3.1 NR2 Peptide Enzyme-Linked Immunoassay (ELISA)

The NR2 Peptide test is a magnetic particle-based enzyme-linked immunosorbent assay (MP-ELISA) intended for the quantitative determination of NR2 peptide fragment of NMDA receptors in plasma. The test is intended to be used in conjunction with clinical evaluation and radiological methods for diagnosis of acute ischemic stroke.

Concentrations of NR2 peptide are determined immunochemically in a blood assay.

In the first incubation step, NR2 peptides in the sample react with immobilized NR2 antibodies. In the second incubation step, horseradish peroxidase-labeled antibodies against NR2 peptides are added to the reagent mixture. Formed immunocomplex is determined using 3,3′,5,5′-tetramethylbenzidine (TMB) detection reaction. An acidic stopping solution is then added. The color converts from blue to yellow. The intensity of the color is directly proportional to the concentration of NR2 peptide in the sample. A dose response curve of the absorbance measured at 450 nm or using dual wave measurement at 450 nm and 630 nm vs. concentration is generated. NR2 peptide concentrations in the plasma samples are determined directly from this calibration curve. Results of a typical standard calibration curve with optic density (OD) readings at 450 vs. 630 nm are shown on the x-axis against NR2 peptide concentrations (ng/mL) shown on the y-axis (Fig. 3a).

3.2 NR2 Peptide Automatic Analyzer (AA) Assay

The assay transfer to automated analyzer platform employed paramagnetic particles and chemiluminescent technology. A modification is made to accommodate unique reagents for “on-board” detection using ready-to-use reagent packs. Preformed with streptavidin supermagnetic particles (0.8 μm) are bound to carrier antibodies (Ab) for the quantitative detection of NR2 peptide (Fig. 3b). The analyte in plasma competes with chemiluminescent labeled synthetic peptide for preformed with microparticles (MP)-polyclonal Ab (competitive assay). Translation from flat surface of microplate to microparticles increases reaction surface with simultaneous reduction in reagents consumption. That allows to speed up the turnaround time of laboratory testing from to 30 min.

Programmed AA separates, aspirates, and washes three times the cuvettes using buffer (PBS/Tween 20). Then it dispenses Base reagent to initiate the chemiluminescent reaction that automatically measured and reported in relative light units (RLU) according to software to the selected option, as described in the system operating instructions. Results of a standard calibration curve with RLU readings are shown on the y-axis against NR2 peptide concentrations (ng/mL) shown on the x-axis (Fig. 3c).

The anti-NR2 peptide assay kit uses a ready-to-use pack consisting solid phase compartment filled with 100 μg/mL latex paramagnetic MP preformed with biotinylated pAb. The second compartment of ready-to-use pack contains 10 ng/mL NR2—acridinium ester (AE) Lite reagent. The separate ancillary pack holds PBS-T buffer is necessary for reagent dilutions and pack for base reagents used to initiate the chemiluminescent reaction should be presented on-board of AA during the testing. Reagents in both packs are sufficient for 100 tests.

3.3 Nanoparticles Lateral Flow (nLF) Procedure for NR2 Peptide Detection

Recognizing the medical need of bedside testing for acute stroke, largely for neuro-critical care, point-of-care (POC) testing platform development has been initiated. Lateral flow based immunostrips engage gold nanoshells (150 nm) that dramatically increase sensitivity of detection reagents. The use of nanoshells results in a large visible signal even at low analyte concentrations.

The test principle is based on nanoparticles lateral flow technique and is a direct immunoassay [12]. The conjugate consists of nanoshells labeled with specific capture antibodies. NR2 peptide in the sample interacts with the conjugate to form a complex that migrates along the strip to the next section, which is the reaction matrix [12]. This reaction matrix is a nitrocellulose membrane, onto which the detection antibodies have been immobilized in bands to create test (T) and control (C) lines that serve to capture the NR2 peptide-conjugate complex and unbound conjugate, respectively. Excess reagents move past the capture lines and are entrapped in the wick or absorbent pad. Results are interpreted on the reaction matrix as the presence or absence of lines of captured conjugate, read either by eye or using a reader [12].

4.1 NR2 Ab ELISA Assay

The detection of the NR2 Ab on the microplate (MTP) loaded with synthetic NR2 peptide (antigen) is performed with horseradish peroxidase (HRP)-labeled Protein A (PrA) that reacts with the heavy chain of the human IgG from sample that is captured by antigen (Fig. 4a).

The detection of the formed immunocomplex—fixed on the MTP—is then determined by the HRP/tetramethylbenzidine (TMB)/H₂O₂-enzymatic reaction which is stopped by diluted acid. A color change from blue to yellow occurs after the addition of acid and the obtained color is measured photometrically at 450 nm referenced against 630 nm. Results of a typical standard calibration curve with optical density (OD) readings at 450 nm vs. 630 nm are shown on the x-axis against NR2 Ab concentrations (ng/mL) shown on the y-axis (Fig. 4b).

4.2 Automatic Analyzer Platform for NR2 Ab Assay

The automated analyzer platform for NR2 Ab also employs paramagnetic particles and chemiluminescent technology. Free antibodies from serum sample react with biotinylated NR2 peptide from ancillary pack then on-board coupling allowed before solid-phase streptavidin supermagnetic particles (0.8 μm) is added (Fig. 5a). Then anti-human antibodies or PrA labeled with acridinium ester bind in the first pass complex (direct sandwich assay). Programmed analyzer separates, aspirates, and washes three times the cuvettes using buffer. Then it dispenses base reagent to initiate the chemiluminescent reaction that automatically measured and reported in RLU according to software to the selected option. Results of a standard calibration curve with RLU readings are shown on the y-axis against NR2 Ab concentrations (ng/mL) shown on the x-axis (Fig. 5b).

The NR2 Ab assay kit uses a ready-to-use pack consisting solid phase compartment filled with 100 μg/mL latex paramagnetic MP. The second compartment of ready pack contains 0.5 μg/mL biotinylated NR2 peptide. The separate three ancillary packs hold (1) 10 ng/mL Protein A reagent, (2) buffer that is necessary for reagent dilutions, and (3) pack for base reagents used to initiate the chemiluminescent reaction. Reagents in all packs are sufficient for 100 on-board tests.

4.3 NR2 Ab LF Design

Figure 5c shows the configuration of nLF immunoassay for NR2 Ab detection in serum and whole blood. The assay consists of several overlapping zones, constituted by segments of various filters of different material and nitrocellulose mounted on a plastic card using an adhesive. To run a test, sample (20–30 μL) is added to the proximal end of strip, the filter pad where sample is treated making it compatible with the rest of test. Then sample migrates through this region to the conjugate pad containing gold nanospheres (80 nm) with attached modified NR2 peptide. The sample remobilizes the dried conjugate, and the NR2 Ab in the serum interacts with the conjugate as both migrate into the next section of the nitrocellulose strip or the reaction matrix. This reaction matrix contains one band of Protein A (test) and the second one of specific polyclonal or monoclonal antibodies against NR2 peptide (control band) where they serve to capture the analyte and the conjugate as they migrate by the capture lines. Excess reagents move past the capture lines and are entrapped in the wick or absorbent pad. Results are interpreted on the reaction matrix as the presence or absence of lines of captured conjugate, read either by eye or using a reader.

5.1 Original NR2 Peptide and Antibody Stability in Blood Samples

The recovery study of neurotoxicity biomarkers in blood samples (plasma and serum) from individuals is performed at approximate storage times spent by: (1) phlebotomist as a daily routine of blood draws in in-patient wards with storage on cold pack (+4 °C, 4 h), (2) reference laboratory representative for regular samples pick up at medical office’s outdoor storage containers. There is an adjustment of time of storage (24 h) made at room temperature (RT) accounting summer temperatures on South East of USA (35–40 °C) and 12-h shift. (3) The point of storage at −20 °C for 2 months is chosen as an average time of temporary refrigerated storage for research purposes.

On the basis of these results for NR2 peptide biomarker, it is recommended specimen collection is made in tubes containing EDTA (not heparinized) with immediate storage on cold packs. Samples should be centrifuged (3500 × g, 4 °C for 5 min) within 1 h of blood drawing. For storage up to a week, processed samples are stored in an aliquot tube at or below −20 °C. For longer storage, freeze samples at –80 °C.

Processed plasma samples may be frozen and thawed only once without significantly affecting the amount of NR2 peptide. To transport samples, shipment boxes in bigger Styrofoam box filled with dry ice (−78.5 °C, pellets or slabs not less than 3 lb per box) should be prepared.

NR2 antibodies in samples are stable for up to 3 h at room temperature. Serum samples may be tested within 3 days of collection when stored at 2–8 °C. It is recommended collecting blood samples in serum collection tube (e.g., gel separation). Samples should be centrifuged and separated at 2500 × g for 5 min within 3 h of venipuncture. Store all serum samples refrigerated (2–8 °C) immediately upon collection; however, for storage up to 3 months, processed aliquoted serum samples should be frozen at or below −20 °C and for longer storage at –80 °C.

Processed serum samples may be frozen and thawed up to two times without significantly affecting the amount of NR2 antibodies. To transport samples domestically, samples could be shipped at 2–8 °C on ice pack. For international delivery, frozen samples should be shipped on dry ice.

5.2 NR2 Peptide/Antibodies Assays Cut Offs, Turnaround Time, and Stability

6.1 NR2 Peptide MP-ELISA and AA Performance Characteristics

Bench testing has been conducted to evaluate the performance of the NR2 peptide and antibody assays before use for clinical studies. The results presented below showed that devices satisfy the diagnostic test performance requirements. Sensitivity, assay precision, linearity, interfering substances, interfering antibodies, cross-reactivity, spiking recovery, dilution recovery, hook effect, and reference interval were assessed according to corresponding NCCLS guidelines EP5-A (http://​www.​zxyjhjy.​com/​upload/​attached/​file/​20170406/​20170406154608_​8336.​pdf).

6.1.1 Assay Precision

For NR2 peptide ELISA assay each calibrator (n = 3) and control (n = 2) sample is tested in duplicate, twice a day for at least 20 consecutive days. Runs performed on daily basis are separated by 2 h. Within-run, between-run, between-day, and total precision were determined by testing calibrators containing NR2 peptide, negative and positive controls, negative serum samples, and positive serum samples. Calibrator performance data showed good standard deviations (<0.1), and coefficient variations (<10%) in all assay characteristics (Table 3).

Table 3

NR2 peptide ELISA assay precision results

Precision	Calibrator 1			Calibrator 2			Calibrator 3			Negative control			Positive control
	SD	Variance	CV	SD	Variance	CV	SD	Variance	CV	SD	Variance	CV	SD	Variance	CV
Repeatability (within-run)	0.045	0.002	3.80	0.074	0.005	3.37	0.098	0.010	2.70	0.019	0.002	4.14	0.104	0.011	3.17
Between-run, within day	0.018	0.000	1.55	0.072	0.005	3.31	0.106	0.011	2.92	0.008	0.001	1.73	0.051	0.003	1.55
Between-day	0.100	0.010	8.45	0.016	0.000	0.72	0.040	0.002	1.11	0.009	0.000	2.00	0.047	0.002	1.43
Within device	0.111	0.012	9.39	0.105	0.011	4.78	0.150	0.022	4.13	0.022	0.001	4.91	0.125	0.016	3.81

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