Enzyme amplified immunoassay for steroids in biosamples at low picomolar concentrations
✍ Scribed by Ulf Lövgren; Karin Kronkvist; Gillis Johansson; Lars-Erik Edholm
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 854 KB
- Volume
- 288
- Category
- Article
- ISSN
- 0003-2670
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✦ Synopsis
AhtraCt
A competitive enzyme amplified ELBA for steroids was developed using recycling of NADH/NAD+ between the enzymes diaphorase and alcohol dehydrogenase. The substrate was generated from the steroid-bound enzyme label alkaline phosphatase, which dephosphorylated NADPH or NADP+. Secondary antibodies were partially denatured and adsorbed to the microtitre plates to overwme the inhomogeneity of the plastic material. In the amplified ELISA reported here, amounts down to 1 femtomol per well of the steroid budesonide could be quantified with a relative standard deviation of 30%. Plasma samples were pretreated using a solid phase extraction and a subsequent column liquid chromatography fractionation. Concentrations in blood plasma could be quantified down to 8 pM (5 fmol per well) with a precision of better than 20%. Different detection principles for the alkaline phosphatase label were compared, and the proposed double amplification procedure was found to give a substantial increase in detectability of the enzyme conjugate compared to the conventional detection of p-nitrophenol.