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1.
Rinsho Ketsueki ; 63(5): 454-462, 2022.
Article in Japanese | MEDLINE | ID: covidwho-1879648

ABSTRACT

Antiplatelet factor 4 (PF4) antibodies, also known as anti-PF4/heparin complex antibodies, are measured to diagnose heparin-induced thrombocytopenia (HIT). In HIT, anti-PF4 antibodies induced by heparin exposure cause thrombocytopenia and thrombosis. However, in recent years, autoimmune HIT (aHIT) that develops without heparin exposure has been getting attention. In 2021, anti-PF4 antibodies were reported to cause the fatal vaccine-induced immune thrombotic thrombocytopenia (VITT) that developed after adenoviral vector vaccination for COVID-19. HIT, aHIT, and VITT are considered to be caused by anti-PF4 antibodies, and their pathological conditions are similar. However, they have different levels of severity, and the detection sensitivity of their antibodies varies depending on the assay. Herein, we review three pathologies, namely, HIT, aHIT, and VITT, associated with anti-PF4 antibodies.


Subject(s)
COVID-19 , Purpura, Thrombocytopenic, Idiopathic , Thrombocytopenia , Thrombosis , Vaccines , Antibodies , Heparin/adverse effects , Humans , Platelet Factor 4/immunology , Thrombocytopenia/chemically induced , Thrombocytopenia/diagnosis , Thrombosis/etiology , Vaccines/adverse effects
2.
Blood Adv ; 6(11): 3315-3320, 2022 06 14.
Article in English | MEDLINE | ID: covidwho-1879217

ABSTRACT

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) adenoviral vector vaccination. In British Columbia (BC), Canada, a provincial clinical care pathway was developed to guide clinicians in evaluating for VITT among patients who present with thrombocytopenia or thrombosis symptoms within 4 to 28 days after adenoviral vector vaccine exposure. All patients had enzyme-linked immunosorbent assay (ELISA) testing for platelet factor 4 (PF4) antibodies, and all cases with positive PF4-ELISA or d-dimer levels ≥2.0 mg/L fibrinogen equivalent units (FEU) had further testing for platelet-activating PF4 antibodies using a modified serotonin release assay (SRA). Between 1 May and 30 June 2021, 37% of 68 patients investigated for VITT had thrombosis, but only 3 had VITT confirmed by PF4-ELISA and SRA. Platelet counts, d-dimer levels, and ELISA optical density values were significantly different between those with and without VITT. Three patients had thrombocytopenia and thrombosis with d-dimer levels >4.0 mg/L FEU but had negative PF4-ELISA and SRA results. Patients with VITT were treated successfully with IV immunoglobulin, nonheparin anticoagulants, and corticosteroids. Our pathway demonstrated that thrombosis is common among patients investigated for VITT and that PF4-ELISA testing is necessary to confirm VITT in those presenting with thrombosis and thrombocytopenia.


Subject(s)
COVID-19 , Purpura, Thrombocytopenic, Idiopathic , Thrombocytopenia , Thrombosis , Vaccines , Antibodies , COVID-19/diagnosis , COVID-19/prevention & control , COVID-19 Vaccines/adverse effects , Critical Pathways , Humans , Platelet Factor 4 , Purpura, Thrombocytopenic, Idiopathic/etiology , SARS-CoV-2 , Thrombocytopenia/chemically induced , Thrombocytopenia/etiology , Vaccination , Vaccines/adverse effects
3.
Clin Chem Lab Med ; 60(7): 959-960, 2022 May 03.
Article in English | MEDLINE | ID: covidwho-1875156
4.
Viruses ; 14(5)2022 May 12.
Article in English | MEDLINE | ID: covidwho-1869809

ABSTRACT

A new antibody diagnostic assay with more rapid and robust properties is demanded to quantitatively evaluate anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunity in a large population. Here, we developed a nanometer-scale fluorescent biosensor system consisting of CdSe-ZnS quantum dots (QDs) coupled with the highly sensitive B-cell epitopes of SARS-CoV-2 that could remarkably identify the corresponding antibody with a detection limit of 100 pM. Intriguingly, we found that fluorescence quenching of QDs was stimulated more obviously when coupled with peptides than the corresponding proteins, indicating that the energy transfer between QDs and peptides was more effective. Compared to the traditional enzyme-linked immunosorbent assay (ELISA), the B-cell-epitope-based QD-biosensor could robustly distinguish coronavirus disease 2019 (COVID-19) antibody-positive patients from uninfected individuals with a higher sensitivity (92.3-98.1% positive rates by QD-biosensor vs. 78.3-83.1% positive rates by ELISAs in 207 COVID-19 patients' sera) in a more rapid (5 min) and labor-saving manner. Taken together, the 'QD-peptides' biosensor provided a novel real-time, quantitative, and high-throughput method for clinical diagnosis and home-use tests.


Subject(s)
Biosensing Techniques , COVID-19 , Quantum Dots , Antibodies , COVID-19/diagnosis , Epitopes, B-Lymphocyte , Humans , Peptides , SARS-CoV-2
6.
Front Immunol ; 13: 856906, 2022.
Article in English | MEDLINE | ID: covidwho-1834405

ABSTRACT

Tuberculosis (TB) is among the leading causes of death worldwide from a single infectious agent, second only to COVID-19 in 2020. TB is caused by infection with Mycobacterium tuberculosis (Mtb), that results either in a latent or active form of disease, the latter associated with Mtb spread. In the absence of an effective vaccine, epidemiologic modeling suggests that aggressive treatment of individuals with active TB (ATB) may curb spread. Yet, clinical discrimination between latent (LTB) and ATB remains a challenge. While antibodies are widely used to diagnose many infections, the utility of antibody-based tests to diagnose ATB has only regained significant traction recently. Specifically, recent interest in the humoral immune response to TB has pointed to potential differences in both targeted antigens and antibody features that can discriminate latent and active TB. Here we aimed to integrate these observations and broadly profile the humoral immune response across individuals with LTB or ATB, with and without HIV co-infection, to define the most discriminatory humoral properties and diagnose TB disease more easily. Using 209 Mtb antigens, striking differences in antigen-recognition were observed across latently and actively infected individuals that was modulated by HIV serostatus. However, ATB and LTB could be discriminated, irrespective of HIV-status, based on a combination of both antibody levels and Fc receptor-binding characteristics targeting both well characterized (like lipoarabinomannan, 38 kDa or antigen 85) but also novel Mtb antigens (including Rv1792, Rv1528, Rv2435C or Rv1508). These data reveal new Mtb-specific immunologic markers that can improve the classification of ATB versus LTB.


Subject(s)
COVID-19 , HIV Infections , Latent Tuberculosis , Tuberculosis , Antibodies , HIV Infections/complications , Humans
7.
Nanoscale ; 14(19): 7110-7122, 2022 May 19.
Article in English | MEDLINE | ID: covidwho-1830194

ABSTRACT

The increasing demand for convenient, miniaturized and multifunctional antibodies necessitates the development of novel antigen-recognition molecules for biological and medical studies. Nanobodies, the functional variable regions of camelid heavy-chain-only antibodies, as a new tool, complement the conventional antibodies and are in the stage of rapid development. The outstanding advantages of nanobodies include a stable structure, easy production, excellent water solubility, high affinity toward antigens and low immunogenicity. With promising application potential, nanobodies are now increasingly applied to various studies, including protein structure analysis, microscopic imaging, medical diagnosis, and drug development. The approval of the first nanobody drug Caplacizumab by the FDA disclosed the therapeutic potential of nanobodies. The outbreak of COVID-19 accelerated the development of nanobody drugs in non-injectable and bispecific biotherapeutic applications. Herein, we reviewed recent studies on the nanobody structure, screening and their applications in protein structure analysis and nanobody drugs, especially on non-injectable nanobody and bispecific nanobody development.


Subject(s)
COVID-19 , Single-Domain Antibodies , Antibodies , Antigens , COVID-19/drug therapy , Diagnostic Imaging , Humans , Single-Domain Antibodies/chemistry
8.
Nature ; 605(7909): 207, 2022 05.
Article in English | MEDLINE | ID: covidwho-1830020
9.
ACS Appl Bio Mater ; 5(5): 2421-2430, 2022 05 16.
Article in English | MEDLINE | ID: covidwho-1829968

ABSTRACT

In this work, we report a facile synthesis of graphene oxide-gold (GO-Au) nanocomposites by electrodeposition. The fabricated electrochemical immunosensors are utilized for the dual detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen and SARS-CoV-2 antibody. The GO-Au nanocomposites has been characterized by UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectroscopy (EIS) for its biosensing properties. The linear detection range of the SARS-CoV-2 antigen immunosensor is 10.0 ag mL-1 to 50.0 ng mL-1, whereas that for the antibody immunosensor ranges from 1.0 fg mL-1 to 1.0 ng mL-1. The calculated limit of detection (LOD) of the SARS-CoV-2 antigen immunosensor is 3.99 ag mL-1, and that for SARS-CoV-2 antibody immunosensor is 1.0 fg mL-1 with high sensitivity. The validation of the immunosensor has also been carried out on patient serum and patient swab samples from COVID-19 patients. The results suggest successful utilization of the immunosensors with a very low detection limit enabling its use in clinical samples. Further work is needed for the standardization of the results and translation in screen-printed electrodes for use in portable commercial applications.


Subject(s)
Biosensing Techniques , COVID-19 , Metal Nanoparticles , Nanocomposites , Antibodies , Biosensing Techniques/methods , COVID-19/diagnosis , Gold/chemistry , Graphite , Humans , Immunoassay/methods , Metal Nanoparticles/chemistry , Nanocomposites/chemistry , SARS-CoV-2
10.
HLA ; 100(1): 52-58, 2022 Jul.
Article in English | MEDLINE | ID: covidwho-1816658

ABSTRACT

The effects of COVID-19 vaccination on alloimmunization and clinical impact in transplant candidates remain largely unknown. In a 61-year-old man who had no donor-specific antibodies (DSA) and was planned to undergo ABO-incompatible kidney transplantation (ABOi KT), DSAs (anti-A24, anti-B51, and anti-Cw14) developed after COVID-19 vaccination. After desensitization therapy, antibody level was further increased, leading to flow cytometric crossmatch-positive status. Donor-specific T cell immunity using interferon-gamma ELISPOT was continuously negative, whereas SARS-CoV-2 specific T cell immunity was intact. After confirming the C1q-negative status of DSA, the patient received ABOi KT. The patient had stable graft function and suppressed alloimmunity up to 2 months after KT. COVID-19 vaccination might relate to alloimmunization in transplant candidates, and desensitization through immune monitoring can help guide transplantation.


Subject(s)
COVID-19 , Kidney Transplantation , Alleles , Antibodies , COVID-19 Vaccines , Flow Cytometry , Graft Rejection , Graft Survival , HLA Antigens , Humans , Living Donors , Male , Middle Aged , SARS-CoV-2 , Vaccination
11.
STAR Protoc ; 3(2): 101406, 2022 Jun 17.
Article in English | MEDLINE | ID: covidwho-1815284

ABSTRACT

Elucidating the molecular interactions between virus and host is fundamental to understanding the mechanism of viral pathogenesis. Here, we present a protocol to screen SARS-CoV-2 protein interactors using an antibody-based TurboID proximity labeling approach. This technique directly identifies biotinylated peptides labeled by the TurboID-tagged viral proteins. We describe the steps to prepare biotinylated peptide samples for mass spectrometry analysis and a stringent workflow to identify biotinylated high-confidence interactors of the virus by filtering out non-specific co-purified proteins. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2022).


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies , COVID-19/diagnosis , Humans , Mass Spectrometry , Viral Proteins
12.
Biosensors (Basel) ; 12(5)2022 Apr 22.
Article in English | MEDLINE | ID: covidwho-1809707

ABSTRACT

The timely detecting of SARS-CoV-2 coronavirus antigens for infection validation is an urgent request for COVID-19 pandemic control. This study constructed label-free electrochemical impedance spectroscopy (EIS)-based immunosensors based on gold nanostructured screen-printed carbon electrodes (AuNS/SPCEs) to detect the SARS-CoV-2 nucleocapsid protein (N-protein) in saliva. Using short-chain 3-mercaptopropionic acid (MPA) as a linker to covalently bond streptavidin (SA) and bovine serum albumin (BSA) for controlling the oriented immobilization of the biotinylated anti-N-protein antibody (BioAb) can offer a greater sensitivity, a lower limit of detection (LOD), and better reproducibility of immunosensors (defined as BioAb/SA-BSA/MPA/AuNS/SPCEs) than the antibody randomly immobilized immunosensors and the long-chain 11-mercaptoundecanoic acid (MUA)-modified immunosensors (BioAb/SA-BSA/MUA/AuNS/SPCEs). The BioAb/SA-BSA/MPA/AuNS/SPCE-based immunosensors presented good linearity from 0.01 ng/mL to 100 ng/mL and a low LOD of 6 pg/mL in a phosphate buffer solution (PBS) and PBS-diluted saliva. Moreover, the immunosensor exhibited little cross-activity with other viral antigens such as MERS-CoV N-protein, influenza A N-protein, influenza B N-protein, and SARS-CoV-2 spike protein, indicating the high specificity of the immunosensors. The disposable label-free EIS-based immunosensors have promising potential in facilitating the rapid and sensitive tests of saliva-based COVID-19 diagnostics.


Subject(s)
Biosensing Techniques , COVID-19 , Influenza, Human , Antibodies , COVID-19/diagnosis , Humans , Immunoassay , Nucleoproteins , Pandemics , Reproducibility of Results , SARS-CoV-2 , Saliva , Spike Glycoprotein, Coronavirus , Streptavidin
13.
BMJ ; 377: o1009, 2022 04 20.
Article in English | MEDLINE | ID: covidwho-1807366
14.
ACS Sens ; 7(4): 1122-1131, 2022 04 22.
Article in English | MEDLINE | ID: covidwho-1788266

ABSTRACT

Rapid antigen tests are currently used for population screening of COVID-19. However, they lack sensitivity and utilize antibodies as receptors, which can only function in narrow temperature and pH ranges. Consequently, molecularly imprinted polymer nanoparticles (nanoMIPs) are synthetized with a fast (2 h) and scalable process using merely a tiny SARS-CoV-2 fragment (∼10 amino acids). The nanoMIPs rival the affinity of SARS-CoV-2 antibodies under standard testing conditions and surpass them at elevated temperatures or in acidic media. Therefore, nanoMIP sensors possess clear advantages over antibody-based assays as they can function in various challenging media. A thermal assay is developed with nanoMIPs electrografted onto screen-printed electrodes to accurately quantify SARS-CoV-2 antigens. Heat transfer-based measurements demonstrate superior detection limits compared to commercial rapid antigen tests and most antigen tests from the literature for both the alpha (∼9.9 fg mL-1) and delta (∼6.1 fg mL-1) variants of the spike protein. A prototype assay is developed, which can rapidly (∼15 min) validate clinical patient samples with excellent sensitivity and specificity. The straightforward epitope imprinting method and high robustness of nanoMIPs produce a SARS-CoV-2 sensor with significant commercial potential for population screening, in addition to the possibility of measurements in diagnostically challenging environments.


Subject(s)
COVID-19 , Molecular Imprinting , Nanoparticles , Antibodies , COVID-19/diagnosis , Humans , Molecularly Imprinted Polymers , Nanoparticles/chemistry , Point-of-Care Systems , SARS-CoV-2
15.
Molecules ; 27(7)2022 Mar 28.
Article in English | MEDLINE | ID: covidwho-1785836

ABSTRACT

Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.


Subject(s)
Single-Domain Antibodies , Animals , Antibodies
16.
Int J Mol Sci ; 23(7)2022 Apr 02.
Article in English | MEDLINE | ID: covidwho-1785742

ABSTRACT

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.


Subject(s)
Antibodies , Receptor, Angiotensin, Type 1 , Alanine , Angiotensin II , Antibodies/pharmacology , Cell Proliferation , HEK293 Cells , Humans , Receptor, Angiotensin, Type 1/genetics , Receptor, Angiotensin, Type 1/metabolism
17.
Int J Mol Sci ; 23(7)2022 Mar 28.
Article in English | MEDLINE | ID: covidwho-1785734

ABSTRACT

VHH, i.e., VH domains of camelid single-chain antibodies, are very promising therapeutic agents due to their significant physicochemical advantages compared to classical mammalian antibodies. The number of experimentally solved VHH structures has significantly improved recently, which is of great help, because it offers the ability to directly work on 3D structures to humanise or improve them. Unfortunately, most VHHs do not have 3D structures. Thus, it is essential to find alternative ways to get structural information. The methods of structure prediction from the primary amino acid sequence appear essential to bypass this limitation. This review presents the most extensive overview of structure prediction methods applied for the 3D modelling of a given VHH sequence (a total of 21). Besides the historical overview, it aims at showing how model software programs have been shaping the structural predictions of VHHs. A brief explanation of each methodology is supplied, and pertinent examples of their usage are provided. Finally, we present a structure prediction case study of a recently solved VHH structure. According to some recent studies and the present analysis, AlphaFold 2 and NanoNet appear to be the best tools to predict a structural model of VHH from its sequence.


Subject(s)
Camelids, New World , Immunoglobulin Heavy Chains , Amino Acid Sequence , Animals , Antibodies , Immunoglobulin Heavy Chains/chemistry , Models, Structural
18.
Genome Biol ; 23(1): 55, 2022 02 16.
Article in English | MEDLINE | ID: covidwho-1785167

ABSTRACT

BACKGROUND: Multiplexing of samples in single-cell RNA-seq studies allows a significant reduction of the experimental costs, straightforward identification of doublets, increased cell throughput, and reduction of sample-specific batch effects. Recently published multiplexing techniques using oligo-conjugated antibodies or -lipids allow barcoding sample-specific cells, a process called "hashing." RESULTS: Here, we compare the hashing performance of TotalSeq-A and -C antibodies, custom synthesized lipids and MULTI-seq lipid hashes in four cell lines, both for single-cell RNA-seq and single-nucleus RNA-seq. We also compare TotalSeq-B antibodies with CellPlex reagents (10x Genomics) on human PBMCs and TotalSeq-B with different lipids on primary mouse tissues. Hashing efficiency was evaluated using the intrinsic genetic variation of the cell lines and mouse strains. Antibody hashing was further evaluated on clinical samples using PBMCs from healthy and SARS-CoV-2 infected patients, where we demonstrate a more affordable approach for large single-cell sequencing clinical studies, while simultaneously reducing batch effects. CONCLUSIONS: Benchmarking of different hashing strategies and computational pipelines indicates that correct demultiplexing can be achieved with both lipid- and antibody-hashed human cells and nuclei, with MULTISeqDemux as the preferred demultiplexing function and antibody-based hashing as the most efficient protocol on cells. On nuclei datasets, lipid hashing delivers the best results. Lipid hashing also outperforms antibodies on cells isolated from mouse brain. However, antibodies demonstrate better results on tissues like spleen or lung.


Subject(s)
COVID-19/blood , Sequence Analysis, RNA/methods , Single-Cell Analysis/methods , Animals , Antibodies/chemistry , Case-Control Studies , Cell Line, Tumor , Cell Nucleus/chemistry , Humans , Lipids/chemistry , Mice, Inbred BALB C , Mice, Inbred C57BL , Neutrophils/chemistry , Neutrophils/immunology , Neutrophils/virology
19.
Int J Mol Sci ; 23(7)2022 Mar 28.
Article in English | MEDLINE | ID: covidwho-1780038

ABSTRACT

Antibody-based therapeutics have achieved unprecedented success in treating various diseases, including cancers, immune disorders, and infectious diseases [...].


Subject(s)
Antibodies , Neoplasms , Antibodies/therapeutic use , Humans , Neoplasms/drug therapy
20.
J Glob Health ; 12: 05009, 2022.
Article in English | MEDLINE | ID: covidwho-1737332

ABSTRACT

Background: The COVID-19 pandemic caused by SARS-CoV-2 exposed a global problem, as highly effective vaccines are challenging to produce and distribute, particularly in regions with limited resources and funding. As an alternative, immunoglobulins produced in eggs of immunized hens (IgY) can be a simple and inexpensive source for a topical and temporary prophylaxis. Here, we developed a method to extract and purify IgY antibodies from egg yolks of hens immunized against viral pathogen-derived proteins using low-cost, readily available materials, for use in resource-limited settings. Methods: Existing protocols for IgY purification and equipment were modified, including extraction from yolks and separation of water-soluble IgY using common household reagents and tools. A replacement for a commercial centrifuge was developed, using a home food processor equipped with a 3D printed adapter to enable IgY precipitation. IgY purification was verified using standard gel electrophoresis and Western blot analyses. Results: We developed a step-by-step protocol for IgY purification for two settings in low- and middle-income countries (LMIC): a local laboratory, where commercial centrifuges are available, or a more rural setting, where an alternative for expensive centrifuges can be used. Gel electrophoresis and Western blot analyses confirmed that the method produced highly enriched IgY preparation; each commercial egg produced ~ 90 mg of IgY. We also designed a kit for IgY production in these two settings and provided a cost estimate of the kit. Conclusion: IgY purified from eggs of immunized local hens can offer a fast and affordable prophylaxis, provided that purification can be performed in a resource-limited setting. Here, we created a low-cost method that can be used anywhere where electricity is available using inexpensive, readily available materials in place of costly, specialized laboratory equipment and chemicals. This procedure can readily be used now to make an anti-SARS-CoV-2 prophylaxis in areas where vaccines are unavailable, and can be modified to combat future threats from viral epidemics and pandemics.


Subject(s)
COVID-19 , Pandemics , Animals , Antibodies , Antiviral Agents , COVID-19/prevention & control , Chickens , Female , Humans , Immunoglobulins , SARS-CoV-2
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