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 in some biological fluids, and are restricted to the measurement of only one analyte per ELISA².

Recently, a new immunoassay technique was introduced that circumvents many of these drawbacks. This modified assay, developed by MSD, uses electrochemiluminescence (ECL) for signal detection, which allows for a very low background and an increased sensitivity, enabling the use of small sample volumes. Electrochemiluminescence is based on the reporter molecule ruthenium (II) trisbipyridal, which is attached to the detection antibodies. This reporter molecule emits light at 620 nm upon the electrical stimulation of the bottom of the 96-well plate which has carbon electrodes integrated in them³. Also, by using spot coating, multiple capture antibodies can be coated into one well (up to 10 on a 96-well plate), allowing for simultaneous quantification of different proteins in a single sample⁴. This technique has recently been used to measure proinflammatory cytokine profiles in serum^5,6. The multiplex plates from MSD compare favorably to other multiplex assay platforms⁷.

Using MSD as the primary assay platform, we further developed a custom made 3-plex plate that can simultaneously quantify the level of cardiac myosin binding protein-C (cMyBP-C), creatine-kinase MB (CK-MB), and cardiac troponin I (cTnI), and the results were compared with monoplex detection of cMyBP-C. CK-MB and cTnI are well-established biomarkers for MI. However, increases of these biomarkers can be caused by pathologies other than MI, e.g. myocarditis or renal failure⁸. This argues for the addition of additional biomarkers to increase the specificity of MI diagnosis. We have recently shown that cMyBP-C is also a potential biomarker for MI⁹. cMyBP-C is a thick filament associated protein that is expressed in the heart,^10-12 but not in skeletal or smooth muscles. Thus, the increased level of cMyBP-C in the circulatory system is a specific indicator of cardiac damage¹³.

In this study, we compared uniplex detection of cMyBP-C with the use of a custom 3-plex assay to measure serum levels of cMyBP-C, CK-MB, and cTnI in serum of patients with MI. In the future, this signature technique might be used to diagnose MI in patients presenting with chest pain in the emergency room.

The institution review board (IRB) of the Loyola University Chicago approved the study for use of deidentified human samples and the use of the immunoassay (LU# 20392).

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 stack of paper towels. Non-specific binding to the plate is blocked by adding 150 μl of a 5% (w/v) blocker A (high-grade BSA) solution in PBS to each well.
5. Seal the plate and incubate for 1 hr at RT while shaking at 700 rpm.
6. During the blocking step, prepare the standards and samples. Make the standard series by diluting recombinant cMyBP-C protein fragment (amino acids 1 - 271) to a starting concentration of 2,000 ng/ml in 1% (w/v) blocker A/PBS. Then serially dilute by a factor of 5 in 1% (w/v) blocker A/PBS. Total of 7 standards + 1 blank (1% (w/v) blocker A/PBS alone) were used.
7. Remove the blocking solution and wash the plate 3x with 150 μl 0.05% (v/v) Tween-20/PBS. Each time, remove the wash solution by inverting the plate above a sink. After the third wash step, vigorously flick the plate over a sink and pat the plate vigorously on a layer of paper towels until it is completely dry. This is a crucial step, as incubation volumes are small, and any remaining wash solution will significantly dilute the next incubation.
8. Pipette 25 μl of standards and samples into the wells. Seal the plate and incubate at RT, while shaking at 700 rpm for 1 hr.
9. Prepare the detection antibody solution at 1 μg/ml in 1% blocker A/PBS. A custom made cMyBP-C antibody (epitope amino acids 2 - 14) with MSD SULFO-TAG labeling serves as the detection antibody. Kits are available for SULFO-TAG labeling, making it a relatively simple procedure to label any antibody.
10. Repeat the wash step as described in step 1.4.
11. Add 25 μl of detection antibody solution to each well, seal the plate, and incubate at RT for 1 hr on a plate shaker set at 700 rpm.
12. During the incubation period, run MSD's demo-plate (plate with LED lights) to ensure proper function of the Sector Imager and software (refer Reagent section). Also, dilute the read-buffer, which is provided by MSD, by adding 5 ml of 4x read-buffer to 15 ml distilled water.
13. Repeat the wash step as described in step 1.4.
14. Add 150 μl of read-buffer to each well by using a multichannel pipette. Be sure to avoid air bubbles (reverse pipetting is a suitable method). After addition of the read-buffer, immediately scan the plate in the Sector Imager.

2. 3-plex cMyBP-C, CK-MB, and cTnI Assay

1. A 96-well 3-plex plate and diluent solutions are allowed to equilibrate to RT before use. This plate is pre-coated with capture antibodies for cMyBP-C, CK-MB, and cTnI by MSD.
2. Add 25 μl of 1% (w/v) blocker A/PBS to each well. Tap the sides of the plate to make sure that the entire well is covered by solution.
3. Seal the plate with a plate sealer, place the plate on a plate shaker, and shake at 700 rpm for 30 min.
4. In the meantime, a dilution series of individual calibrators is prepared. Because each well has three capture antibodies three calibrators need to be mixed and serially diluted before use. Dilute recombinant cMyBP-C, CK-MB (MSD), and cTnI (MSD) together in 1% (w/v) blocker A/PBS to create one sample with 2,000 ng/ml cMyBP-C, 100 ng/ml CK-MB, and 25 ng/ml cTnI (sample A).
5. A final volume of at least 100 μl is prepared for each standard. To complete the standard series, serially dilute the samples are by a factor of 5. Use 25 μl of sample A in 100 μl 1% (w/v) blocker A/PBS to create sample B. Then use 25 μl of sample B in 100 μl 1% (w/v) blocker A/PBS to create sample C, and so forth. Human serum samples from 16 subjects with MI and 16 control subjects were used to measure cMyBP-C, CK-MB, and cTnI levels. These samples were diluted 2x in 1% (w/v) blocker A/PBS.
6. Add 25 μl of the standards and diluted samples to the 25 μl already present in the wells of the 3-plex plate. It is important to also include a blank (diluent only). To establish the technique, samples are loaded in technical triplicates. Otherwise, duplicates are sufficient.
7. Seal the plate again (plate sealer can be reused) and incubate on a plate shaker (700 rpm) at RT for 2 hr.
8. Just before the incubation is finished, the detection antibody solution is prepared. The diluent used is 1% (w/v) blocker A in PBS. All three SULFO-tagged detection antibodies are diluted together into a final concentration of 1 μg/ml each. Detection antibodies are typically provided at a 50x stock concentration.
9. For a full 96-well plate, 2,700 μl of total diluted solution is needed (with pipetting error included). Add 54 μl of each detection antibody to 2,538 μl 1% (w/v) blocker A/PBS.
10. After 2 hr, remove solutions by vigorously flicking the plate above a sink. Wash the wells 3x with 150 μl of 0.05% Tween-20 in PBS. Add 25 μl of detection antibody solution to each well using a multichannel pipette, and the sides of the plate are tapped to ensure that the entire well is covered by solution.
11. Seal the plate and incubate for 1 hr while shaking at 700 rpm.
12. During the incubation period, run MSD's demo-plate (plate with LED lights) to ensure proper function of the Sector Imager and software. Also dilute the read-buffer, which is provided by MSD, by adding 5 ml of 4x read-buffer to 15 ml distilled water.
13. Repeat the wash step described in step 2.8.
14. Add 150 μl of read-buffer to each well using a multichannel pipette. Avoiding air bubbles is critical (reverse pipetting is a suitable method). After addition of the read-buffer, immediately scan the plate in the Sector Imager.

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