This document provides a detailed general ELISA protocol, including step‑by‑step procedures, recommended reagents, and optimization tips.
The protocol describes three ELISA formats—colorimetric, chemiluminescent, and fluorescent sandwich ELISA—as well as direct and indirect detection methods. Each format includes detailed steps for plate coating, blocking, sample addition, antibody incubation, washing, substrate development, and signal measurement.
A comprehensive list of recommended Thermo Scientific reagents and consumables is provided, together with recommended concentrations for capture and detection antibodies, substrate choices, and optimization guidelines for different detection systems. This manual is written in Chinese.
| Capture antibody coating concentration | 5-15 µg/mL |
|---|---|
| Detection antibody concentration (HRP colorimetric) | 20-200 ng/mL |
| Detection antibody concentration (fluorescent) | 25-50 ng/mL |
| Detection antibody concentration (chemiluminescent) | 10-100 ng/mL |
Incubate the coated plate at room temperature for 1 hour, or overnight (12–18 hours) at 2–8 °C.
The protocol uses TMB (3,3',5,5'-tetramethylbenzidine) substrate solution.
The recommended concentration is 20–200 ng/mL.
A microplate reader such as the Thermo Scientific Multiskan FC is suggested.