The ECLIA is a simple yet efficient method to quantify cellular levels of MeCP2. The ECLIA is fast, more sensitive, and easier to handle compared to other quantification methods such as the Western blot and the ELISA. To prepare the washing solution, which is 0.5%Tween 20 in PBS, add 500 microliters of Tween 20 to one liter of PBS and mix vigorously.
Prepare a blocking solution of 3%Blocker A and PBS and stir gently. Sterilize the blocking solution by filtration. To prepare the assay diluent solution, 1%Blocker A, and PBS, add five milliliters of blocking solution to 10 milliliters of PBS.
Next thaw the monoclonal mouse anti-MeCP2 antibody on ice. Mix 0.67 microliters of the antibody with four milliliters of PBS, then vortex the solution. To solution coat the 96-well high bind plate, carefully dispense 25 microliters of coating solution into the bottom corner of each well, using a multichannel pipetter.
Dab the 96-well plate gently on each side to ensure that the coating solution covers the bottom of each well. Seal the plate with adhesive foil, and incubate overnight at four degrees Celsius. Retrieve the 96-well plate from the refrigerator and remove the foil.
Remove the antibody coating solution in the wells by flicking it into a waste container. Then tap the plate on a paper towel to remove any remaining solution. Next, add 125 microliters of blocking solution to each well.
Then seal the plate again with adhesive foil. Place the plate on an orbital microplate shaker, set at 800 RPM, and incubate it for 90 minutes at room temperature. Thaw on ice one vial of MeCP2 or Tat-MeCP2 protein stock solution, mouse brain lysates, and HDF lysates.
Dilute the protein stock solution in clean tubes as described in table two of the manuscript. Next dilute the lysate samples. For the mouse brain lysates, use one to 20 micrograms per 25 microliters of lysis buffer.
For the HDF lysate, add 0.25 to one microgram per 25 microliters of lysis buffer. After the 96-well plate has been incubating for 90 minutes, remove the blocking solution from the wells by flicking it into a waste container. Then tap the plate on a paper towel to remove any remaining solution.
Then wash the plate three times with 150 microliters of washing solution, by adding the washing solution and immediately removing it. Add 25 microliters of standards and samples to the wells of the 96-well plate. Seal the plate and incubate it for another four hours at room temperature with constant 800 RPM shaking.
Thaw the unlabeled detection antibody, polyclonal rabbit anti-MeCP2, on ice. Using assay diluent solution, dilute the antibody at a ratio of one to 6, 000. When the 96-well plate is finished incubating on the shaker, remove the standards and samples by flicking the plate into a waste container.
Then tap the plate on a paper towel to remove any remaining solution. Wash the plate three times with 150 microliters of washing solution by adding the washing solution and immediately removing it. Add 25 microliters of unlabeled detection antibody solution to each well with the multichannel pipetter.
Seal the plate and incubate it for one hour at room temperature with constant shaking at 800 RPM. Obtain the specific secondary antibody from the refrigerator and place it one ice. Dilute the secondary body in assay diluent solution and mix gently.
To remove the free unlabeled detection antibody from the 96-well plate, flick in into the waste container and then tap the plate on a paper towel. After washing the plate three times as perviously described, add 25 microliters of secondary antibody to each well with the multichannel pipetter. Seal the plate and incubate it for one hour at room temperature with constant shaking at 800 RPM.
Remove the free secondary antibody by flicking in into the waste container and tapping the plate on a paper towel and then wash the plate three times, as previously described. To each well of the plate, add 150 microliters of 1X Tris base Gold Read Buffer with surfactant, containing tripropylamine as a coreactant for light generation. To avoid producing air bubbles, use reverse pipetting techniques.
Place the 96-well plate on the microplate platform with electrochemiluminescence detection system. Using the built in CCD camera, immediately begin capturing data. Record signal counts which correspond to relative light units and are directly proportional to the intensity of light.
MeCP2 standard curves were generated from human MeCP2 in multiple measurements. The ECLIA can accurately quantify recombinant human MeCP2 over a range from one nanogram per milliliter to 1800 nanograms per milliliter. Using ECLIA, MeCP2 protein levels were measured in brain nuclear lysates from heterozygous and female wild-type mice and from one MeCP2 knockout mouse.
ECLIA was conducted on cell lysates from a healthy control and from the c. 806delG cell line used as a model for Rett syndrome. No MeCP2 protein was detected in the mutant cell lines by ECLIA or by immunofluorescence.
ECLIA was also used to investigate the uptake of Tat-MeCP2 by the MeCP2 deficient cell line overtime. ECLIA inter and intra assay precision was determined over three consecutive days. For future protein replacement therapy, MeCP2 delivery can be repeatedly optimized following protein level assessment by the ECLIA.
