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上海乾思生物科技有限公司
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ELISA試劑盒的包被方式

時間:2011/11/14閱讀:719
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     將抗原或抗體固定在過程稱為包被(coating)。換言之,包被即是抗原或抗體結(jié)合到固相載體表面的過程。蛋白質(zhì)與聚苯乙烯固相載體是通過物理吸附結(jié)合的,靠的是蛋白質(zhì)分子結(jié)構(gòu)上的疏水基團與固相載體表面的疏水基團間的作用于。這種物理吸附是非特異性的,受蛋白質(zhì)的分子量、等電點、濃度等的影響。載體對不同蛋白質(zhì)的吸附能力是不相同的,大分子蛋白質(zhì)較小分子蛋白質(zhì)通常含有更多的疏水基團,故更易吸附到固相載體表面。IgG對聚苯乙烯等固相具有較強的吸附力,其聯(lián)結(jié)多發(fā)生在Fc段上,抗體結(jié)合點暴露于外,因此抗體的包被一般均采用直接吸附法。蛋白質(zhì)抗原大多也可采用與抗體相似的方法包被。當抗原決定簇存在于或鄰近于疏水區(qū)域時,抗原與固相載體的直接吸附可使抗原決定簇不能充分暴露,在這種情況下,直接包被效果不佳,可以采用間接的捕獲包被法,即先將針對該抗原的特異抗體作預包被,其后通過抗原抗體反應使抗原固相化。此間接結(jié)合在固相上的抗原遠離載體表面,其抗原決定簇也得以充分暴露。間接包被的抗原經(jīng)固相抗體的親和層析作用,包被在固相上的抗原純度大大提高,因此含雜質(zhì)較多的抗原也可采用捕獲包被法,試驗的特異性、敏感性均由此得以改善,重復性亦佳。間接包被的另一優(yōu)點是抗原用量少,僅為直接包被的1/10乃至于/100。不易吸附在聚苯乙烯載體上的非蛋白質(zhì)抗原可采用特殊的包被方式。例如,在檢測抗DNA抗體時,需用DNA作為包被抗原,而普遍的固相載體一般不能直接與核酸結(jié)合??蓪⒕郾揭蚁┌逑冉?jīng)紫外線照射(例如30W紫外燈,75cm照射12小時),以增加其吸附性能。固相載體先用堿性蛋白質(zhì),如聚賴氨酸、魚精蛋白等作預包被,也可提高核酸的結(jié)合力。也可用親和素生物素系統(tǒng)作間接包被,即用親和素先包被載體,然后加入生物素化的DNA,這種包被方法均勻、牢固,已擴大應用于各種抗原物質(zhì)的定量測定。

    脂類物質(zhì)無法與固相載體結(jié)合,可將其在有機溶劑(例如乙醇)中溶解后加入ELISA板孔中,開蓋置冰箱過夜或冷風吹干,待酒精揮發(fā)后,讓脂質(zhì)自然干固在固相表面??剐牧字贵w的ELISA試劑一般采用這種包被方式。

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