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

Summary

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.

Abstract

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.

Introduction

The measurement of low amounts of protein in complex biological samples, such as serum, is of growing clinical importance for patient management, as well as basic science. For instance, an increase in serum levels of cardiac biomarkers, such as troponin I, in a clinical setting is consistent with acute myocardial infarction (MI)¹. To detect proteins in serum samples, standard enzyme-linked immunosorbent assay (ELISA) is the most often used technique as it has high sensitivity and allows for absolute quantification of the analyte. However, traditional ELISAs require a relatively large amount of sample (typically 100 μl), have high background signal....

Protocol

1. Uniplex cMyBP-C Assay

1. The day before the experiment, coat the 96-well MSD bare standard plate with capture antibody. For cMyBP-C, use a 30 μl volume of mouse monoclonal anti-cMyBP-C antibody (gelatin free) at a concentration of 5 μg/ml diluted in phosphate buffered saline (PBS).
2. Since the well is hydrophobic, pipette the solution into the bottom corner of the well; then tap the sides of the plate to spread the solution over the entire well.
3. The plate is covered with a plate sealer and incubated O/N at 4 °C without shaking.
4. Remove the capture antibody solution by tapping the solution out over a sink and then on a s....

Results

The basic principles and workflow of the 3-plex assay are shown in Figure 1, and the overall workflow is described in Table 1. Uniplex assays work along the same principle except that the entire bottom well is coated with one capture antibody. Signal detection is done by ECL in which an electrical signal is applied to the bottom of the well. This, in turn, initiates a local production of light through a chemical reaction of the read-buffer with the SULFO-TAG label on the detection antibo.......

Discussion

In this study, we show the applicability of a multiplex immunoassay for the detection of multiple cardiac biomarkers in serum from patients. This technique has numerous advantages over traditional ELISA. First, ECL in the assay kit is used instead of colorimetric detection. In ECL, an electrical signal stimulates the local production of the measured property (light), thus uncoupling the stimulation event from the signal, which reduces background¹⁴. This allows for high sensitivity, as can be seen in Ta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
MSD Read-Buffer T (4X) with surfactant	Meso Scale Discovery	R92TC-2
Tween-20	Acros	9005-64-5
MSD Blocker A	Meso Scale Discovery	R93BA-1
Phosphate buffered saline, 10X	Fisher BioReagents	BP399-4	Filter before use
Myosin binding protein C antibody, mouse monoclonal (E7), gelatin free	Santa Cruz	SC-137180L	For coating, antibody has to be gelatin free
Plates and Equipment
Multi-Array 96-well standard plate	Meso Scale Discovery	L15XA-3
A prototype 3-plex, four-spot, 96-well plate with cMyBP-C, cTnI and CK-MB	Meso Scale Discovery	N452A-1
ThermaSeal Sealing Film	Life Science Products Inc.	ST-3098
MSD SECTOR Imager 2400A	Meso Scale Discovery
MTS 2/4 digital microtiter shaker	IKA	3208001