Human Blood Samples. Human blood plasma samples were used in this study. The blood samples collected in BD Vacutainer Plus Blood Collection Tubes containing K2EDTA were centrifuged at 5K RPM for separation into cells and plasma as supernatant. The blood plasma samples were obtained from two different sources. Plasma samples of deidentified healthy controls (n = 80) were previously obtained and archived from Boca Biolistics (Boca Raton, Florida) in December 2018 for a different study. According to NIH human subject research guidelines, the use of these samples is not considered as human subject research, therefore Institutional Research Board (IRB) approval was not required. Among the 80 samples, 42 were collected in the United States and 38 samples were collected in Dominican Republic. All donors were healthy donors without any reported or known infectious diseases when the samples were collected. The samples were aliquoted and stored at -80oC upon receipt until used for this study. Prior to the use for D2Dx™ assay, the samples were thawed for overnight at 4oC, and then left to equilibrate at room temperature for two hours before testing. The samples were tested without any dilution or other treatment.
We collected blood samples from COVID-19 patients and healthy volunteer donors at Orlando Health. The separated plasma supernatants were aliquoted and stored in freezer (-30 oC). Of which, 62 were from healthy donors and 153 were from COVID-19 positive cases. The study (OH IRB # 20.095.06) was approved by Orlando Health IRB#2. Informed consent was obtained from each patient. COVID-19 patients were treated at various hospitals within the Orlando Health hospital system as in-patients or out-patients. The IRB also approved to use remnant blood plasma or serum samples from patients and volunteers from a previous serology validation study. The clinical statuses of the study patients were obtained from the patient’s medical record, or by self-reporting by volunteer donors.
D2Dx™ immunity test of blood plasma samples. D2Dx™ immunity test kits (catalog D2Dx-hu-500, lot number hu08012020) were received from Nano Discovery Inc. (Orlando, Florida). Each kit contains the AuNP reagent and cuvettes for 500 tests. A handheld colorimeter reader device, CT-100 from Nano Discovery Inc. was used to read the test result. The specific composition and chemical structure of the AuNP reagent is proprietary information of Nano Discovery Inc. The AuNP reagent was manufactured, formulated, and calibrated using the CT-100 reader according to an internal quality control standard established by Nano Discovery Inc.
A 50 µL of the AuNP reagent solution was first placed into a cuvette using a micropipette. Then 10 µL of an undiluted blood plasma sample was added. After mixing the assay solution for 5 seconds using a mini-vortex mixer, the cuvette was placed in the cuvette holder in CT-100, and the result was read automatically in 30 seconds. The response of the test was reported directly as the absorbance change of the assay solution over a 30-second of reaction time.
Kinetic interaction study of AuNP with IgG subclasses. The study of the interaction between the AuNP reagents and IgG subclasses from bovine, human and murine was conducted using the following materials: Bovine IgG1 (pep003, Bio-Rad, 1 mg/mL); bovine IgG2 (pep004, Bio-Rad, 1 mg/mL); human IgG1 (ab90283, Abcam, 3 mg/mL); human IgG2 (ab90284, Abcam, 2.2 mg/mL); human IgG3 (ab118462, Abcam, 2.2 mg/mL); human IgG4 (ab183266, Abcam, 1.5 mg/mL); mouse IgG1 (02-6100, Thermofisher, 1 mg/mL); mouse IgG2a (02-6200, Thermofisher, 1 mg/mL); mouse IgG2b (02-6300, Thermofisher, 1 mg/mL); mouse IgG3 (IMG5119A, Novus Biological, 0.5 mg/mL).
The kinetic study was conducted using a LaMotte model 3250 colorimeter. To an optical cuvette, 100 µL AuNP reagent from the D2Dx immunity test kit (D2Dx-hu-500) was added. Then 10 µL of the IgG subclass protein solution was added. Following mixing for 5 seconds using a mini vortex mixer, the cuvette was placed in the colorimeter, and the absorbance change of the assay solution was recorded every 30 seconds for a total reaction time of 3 minutes.
SARS-CoV-2 specific antibody measurements: An enzyme-linked immunosorbent assay (ELISA) was used to detect SARS-CoV-2 specific IgG and IgM antibodies in plasma samples from patients (positive by PCR test with nasopharyngeal samples) and from donors with or without symptoms, who were tested positive or negative by PCR. Qualitative serology ELISA kits from Creative Diagnostics (Shirley, NY, USA) were used to measure anti-SARS-CoV-2 IgG (product # DEIASL019) and IgM (product # DEIASL020) levels in plasma samples. Briefly, 100 µL of negative and positive control were added to the positive and negative control wells without dilution. Ten microliters (10 µL) of plasma samples were added to the wells containing 100 ul of dilution buffer, mixed thoroughly, sealed with cover film, and incubated at 37°C for 30 minutes. The plate was washed five times using 300 microliters of 1x diluted wash buffer. After completing all the assay steps according to the product instructions, the absorbance of the assay solutions at 450 nm was measured using a plate reader (Synergy H1 Hybrid Reader, BioTek, USA). An OD value of ≥ 0.50 for the positive control and ≤ 0.10 for the negative control sample were obtained, confirming the validity of the assay, per product instruction. The following cutoff values, as provided in the product manual, were used for results interpretation: <0.3, negative; ≥ 0.3 to < 0.5, intermediate; ≥ 0.50, positive.
Statistical Analysis. Statistical differences of test results between different cohorts were analyzed using student t test, two-sample assuming unequal variances. P values < 0.05 were considered as significant difference. The numbers of asterisks indicate significance levels of P values, for example, the symbols of *, **, ***, and **** represent P values of ≤ 0.05, ≤ 0.01, ≤ 0.001, and ≤ 0.0001, respectively. If there is no significant difference (P > 0.05) between the groups, the results are presented as “ns”, namely, not significant.
Spearman’s rank-order correlation was used to analyze the correlation between the D2Dx immunity test scores of COVID-19 patients in the severe symptom cohort and the days from symptom onset to blood draw. The strength of the correlation was interpreted according to the scale suggest by Akoglu40: correlation coefficient 1 – perfect; 0.7–0.9 – strong positive correlation; 0.4–0.6 – moderate positive correlation; 0.1–0.3 – weak positive correlation; and 0 – zero correlation. Both student t test and Spearman’s rank-order correlation was conducted using the data analysis function in Microsoft Office 2010 Excel software.