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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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.
FunctionNameCloneDilution
PrimaryMouse, anti-MeCP2Mec-1681:500−1:10,000
PrimaryMouse, anti-MeCP24B61:250−1:4,000
PrimaryMouse, anti-MeCP2Men-81:500−1:4,000
PrimaryMouse, anti-MeCP21B111:500−1:4,000
PrimaryRabbit, anti-MeCP2D4F31:500−1:4,000
SecondaryRabbit, anti-MeCP2polyclonal1:2,000−1:20,000
SecondaryRabbit, anti-MeCP2polyclonal1:2,000−1:20,000
DetectionSULFO-TAG labeled anti-rabbitpolyclonal1:500−1:1,000

Table 1: List of antibodies and used working dilutions.

  1. 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)-Mecp2tm1.1Bird/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.
StandardConcentrationDilution
Standard 11,800 ng/mL1.08 µL Standard stock solution + 148.92 µL Lysis buffer
Standard 2600 ng/mL50 µL Standard 1 + 100 µL Lysis buffer
Standard 3200 ng/mL50 µL Standard 2 + 100 µL Lysis buffer
Standard 466.67 ng/mL50 µL Standard 3 + 100 µL Lysis buffer
Standard 522.22 ng/mL50 µL Standard 4 + 100 µL Lysis buffer
Standard 67.41 ng/mL50 µL Standard 5 + 100 µL Lysis buffer
Standard 72.47 ng/mL50 µL Standard 6 + 100 µL Lysis buffer
Standard 80.82 ng/mL50 µL Standard 7 + 100 µL Lysis buffer
Standard 90.27 ng/mL50 µL Standard 8 + 100 µL Lysis buffer
Standard 100 ng/mL150 µL Lysis buffer

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

  1. 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.
  2. 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.
  3. 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.
  4. 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).

Results

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...

Discussion

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,

Disclosures

The authors have nothing to disclose.

Acknowledgements

We are very grateful to Dr. Brigitte Sturm for her support with the ECLIA instrument.

Materials

NameCompanyCatalog NumberComments
1,4-Dithiothreitol (DTT)Sigma-AldrichD9779Hypotonic lysis reagent, Extraction Buffer
Bio-Rad Protein Assay Dye Reagent ConcentrateBio-Rad Laboratories Inc.500-0006Sample preperation
Detection AB SULFO-TAG labeled anti-rabbitMeso Scale DiagnosticsR32AB-1Antibody
Discovery workbench 4.0Meso Scale DiscoverySoftware
DMEM (1X)gibco by Life Technologies41966-029Sample preperation
Dulbecco’s PBS (sterile)Sigma-AldrichD8537-500MLSample preperation, Washing solution, Coating solution
EDTASigma-AldrichEDSExtraction Buffer
Fetal Bovine SerumSigma-AldrichF9665Sample preperation
GlycerolSigma-AldrichG2025Extraction Buffer
Gold Read Buffer T (1x) with surfactantMeso Scale DiagnosticsR92TGMeCP2 ECLIA protocol
HEPESSigma-AldrichH3375Hypotonic lysis reagent, Extraction Buffer
KIMBLE Dounce tissue grinder setSigma-AldrichD8938Sample preperation
Laboratory Shaker, rocking motion (low speed)GFL3014MeCP2 ECLIA protocol
Magnesium chloride hexahydrate (MgCl*6H20)Sigma-AldrichM2670Hypotonic lysis reagent, Extraction Buffer
MeCP2 (Human) Recombinant Protein (P01)Abnova CorporationH00004204-P01Cell treatment
Microseal B sealBio-Rad Laboratories Inc.MSB1001for plate sealing
Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulinSigma-AldrichM6818-100UL; RRID:AB_262075Antibody, Coating solution
MSD Blocker AMeso Scale DiagnosticsR93BA-4Blocker
MSD SECTOR Imager 2400Meso Scale DiagnosticsI30AA-0MeCP2 ECLIA protocol
Multi-Array 96-well PlateMeso Scale DiagnosticsL15XB-3/L11BX-3MeCP2 ECLIA protocol
Penicillin-Streptomycingibco by Life Technologies15140122Sample preperation
Polyclonal Anti-MeCP2, produced in rabbitEurogentec S.A.custom-designedAntibody
Potassium chloride (KCl)Merck KGaA1049361000Hypotonic lysis reagent
Primary AB Mouse, anti-MeCP2 (1B11)Sigma-AldrichSAB1404063; RRID:AB_10737296Antibody
Primary AB Mouse, anti-MeCP2 (4B6)Sigma-AldrichWH0004204M1; RRID:AB_1842411Antibody
Primary AB Mouse, anti-MeCP2 (Mec-168)Sigma-AldrichM6818; RRID:AB_262075Antibody
Primary AB Mouse, anti-MeCP2 (Men-8)Sigma-AldrichM7443; RRID:AB_477235Antibody
Primary AB Rabbit, anti-MeCP2 (D4F3)Cell Signaling Technology3456S; RRID:AB_2143849Antibody
Protease Inhibitor Cocktail (100X)Sigma-Aldrich8340Hypotonic lysis reagent, Extraction Buffer
Secondary AB, Rabbit, anti-MeCP2Eurogentec S.A.customAntibody
Secondary AB, Rabbit, anti-MeCP2Merck07-013Antibody
Sodium chloride (NaCl)Sigma-AldrichS3014Extraction Buffer
SULFO-TAG Labeled Anti-Rabbit Antibody (goat)Meso Scale DiagnosticsW0015528SAntibody
TAT-MeCP2 fusion proteinin-house productionCell treatment
Trypsin EDTA 0.25% (1X)gibco by Life Technologies25200-056Cell treatment
Tween 20Sigma-AldrichP9416Washing solution

References

  1. Debad, J. D., Glezer, E. N., Wohlstadter, J., Sigal, G. B., Leland, J. K., Bard, A. J. Clinical and biological applications of ECL. Electrogenerated Chemiluminescence. , 359-383 (2004).
  2. Yuzaburo, N., Michinori, U., Osamu, S. Highly Sensitive Electrochemiluminescence Immunoassay Using the Ruthenium Chelate-Labeled Antibody Bound on the Magnetic Micro Beads. Analytical Sciences. 15 (11), 1087-1093 (1999).
  3. Liu, W., Hu, Y., Yang, Y., Hu, T., Wang, X. Comparison of two immunoassays for quantification of hepatitis B surface antigen in Chinese patients with concomitant hepatitis B surface antigen and hepatitis B surface antibodies. Archives of Virology. 160 (1), 191-198 (2015).
  4. Shah, R. R., Bird, A. P. MeCP2 mutations: progress towards understanding and treating Rett syndrome. Genome Medicine. 9 (1), 17 (2017).
  5. Chahrour, M., et al. MeCP2, a key contributor to neurological disease, activates and represses transcription. Science. 320 (5880), 1224-1229 (2008).
  6. Lyst, M. J., Bird, A. Rett syndrome: a complex disorder with simple roots. Nature Reviews Genetics. 16 (5), 261-275 (2015).
  7. Katz, D. M., et al. Rett Syndrome: Crossing the Threshold to Clinical Translation. Trends in Neurosciences. 39 (2), 100-113 (2016).
  8. Steinkellner, H., et al. An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants. Scientific Reports. 9 (1), 7929 (2019).
  9. Laccone, F. A. . Synthetic MeCP2 sequence for protein substitution therapy. , (2007).
  10. Koutsokeras, A., Kabouridis, P. S. Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells. Biochimica et Biophysica Acta (BBA) - General Subjects. 1790 (2), 147-153 (2009).
  11. Suzuki, K., Bose, P., Leong-Quong, R. Y., Fujita, D. J., Riabowol, K. R. E. A. P. REAP: A two minute cell fractionation method. BMC Research Notes. 3, 294 (2010).
  12. Xia, H., Mao, Q., Davidson, B. L. The HIV Tat protein transduction domain improves the biodistribution of beta-glucuronidase expressed from recombinant viral vectors. Nature Biotechnology. 19 (7), 640-644 (2001).
  13. Schwarze, S. R., Ho, A., Vocero-Akbani, A., Dowdy, S. F. In vivo protein transduction: delivery of a biologically active protein into the mouse. Science. 285 (5433), 1569-1572 (1999).
  14. Nagahara, H., et al. Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p27Kip1 induces cell migration. Nature Medicine. 4 (12), 1449-1452 (1998).
  15. Trazzi, S., et al. CDKL5 protein substitution therapy rescues neurological phenotypes of a mouse model of CDKL5 disorder. Human Molecular Genetics. 27 (9), 1572-1592 (2018).
  16. Van Esch, H. MECP2 Duplication Syndrome. Molecular Syndromology. 2 (3-5), 128-136 (2012).
  17. Collins, A. L., et al. Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Human Molecular Genetics. 13 (21), 2679-2689 (2004).
  18. Luikenhuis, S., Giacometti, E., Beard, C. F., Jaenisch, R. Expression of MeCP2 in postmitotic neurons rescues Rett syndrome in mice. Proceedings of the National Academy of Sciences of the United States of America. 101 (16), 6033-6038 (2004).

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