The electrochemiluminescence immunoassay (ECLIA) is a novel approach for quantitative detection of endogenous and exogenously applied MeCP2 protein variants, which produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error over a wide working range. Here, the protocol for the MeCP2-ECLIA in a 96-well format is described.
The ECLIA is a versatile method which is able to quantify endogenous and recombinant protein amounts in a 96-well format. To demonstrate ECLIA efficiency, this assay was used to analyze intrinsic levels of MeCP2 in mouse brain tissue and the uptake of TAT-MeCP2 in human dermal fibroblasts. The MeCP2-ECLIA produces highly accurate and reproducible measurements with low intra- and inter-assay error. In summary, we developed a quantitative method for the evaluation of MeCP2 protein variants that can be utilized in high-throughput screens.
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 expression4,5. 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 syndrome6,7.
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-barrier8,9 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
Function | Name | Clone | Dilution |
Primary | Mouse, anti-MeCP2 | Mec-168 | 1:500−1:10,000 |
Primary | Mouse, anti-MeCP2 | 4B6 | 1:250−1:4,000 |
Primary | Mouse, anti-MeCP2 | Men-8 | 1:500−1:4,000 |
Primary | Mouse, anti-MeCP2 | 1B11 | 1:500−1:4,000 |
Primary | Rabbit, anti-MeCP2 | D4F3 | 1:500−1:4,000 |
Secondary | Rabbit, anti-MeCP2 | polyclonal | 1:2,000−1:20,000 |
Secondary | Rabbit, anti-MeCP2 | polyclonal | 1:2,000−1:20,000 |
Detection | SULFO-TAG labeled anti-rabbit | polyclonal | 1:500−1:1,000 |
Table 1: List of antibodies and used working dilutions.
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.
3. Sample preparation
4. MeCP2-ECLIA protocol
Standard | Concentration | Dilution |
Standard 1 | 1,800 ng/mL | 1.08 µL Standard stock solution + 148.92 µL Lysis buffer |
Standard 2 | 600 ng/mL | 50 µL Standard 1 + 100 µL Lysis buffer |
Standard 3 | 200 ng/mL | 50 µL Standard 2 + 100 µL Lysis buffer |
Standard 4 | 66.67 ng/mL | 50 µL Standard 3 + 100 µL Lysis buffer |
Standard 5 | 22.22 ng/mL | 50 µL Standard 4 + 100 µL Lysis buffer |
Standard 6 | 7.41 ng/mL | 50 µL Standard 5 + 100 µL Lysis buffer |
Standard 7 | 2.47 ng/mL | 50 µL Standard 6 + 100 µL Lysis buffer |
Standard 8 | 0.82 ng/mL | 50 µL Standard 7 + 100 µL Lysis buffer |
Standard 9 | 0.27 ng/mL | 50 µL Standard 8 + 100 µL Lysis buffer |
Standard 10 | 0 ng/mL | 150 µL Lysis buffer |
Table 2: Standard series from 0 to 1,800 ng/mL.
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.
INTRA ASSAY | INTER ASSAY | ||||||||||
ng MeCP2 per mL protein | ng MeCP2 per mL protein | ||||||||||
Well 1 | Well 2 | Well 3 | Mean | SDa | SEMb | %CVc | Mean | SDa | SEMb | %CVc | |
Human fibroblasts | 6.42 | 6.30 | 6.45 | 6.39 | 0.08 | 0.05 | 1.24 | 6.61 | 0.64 | 0.37 | 9.71 |
Mouse brain lysate | 9.92 | 10.16 | 10.36 | 10.14 | 0.22 | 0.13 | 2.17 | 11.22 | 0.94 | 0.54 | 8.33 |
aStandard Deviation, bStandard Error Mean, cCoefficient of Variation |
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 concentration12,13,14,15. The potential of TAT-fusion proteins to cross the blood-brain barrier has proven to be successful over the last two decades12,13,14,15. 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 syndrome16,17,18. As such, this method can be of integral importance in optimizing the amount of exogenously introduced MeCP2 during protein replacement therapy.
The authors have nothing to disclose.
We are very grateful to Dr. Brigitte Sturm for her support with the ECLIA instrument.
1,4-Dithiothreitol (DTT) | Sigma-Aldrich | D9779 | Hypotonic lysis reagent, Extraction Buffer |
Bio-Rad Protein Assay Dye Reagent Concentrate | Bio-Rad Laboratories Inc. | 500-0006 | Sample preperation |
Detection AB SULFO-TAG labeled anti-rabbit | Meso Scale Diagnostics | R32AB-1 | Antibody |
Discovery workbench 4.0 | Meso Scale Discovery | Software | |
DMEM (1X) | gibco by Life Technologies | 41966-029 | Sample preperation |
Dulbecco’s PBS (sterile) | Sigma-Aldrich | D8537-500ML | Sample preperation, Washing solution, Coating solution |
EDTA | Sigma-Aldrich | EDS | Extraction Buffer |
Fetal Bovine Serum | Sigma-Aldrich | F9665 | Sample preperation |
Glycerol | Sigma-Aldrich | G2025 | Extraction Buffer |
Gold Read Buffer T (1x) with surfactant | Meso Scale Diagnostics | R92TG | MeCP2 ECLIA protocol |
HEPES | Sigma-Aldrich | H3375 | Hypotonic lysis reagent, Extraction Buffer |
KIMBLE Dounce tissue grinder set | Sigma-Aldrich | D8938 | Sample preperation |
Laboratory Shaker, rocking motion (low speed) | GFL | 3014 | MeCP2 ECLIA protocol |
Magnesium chloride hexahydrate (MgCl*6H20) | Sigma-Aldrich | M2670 | Hypotonic lysis reagent, Extraction Buffer |
MeCP2 (Human) Recombinant Protein (P01) | Abnova Corporation | H00004204-P01 | Cell treatment |
Microseal B seal | Bio-Rad Laboratories Inc. | MSB1001 | for plate sealing |
Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulin | Sigma-Aldrich | M6818-100UL; RRID:AB_262075 | Antibody, Coating solution |
MSD Blocker A | Meso Scale Diagnostics | R93BA-4 | Blocker |
MSD SECTOR Imager 2400 | Meso Scale Diagnostics | I30AA-0 | MeCP2 ECLIA protocol |
Multi-Array 96-well Plate | Meso Scale Diagnostics | L15XB-3/L11BX-3 | MeCP2 ECLIA protocol |
Penicillin-Streptomycin | gibco by Life Technologies | 15140122 | Sample preperation |
Polyclonal Anti-MeCP2, produced in rabbit | Eurogentec S.A. | custom-designed | Antibody |
Potassium chloride (KCl) | Merck KGaA | 1049361000 | Hypotonic lysis reagent |
Primary AB Mouse, anti-MeCP2 (1B11) | Sigma-Aldrich | SAB1404063; RRID:AB_10737296 | Antibody |
Primary AB Mouse, anti-MeCP2 (4B6) | Sigma-Aldrich | WH0004204M1; RRID:AB_1842411 | Antibody |
Primary AB Mouse, anti-MeCP2 (Mec-168) | Sigma-Aldrich | M6818; RRID:AB_262075 | Antibody |
Primary AB Mouse, anti-MeCP2 (Men-8) | Sigma-Aldrich | M7443; RRID:AB_477235 | Antibody |
Primary AB Rabbit, anti-MeCP2 (D4F3) | Cell Signaling Technology | 3456S; RRID:AB_2143849 | Antibody |
Protease Inhibitor Cocktail (100X) | Sigma-Aldrich | 8340 | Hypotonic lysis reagent, Extraction Buffer |
Secondary AB, Rabbit, anti-MeCP2 | Eurogentec S.A. | custom | Antibody |
Secondary AB, Rabbit, anti-MeCP2 | Merck | 07-013 | Antibody |
Sodium chloride (NaCl) | Sigma-Aldrich | S3014 | Extraction Buffer |
SULFO-TAG Labeled Anti-Rabbit Antibody (goat) | Meso Scale Diagnostics | W0015528S | Antibody |
TAT-MeCP2 fusion protein | in-house production | Cell treatment | |
Trypsin EDTA 0.25% (1X) | gibco by Life Technologies | 25200-056 | Cell treatment |
Tween 20 | Sigma-Aldrich | P9416 | Washing solution |