Samples and reference assays
WHO Third International Standard for HBsAg (12/226) was purchased from the National Institute for Biological Standards and Controls (NIBSC, United Kingdom). The 1st WHO International Reference Panel for Hepatitis B Virus (HBV) Genotypes for Hepatitis B Surface Antigen (HBsAg) Assays (6100/09) was purchased from Paul-Ehrlich Institute (Germany).
The following commercial sample panels were purchased from SeraCare Life Sciences Inc. (MA, USA): AccuSet™ HBsAg Performance Panel (0805-0340), AccuSet™ HBsAg Mixed Titer Performance Panel PHA207(M) (0805-0217), AccuSet™ HBV Worldwide Performance Panel (0805-0313), HBV Seroconversion Panel PHM941 (0606-0060) and 24 HBsAg-positive disease-state samples. These panels consist of undiluted, naturally occurring plasma samples. Fifty HBsAg-positive serum samples were purchased from Biomex GmbH (Germany). Eighteen HBsAg-positive plasma samples were purchased from Labquality Oy (Finland). Whole blood and serum used for international standard’s dilutions, as well as a sample panel consisting of 100 presumed HBsAg-negative serum samples, were purchased from Turku University of Applied Sciences (Finland).
Apart from commercially available sample panels, 215 clinical serum and plasma samples that tested negative for HBsAg and 16 samples that tested positive for HBsAg were obtained from the Department of Virology, University of Turku (Finland). All the patient data, except for the status for the presence or absence of HBsAg, were anonymized and no clinical data of the patients were handled. Use of these samples in this study was approved by the Ethical Committee of the Hospital District of Southwest Finland (Decision T012/011/18). The details of the reference tests used for the characterization of these samples are summarized in Table S1 (see Electronic Supplementary Material, ESM). Assay principle for the central laboratory reference tests is described in the ESM.
Apart from using central laboratory tests as reference, a commercially available conventional rapid LFIA (Alere DetermineTM HBsAg (code # 7D2543), Abbott Laboratories, USA) based on visual detection of signals was used for comparison.
Mouse monoclonal antibodies against HBsAg were generated using hybridoma technology .
Bioconjugation of UCNP reporters
Carboxylated Upcon™ UCNP reporter particles of 68-nm diameter with a hydrophilic coating (Kaivogen Oy, Finland) were covalently coupled with an in-house mouse anti-HBsAg monoclonal antibody (mAb) 3D3. A solution containing 1 mg UCNPs was centrifuged, the supernatant was removed, and the particle surface was activated by suspending the pellet into particle activation buffer (20 mM MES pH 6.1, 2 mM KF, 20 mM EDC, and 30 mM sulfo-NHS). The incubation was performed with rotation for 15 min at room temperature. The UCNPs were washed by centrifugation, removing the supernatant and suspending the UCNPs to 20 mM MES, pH 6.1. The UCNPs were centrifuged as before and resuspended into 20 mM MES, pH 6.1 containing 84 μg of mAb 3D3. This conjugation reaction was incubated for 2.5 h at room temperature with rotation and the reaction was stopped by adding glycine, pH 11. To remove non-covalently associated mAb, the UCNP conjugates were washed twice by centrifugation and the pellet was suspended in 500 μl of storage buffer (5 mM Tris, 0.05% NaN3, 0.05% Tween-85, pH 8.5). This step was repeated twice, and finally, the pellet was suspended to 250 μl of storage buffer and BSA was added to a final concentration of 0.5%.
Preparation of LFIA strips
A lateral flow card was assembled on a plastic backing by pasting nitrocellulose membrane 200CNPH-N-SS60 (Advanced Microdevices Pvt. Ltd., India), a cellulose absorbent pad (CFSP223000, Merck Millipore, MA, USA), a glass fiber conjugate pad (8951, Ahlstrom-Munksjö Oyj, Finland), and a red blood cell separator sample pad (FR1 0.6, Advanced Microdevices Pvt. Ltd., India). The sample pad was pre-treated with blocking buffer solution (10 mM Tris-HCl pH 8.5, 135 mM NaCl, 0.5% Tween-20, 0.1% Triton-X-100, 0.8 mg/ml mouse IgG, 0.2% denatured mouse IgG, 0.24% bovine IgG). The test line solution consisted of equal proportions of three anti-HBsAg mAbs. Two of these mAbs were in-house (mAb 3G8 and mAb 4G9) and one was procured from a commercial source (mAb 2508 SPTN-5, Medix Biochemica Oy Ab, Finland). The line dispensing was performed in 10 mM Tris-HCl, pH 8.0 buffer on the nitrocellulose membrane at a concentration of 300 ng/cm. The control line was printed 4 mm from the test line with 300 ng/cm of rabbit anti-mouse IgG (Dako Products, Agilent Technologies Inc., USA). After printing test and control lines, the cards were dried overnight at + 35 °C, other components of the test strips were assembled, and the cards were stored at room temperature protected from humidity. The cards were covered with transparent cover tape (KN-CPP1-Clear Kenosha cover plastic, Kenosha, Netherlands) starting from the conjugate pad to the end of the absorbent pad. Before the use, the cards were cut into 4.8-mm-wide strips. Twenty nanograms of UCNP reporters were dried onto the conjugate pad of each of the strip in 10 mM Tris-HCl buffer, pH 8.5 containing 135 mM NaCl, 0.5% Tween-20, 1% BSA, 5% sucrose. The strips were dried protected from humidity. The strips were placed in a suitable plastic housing with a common inlet for sample and buffer.
To start the procedure, 50 μl of the sample was added into the sample inlet, followed by applying 50 μl of chase buffer (10 mM Tris-HCl pH 8.5, 135 mM NaCl, 0.5% Tween-20, 1% BSA, and 0.1% Triton-X-100). After 30 min, the test and control line upconversion photoluminescence signals were measured with an Upcon reader device (Labrox Oy, Finland) with excitation at 976 nm and emission at 550 nm. Details of the benchtop UCNP reader and a battery-operated portable UCNP reader are provided in the ESM (Fig. S10).
The interpretation of the results was based on the maximum signal measured at the test line position. The overall baseline signal measured along the strip was subtracted from the test line maximum signal. Thus, only the test line peak signal was considered the outcome of the measurement. The control line signals were interpreted as qualitative control.
Limit of detection
Two methods were used for determining the limit of detection (LoD), both in serum and in whole blood. In the first method, dilutions of the WHO Third International Standard for HBsAg between 0.01 and 12.8 IU/ml were used for plotting standard curves. For the standard curve, 20 replicates were used for each of the three low concentration dilutions (0.05, 0.1, and 0.2 IU/ml) near the pre-estimated detection limit. Four replicate strips were used for the other concentrations.
The cutoff level for the LoD was determined by using 60 replicates of the blank sample and selecting the highest measured signal value from these replicates as the cutoff value. The LoD (IU/ml) was calculated from the cutoff and the equation obtained with linear regression of the standard curve.
The LoD was further confirmed with a second method by performing an additional test using 80 replicates of the three concentrations below, above, and at the same level (0.05, 0.1, and 0.2 IU/ml) as the previously calculated LoD. The sample size of 80 was based on a statistical sampling plan (ISO 2859-1:1999) previously used by Das et al. . Sample size code J was determined by using general inspection level II and was based on a lot size of 501–1200. A single sampling plan for normal inspection was used, which resulted in a sample size of 80.
The LoD of conventional HBsAg LFIA (Alere DetermineTM HBsAg) was determined similarly with the WHO third international standard for HBsAg. The standard was diluted in serum in concentrations of 0.8, 1.6, 3.2, 4.8, 6.4, and 12.8 IU/ml. Dilutions were run on the commercial RDT, in 4 replicates for concentrations 0.8–6.4 IU/ml, and two replicates for concentration 12.8 IU/ml, according to the manufacturer’s instructions. The visual results were interpreted by two individuals.
Assay performance evaluation
All the clinical samples and sample panels were evaluated with the developed assay according to the UCNP-LFIA procedure. All the clinical serum and plasma samples from different individuals not belonging to a commercial fixed sample panel (e.g., performance panel, mixed titer panel) were used for determining the sensitivity and specificity of the assay. The total sample numbers used for the calculation of the assay performance were 108 HBsAg-positive and 315 HBsAg-negative samples.
The optimal clinical cutoff value was determined based on receiver operating characteristic (ROC) analysis executed by SAS JMP Pro 14 statistics software. To calculate the signal-to-cutoff (S/Co), photoluminescence signal obtained from each sample with peak detection was divided by the cutoff. Samples with S/Co values ≥ 1 were considered reactive.
In order to compare the performance of the developed assay with a conventional HBsAg rapid LFIA, all the clinical samples were tested with commercial RDT according to the manufacturer’s instructions. The visual results were interpreted by two individuals who were blinded for the reference assay results.
UCNP-LFIA strip stability study
The strips were stored in sealed aluminum foil pouches with a desiccant at + 37 °C for 50 days. The strip stability was studied at 7 time points during this period by testing the strips with the UCNP-LFIA procedure. Native HBsAg protein (Yashraj Biotechnology Ltd., India) was diluted in HBsAg-negative human serum in concentrations of 0, 0.5, and 5 ng/ml and used as a sample.
Photoluminescence measurement reproducibility
Dry strips assayed with human serum spiked with 0, 0.5, and 5 ng/ml HBsAg were measured 15 times. The measurement reproducibility was determined by calculating the coefficient of variation between repeat measurements of the same strip.