A Sensitive phage‐based capture ELISA for sub‐femtomolar detection of protein variants directly from biological samples

Abstract

To determine the role of proteins, and in particular protein variants, in human health, it may often be necessary to quantitatively determine the concentration of a specific protein variant present in complex biological samples such as blood, cerebral spinal fluid (CSF), or tissue. Many protein variants are present only at trace levels and therefore a simple assay with very high sensitivity and reliability would greatly facilitate correlation of the presence of particular protein variants with the progression of specific diseases. We have developed a simple phage based capture ELISA system that enables femtomolar or better detection of individual protein variants directly from complex biological samples. The protocol utilizes a capture reagent that selectively recognizes a unique epitope of the protein variant and a phage based detection reagent that binds to a second epitope present in all forms of the target protein. The phage based detection reagent is essentially a self-assembling nanoparticle consisting of several thousand coat proteins that can each be labeled to amplify the detection signal by several orders of magnitude. Here we demonstrate that we can achieve subfemtomolar detection of individual protein variants that have been implicated in neurodegenerative disease directly from complex tissue homogenates and sera. The ELISA system should facilitate identification of disease specific protein variants or other compounds even when present at trace amounts in samples including blood, CSF, saliva and urine.

TDP-43 protein variants as biomarkers in amyotrophic lateral sclerosis

“…To produce phage particles from the ALS TDP-43 clones isolated earlier, we essentially follow the previously described protocol (http://www.lifesciences.sourcebioscience.com/media/143421/tomlinsonij.pdf) [28, 32, 38]. E. coli TG1 cells containing the plasmids for our clones were cultured in 2xYT containing 100 μg/ml ampicillin and 1% glucose until OD 600 was 0.4–0.6.…”

“…We previously demonstrated a simple capture ELISA that utilizes a phage-displayed detection antibody with sub-femtomolar sensitivity in conjunction with capture scFvs similar to those examined here [28, 38]. Here we generated a phage based detection antibody against TDP-43 for use in a capture ELISA in conjunction with the 23 scFvs (Stages 1B and 2 from Additional file 1: Fig.…”

Isolation and characterization of antibody fragments selective for toxic oligomeric tau

“…Phage was biotinylated for enhanced signal detection in ELISA using the EZ-Link Pentylamine-biotin kit (Thermo Scientific) as described (Williams et al, 2014). A 10 11 pfu/mL aliquot of phage stock (0.729 mg/ml) was incubated with Pentylamine-Biotin (4.86 mM final concentration) and 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) of 0.1M final concentration at room temperature for 2 hours with stirring.…”

“…To validate that scFvs were targeting tau and to quantify oligomeric tau present in mouse brain samples, we utilized a capture ELISA essentially as described (Williams et al, 2014). Basically high affinity polystyrene microtiter 96-well plates were first coated with 100 μL/well of 0.3 mg/ml purified scFv (the capture antibody) and incubated at 37 °C for two hours.…”

“…To quantitatively compare the levels of oligomeric tau in the transgenic tau mouse brain tissue samples we used a sandwich ELISA protocol with F9T scFv as the capture antibody and a phage displayed pan-specific tau scFv (F3A) as the detection antibody essentially as described (Williams et al, 2014). The F3A scFv was obtained from the Sheets library by panning against monomeric tau.…”