An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

The electrochemiluminescence immunoassay (ECLIA) is based on a process that utilizes labels designed to emit luminescence when electrochemically stimulated. It is a broadly applicable technique for the quantitative detection of biological analytes in basic industry and academic research, food industry as well as in clinical diagnostics¹. Commonly, a disposable 96-well plate with carbon ink electrodes is used. These electrodes act as a solid-phase carrier for the immunoassay. A secondary antibody is conjugated to an electrochemiluminescent label and when electricity is applied to the system, light emission of the chemical label is triggered. An ultra-low noise charge-coupled device (CCD) records the light intensity which is directly proportional to the antigen bound to the capture antibody resulting in the quantification of the targeted analyte of the sample². Compared to the enzyme-linked immunosorbent assay (ELISA), ECLIA is considered to be advantageous as it offers higher sensitivity and reproducibility as well as better automation and consistency³.

Here we analyzed the methyl-CpG binding protein 2 (MeCP2) levels in samples of human and murine origin as well as different variants of the recombinant protein using the newly developed ECLIA system. MecP2 is an X-linked nucleic acid-binding protein known to interact with methylated DNA sequences. This protein has been implicated in the regulation of gene expression^4,5. Loss-of-function mutations in the gene which encodes this protein are the main culprits causing Rett syndrome (RTT), a severe neurodevelopmental disorder⁶. Another MeCP2-related disorder, MECP2 duplication syndrome, also leads to neurological symptoms that can overlap with those of RTT⁷. Notably, females are mostly affected by RTT while males are mostly afflicted by MECP2 duplication syndrome^6,7.

These disorders are associated with insufficient or excess MeCP2 levels respectively in the central nervous system (CNS). Hence, treatment options for RTT that involve increasing MeCP2 levels in the CNS would need to avoid the detrimental effects associated with excess of MeCP2⁷. Due to this fact, a highly sensitive and accurate quantification of MeCP2 protein levels, as provided by the ECLIA system, is crucial for the advancement of RTT as well as MECP2 duplication syndrome research. The precise measurements of endogenous and exogenous MeCP2 levels from human cell lines and mouse tissue samples as well as a recombinant protein consisting of human MeCP2 isoform B (also known as isoform e1), and a minimal N-terminal HIV-TAT transduction domain (TAT-MeCP2) that has the potential to cross the blood-brain-barrier^8,9 are presented in this work.

Approval for skin biopsy procurement for research purposes was obtained from the Human Research Ethics Committee of the Children’s Hospital at Westmead, Australia. Consent for animal experiments was obtained from the Austrian Federal Ministry of Science, Research and Economy, which were performed in accordance with local animal welfare regulations (GZ: 66.009/0218-II/3b/2015).

NOTE: The principle of the ECLIA system is depicted in Figure 1.

1. Antibody selection

1. Evaluate the signal-to-noise ratio for a range of various MeCP2 antibodies and identify the best signal strength and highest specificity within the combination of a primary mouse monoclonal MeCP2 antibody (clone Mec-168) and rabbit polyclonal MeCP2 detection antibody (custom made). For the secondary antibody, use a system-specific antibody (Table of Materials), which was also experimentally verified.
   NOTE: Table 1 gives an overview of all verified antibodies during MeCP2-ECLIA development and their tested dilution ranges.

Table 1: List of antibodies and used working dilutions.

2. Alternatively, use each pair of MeCP2-antibodies that works well with conventional ELISA.

2. Treatment of HDFs with TAT-MeCP2 fusion protein

NOTE: TAT-MeCP2 was recombinantly expressed in Escherichia coli and purified using standard chromatographic techniques as previously described⁸ and stored at -80 °C.

1. Plate 1 x 10⁶ human dermal fibroblast (HDF) cells in 100 mm dishes and grow the cells overnight at 37 °C with 5% CO₂ until they reach about 90% confluency.
2. Take out one vial of TAT-MeCP2 fusion protein. Thaw on ice and mix gently.
3. Dilute TAT-MeCP2 to a final concentration of 500 nM in 50 mL of HDF culture media.
4. Remove the media from 100 mm dishes and incubate the cells with 500 nM TAT-MeCP2 at 37 °C for various incubation periods up to 24 h.
5. Remove the TAT-MeCP2 solution from the cells. Wash the cells 2x with pre-warmed Dulbecco’s phosphate-buffered saline (DPBS).
6. Treat the cells with 2 mL of 0.05% trypsin-EDTA solution for 5 min at 37 °C¹⁰.
7. Add 6 mL of HDF culture media to inactivate the trypsin and collect the cells in 15 mL tubes.
8. Pellet the cells by centrifugation at 500 x g for 5 min at room temperature.
9. Remove the supernatant and wash the cells 2x with ice-cold DPBS. Store the pellet on ice and proceed with sample preparation.

3. Sample preparation

1. HDF lysates
   1. Determine the highest protein levels of MeCP2 using the REAP method for subcellular fractionation into the cytoplasmic and nuclear subcellular compartments according to Suzuki et al.¹¹. Limit fractionation to no more than six samples per experiment.
2. Mouse brain lysates
   1. Prepare 100 mL of each hypotonic lysis reagent and extraction buffer.
      1. For the hypotonic lysis reagent, mix well 10 mM HEPES, 1.5 mM magnesium chloride and 10 mM potassium chloride.
      2. For the extraction buffer, mix well 20 mM HEPES, 1.5 mM magnesium chloride, 0.42 M sodium chloride, 0.2 mM EDTA, and 25% (v/v) glycerol.
      3. Adjust the pH of both buffers to 7.9.
      4. Add 1 mM dithiothreitol (DTT) and 1x protease inhibitor cocktail to both buffers freshly. Chill on ice before use.
   2. Suspend 100 mg of total mouse brain (strain: C57BL/6J, wildtype male and female and B6.129P2(C)-Mecp2^tm1.1Bird/J, hemizygous male and heterozygous female; age: 4−8 weeks old) in 1 mL of ice-cold hypotonic lysis reagent.
   3. Homogenize the brain with a pre-chilled Dounce all-glass tissue homogenizer by 15 strokes of homogenizer A (loose, for large clearance) and B (tight, for small clearance), respectively.
   4. Transfer the homogenized cells to a clean tube and centrifuge at 10,000 x g for 20 min at 4 °C. Discard the supernatant (or keep it as cytoplasmatic fraction for further use).
   5. Resuspend the pellet in 140 µL of extraction buffer per 100 mg of starting material. Place the tube in a pre-cooled thermoblock and mix gently for 30 min at 4 °C.
   6. Centrifuge the tube at 16,000 g for 10 min at 4 °C. Transfer the supernatant (i.e., the nuclear fraction) to a new pre-chilled tube and store at -80 °C until use.
      NOTE: The protein concentration of lysates derived from mouse brain and HDFs can be assessed by the BCA protein assay.

4. MeCP2-ECLIA protocol

1. Preparation of washing solution, blocking buffer and assay diluent solution (day 1)
   1. Add 500 µL of Tween 20 to 1 L of PBS to prepare a 0.05% Tween 20 in PBS solution, mix vigorously and label as “washing solution”.
      NOTE: Ensure that only freshly prepared washing buffer is used.
   2. Prepare a blocking solution of 3% blocker A (Table of Materials) in phosphate-buffered saline (PBS). Mix by gentle stirring, filter sterilize and keep them in the fridge until use for a maximum of two weeks.
   3. Add 5 mL of blocking solution to 10 mL of PBS to prepare assay diluent solution (1% blocker A in PBS).
2. Coating of high bind plates
   1. Take out a 96-well multi-array single spot high bind plate.
      NOTE: High bind plates have a greater binding capacity and therefore a larger dynamic range than standard plates with hydrophobic surfaces.
   2. Thaw the monoclonal mouse anti-MeCP2 antibody on ice and mix 0.67 µL of the antibody with 4 mL of PBS (1:6,000 antibody dilution in PBS). Vortex the antibody solution to mix well and label the tube as “coating solution”.
   3. Carefully dispense 25 µL of coating solution in the bottom corner of each well using a multichannel pipettor; this is called the solution coating method. Tap the 96-well plate gently on each side to ensure that the coating solution covers the bottom of each well.
   4. Seal the plate with an adhesive foil and incubate the plate in the fridge at 4 °C overnight (12−16 h).
3. Blocking (day 2)
   1. Take out the plate from the fridge and remove the foil.
   2. Remove the antibody coating solution by flicking it into the waste basket and tap the plate on a paper towel to remove all the coating solution from the wells.
   3. Add 125 µL of blocking solution per well. Seal the plate again and place it on an orbital microplate shaker.
   4. Incubate the plate for 90 min at room temperature with constant shaking at 800 rpm.
4. Preparations of standards and samples
   1. During the incubation time, prepare the MeCP2 and/or TAT-MeCP2 protein standards and various samples.
      NOTE: Lysis buffer used for standard dilution must be the same as that used in the analyzed samples.
   2. Take out one vial of MePC2 and/or TAT-MeCP2 protein stock solution (250 µg/mL), mouse brain lysates and HDF lysates from -80 °C. Thaw them on ice.
   3. Dilute the standard stock solution (MeCP2 and/or TAT-MeCP2) in clean tubes according to Table 2.
   4. Dilute the samples in lysis buffer as follows: 1−20 µg of mouse brain lysate per 25 µL of lysis buffer, and 0.25−1 µg of HDF lysate per 25 µL of lysis buffer. Prepare enough volume of each sample to carry out analysis in triplicate.

Table 2: Standard series from 0 to 1,800 ng/mL.

5. Adding the samples and standard solutions
   1. Remove the blocking solution by flicking it into the waste basket and tap the plate on a paper towel to remove all the blocking solution from the wells.
   2. Wash the plate 3x with 150 µL of washing solution by adding the washing solution and immediately removing it.
   3. Add 25 ul of standards and samples to the bottom corner of the well using a single channel pipette.
   4. Seal the plate and incubate the plate for 4 h at room temperature with constant shaking at 800 rpm.
6. Unlabeled detection antibody
   1. Thaw the polyclonal rabbit anti-MeCP2 antibody on ice. Dilute the antibody 1:6,000 in assay diluent solution.
   2. Remove the standards and samples by flicking it into the waste basket and tap the plate on a paper towel.
   3. Wash the plate 3x with 150 µL of washing solution by adding the washing solution and immediately removing it.
   4. Add 25 µL of unlabeled detection antibody to each well with the multichannel pipettor. Seal the plate and incubate it for 1 h with constant shaking at 800 rpm at room temperature.
7. Specific conjugated antibody
   1. Take out the specific secondary antibody (Table of Materials) from the fridge and place it on ice. Dilute the antibody 1:666.67 in assay diluent solution and mix gently.
   2. Remove the free unlabeled secondary antibody by flicking it into the waste basket and tap the plate on a paper towel.
   3. Wash the plate 3x with 150 µL of washing solution by adding the washing solution and immediately removing it.
   4. Add 25 µL of specific conjugated antibody (Table of Materials) to each well with the multichannel pipettor. Seal the plate and incubate for 1 h with constant shaking at 800 rpm at room temperature.
8. Reading the plate
   1. Remove the free conjugated antibody (Table of Materials) by flicking it into the waste basket and tap the plate on a paper towel.
   2. Wash the plate 3x with 150 µL of washing solution.
   3. Add 150 μL of 1x Tris-based Gold read buffer (Table of Materials) with surfactant containing tripropylamine as a co-reactant for light generation to the plate. Avoid any air bubbles by using reverse pipetting techniques.
   4. Place the plate on the microplate detection platform (Table of Materials) and start the measurement immediately. Use the settings for 96-well plate acquisition.
   5. Capture the electrochemiluminescence signals by a built-in CCD camera in an electrochemiluminescence detection system (Table of Materials) and record the signal counts, which correspond to relative light units (RLU) and are directly proportional to the intensity of light.
      NOTE: Upon electrochemical stimulation, the ruthenium label bound to the carbon electrode emits luminescence light at 620 nm. Analyze data with the instrument-accompanied software (Table of Materials).

The principle of the ECLIA system is described in Figure 1. Standard curves for two MeCP2 variants are shown in Figure 2. Accurate quantification was possible over a wide range of concentrations (1−1,800 ng/mL). In Figure 3, MeCP2 levels of lysates derived from mouse brain and HDFs were analyzed. MeCP2 expression in brain nuclear lysates from heterozygous, wildtype and knockout mice were compared in Figure 3A, while in Figure 3B no MeCP2 protein was detected in the MECP2-deficient human fibroblasts (c.806delG) using the ECLIA. The uptake of TAT-MeCP2 by the MECP2-deficient cell line (c.806delG) was also investigated over time (Figure 4). Finally, inter- and intra-assay precision was demonstrated as shown in Table 3.

Figure 1: Diagram of MeCP2 electrochemiluminescence assay. This figure was adapted from www.meso-scale.com. Please click here to view a larger version of this figure.

Figure 2: MeCP2 standard curve generated from human MeCP2 in multiple measurements. The lower limit of detection (LLOD), defined as 2.5 standard deviations (SDs) above the blank, is 1.00 ng/mL. Recombinant human MeCP2 (Abnova) could be accurately quantified over a range from 1.00 ng/mL (LLOD) to 1,800 ng/mL (upper limit of detection [ULOD]) with R² = 0.996. Error bars represent the standard error of n = 3. The figure has been modified from Steinkellner et al.⁸. Please click here to view a larger version of this figure.

Figure 3: MeCP2 levels in mouse brain and HDFs. (A) MeCP2-protein levels were measured in brain nuclear lysates from heterozygous (grey, HET) and female wild type mice (black, wildtype) (n = 4) and one Mecp2-knockout mouse (RTT); (B) Cell lysates from MeCP2-deficient fibroblast cell line (c.806delG) derived from a male patient with neonatal encephalopathy as model for RTT syndrome were conducted to assess the MeCP2 protein levels in humans and a healthy control (black). The presented data are mean ± SD of triplicate wells (n = 3). No MeCP2 protein was detected (below detection range) in the mutant cell lines by immunofluorescence or using the ECLIA, further demonstrating that it is a highly sensitive system for further uptake studies with TAT-fusion proteins. This figure has been modified from Steinkellner et al.⁸. Please click here to view a larger version of this figure.

Figure 4: Time dependent uptake of TAT-MeCP2. MeCP2 levels of nuclear fractions in c.806delG HDFs were treated with 500 nM recombinant TAT-MeCP2 fusion protein. Analysis was performed with the MeCP2-ECLIA. At stipulated time points, the cells were washed with DPBS and incubated with 0.05% trypsin-EDTA for 5 min to eliminate extracellular-bound TAT-MeCP2. Trypsinization was stopped by adding media with serum. The cell suspension was centrifuged at 500 x g for 5 min. After washing the cell pellet 2x with ice-cold DPBS, the sample was prepared for extraction of nuclear fraction as described in the protocol section. This figure has been modified from Steinkellner et al.⁸. Please click here to view a larger version of this figure.

Table 3: Determination of inter-assay precision on three consecutive days of HDF and wildtype mouse brain lysates. Per well 1−10 µg protein of cell lysate was applied. ^aSD, standard deviation; ^bSEM, standard error mean; ^cCV, coefficient of variation. This table has been modified from Steinkellner et al.⁸.

To measure endogenous MeCP2, recombinant MeCP2 and TAT-MeCP2 levels, a 96-well plate ECLIA was developed. It has been shown that loss of MeCP2 protein function leads to RTT syndrome⁶, for which treatment is currently limited to symptom management and physical therapy. One promising treatment avenue is the so-called protein replacement therapy, where MeCP2 levels can be titrated up to their needed concentration^12,13,14,15. The potential of TAT-fusion proteins to cross the blood-brain barrier has proven to be successful over the last two decades^12,13,14,15. As such, this method for MeCP2 delivery may be useful in context of protein replacement therapy administration. In order to assess the potential of TAT-fusion proteins and other treatments to restore MeCP2 protein levels, developing an efficient and affordable assay that can quantify them is of utmost importance. The assay described in this work, the ECLIA, is able to determine levels of MeCP2 accurately as well as consistently, with favorable intra- and inter-assay values (Table 3).

For the following MeCP2-ECLIA protocol, mouse brain lysates and HDFs were employed as the cells of interest. However, this protocol may be used with all other cell types of at least human and murine origin. Moreover, HDFs were treated with TAT-MePC2 fusion protein to show the capability of this assay to measure various MeCP2 protein variants. During sample preparation, avoiding or minimizing reducing agents in the lysis buffer such as DTT or β-mercaptoethanol, is crucial to preserving the efficiency of the technique. Additional critical steps in this method involve plate coating with a mouse anti-MeCP2 antibody, plate blocking, and sample addition, followed by an 4 h incubation with a rabbit anti-MeCP2 antibody. Subsequent incubation with a specific secondary antibody (Table of Materials) and addition of reagents necessary for the luminescence reaction to take place, comprise the final important steps of this procedure.

In order to optimize ECLIA performance, the following steps can be undertaken. The maximum output for the signal for this assay should not exceed one million counts. In order to prevent the fluid from spreading beyond the electrode, a technique called spot coating can be used to introduce the coating solution into the well. This technique requires high precision pipetting or the use of a pipette robot. In addition, testing various antibody concentrations could be useful to both increase assay specificity and reduce background signal. To address the latter, various blockers (such as MSD, Blocker D-M) can also be used to a final concentration of 0.1%. In order to optimize the signal quality, testing various incubation times (shaking at or above 300 rpm) is recommended. Finally, to increase recovery and dilution linearity in specific media such as serum, plasma, urine or cerebrospinal fluid, several diluents can be tested.

Semi-quantitative western blot and commercially available MeCP2-ELISA are normally used to study MeCP2 protein levels. The working principle behind the ELISA is similar to that of our ECLIA with the notable difference being the detection mode. Compared to these methods, the MeCP2-ECLIA is faster and more convenient. The ECLIA was used to assay for wildtype, heterozygous and Mecp2-knockout mouse brain samples (Figure 3A), with findings compared to MeCP2 amounts from the same samples obtained by western blotting (data not shown). A marked difference was observed in MeCP2 levels of female wildtype and heterozygous mouse brain samples measured by the ECLIA which was not detected as statistically significant by the western blot. This higher ECLIA assay accuracy can be important in the search for novel compounds that can elevate MeCP2 protein levels.

In addition, the MeCP2-ECLIA is less costly than its MeCP2-ELISA counterpart and due to its high dynamic range from 1 ng/mL to 1,800 ng/mL (R² = 0.996), can be used with samples containing low MeCP2 amounts. When compared to all the commercially available ELISA kits, the ECLIA greatly outperforms them. The ECLIA possesses a more favorable dynamic range from 1−1,800 ng/mL compared to its ELISA counterparts, which were found to be 0.312−20 ng/mL (mouse, Cloud-Clone Corp.) and 0.156−10 ng/mL (human, Cloud-Clone Corp.). Due to the absence of an explicit definition of a lower limit of detection, its direct comparison between those two assays is not possible for the purposes of this work.

In summary, it has been shown that the MeCP2-ECLIA can accurately determine MeCP2 amounts in vivo and in vitro. While replenishing MeCP2 protein levels in the neurons of RTT-affected patients is indeed a promising treatment avenue, presence of excessive MeCP2 may also result in severe neurological symptoms, associated with MECP2 duplication syndrome^16,17,18. As such, this method can be of integral importance in optimizing the amount of exogenously introduced MeCP2 during protein replacement therapy.