Human blood sample sources and collections
All methods were carried out in accordance with relevant guidelines and regulations. The blood samples collected in BD Vacutainer Plus Blood Collection Tubes containing K2EDTA were centrifuged at 2000g 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. 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 − 80 °C upon receipt until used for this study. Prior to the use for D2Dx assay, the samples were thawed for overnight at 4 °C, and then left to equilibrate at room temperature for 2 h 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 °C). 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. Informed consent statement from healthy individual donors was obtained as well. 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.
Patient cohorts and demographic information
In this study, we tested 142 negative control samples and 153 COVID-19 positive samples. Table 1 summarizes the clinical status and sample size of each study cohorts. We included three negative control groups. Previously, we collected 80 blood plasma samples from healthy donors in December 2018, about 1 year before the COVID-19 outbreak. The samples were collected at two geographic locations. A total of 42 samples were from the United States (Normal-USA cohort) and 38 samples were from Dominican Republic (Normal-DR cohort). A third cohort of 62 samples were collected at Orlando Health from healthy volunteer donors (Normal-OH cohort) from April to August 2020, in the same time period when we collected COVID-19 positive samples. These volunteer donors were tested negative in anti-SARS-CoV-2 IgG and IgM serology test, and never reported any clinical symptoms associated with COVID-19.
The 153 COVID-19 positive samples were collected between April to August 2020 in Orlando Health (Orlando, Florida). In this study, we used the World Health Organization (WHO) Eight Category Ordinal Scale for Clinical Improvement45, as used in the seminal Remdesivir trial46, to rank the clinical severity of the patients and group the patients into different cohorts. The uninfected controls are assigned with a 0 score. Patients who were tested positive but exhibited no or only mild symptoms were assigned with a score of 1 (ambulatory with no limitation of activities) or 2 (ambulatory with limitation of activities). These patients were either not hospitalized or were hospitalized for unrelated conditions and found to be positive. From score 3 and above, all patients were hospitalized and treated in various hospitals at Orlando Health. Blood samples were collected during patients’ stay in the hospital. The symptoms exhibited by the patient at the time of blood draw were documented. According to the clinical symptom severity, the patient was assigned with the Ordinal Scale from 3 to 7: 3—patients were hospitalized, but no oxygen therapy; 4—patients require oxygen by mask or nasal prongs; 5—patients require non-invasive ventilation or high flow mask; 6—patients require intubation and mechanical ventilation; 7—patients require ventilation and additional organ support such as vasopressors, RRT, and ECMO. We did not include samples from patients who died of COVID-19 in our study, which would be scale 8. In keeping with the Eight Category Ordinal Scale, patients with scale 1–2 were further grouped as asymptomatic/mild cohort (38 samples); patients with scale 3–4 are grouped as moderate cohort (54 samples); and patients with scale of 5 and above are group as severe cohort (61 samples).
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 s using a mini-vortex mixer, the cuvette was placed in the cuvette holder in CT-100, and the result was read automatically in 30 s. The response of the test was reported directly as the absorbance change of the assay solution over a 30-s 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 s using a mini vortex mixer, the cuvette was placed in the colorimeter, and the absorbance change of the assay solution was recorded every 30 s for a total reaction time of 3 min.
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 µL of dilution buffer, mixed thoroughly, sealed with cover film, and incubated at 37 °C for 30 min. The plate was washed five times using 300 µL of 1 × 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 Akoglu47: 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.
