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

Approval for skin biopsy procurement for research purposes was obtained from the Human Research Ethics Committee of the Children&#8217;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
<ol>
	<li>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.</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Function</td>
			<td>Name</td>
			<td>Clone</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Primary</td>
			<td>Mouse, anti-MeCP2</td>
			<td>Mec-168</td>
			<td>1:500&#8722;1:10,000</td>
		</tr>
		<tr>
			<td>Primary</td>
			<td>Mouse, anti-MeCP2</td>
			<td>4B6</td>
			<td>1:250&#8722;1:4,000</td>
		</tr>
		<tr>
			<td>Primary</td>
			<td>Mouse, anti-MeCP2</td>
			<td>Men-8</td>
			<td>1:500&#8722;1:4,000</td>
		</tr>
		<tr>
			<td>Primary</td>
			<td>Mouse, anti-MeCP2</td>
			<td>1B11</td>
			<td>1:500&#8722;1:4,000</td>
		</tr>
		<tr>
			<td>Primary</td>
			<td>Rabbit, anti-MeCP2</td>
			<td>D4F3</td>
			<td>1:500&#8722;1:4,000</td>
		</tr>
		<tr>
			<td>Secondary</td>
			<td>Rabbit, anti-MeCP2</td>
			<td>polyclonal</td>
			<td>1:2,000&#8722;1:20,000</td>
		</tr>
		<tr>
			<td>Secondary</td>
			<td>Rabbit, anti-MeCP2</td>
			<td>polyclonal</td>
			<td>1:2,000&#8722;1:20,000</td>
		</tr>
		<tr>
			<td>Detection</td>
			<td>SULFO-TAG labeled anti-rabbit</td>
			<td>polyclonal</td>
			<td>1:500&#8722;1:1,000</td>
		</tr>
	</tbody>
</table>
Table 1: List of antibodies and used working dilutions.
<ol start="2">
	<li>Alternatively, use each pair of MeCP2-antibodies that works well with conventional ELISA.</li>
</ol>
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 &#176;C.
<ol>
	<li>Plate 1 x 106 human dermal fibroblast (HDF) cells in 100 mm dishes and grow the cells overnight at 37 &#176;C with 5% CO2 until they reach about 90% confluency.</li>
	<li>Take out one vial of TAT-MeCP2 fusion protein. Thaw on ice and mix gently.</li>
	<li>Dilute TAT-MeCP2 to a final concentration of 500 nM in 50 mL of HDF culture media.</li>
	<li>Remove the media from 100 mm dishes and incubate the cells with 500 nM TAT-MeCP2 at 37 &#176;C for various incubation periods up to 24 h.</li>
	<li>Remove the TAT-MeCP2 solution from the cells. Wash the cells 2x with pre-warmed Dulbecco&#8217;s phosphate-buffered saline (DPBS).</li>
	<li>Treat the cells with 2 mL of 0.05% trypsin-EDTA solution for 5 min at 37 &#176;C10.</li>
	<li>Add 6 mL of HDF culture media to inactivate the trypsin and collect the cells in 15 mL tubes.</li>
	<li>Pellet the cells by centrifugation at 500 x g for 5 min at room temperature.</li>
	<li>Remove the supernatant and wash the cells 2x with ice-cold DPBS. Store the pellet on ice and proceed with sample preparation.</li>
</ol>
3. Sample preparation
<ol>
	<li>HDF lysates
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Mouse brain lysates
	<ol>
		<li>Prepare 100 mL of each hypotonic lysis reagent and extraction buffer.
		<ol>
			<li>For the hypotonic lysis reagent, mix well 10 mM HEPES, 1.5 mM magnesium chloride and 10 mM potassium chloride.</li>
			<li>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.</li>
			<li>Adjust the pH of both buffers to 7.9.</li>
			<li>Add 1 mM dithiothreitol (DTT) and 1x protease inhibitor cocktail to both buffers freshly. Chill on ice before use.</li>
		</ol>
		</li>
		<li>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&#8722;8 weeks old) in 1 mL of ice-cold hypotonic lysis reagent.</li>
		<li>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.</li>
		<li>Transfer the homogenized cells to a clean tube and centrifuge at 10,000 x g for 20 min at 4 &#176;C. Discard the supernatant (or keep it as cytoplasmatic fraction for further use).</li>
		<li>Resuspend the pellet in 140 &#181;L 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 &#176;C.</li>
		<li>Centrifuge the tube at 16,000 g for 10 min at 4 &#176;C. Transfer the supernatant (i.e., the nuclear fraction) to a new pre-chilled tube and store at -80 &#176;C until use. 
		NOTE: The protein concentration of lysates derived from mouse brain and HDFs can be assessed by the BCA protein assay.</li>
	</ol>
	</li>
</ol>
4. MeCP2-ECLIA protocol
<ol>
	<li>Preparation of washing solution, blocking buffer and assay diluent solution (day 1)
	<ol>
		<li>Add 500 &#181;L of Tween 20 to 1 L of PBS to prepare a 0.05% Tween 20 in PBS solution, mix vigorously and label as &#8220;washing solution&#8221;. 
		NOTE: Ensure that only freshly prepared washing buffer is used.</li>
		<li>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.</li>
		<li>Add 5 mL of blocking solution to 10 mL of PBS to prepare assay diluent solution (1% blocker A in PBS).</li>
	</ol>
	</li>
	<li>Coating of high bind plates
	<ol>
		<li>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.</li>
		<li>Thaw the monoclonal mouse anti-MeCP2 antibody on ice and mix 0.67 &#181;L 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 &#8220;coating solution&#8221;.</li>
		<li>Carefully dispense 25 &#181;L 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.</li>
		<li>Seal the plate with an adhesive foil and incubate the plate in the fridge at 4 &#176;C overnight (12&#8722;16 h).</li>
	</ol>
	</li>
	<li>Blocking (day 2)
	<ol>
		<li>Take out the plate from the fridge and remove the foil.</li>
		<li>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.</li>
		<li>Add 125 &#181;L of blocking solution per well. Seal the plate again and place it on an orbital microplate shaker.</li>
		<li>Incubate the plate for 90 min at room temperature with constant shaking at 800 rpm.</li>
	</ol>
	</li>
	<li>Preparations of standards and samples
	<ol>
		<li>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.</li>
		<li>Take out one vial of MePC2 and/or TAT-MeCP2 protein stock solution (250 &#181;g/mL), mouse brain lysates and HDF lysates from -80 &#176;C. Thaw them on ice.</li>
		<li>Dilute the standard stock solution (MeCP2 and/or TAT-MeCP2) in clean tubes according to Table 2.</li>
		<li>Dilute the samples in lysis buffer as follows: 1&#8722;20 &#181;g of mouse brain lysate per 25 &#181;L of lysis buffer, and 0.25&#8722;1 &#181;g of HDF lysate per 25 &#181;L of lysis buffer. Prepare enough volume of each sample to carry out analysis in triplicate.</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Standard</td>
			<td>Concentration</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>Standard 1</td>
			<td>1,800 ng/mL</td>
			<td>1.08 &#181;L Standard stock solution + 148.92 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 2</td>
			<td>600 ng/mL</td>
			<td>50 &#181;L Standard 1 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 3</td>
			<td>200 ng/mL</td>
			<td>50 &#181;L Standard 2 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 4</td>
			<td>66.67 ng/mL</td>
			<td>50 &#181;L Standard 3 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 5</td>
			<td>22.22 ng/mL</td>
			<td>50 &#181;L Standard 4 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 6</td>
			<td>7.41 ng/mL</td>
			<td>50 &#181;L Standard 5 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 7</td>
			<td>2.47 ng/mL</td>
			<td>50 &#181;L Standard 6 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 8</td>
			<td>0.82 ng/mL</td>
			<td>50 &#181;L Standard 7 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 9</td>
			<td>0.27 ng/mL</td>
			<td>50 &#181;L Standard 8 + 100 &#181;L Lysis buffer</td>
		</tr>
		<tr>
			<td>Standard 10</td>
			<td>0 ng/mL</td>
			<td>150 &#181;L Lysis buffer</td>
		</tr>
	</tbody>
</table>
Table 2: Standard series from 0 to 1,800 ng/mL.
<ol start="5">
	<li>Adding the samples and standard solutions
	<ol>
		<li>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.</li>
		<li>Wash the plate 3x with 150 &#181;L of washing solution by adding the washing solution and immediately removing it.</li>
		<li>Add 25 ul of standards and samples to the bottom corner of the well using a single channel pipette.</li>
		<li>Seal the plate and incubate the plate for 4 h at room temperature with constant shaking at 800 rpm.</li>
	</ol>
	</li>
	<li>Unlabeled detection antibody
	<ol>
		<li>Thaw the polyclonal rabbit anti-MeCP2 antibody on ice. Dilute the antibody 1:6,000 in assay diluent solution.</li>
		<li>Remove the standards and samples by flicking it into the waste basket and tap the plate on a paper towel.</li>
		<li>Wash the plate 3x with 150 &#181;L of washing solution by adding the washing solution and immediately removing it.</li>
		<li>Add 25 &#181;L 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.</li>
	</ol>
	</li>
	<li>Specific conjugated antibody
	<ol>
		<li>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.</li>
		<li>Remove the free unlabeled secondary antibody by flicking it into the waste basket and tap the plate on a paper towel.</li>
		<li>Wash the plate 3x with 150 &#181;L of washing solution by adding the washing solution and immediately removing it.</li>
		<li>Add 25 &#181;L 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.</li>
	</ol>
	</li>
	<li>Reading the plate
	<ol>
		<li>Remove the free conjugated antibody (Table of Materials) by flicking it into the waste basket and tap the plate on a paper towel.</li>
		<li>Wash the plate 3x with 150 &#181;L of washing solution.</li>
		<li>Add 150 &#956;L of 1x Tris-based Gold read buffer&#160;(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.</li>
		<li>Place the plate on the microplate detection platform (Table of Materials) and start the measurement immediately. Use the settings for 96-well plate acquisition.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

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

The authors have nothing to disclose.

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,<sup class="x...

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.

<table><tbody><tr><td>1,4-Dithiothreitol (DTT)</td><td>Sigma-Aldrich</td><td>D9779</td><td>Hypotonic lysis reagent, Extraction Buffer</td></tr><tr><td>Bio-Rad Protein Assay Dye Reagent Concentrate</td><td>Bio-Rad Laboratories Inc.</td><td>500-0006</td><td>Sample preperation</td></tr><tr><td>Detection AB SULFO-TAG labeled anti-rabbit</td><td>Meso Scale Diagnostics</td><td>R32AB-1</td><td>Antibody</td></tr><tr><td>Discovery workbench 4.0</td><td>Meso Scale Discovery</td><td></td><td>Software</td></tr><tr><td>DMEM (1X)</td><td>gibco by Life Technologies</td><td>41966-029</td><td>Sample preperation</td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (sterile)</td><td>Sigma-Aldrich</td><td>D8537-500ML</td><td>Sample preperation, Washing solution, Coating solution</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>EDS</td><td>Extraction Buffer</td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma-Aldrich</td><td>F9665</td><td>Sample preperation</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G2025</td><td>Extraction Buffer</td></tr><tr><td>Gold Read Buffer T (1x) with surfactant</td><td>Meso Scale Diagnostics</td><td>R92TG</td><td>MeCP2 ECLIA protocol</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td>Hypotonic lysis reagent, Extraction Buffer</td></tr><tr><td>KIMBLE Dounce tissue grinder set</td><td>Sigma-Aldrich</td><td>D8938</td><td>Sample preperation</td></tr><tr><td>Laboratory Shaker, rocking motion (low speed)</td><td>GFL</td><td>3014</td><td>MeCP2 ECLIA protocol</td></tr><tr><td>Magnesium chloride hexahydrate (MgCl*6H20)</td><td>Sigma-Aldrich</td><td>M2670</td><td>Hypotonic lysis reagent, Extraction Buffer</td></tr><tr><td>MeCP2 (Human) Recombinant Protein (P01)</td><td>Abnova Corporation</td><td>H00004204-P01</td><td>Cell treatment</td></tr><tr><td>Microseal B seal</td><td>Bio-Rad Laboratories Inc.</td><td>MSB1001</td><td>for plate sealing</td></tr><tr><td>Monoclonal Anti-MeCP2, produced in mouse, clone Mec-168, purified immunoglobulin</td><td>Sigma-Aldrich</td><td>M6818-100UL; RRID:AB_262075</td><td>Antibody, Coating solution</td></tr><tr><td>MSD Blocker A</td><td>Meso Scale Diagnostics</td><td>R93BA-4</td><td>Blocker</td></tr><tr><td>MSD SECTOR Imager 2400</td><td>Meso Scale Diagnostics</td><td>I30AA-0</td><td>MeCP2 ECLIA protocol</td></tr><tr><td>Multi-Array 96-well Plate</td><td>Meso Scale Diagnostics</td><td>L15XB-3/L11BX-3</td><td>MeCP2 ECLIA protocol</td></tr><tr><td>Penicillin-Streptomycin</td><td>gibco by Life Technologies</td><td>15140122</td><td>Sample preperation</td></tr><tr><td>Polyclonal Anti-MeCP2, produced in rabbit</td><td>Eurogentec S.A.</td><td>custom-designed</td><td>Antibody</td></tr><tr><td>Potassium chloride (KCl)</td><td>Merck KGaA</td><td>1049361000</td><td>Hypotonic lysis reagent</td></tr><tr><td>Primary AB Mouse, anti-MeCP2 (1B11)</td><td>Sigma-Aldrich</td><td>SAB1404063; RRID:AB_10737296</td><td>Antibody</td></tr><tr><td>Primary AB Mouse, anti-MeCP2 (4B6)</td><td>Sigma-Aldrich</td><td>WH0004204M1; RRID:AB_1842411</td><td>Antibody</td></tr><tr><td>Primary AB Mouse, anti-MeCP2 (Mec-168)</td><td>Sigma-Aldrich</td><td>M6818; RRID:AB_262075</td><td>Antibody</td></tr><tr><td>Primary AB Mouse, anti-MeCP2 (Men-8)</td><td>Sigma-Aldrich</td><td>M7443; RRID:AB_477235</td><td>Antibody</td></tr><tr><td>Primary AB Rabbit, anti-MeCP2 (D4F3)</td><td>Cell Signaling Technology</td><td>3456S; RRID:AB_2143849</td><td>Antibody</td></tr><tr><td>Protease Inhibitor Cocktail (100X)</td><td>Sigma-Aldrich</td><td>8340</td><td>Hypotonic lysis reagent, Extraction Buffer</td></tr><tr><td>Secondary AB, Rabbit, anti-MeCP2</td><td>Eurogentec S.A.</td><td>custom</td><td>Antibody</td></tr><tr><td>Secondary AB, Rabbit, anti-MeCP2</td><td>Merck</td><td>07-013</td><td>Antibody</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S3014</td><td>Extraction Buffer</td></tr><tr><td>SULFO-TAG Labeled Anti-Rabbit Antibody (goat)</td><td>Meso Scale Diagnostics</td><td>W0015528S</td><td>Antibody</td></tr><tr><td>TAT-MeCP2 fusion protein</td><td>in-house production</td><td></td><td>Cell treatment</td></tr><tr><td>Trypsin EDTA 0.25% (1X)</td><td>gibco by Life Technologies</td><td>25200-056</td><td>Cell treatment</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P9416</td><td>Washing solution</td></tr></tbody></table>

an electrochemiluminescence-based assay for mecp2 protein variants

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 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<...

Watch this Scientific Journal Video about An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants at JoVE.com

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

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, ...

an-electrochemiluminescence-based-assay-for-mecp2-protein-variants

Research

JoVE Journal

Biochemistry

27.1K Views.  Medical University of Vienna (MUV). De ECLIA is een eenvoudige maar efficiënte methode om cellulaire niveaus van MeCP2 te kwantificeren. De ECLIA is snel, gevoeliger en gemakkelijker te hanteren in vergelijking met andere kwantificeringsmethoden zoals de westelijke vlek en de ELISA. Om de wasoplossing, die 0,5%Tween 20 in PBS is, voor te bereiden, voeg 500 microliter tween 20 toe aan een liter PBS en meng krachtig.Bereid een blokkeringsoplossing van 3% Blocker A en PBS voor en roer voorzichtig. Steriliseren van de blokk...

Video: An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. Radioactivity has been the predominant method of DNA labeling in EMSAs. However, recent advances in fluorescent dyes and scanning methods have prompted the use of fluorescent tagging of DNA as an alternative to radioactivity for the advantages of easy handling, saving time, reducing cost, and improving safety. We have recently used fluorescent EMSA (fEMSA) to successfully address an important biological question. Our fEMSA analysis provides mechanistic insight into the effect of a missense mutation, G73E, in the highly conserved HMG transcription factor SOX-2 on olfactory neuron type diversification. We found that mutant SOX-2G73E protein alters specific DNA binding activity, thereby causing olfactory neuron identity transformation. Here, we present an optimized and cost-effective step-by-step protocol for fEMSA using infrared fluorescent dye-labeled oligonucleotides containing the LIM-4/SOX-2 adjacent target sites and purified SOX-2 proteins (WT and mutant SOX-2G73E proteins) as a biological example.

We describe here an optimized protocol of fluorescent Electrophoretic Mobility Shift Assays (fEMSA) using purified SOX-2 proteins together with infrared fluorescent dye-labeled DNA probes as a case study to tackle an important biological question.

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Pooled shRNA Screen for Reactivation of MeCP2 on the Inactive X Chromosome

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control in model organisms. Technologies including short hairpin RNAs (shRNAs) and clustered regularly interspaced short palindromic repeats (CRISPR) have enabled such screens in diploid mammalian cells. Here we describe a large-scale shRNA screen for regulators of X-chromosome inactivation (XCI), using a murine cell line with firefly luciferase and hygromycin resistance genes knocked in at the C-terminus of the methyl CpG binding protein 2 (MeCP2) gene on the inactive X-chromosome (Xi). Reactivation of the construct in the reporter cell line conferred survival advantage under hygromycin B selection, enabling us to screen a large shRNA library and identify hairpins that reactivated the reporter by measuring their post-selection enrichment using next-generation sequencing. The enriched hairpins were then individually validated by testing their ability to activate the luciferase reporter on Xi.

We report a small hairpin RNA (shRNA) and next generation sequencing-based protocol for identifying regulators of X-chromosome inactivation in a murine cell line with firefly luciferase and hygromycin resistance genes fused to the methyl CpG binding protein 2 (MeCP2) gene on the inactive X chromosome.

Forward genetic screens using reporter genes inserted into the heterochromatin have been extensively used to investigate mechanisms of epigenetic control ...

Genetics

Efficient Purification and LC-MS/MS-based Assay Development for Ten-Eleven Translocation-2 5-Methylcytosine Dioxygenase

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these epigenetic marks is accomplished by sequential oxidation by ten-eleven translocation dioxygenases (TET1-3), followed by the thymine-DNA glycosylase-dependent base excision repair. Inactivation of the TET2 gene due to genetic mutations or by other epigenetic mechanisms is associated with a poor prognosis in patients with diverse cancers, especially hematopoietic malignancies. Here, we describe an efficient single step purification of enzymatically active untagged human TET2 dioxygenase using cation exchange chromatography. We further provide a liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach that can separate and quantify the four normal DNA bases (A, T, G, and C), as well as the four modified cytosine bases (5-methyl, 5-hydroxymethyl, 5-formyl, and 5-carboxyl). This assay can be used to evaluate the activity of wild type and mutant TET2 dioxygenases.

Here, we present a protocol for an efficient single step purification of the active untagged human ten-eleven translocation-2 (TET2) 5-methylcytosine dioxygenase using ion-exchange chromatography and its assay using a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach.

The epigenetic transcription regulation mediated by 5-methylcytosine (5mC) has played a critical role in eukaryotic development. Demethylation of these ...

Non-Radioactive In Vitro Cardiac Myosin Light Chain Kinase Assays

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular isoform of the myosin regulatory light chain (MLC2v). MLC2v phosphorylation levels are significantly reduced in failing hearts, indicating the clinical importance of assessing the activity of cMLCK and the phosphorylation level of MLC2v to elucidate the pathogenesis of heart failure. This paper describes nonradioactive methods to assess both the activity of cMLCK and MLC2v phosphorylation levels. In vitro kinase reactions are performed using recombinant cMLCK with recombinant calmodulin and MLC2v in the presence of ATP and calcium at 25 &#176;C, which are followed by either a bioluminescent ADP detection assay or a phosphate-affinity SDS-PAGE. In the representative study, the bioluminescent ADP detection assay showed a strict linear increase of the signal at cMLCK concentrations between 1.25 nM to 25 nM. Phosphate-affinity SDS-PAGE also showed a linear increase of phosphorylated MLC2v in the same cMLCK concentration range. Next, the time-dependency of the reactions was examined at the concentration of 5 nM cMLCK. A bioluminescent ADP detection assay showed a linear increase in the signal during 90 min of the reaction. Similarly, phosphate-affinity SDS-PAGE showed a time-dependent increase of phosphorylated MLC2v. The biochemical parameters of cMLCK for MLC2v were determined by a Michaelis-Menten plot using the bioluminescent ADP detection assay. The Vmax was 1.65 &#177; 0.10 mol/min/mol kinase and the average Km was around 0.5 USA &#181;M at 25 &#176;C. Next, the activity of wild type and the dilated cardiomyopathy-associated p.Pro639Valfs*15 mutant cMLCK were measured. The bioluminescent ADP detection assay and phosphate-affinity SDS-PAGE correctly detected defects in cMLCK activity and MLC2v phosphorylation, respectively. In conclusion, a combination of the bioluminescent ADP detection assay and the phosphate-affinity SDS-PAGE is a simple, accurate, safe, low-cost, and flexible method to measure cMLCK activity and the phosphorylation level of MLC2v.

This study describes protocols for nonradiometric methods, a bioluminescent ADP detection assay and a phosphate-affinity SDS-PAGE, to determine the kinase activity of cardiac myosin light chain kinase (cMLCK) and the phosphorylation level of its substrate, myosin regulatory light chain (MLC2v).

Cardiac-specific myosin regulatory light chain kinase (cMLCK) regulates cardiac sarcomere structure and contractility by phosphorylating the ventricular ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction followed by probe hybridization on a nylon membrane, which is re-usable. Probes are 5' amine modified to allow fixation to the membrane. Primers are 5' biotin modified which allows detection of hybridized PCR products using streptavidin-peroxidase and a chemiluminescent substrate via photosensitive film. With low setup and consumable costs, this technique is inexpensive (approximately US$2 per sample), high throughput (multiple membranes can be processed simultaneously) and has a short turnaround time (approximately 10 hours). 
The technique can be utilized in a number of ways. Multiple probes can be designed to detect sequence variation within a single amplified product, or multiple products can be amplified
simultaneously, with one (or more) probes used for subsequent detection. A combination of both approaches can also be used within a single assay. The ability to include multiple probes for a single target sequence makes the assay highly specific.
Published applications of mPCR/RLB include detection of antibiotic resistance genes1,2, typing of methicillin-resistant Staphylococcus aureus3-5 and Salmonella sp6, molecular serotyping of Streptococcus pneumoniae7,8, Streptococcus agalactiae9 and enteroviruses10,11, identification of Mycobacterium sp12, detection of genital13-15 and respiratory tract16 and other17 pathogens and detection and identification of mollicutes18. However, the versatility of the technique means the applications are virtually limitless and not restricted to molecular analysis of micro-organisms.
The five steps in mPCR/RLB are a) Primer and Probe design, b) DNA extraction and PCR amplification c) Preparation of the membrane, d) Hybridization and detection, and e) Regeneration of the Membrane.

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

An inexpensive, high throughput method for simultaneous detection of up to 43 molecular targets is described. Applications of mPCR/RLB include microbial typing and detection of multiple pathogens from clinical samples.

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

Biology

Electrochemiluminescence Assays for Human Islet Autoantibodies

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ECL assay is a nonradioactive fluid phase assay for islet autoantibodies with higher sensitivity and specificity than the current 'gold' standard radio-binding assay (RBA). ECL assays can more precisely define the onset of presymptomatic T1D by distinguishing the high-risk, high-affinity autoantibodies from the low-risk, low-affinity autoantibodies generated in RBAs, and conventional enzyme-linked immunosorbent assays (ELISA). The antigen protein used in this ECL assay is labeled with Sulfo-tag and Biotin, respectively. Each ECL autoantibody assay that uses a particular antigen protein needs an optimization step before it can be used for laboratory application. This step is especially vital in determining the requirements for serum acid treatments, concentrations, and ratios of the two different antigens labeled with Sulfo-tag and Biotin. To perform the assay, serum samples are mixed with Sulfo-tag-conjugated and biotinylated capture antigen protein in phosphate buffered solution (PBS), containing 5% Bovine Serum Albumin (BSA). Afterwards, the samples are incubated overnight at 4 &#176;C. The same day, a streptavidin-coated plate is prepared with blocker buffer and incubated overnight at 4 &#176;C. On the second day, wash the streptavidin plate and transfer the serum-antigen mixture onto the plate. Place the plate on the plate shaker, set it at low speed, and incubate at room temperature for 1 h. Subsequently, the plate is washed again, and reader buffer is added. The plate is then counted on the plate reader machine. The results are conveyed through an index, which is generated from internal standard positive and negative control serum samples.

Here, we presented the methods to detect human islet autoantibodies using electrochemiluminescence (ECL) assays. The protocol, used to predict type 1 diabetes, can be expanded upon to detect autoantibodies for other autoimmune diseases.

Pinpointing islet autoantibodies associated with type 1 diabetes (T1D) leads the way to project and deter this disease in the general population. A novel ...

Immunology and Infection

Use of Capillary Electrophoresis Immunoassay to Search for Potential Biomarkers of Amyotrophic Lateral Sclerosis in Human Platelets

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant proteins and drugs in clinical trials. Reproducibility, sensitivity, small sample volume requirement, multiplexing antibodies for multiple protein labeling in the same sample, automated high-throughput ability to analyze up to 24 individual samples, and short time requirement make CEI advantageous over the classical western blot immunoassay. There are some limitations of this method, such as the inability to utilize a gradient gel (4%&#8211;20%) matrix, high background with unrefined biological samples, and commercial unavailability of individual reagents. This paper describes an efficient method for running CEI in a multiple assay setting, optimizing protein concentration and primary antibody titration in one assay plate, and providing user-friendly templates for sample preparation. Also described are methods for measuring pan TDP-43 and phosphorylated TDP-43 derivative in platelet lysate cytosol as part of the initiative in blood-based biomarker development for neurodegenerative diseases.

Blood-based biomarkers for neurodegenerative diseases are essential for implementing large-scale clinical studies. A reliable and validated blood test should require a small sample volume as well as be a less invasive sampling method, affordable, and reproducible. This paper demonstrates that high-throughput capillary electrophoresis immunoassay satisfies criteria for potential biomarker development.

Capillary electrophoresis immunoassay (CEI), also known as capillary western technology, is becoming a method of choice for screening disease relevant ...

Neuroscience

A Sensitive and Specific Quantitation Method for Determination of Serum Cardiac Myosin Binding Protein-C by Electrochemiluminescence Immunoassay

Biomarkers are becoming increasingly more important in clinical decision-making, as well as basic science. Diagnosing myocardial infarction (MI) is largely driven by detecting cardiac-specific proteins in patients' serum or plasma as an indicator of myocardial injury. Having recently shown that cardiac myosin binding protein-C (cMyBP-C) is detectable in the serum after MI, we have proposed it as a potential biomarker for MI. Biomarkers are typically detected by traditional sandwich enzyme-linked immunosorbent assays. However, this technique requires a large sample volume, has a small dynamic range, and can measure only one protein at a time.	
Here we show a multiplex immunoassay in which three cardiac proteins can be measured simultaneously with high sensitivity. Measuring cMyBP-C in uniplex or together with creatine kinase MB and cardiac troponin I showed comparable sensitivity. This technique uses the Meso Scale Discovery (MSD) method of multiplexing in a 96-well plate combined with electrochemiluminescence for detection. While only small sample volumes are required, high sensitivity and a large dynamic range are achieved. Using this technique, we measured cMyBP-C, creatine kinase MB, and cardiac troponin I levels in serum samples from 16 subjects with MI and compared the results with 16 control subjects. We were able to detect all three markers in these samples and found all three biomarkers to be increased after MI. This technique is, therefore, suitable for the sensitive detection of cardiac biomarkers in serum samples.

Measuring biomarkers in complex biological samples is increasingly guiding clinical decision-making. We describe a highly sensitive method to simultaneously measure cardiac myosin binding protein-C, creatine kinase MB, and cardiac troponin I in serum samples from subjects with myocardial infarction and healthy control subjects.

Biomarkers are becoming increasingly more  important in clinical decision-making, as well as basic science. Diagnosing  myocardial infarction (MI) is ...

Electrochemiluminescence Assay for Quantification of Target Protein Levels in Brain Lysate

Source: Steinkellner, H., et al., <a href="https://app.jove.com/v/61054">An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants.</a> J. Vis. Exp. (2020).
In this video, we demonstrate the electrochemiluminescence immunoassay for the quantitation of target protein levels in different samples. This assay is based on the luminescence produced in the electrochemical reaction. The intensity of light emitted is proportional to the quantity of the target protein in the sample.

Source: Steinkellner, H., et al., An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants. J. Vis. Exp. (2020).
In this video, we demonstrate ...

An Electrochemiluminescence-Based Assay for MeCP2 Protein Variants

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