抗壞血酸、枸櫞酸鈉、鞣酸等）的作用下，聚合成特定大小的金顆粒，并由于靜電作用形成一種穩(wěn)定的膠體狀態(tài)。膠體金在弱堿環(huán)境下帶負(fù)電荷，可與蛋白質(zhì)分子的正電荷基團(tuán)形成牢固的結(jié)合，由于這種結(jié)合只是 靜電結(jié)合，所以蛋白質(zhì)的生物學(xué)特性不受影響。膠體金除了可以與蛋白質(zhì)分子結(jié)合以外，還可以與許多 其它生物大分子（如：SPA、PHA、ConA等）結(jié)合。利用膠體金的一些物理性質(zhì)（如：高電子密度、 顆粒大小、形狀及顏色反應(yīng)等）以及結(jié)合物的免疫和生物學(xué)特性，膠體金被廣泛地應(yīng)用于免疫學(xué)、組織 學(xué)、病理學(xué)和細(xì)胞生物學(xué)等許多領(lǐng)域，并且發(fā)展出了一系列實(shí)驗(yàn)和檢測(cè)技術(shù)。1.2反應(yīng)機(jī)理利用免疫膠體金技術(shù)進(jìn)行液相免疫測(cè)定是基于金溶膠

3、的重要特性：金顆粒與抗原（抗體）形成的結(jié) 合物在溶膠狀態(tài)顏色基本上保持不變，當(dāng)與相應(yīng)抗原或抗體發(fā)生專一性反應(yīng)后出現(xiàn)凝聚，溶膠顆粒極度 增大，光散射隨之發(fā)生變化，顆粒也會(huì)沉降，溶液的顏色變淡甚至變成無(wú)色或藍(lán)色（圖1、圖2）。這一原理可應(yīng)用于定性或定量的免疫反應(yīng)。Assay PrincipleColloidal gold particles combined with antibodies (Red-purple)0.005101520Points253035Time Course (Hitachi 7170)O 5 O- 52 L LO.Doucuqosq圖2： Cys C反應(yīng)曲線圖1：膠體金液

4、相免疫測(cè)定原理免疫膠體金技術(shù)與膠乳增強(qiáng)免疫比濁法相比，該方法檢測(cè)的靈敏度有了很大的提高。1.3與同類檢測(cè)產(chǎn)品（膠乳加強(qiáng)免疫比濁法）比較:取*公司的試劑與B公司的試劑同在日立7060上對(duì)比檢測(cè)，測(cè)試結(jié)果如下: 【定標(biāo)數(shù)據(jù)對(duì)比】 校準(zhǔn)液：B公司校準(zhǔn)液，濃度為：0.00； 0.50； 1.00； 2.00； 4.00 mg/L共計(jì)五點(diǎn)校準(zhǔn)校準(zhǔn)液：*公司校準(zhǔn)液，濃度為：0.00； 0.50； 1.00； 2.00； 4.00 ； 8.00 mg/L共計(jì)六點(diǎn)校準(zhǔn)B公司：Cyscl定標(biāo)數(shù)據(jù)如下:S1S2S3S4S5Cysc1-2425527427354424711443572754457-14209244

5、27055428211743112624387*： Cysc定標(biāo)數(shù)據(jù)如下:S1S2S3S4S5S6CYSC-22220244-47419852-125320187-417318492-848716932-1053115509-20820274-49619972-121419461-414218862-833316733-10539155921）通過(guò)實(shí)驗(yàn)測(cè)試以吸光度的變化率來(lái)看：從標(biāo)準(zhǔn)液各點(diǎn)的吸光度變化上看，B公司試劑的變化遠(yuǎn)遠(yuǎn)小于*試劑的變化。B公司標(biāo)準(zhǔn)液濃度由0.5mg/L到1.0mg/L,吸光度變化率為27即：0.0027。*公司標(biāo)準(zhǔn)液濃度由0.5mg/L到1.0mg/L,吸光度變化率為7

6、50，即：0.0750。*公司的試劑吸光度 變化的敏感程度遠(yuǎn)遠(yuǎn)高于B公司的產(chǎn)品。2）通過(guò)實(shí)驗(yàn)測(cè)試以吸光度的重復(fù)性來(lái)看：B公司標(biāo)準(zhǔn)液濃度0.5mg/L，S2變化率分別為：27/24，重復(fù)性很好，差異僅有3,但是對(duì)比與其自 身的偏差達(dá)到：3/27=11%。*公司標(biāo)準(zhǔn)液濃度0.5mg/L，S2變化率分別為：474/496，重復(fù)性很好，差異為22，但是對(duì)比與其 自身的偏差為：22/496=4.4%。附錄2：試劑精密度數(shù)據(jù)-1胱抑素 C Between-day 和 Within-day 數(shù)據(jù)Serum 11st run2nd run3rd run121212Mean10.800.790.810.810.

7、820.810.8120.780.800.800.810.800.830.8030.800.790.810.800.810.800.8040.810.790.800.800.800.770.8050.800.820.830.800.820.820.8260.800.810.790.790.800.790.8070.790.800.820.830.790.790.8080.800.840.820.800.810.800.8190.810.790.780.780.770.770.78100.790.800.820.790.820.810.81110.810.810.820.810.800.820.

8、81120.800.790.770.770.810.770.79130.800.800.790.790.790.800.80140.810.810.780.780.820.780.80150.790.810.810.810.830.790.81160.810.780.800.790.790.790.79170.820.820.810.800.810.800.81180.790.810.800.780.800.780.79190.820.800.820.810.810.830.82200.800.810.790.800.800.810.80Mean:0.80項(xiàng)目變動(dòng)自由度分散SDCVBetwee

9、n-day0.009497190.0005000.010.9%Within-day0.0164331000.0001640.011.6%合計(jì)0.025930Serum 21st run2nd run3rd run121212Mean1.461.461.481.471.491.501.481.461.461.461.441.481.451.461.471.461.471.481.451.491.471.451.441.471.471.431.471.461.451.441.461.461.481.471.461.451.421.431.441.401.411.431.481.441.451.48

10、1.461.471.461.491.461.431.461.471.481.471.471.441.441.491.481.481.471.461.431.391.441.441.421.431.431.421.431.431.451.461.441.411.431.391.451.431.431.421.471.441.451.441.451.471.451.461.451.431.451.441.471.451.461.431.461.471.471.461.461.461.421.451.461.451.461.451.481.451.461.441.461.481.461.461.43

11、1.441.421.441.451.441.481.461.481.481.481.481.481.491.451.471.451.441.451.46Mean:1.45分散分析表項(xiàng)目變動(dòng)自由度分散SDCVBetween-day0.028143190.0014810.011.0%Within-day0.0279171000.0002790.021.1%合計(jì)0.056059Serum 31st run2nd run3rd run121212Mean1.961.972.032.032.022.012.002.011.951.981.981.992.001.991.981.972.031.951.9

12、91.921.971.971.942.001.951.991.931.961.981.922.001.971.991.961.971.991.941.981.921.941.921.952.011.941.991.951.991.971.982.001.951.961.942.001.961.971.991.942.001.942.011.981.981.961.901.981.971.951.921.951.951.931.981.951.981.931.951.961.971.951.941.941.961.951.971.982.012.011.991.941.982.021.981.9

13、81.951.961.921.971.981.932.001.972.021.961.981.981.952.001.971.971.961.971.961.961.981.961.981.991.972.001.921.951.911.991.941.952.031.971.942.011.991.951.982.021.961.991.961.971.931.97Mean:1.97分散分析表項(xiàng)目變動(dòng)自由度分散SDCVBetween-day0.022420190.0011800.010.4%Within-day0.0795671000.0007960.031.4%合計(jì)0.101987Seru

14、m 41st run2nd run3rd run121212Mean3.944.064.073.994.074.054.033.893.873.853.913.993.863.904.034.034.053.993.944.044.014.004.024.074.094.033.974.033.973.914.014.074.034.074.013.913.904.054.034.003.943.973.943.893.994.013.943.843.944.104.004.033.964.023.964.013.943.894.034.013.993.953.973.983.803.933.

15、883.943.953.913.923.924.024.023.984.043.983.963.973.993.963.974.013.984.024.024.033.974.014.044.023.953.973.913.853.963.813.913.993.973.953.914.023.893.963.963.803.943.924.043.943.934.034.044.083.914.043.954.013.993.983.903.823.973.943.934.034.013.973.943.953.933.974.033.984.083.973.994.034.01Mean:3

16、.97分散分析表項(xiàng)目變動(dòng)自由度分散SDCVBetween-day0.207003190.0108950.040.9%Within-day0.2928331000.0029280.051.4%會(huì)耳 合口 10.499837附錄3： *公司胱抑素試劑與DadeBehring相關(guān)性比較DadecyscDadecysc10.981.12415.946.3320.570.56421.721.8930.790.85433.343.5740.770.86446.236.9251.381.51451.231.2960.951.02461.821.9370.991.02473.163.1681.001.0348

17、4.564.9790.991.05496.637.01101.171.28501.391.52111.241.39511.481.59122.412.68521.171.28130.941.05531.461.56141.681.85543.643.71152.232.48552.182.40163.183.42563.243.27171.982.17574.975.26183.033.27582.082.14191.791.98593.313.50200.931.04601.671.76212.312.42611.591.71223.623.77621.952.16231.982.09630.921.06245.055.30646.076.23251.862.05651.641.70260.930.96661.441.49272.072.15673.183.35282.192.34682.272.47293.533.48693.103.20301.941.97703.633.49312.402.54711.891.99322.042.20721.351.45332.18