ELISA

This protocol automates colorimetric protein detection from liver tissue extracts or plasma samples using enzyme-linked immunosorbent assay (ELISA) technology on the Transcriptic platform.

OVERVIEW

Enzyme-linked immunosorbent assay (ELISA) is a form of ligand binding assay that is commonly used for detection of proteins, particularly antigens. Control standards and samples such as mammalian tissue extracts, plasma, or serum are applied to microplates pre-coated and patterned with capture antibodies. Plates are incubated, treated with a detection antibody, and developed with the addition of an enzymatic substrate to produce a colorimetric signal detectable by optical density readings in a microplate reader. This protocol takes in tissue extracts or plasma and automates high-throughput ELISAs on the Transcriptic platform.

METHODOLOGY

96 or 384-well flat-bottomed plates were patterned and incubated with selected capture antibodies. Per manufacturer’s protocol, standards and samples (murine liver tissue extracts and plasma) were transferred to coated ELISA plates. Following washes, incubations and application of detection antibodies, the optical density of each well at 450 nm was detected in a microplate reader. Plates were also read at 540 nm to correct for background. This protocol captures the entire ELISA workflow from sample prep to protein detection via an absorbance output on the Transcriptic platform.

RESULTS

To generate liver tissue extracts, 50 mg biopsies of whole mouse livers were homogenized and collected for protein analysis. Experimental samples were tested in triplicates alongside standards processed in duplicates in a 96-well plate format ELISA patterned with a selected capture antibody.

 

Figure 1. Standards (A) and homogenized mouse liver tissue extracts (B) were processed on the Transcriptic platform in 96-well microplates patterned with a commercial ELISA capture antibody. The optical density of each well at 450 nm and 540 nm were measured in a microplate reader. Data was plotted after subtracting out the background reading at 540 nm. Samples were processed in triplicates. 

Figure 1. Standards (A) and homogenized mouse liver tissue extracts (B) were processed on the Transcriptic platform in 96-well microplates patterned with a commercial ELISA capture antibody. The optical density of each well at 450 nm and 540 nm were measured in a microplate reader. Data was plotted after subtracting out the background reading at 540 nm. Samples were processed in triplicates. 

 

VALIDATION DATE:  September 2016