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The breakdown of the N-methyl-D-aspartate (NMDA) produces the NR2 peptide, which in turn can be detected in the blood. The general ELISA method has been modified to ensure specific binding and stability by using magnetic particles. This enables the measurement of the NR2 peptide and is analyzed based on the concentrations and compared to that of a healthy person. To conduct this test a 1-step bridging method is employed in the MP ELISA that can measure the NR2 peptide levels in 20 ul of EDTA plasma samples. A set of recombinant NR2 peptide calibrators (0-10 ug/L) are used. The assay measures the free NR2 peptide in equal concentrations with the antibody and there is no cross reaction with the other types of neuroreceptors. The experimental results for the within-run and precision for the NR2 peptide was about 4.2% and 4.9% for the low concentration (0.75 ug/L); 3.8% and 4.0% for mid-level (6.3 ug/L); and 3.1% and 3.8% for the high concentration (16.0 ug/L). The results for lot to lot were very reproducible with a slope of 0.87, intercept of 0.03 and an R2 value of 0.99 when tested with 14 spiked plasma samples. The sensitivity of the assay with a 20% CV was 0.23 ug/L and the dilution studies showed a recovery of 93%. The cross-reactivity experiment showed that at 2X physiological concentrations, there was no reaction with triglycerides, creatinine, cholesterol, aspirin, bilirubin, and human serum albumin; but there was a significant reaction with hemoglobin. In conclusion, the development of a sensitive and reliable MP based ELISA assay to detect NR2 peptide levels has been successful. This assay can measure NR2 peptide levels in health population with <10% CV, thus the results suggest that this assay is a good investigational tool for the NR2 peptide as a biomarker for the cerebral ischemic events.
In an ischemic event, there is restricted flow of oxygen and glucose to the brain and this leads to the release of glutamate. The release of glutamate is caused by the overactivation of the NMDA receptors. This leads to the cleaving of the NR2 subunit from the NMDA receptors by serine proteases. The goal of this study is to quantitatively measure the peptide fragments in the blood and establish a working model. The Magnetic Particle ELISA is going to be evaluated and analyzed. This study will help test the assay's performance characteristics and discover various ways to improve and further the assays ability.
Methods & Materials:
Magnetic Particles ELISA
The concentrations of NR2 peptide is determined immunochemically in a plasma samples using Gold Dot MP-ELISA test kit (CIS, Biotech, Inc). The unlabeled antibodies against NR2A/2B peptide immobilized onto the magnetic particles react with the sample containing NR2A peptide and HRP-conjugated antibodies against NR2A/2B peptide, forming the bridge. Colorimetric substrate is then added to visually detect the presence of NR2 peptide. The intensity of the color is directly proportional to the concentration of NR2 peptide. The basic procedure for MP-ELISA is to first thaw the plasma samples from the -20oC or -80oC to 4oC. In the meantime, PBST needs to be prepared by dissolving the ready to go tablet in 500 milliliter of distilled water. The TMB is set out from 4oC to room temperature. Next, the dry stop reagent is also dissolved in distilled water and allowed to sit for 5 - 10 minutes. Once samples are thawed, the reagent mixture can be prepared. The mixture consists of 78 microliters of PBST, 1 microliters of MP 280 IgY and 1 microliters of anti NR2 IgY-HRP per well. Next, 80 microliters of reagent mixture and 20 microliters of the thawed sample are placed in the well along with a set of calibrators and placed in the incubator on a shaker at 37oC for 30 minutes. After the incubation, 3 washes are performed by placing the microtiter plate onto a magnet that will separate the magnetic particles from the liquid. The liquid is then suctioned out or pipetted out and 200 microliters of PBST buffer is added after the microtiter plate is removed from the magnet. After the 3 washes with PBST are complete, a final wash with deionized water is performed. Then, 100 microliters of TMB substrate is added to each well and allowed to incubate at room temperature for 10 minutes in the dark. One the 10 minutes is up, the microtiter plate is placed on the magnet to allow for the particles to aggregate together so that the stop reagent can be added. The plate is placed on the magnet after the TMB because it was noted that the addition of stop reagent causes the particles to cloud and become foggy thus possible interfering with the optical density reading. Finally, 100 microliters of stop reagent is added and placed in the reader and optical density is measured between 450nm and 630nm.
Table 1. Within-Run and Total Precision
The test was performed according to CLSI guidelines EP5-A2. Each sample was tested in duplicate, twice a day for at least 20 consecutive days. Runs performed each day were separated by 2 hours. Within-run and total precision were determined by testing calibrators containing low, medium and high NR2 peptide concentrations. Calibrator performance data showed good standard deviations, variance (SD2), and coefficient variations in all assay characteristics.
Figure 1. Lot-to-Lot Reproducibility
The lot-to-lot reproducibility was tested by analyzing 14 spiked plasma samples with two different reagent lots over 20 runs, in quadruplets (n=80). This lot-to-lot comparison showed excellent correlation, with a coefficient of 0.99.
Table 2. Analytical Sensitivity
The standard first calibrator was tested on 20 replicates of runs of two 2 independent lots. The minimal detectable concentration of NR2 peptide corresponds to concentration given by two standard deviations above mean concentration and equals to 0.18 µg/L.
Figure 2. Functional Sensitivity
The functional sensitivity of the assay at 20% CV was 0.23 µg/L.
Table 3. Dilution Recovery
Example of dilution recovery of plasma sample with high NR2 peptide concentration.
Figure 3. Linearity
Linearity was conducted according to CLSI guidelines EP6-A. A normal human plasma (NHP) and human plasma with high titer of NR2 peptide were mixed to obtain 6 serial dilutions from low to high range of NR2 peptide. Four replicates were analyzed per dilution. Regression analysis yielded a good linearity with R2=0.997.
Table 4. NR2 Peptide Reference Value
The distributions of NR2 peptide values in plasma samples from apparently healthy males (n=52) and apparently healthy females (n=102), in the clinically relevant age range of 30-70 years, were evaluated. The reference population was represented by the following ethnic backgrounds: African-American (n=84), Caucasian (n=67), and Asian (n=3). The median NR2 peptide value for a healthy male and female population (n=154) was 0.5 µg/L.
Table 5. NR2 Peptide in Patients with Pre-existing Conditions
Figure 4. Interfering Substances
The Interfering Substances were tested in a pooled normal human plasma (pNHP) according to CLSI guidelines EP7-A2. Commercially available substances were diluted in distilled water and spiked into pNHP. Interference tests were performed using spiked levels of albumin 5 g/dL, bicarbonate 130 mg/dL, bilirubin 15 mg/dL, cholesterol 250 mg/dL, creatinine 5 ng/mL, hemoglobin 20 g/dL. No interference was observed in spiked samples containing, triglycerides, cholesterol, bilirubin, and albumin. No interference was observed in pNHP.
A significant interference was detected with hemoglobin.
The NR2 peptide assay showed good within-run and total precision results. The lot-to-lot comparison showed excellent correlation, with a coefficient of 0.99. The NR2 peptide assay demonstrated analytical sensitivity of <0.18 µg/L and functional sensitivity of 0.23 µg/L at 20% CV. The method demonstrated to be linear from minimum to maximum NR2 peptide serum concentrations. The NR2 peptide assay showed no interference from elevated levels of protein, bicarbonate, bilirubin, cholesterol, creatinine. Significant interference was detected only with hemoglobin. NR2 peptide assay is a precise method for measurement of NR2 peptide (%CV<10%). There currently is no simple, rapidly available blood test that can assess the near- and long-term risks of transient ischemic attack (TIA) and stroke and distinguish them from the non-ischemic conditions that can mimic stroke. In conjunction with clinical assessment and brain imaging, these biomarkers may provide greater diagnostic certainty in the evaluation of patients presenting with stroke-like symptoms. The in vitro device (IVD) Gold Dot NR2 Peptide is a diagnostic test that identifies plasma NR2 peptide, a subtype of NMDA receptors. The Gold Dot NR2 Peptide test has the ability to diagnose near-term cerebrovascular events, transient ischemic attack (TIA) and acute ischemic stroke and distinguish it from intracerebral hemorrhage (ICH) and stroke mimics. Thus, this biomarker has the potential to save many lives. It can save hospitals money and time. Overall, this diagnostic tool can become the standard of care in the diagnosis of stroke.