TITLE: Cell-ELONA
AUTHORS: Guillermo Fernández Rodríguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">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. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">(We used 5 replicates per dilution)</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Pre-Coating]
Inoculate a single colony of E.Coli DH5α from LB agar plate  in   of LB. Use a sterile pipette tip, selecting a single colony from  LB agar plate. The liquid culture is incubated overnight
10 ml
37 °C
?. [Pre-Coating]
Spin at 4000 rpm for  . Discard the supernatant, collect pellet and re-suspend in  of NaHCO3-Na2CO3, 50mM, pH 9,6. Mix by inverting the tube.
10 ml
?. [Pre-Coating]
Spin at 4000 rpm for . Discard the supernatant, collect pellet and re-suspend in   of NaHCO3-Na2CO3, 50mM, pH 9,6.  Mix by inverting the tube.
8 ml
?. [Pre-Coating]
Read the absorbance (600nm). Dilute the sample with NaHCO3-Na2CO3, 50mM, pH 9,6, and adjust the absorbance to 1.
?. [Coating (Automated)]
Dilute the sample 1:4 with NaHCO3-Na2CO3, 50mM, pH 9,6 in a eppendorf tube.
?. [Coating (Automated)]
Add  of the bacterial sample into Nunc MaxiSorp 96-well plate and Coating buffer for the negative control.  Incubate overnight at  with agitation (260rpm).
200 µl
4 °C
?. [Cell-ELONA (Automated)]
Remove Coating buffer. CAUTION: Be careful to do not touch the well and remove the bacteria.
?. [Cell-ELONA (Automated)]
Wash 3x  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.
200 µl
?. [Cell-ELONA (Automated)]
Block the plate with   PBS 1x BSA 5% for  (260 rpm).
200 µl
?. [Cell-ELONA (Automated)]
Structure digoxigenin-labelled aptamers (denatured for 10 min at 95°C and then cooled for 10 min on ice), previously prepared in different concetrations.
?. [Cell-ELONA (Automated)]
Wash 3x 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.
200 µl
?. [Cell-ELONA (Automated)]
Add /well of the structured aptamers. Incubated for .
100 µl
?. [Cell-ELONA (Automated)]
Wash 3x with PBS 1x tween 0,1%. Remove the drops after the last wash.
200 µl
?. [Cell-ELONA (Automated)]
Add anti-body antidigoxigenin ( /well) preparing 1:1000 dilution in Aptamer buffer-BSA 0,2%. Incubate at room temperature for  .
100 µl
?. [Cell-ELONA (Automated)]
Wash 3 x   with PBS 1x Tween 0,1%.
200 µl
?. [Cell-ELONA (Automated)]
Add  100 µL/wall of ABTS solution. Read the absorbance (405 nm) every  for . ADVICE: We recomend you to buy the ABTS than comes diluted and with the oxygene peroxide.