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

The overall goal of this procedure is to simultaneously quantify cardiac myosin binding protein C and cardiac troponin I in serum samples. This is accomplished by first coating a 96 well plate with cardiac myosin binding protein C antibody for a plex assay, or by using a pre-coded threeplex assay. In the second step, the coated plates are incubated with calibrators and serum samples.

Next labeled detection antibodies are added in the final step, an chemiluminescence signal is produced that is detected by the plate reader. Ultimately, the levels of the cardiac proteins of interest present in serum samples can be determined by single or multiplex immunoassays. The day before the experiment pipette 30 microliters of gelatin free mouse monoclonal anti cardiac myosin binding protein C antibody into the bottom corner of each well of a 96 Well miso scale discovery bare standard plate.

Then tap the sides of the plate to spread the antibody solution over the bottom of each well. After sealing, incubate the plate overnight at four degrees Celsius without shaking. The next morning, tap the solution out over a sink and then onto a stack of paper towels.

Then add 150 microliters of 1%blocker A in PBS to each well to block nonspecific binding. Incubate the plate sealed for one hour at room temperature while shaking at 700 RPM during the blocking step, dilute the recombinant cardiac myosin binding protein C fragment to a starting concentration of 2000 nanograms per milliliter in 1%blocker A in PBS. Then serially dilute this solution by a factor of five for a total of seven standards.

Now remove the blocking solution as just demonstrated, and then wash the plate three times with 150 microliters of 0.05%tween 20 in PBS. After the third wash, step vigorously flick the plate over a sink and then firmly pat the plate on a layer of paper towels until it is completely dry. Next, pipette 25 microliters of each standard and sample into the appropriate wells.

Then while the sealed plate is incubating on the shaker for one hour at room temperature dilute custom made cardiac myosin binding protein C detection antibody labeled with sul oag to one microgram per milliliter in 1%blocker A in PBS. After carefully washing the plate as just demonstrated, add 25 microliters of detection antibody solution to each well and then incubate the sealed plate for one hour at room temperature while shaking during the incubation period, run the demo plate with LED lights to ensure the proper function of the sector imager and software dilute the Reid Buffer at this time as well. After carefully washing the plate as previously demonstrated, use a multi-channel pipette to add 150 microliters of Reid Buffer to each well taking care to avoid air bubbles, and then immediately scan the plate in the sector imager before beginning the threeplex assay.

Allow the 96 well threeplex plate and diluent solutions to equilibrate to room temperature. Then add 25 microliters of 1%blocker A in PBS to the plate, making sure that the entire well is covered by solution and incubate the sealed plate at room temperature for 30 minutes while shaking. In the meantime, dilute recombinant cardiac myosin binding protein C-C-K-M-B and cardiac troponin I together in 1%blocker A in PBS to create a standard containing 2000 nanograms per milliliter of cardiac myosin binding protein C 100 nanograms per milliliter of CKMB and 25 nanograms per milliliter of cardiac troponin.

I then serially dilute this mixture by a factor of five to a final volume of at least 100 microliters for each standard dilute samples two times with 1%blocker A in PBS. Next, add 2 25 microliter technical replicates of the standards as well as the samples to the 25 microliters of blocking buffer already present in the wells of the three plex plate, include a blank of diluent only after incubating the sealed plate while shaking, dilute the three SUL oag detection antibodies together to a final concentration of one microgram per milliliter each in 1%solution blocker A and PBS after two hours. Wash the plate as just demonstrated, and then use a multi-channel pipette to add 25 microliters of detection antibody solution to each.

Well incubate the sealed plate for one hour while shaking at 700 RPM during the incubation period. Run the demo plate and dilute the Reid buffer as just demonstrated. Then after washing the plate with 0.05%tween 20 in PBS, use the multichannel pipette to add 150 microliters of Reid Buffer to each well avoiding air bubbles and immediately scan the plate in the sector imager.

In this figure, the standard curve of cardiac myosin binding protein C in the plex assay is compared to that of cardiac myosin binding protein C in the three plex assay. Both assays show a very high sensitivity and a high dynamic range. The detection area of the assay is displayed in gray, both plex and three plex.

Cardiac myosin binding protein C standard curves are similar. The lower limit of detection, lower limit of quantification and upper limit of quantification levels are comparable in both the plex and threeplex assays. These graphs show cardiac troponin I and CKMB standard curves of the threeplex plate.

Both calibrators showed high sensitivity and a large dynamic range. The detection area of the assay is displayed in gray. The cross reactivity of the detection antibodies with the other two calibrators was studied by incubating the individual standard curves of all three calibrators with single detection antibodies only a low amount of cross reactivity was seen between the CKMB calibrator and both cardiac troponin I and cardiac myosin binding protein C detection antibodies.

While no cross reactivity was observed between the other calibrators and detection antibodies as proof of the applicability of the assay for the detection of cardiac injury, serum levels of 16 subjects with myocardial infarction were compared with a control group using the three plex plate serum levels of cardiac myosin binding protein C-C-K-M-B and cardiac troponin. I were determined all three biomarkers were increased in the patients with myocardial infarction compared to the control group.