Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers

Serologic tests to detect specific IgGs to antigens related to viral infections are urgently needed for diagnostics and therapeutics. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers that recognize the four categorized serotypes. Using UB-DNA aptamers specific to each serotype of dengue NS1 proteins (DEN-NS1), we developed our aptamer–antibody sandwich ELISA for dengue diagnostics. Furthermore, IgGs highly specific to DEN-NS1 inhibited the serotype-specific NS1 detection, inspiring us to develop the competitive ELISA format for dengue serotype-specific IgG detection. Blood samples from Singaporean patients with primary or secondary dengue infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than the recent infection. Using this dengue competitive ELISA format, cross-reactivity tests of 21 plasma samples from Singaporean Zika virus-infected patients revealed two distinct patterns: 8 lacked cross-reactivity, and 13 were positive with unique dengue serotype specificities, indicating previous dengue infection. This antigen-detection ELISA and antibody-detection competitive ELISA combination using the UB-DNA aptamers identifies both past and current viral infections and will facilitate specific medical care and vaccine development for infectious diseases.

Competitive ELISA for a serologic test to detect dengue serotype-specific anti-NS1 IgGs using high-affinity UB-DNA aptamers

Serologic tests to detect IgGs specific to antigens related to viral infections are urgently needed for diagnostics and therapeutics in endemic, epidemic, and pandemic situations. We present a diagnostic method for serotype-specific IgG identification of dengue infection by a competitive enzyme-linked immunosorbent assay (ELISA), using high-affinity unnatural-base-containing DNA (UB-DNA) aptamers. Dengue is a widespread mosquito-borne viral disease, with four categorized serotypes. Using UB-DNA aptamers that bind specifically to each serotype or sub-serotype dengue NS1 protein (DEN-NS1), we developed an ELISA format with an aptamer − antibody sandwich system for dengue diagnostics. We found that IgGs highly specific to DEN-NS1 inhibit the serotype-specific NS1 detection by the ELISA format, inspiring us to develop another competitive ELISA format for serotype-specific IgG detection of dengue infection. Analyses of clinical blood samples from Singaporean patients with primary or secondary infections confirmed the highly specific IgG detection of this format, and the IgG production initially reflected the serotype of the past infection, rather than that of the recent infection. The combination of the ELISA and competitive ELISA using the UB-DNA aptamers allows the diagnosis of both past and current viral infections and will facilitate prompt and specific medical care and vaccine development for infectious diseases.

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).

Dual-Antigen System Allows Elimination of False Positive Results in COVID-19 Serological Testing

Determining the presence of antibodies in serum is important for epidemiological studies, to be able to confirm whether a person has been infected, predicting risks of them getting sick and spreading the disease. During the ongoing pandemic of COVID-19, a positive serological test result can suggest if it is safe to return to work and re-engage in social activities. Despite a multitude of emerging tests, the quality of respective data often remains ambiguous, yielding a significant fraction of false positive results. The human organism produces polyclonal antibodies specific to multiple viral proteins, so testing simultaneously for multiple antibodies appeared a practical approach for increasing test specificity. We analyzed immune response and testing potential for a spectrum of antigens derived from the spike and nucleocapsid proteins of SARS-CoV-2, developed a dual-antigen testing system in the ELISA format and designed a robust algorithm for data processing. Combining nucleocapsid protein and receptor-binding domain for analysis allowed us to completely eliminate false positive results in the tested cohort (achieving specificity within a 95% confidence interval of 97.2–100%). We also tested samples collected from different households, and demonstrated differences in the immune response of COVID-19 patients and their family members; identifying, in particular, asymptomatic cases showing strong presence of studied antibodies, and cases showing none despite confirmed close contacts with the infected individuals.

Evaluation of the sensitivity and specificity of GST-tagged recombinant antigens 2B2t, Ag5t and DIPOL in ELISA for the diagnosis and follow up of patients with cystic echinococcosis

Cystic echinococcosis (CE) is a neglected zoonotic disease caused by Echinococcus granulosus sensu lato. Diagnosis and monitoring of CE rely primarily on imaging while serology is used as a confirmatory test. However, imaging is not always conclusive and currently available serological assays have suboptimal sensitivity and specificity, lack standardization, and are not useful for patients´ follow-up. Seroassays for CE are usually based on hydatid fluid (HF), a complex, variable antigenic mixture, and cross-reactivity exists especially with alveolar echinococcosis. Recombinant proteins based on immunogenic antigens most abundant in HF, such as AgB1, AgB2 and Ag5, have been used to overcome these limitations. None of them so far showed potential to replace HF; however, their performance have been largely tested on a limited number of samples, and comparison of different antigens using the same cohort has been rarely performed. The combination of several immunogenic epitopes in a single recombinant protein could enhance test sensitivity. For the diagnosis and follow-up of patients with CE, we compared the performance of the crude HF, previously described recombinant 2B2t antigen, and GST-tagged version of 2B2t, and novel designed recombinants (GST-Ag5t and the GST-DIPOL chimera containing AgB1, AgBB2 and Ag5 epitopes) by IgG-ELISA format. Samples belong to a retrospective cohort of 253 well-characterized patients with CE, previously described for the evaluation of the 2B2t antigen, 92 patients with alveolar echinococcosis, and 82 healthy donors. The reference standard for CE diagnosis was the presence of a CE lesion as diagnosed by ultrasonography. The highest sensitivity was obtained with HF [86.7%, 95% confidence interval (CI): 81.2–91.0], followed by GST-2B2t (70.0%, 95% CI: 63.1–76.2), 2B2t (65.5%, 95% CI: 58.5–72.0), GST-Ag5t (64.5%, 95% CI: 57.5–71.1) and GST-DIPOL (63.1%, 95% CI: 56.0–69.7). The GST-2B2t had the best specificity (95.8%, 95% CI: 88.3–99.1) and the lowest cross-reactivity (38.7%, 95% CI: 27.6–50.6). Good response to treatment also correlated to negative test results in the GST-2B2t ELISA. While none of the tested recombinant antigen appears suitable to replace HF for the diagnosis of CE, GST-2B2t should be further explored as a confirmation test, based on its high specificity and low cross-reactivity, and for the follow-up after treatment in those patients with positive serology for this antigen.

De Novo Discovery of High Affinity Peptide Binders for the SARS-CoV-2 Spike Protein

The β-coronavirus SARS-CoV-2 has caused a global pandemic. Affinity reagents targeting the SARS-CoV-2 spike protein, the most exposed surface structure of the virus, are of interest for the development of therapeutics and diagnostics. We used affinity selection-mass spectrometry for the rapid discovery of synthetic high affinity peptide binders for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. From library screening with 800 million synthetic peptides, we identified three sequences with nanomolar affinities (dissociation constants Kd = 80 to 970 nM) for RBD and selectivity over human serum proteins. Picomolar RBD concentrations in biological matrix could be detected using the biotinylated lead peptide in ELISA format. These peptides might associate with the SARS-CoV-2-spike-RBD at a site unrelated to ACE2 binding, making them potential orthogonal reagents for sandwich immunoassays. We envision our discovery as a robust starting point for the development of SARS-CoV-2 diagnostics or conjugates for virus directed delivery of therapeutics.Abstract Figure

Production of Trimeric SARS-CoV-2 Spike Protein by CHO Cells for Serological COVID-19 Testing

We describe scalable and cost-efficient production of full length, His-tagged SARS-CoV-2 spike glycoprotein trimer by CHO cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both HEK and CHO cells mediated by PEI was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32C to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell-derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in ELISA format to detect IgG antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by PCR, including those who had displayed COVID-19 symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.

Detection of C-Reactive Protein Using Histag-HRP Functionalized Nanoconjugate with Signal Amplified Immunoassay

Ultrasensitive detection of biomarkers is highly significant for disease prognosis and public health treatment. Despite wide acceptance in routine laboratory tests, the conventional enzyme-linked immunosorbent assay (ELISA) has been of limited use for early biomarker detection due to insufficient sensitivity and multiple long incubation time. Several nanoprobes have been introduced to circumvent the limitation, however, rapid, simple, and chemical-free nanoprobe synthesis and sensitive detection methods, particularly for ELISA, are still lacking. In this study, we have synthesized a gold nanoprobe, conjugated with multiple 6X-histidine (6X-his) peptide and nickel-horseradish peroxidase (Ni2+-HRP), for enhancing the colorimetric signal in ELISA. The developed nanoprobe has been tested for the detection of immunologically significant C-reactive protein (CRP) in ELISA format. The performance of designed probe is validated by testing standard and serum samples, and the detection limit of 32.0 pg/mL with R2 = 0.98 is confirmed. Furthermore, a comparative analysis of the developed nanoprobe was performed with ELISA developed on conventional guidelines, the proposed immunoassay showed an increase of 12-fold sensitivity for detecting CRP due to the high loading of 6Xhis peptide and binding of multiple Ni2+-HRP on a gold nanoparticle. Additionally, the proposed assay provides a simple, fast, and cost-efficient (not requiring multiple antibodies) detection of CRP with easy nanoprobe synthesis. Moreover, the developed Histag-HRP functionalized nanoconjugate immunoassay is flexible and can be applied to other biomarkers efficiently by using disease specific antibody.

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N=80 samples from persons with RT-PCR confirmed SARS-CoV2 infection), and a specificity of 97.2% (N=106 control samples).

Integration of IgA and IgG Autoantigens Improves Performance of Biomarker Panels for Early Diagnosis of Lung Cancer

Lung cancer (LC) remains the leading cause of mortality from malignant tumors worldwide. In our previous study, we surveyed both IgG and IgM-bound serological biomarkers and validated a panel of IgG-bound autoantigens for early LC diagnosis with 50% sensitivity at 90% specificity. To further improve the performance of these serological biomarkers, we surveyed HuProt arrays, comprised of 20,240 human proteins, for IgA-bound autoantigens because IgAs are a major immunoglobulin isotype in the lung. Integrating with IgG-bound autoantigens, we discovered and validated a combined biomarker panel using ELISA-format tests. Specifically, in Phase I, we obtained IgA-based autoimmune profiles of 69 early stage LC patients, 30 healthy subjects and 25 patients with lung benign lesions (LBL) on HuProt arrays and identified 28 proteins as candidate autoantigens that were significantly associated with early stage LC. In Phase II, we re-purified the autoantigens and converted them into an ELISA-format testing to profile an additional large cohort, comprised of 136 early stage LC patients, 58 healthy individuals, and 29 LBL patients. Integration of IgG autoimmune profiles allowed us to identify and validate a biomarker panel of three IgA autoantigens (i.e. BCL7A, and TRIM33 and MTERF4) and three IgG autoantigens (i.e. CTAG1A, DDX4 and MAGEC2) for diagnosis of early stage LC with 73.5% sensitivity at >85% specificity. In Phase III, the performance of this biomarker panel was confirmed with an independent cohort, comprised of 88 early stage LC patients, 18 LBL patients, and 36 healthy subjects. Finally, a blind test on 178 serum samples was conducted to confirm the performance of the biomarker panel. In summary, this study demonstrates for the first time that an integrated panel of IgA/IgG autoantigens can serve as valuable biomarkers to further improve the performance of early diagnosis of LC.