一种使用3,6 - 荧光素二磷酸作为荧光底物检测2,4 - 二硝基苯酚的灵敏竞争性酶联免疫吸附测定法。

A sensitive competitive ELISA for 2,4-dinitrophenol using 3,6-fluorescein diphosphate as a fluorogenic substrate.

作者信息

Huang Z, Olson N A, You W, Haugland R P

机构信息

Molecular Probes, Inc., Eugene, OR 97402.

出版信息

J Immunol Methods. 1992 May 18;149(2):261-6. doi: 10.1016/0022-1759(92)90258-u.

DOI:10.1016/0022-1759(92)90258-u

PMID:1301064

Abstract

A competitive ELISA for sensitive detection of 2,4-dinitrophenol (DNP) was established. Certain amounts of bovine serum albumin conjugate of DNP were readily coated on a polystyrene microplate. Free DNP was then quantitated by its competition with the coated DNP for binding to anti-DNP (antibody)-alkaline phosphatase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by an enzymatic reaction with a fluorogenic substrate, 3,6-fluorescein diphosphate (FDP), or comparatively with a conventional chromogenic substrate, p-nitrophenyl phosphate (PNPP). The results showed that the ELISA with FDP at the optimal conditions of enzymatic reaction can detect as little as 10 fmol DNP, a detection limit of 50 times less than that with PNPP. The easy and sensitive DNP assay procedures in this work can be generalized to ELISAs for other antigens, particularly small antigens, haptens or drugs.

摘要

建立了一种用于灵敏检测2,4-二硝基苯酚（DNP）的竞争性酶联免疫吸附测定法（ELISA）。一定量的DNP牛血清白蛋白偶联物很容易包被在聚苯乙烯微孔板上。然后通过游离DNP与包被的DNP竞争结合抗DNP（抗体）-碱性磷酸酶偶联物来对游离DNP进行定量。通过与荧光底物3,6-荧光素二磷酸（FDP）的酶促反应，或者与传统显色底物对硝基苯磷酸酯（PNPP）进行比较，来检测因竞争而残留在板表面的酶偶联物。结果表明，在酶促反应的最佳条件下使用FDP的ELISA能够检测低至10飞摩尔的DNP，检测限比使用PNPP时低50倍。本研究中简便且灵敏的DNP检测方法可推广应用于其他抗原的ELISA检测，尤其是小分子抗原、半抗原或药物。