Oct 20, 2019
Cell-ELONA
- 1AEGIS - Madrid iGEM 2019
- AEGIS - Madrid iGEM 2019
Protocol Citation: Guillermo Fernández Rodríguez 2019. Cell-ELONA. protocols.io https://dx.doi.org/10.17504/protocols.io.7umhnu6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: October 01, 2019
Last Modified: October 20, 2019
Protocol Integer ID: 28269
Keywords: Cell-ELONA, ELONA, Aptamer characterization, Biochemistry, cell-SELEX, Automation
Abstract
ELONA (Enzyme-Linked Oligonucleotide Assay), is a biochemical method based on enzyme linked immunosorbent assay (ELISA) that allows to demonstrate that aptamers selected by SELEX can be effective and useful as biorecognition molecules and laboratory tools. The ELONA format choosed uses an anti-digoxigenin antibody to recognize an aptamer previously labelled with digoxigenin. This antibody is conjugated with a peroxidase enzyme, and once it adds ABTS solution, it will be responsible for the colourimetric reaction which will be detected.
(We used 5 replicates per dilution)
Materials
MATERIALS
MGaddgeneCatalog #26528
Anti-Digoxigenin-AP, Fab fragmentsSigma – AldrichCatalog #11093274910
Sodium bicarbonateSigma – AldrichCatalog #S6014
PBST (PBS 1:1000 Tween-20)
BSASigma Aldrich
CentrifugeEppendorf CentrifugeCatalog #5415D
LBResearch Products International (rpi)Catalog #L24400-2000.0
ABTS, solutionThermo FisherCatalog #002024
White 96-Well Immuno Plates, Maxisorp, Flat-Bottom, MaxiSorp, 350μLThermo FisherCatalog #436110
Before start
Clean all the working surface with ethanol.
Pre-Coating
Pre-Coating
Inoculate a single colony of E.Coli DH5α from LB agar plate in 10 mL of LB. Use a sterile pipette tip, selecting a single colony from LB agar plate. The liquid culture is incubated overnight 37 °C
Spin at 4000 rpm for 00:05:00 . Discard the supernatant, collect pellet and re-suspend in 10 mL of NaHCO3-Na2CO3, 50mM, pH 9,6. Mix by inverting the tube.
Spin at 4000 rpm for00:05:00 . Discard the supernatant, collect pellet and re-suspend in 8 mL of NaHCO3-Na2CO3, 50mM, pH 9,6. Mix by inverting the tube.
Read the absorbance (600nm). Dilute the sample with NaHCO3-Na2CO3, 50mM, pH 9,6, and adjust the absorbance to 1.
Coating (Automated)
Coating (Automated)
Dilute the sample 1:4 with NaHCO3-Na2CO3, 50mM, pH 9,6 in a eppendorf tube.
Add 200 µL of the bacterial sample into Nunc MaxiSorp 96-well plate and Coating buffer for the negative control. Incubate overnight at 4 °C with agitation (260rpm).
Cell-ELONA (Automated)
Cell-ELONA (Automated)
Remove Coating buffer. CAUTION: Be careful to do not touch the well and remove the bacteria.
Wash 3x200 µL with PBS 1x-Tween 0,1%. Remove the drops after the last wash. CAUTION: Be careful to do not touch the well and remove the bacteria.
Block the plate with 200 µL PBS 1x BSA 5% for 01:00:00 (260 rpm).
Structure digoxigenin-labelled aptamers (denatured for 10 min at 95°C and then cooled for 10 min on ice), previously prepared in different concetrations.
Wash 3x200 µL with PBS 1x tween 0,1%. Remove the drops after the last wash. CAUTION: Be careful to do not touch the well and remove the bacteria.
Add 100 µL /well of the structured aptamers. Incubated for 01:00:00 .
Wash 3x200 µL with PBS 1x tween 0,1%. Remove the drops after the last wash.
Add anti-body antidigoxigenin (100 µL /well) preparing 1:1000 dilution in Aptamer buffer-BSA 0,2%. Incubate at room temperature for 01:00:00 .
Wash 3 x 200 µL with PBS 1x Tween 0,1%.
Add 100 µL/wall of ABTS solution. Read the absorbance (405 nm) every 00:10:00 for 01:00:00 . ADVICE: We recomend you to buy the ABTS than comes diluted and with the oxygene peroxide.