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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...
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,
The authors have nothing to disclose.
We are very grateful to Dr. Brigitte Sturm for her support with the ECLIA instrument.
Name | Company | Catalog Number | Comments |
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 |
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