An Evaluation of a SARS-CoV-2 Pseudovirus Neutralization Test and A Comparison to a SARS-CoV-2 Surrogate Virus Neutralization Test in a COVID-19 Long-Term Follow-Up Cohort

Microbiology Research, Journal Year: 2024, Volume and Issue: 15(1), P. 422 - 430

Published: March 21, 2024

Background: The detection of neutralizing anti-SARS-CoV-2 antibodies is important since they represent the subset able to prevent virus invade human cells. aim this study evaluate clinical performances an in-house pseudovirus neutralization test (pVNT) versus a commercial surrogate (sVNT). Material and Methods: A total 114 RT-PCR positives samples from 75 COVID-19 patients were analyzed using pVNT sVNT technique. Fifty-six pre-pandemic also assess specificity two techniques. An analysis repeatability reproducibility was performed. Results: coefficient variation (CV) 10.27% for computed. For test, CVs ranged 16.12% low NAbs titer 6.40% high titer. Regarding sensitivity, 90 positive out with (78.94%), 97 (84.21%). About specificity, all 56 negative in both When comparing pVNT, sensibility 66.67% (95%CI: 47.81–85.53%) 98.88% 96.72–99.99%), respectively. Conclusions: results obtained automated technique are consistent those developed in-house. various tests demonstrate good robustness fully manual

Language: Английский

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An unexpected IgE anti-receptor binding domain response following natural infection and different types of SARS-CoV-2 vaccines

Scientific Reports, Journal Year: 2024, Volume and Issue: 14(1)

Published: Aug. 28, 2024

Humoral response to SARS-CoV-2 has been studied, predominantly the classical IgG and its subclasses. Although IgE antibodies are typically specific allergens or parasites, a few reports describe their production in other viruses. Here, we investigated receptor binding domain (RBD) of Brazilian cohort following natural infection vaccination. Samples from 59 volunteers were assessed after (COVID-19), primary immunization with vectored (ChAdOx1) inactivated (CoronaVac) vaccines, booster mRNA (BNT162b2) vaccine. Natural COVID-19 induced IgE, but vaccination increased levels. Subjects vaccinated two doses ChAdOx1 exhibited more robust than those immunized CoronaVac; however, boosting BNT162b2, all groups presented similar showed intermediate-to-high avidity, especially We also found IgG4 antibodies, mainly booster, they moderately correlated IgE. ELISA results confirmed by control assays, using depletion protein G lack reactivity heterologous antigen. In our cohort, no clinical data could be associated response. advocate for further research on role viral immunity, extending beyond allergies parasitic infections.

Language: Английский

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Validation of a SARS-CoV-2 Surrogate Neutralization Test Detecting Neutralizing Antibodies against the Major Variants of Concern

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(19), P. 14965 - 14965

Published: Oct. 6, 2023

SARS-CoV-2 infection and/or vaccination elicit a broad range of neutralizing antibody responses against the different variants concern (VOC). We established new variant-adapted surrogate virus neutralization test (sVNT) and assessed activity ancestral B.1 (WT) VOC Delta, Omicron BA.1, BA.2, BA.5. Analytical performances were compared respective to reference (VNT) two CE-IVD labeled kits using three cohorts collected during COVID-19 waves. Correlation analyses showed moderate strong correlation for sub-variants (Spearman's r = 0.7081 0.7205 0.6042 BA.5), WT (r 0.8458) Delta-sVNT 0.8158), respectively. Comparison WT-sVNT performance with kits, "Icosagen Neutralizing Antibody ELISA kit" "Genscript cPass, revealed an overall good ranging from 0.8673 -0.8773 midway profile between both commercial 87.76% sensitivity 90.48% clinical specificity. The BA.2-sVNT was similar BA.2 Genscript test. Finally, analysis association 0.8583) BA.5-sVNT VNT sVNT double-vaccinated cohort (n 100) Omicron-breakthrough 91). In conclusion, allows efficient prediction immune protection various VOCs.

Language: Английский

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Outpatient COVID-19 convalescent plasma recipient antibody thresholds correlated to reduced hospitalizations within a randomized trial

JCI Insight, Journal Year: 2024, Volume and Issue: 9(8)

Published: March 14, 2024

BACKGROUND. COVID-19 convalescent plasma (CCP) virus-specific antibody levels that translate into recipient posttransfusion sufficient to prevent disease progression are not defined.

Language: Английский

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Neutralizing and binding antibody dynamics following primary and booster COVID-19 vaccination among healthcare workers

BMC Infectious Diseases, Journal Year: 2025, Volume and Issue: 25(1)

Published: Feb. 14, 2025

Abstract Background Vaccine-induced neutralizing antibodies (NAbs) are key for COVID-19 protective-immunity. As the efficacy of SARS-CoV-2 vaccines declines over time and variants virus continue to emerge, need booster doses vaccine remains on agenda. The aim this study was assess NAbs dynamics its correlation with anti-RBD IgG levels during nine-month follow-up period after primary-CoronaVac vaccination vaccinations evaluate strategies. Methods This prospective longitudinal observational followed 226 healthcare workers who received primary (two CoronaVac) (CoronaVac or BNT162b2) immunization. Serum samples were collected at four different points, two booster. Anti-RBD antibody assessed SARS CoV-2 IgG-II-QUANT kit (Abbott, USA) determined ACE2-RBD-Neutralization-Assay (Dia-Pro, Italy) using a surrogate neutralization method. Factors affecting response analyzed. Statistical analysis performed IBM-SPSS-22.0. Results One month second dose CoronaVac, 79.2% participants had NAb, but decreased 49.7% by fourth influenced smoking, BMI chronic diseases. Boosters, regardless type, significantly raised NAb levels. Heterologous yielded higher responses. Both single double-BNT162b2 boosters resulted in similar There strong between following CoronaVac vaccination, leading identification predictive threshold presence NAb. type strength threshold-value. Conclusions rapidly vaccination. Boosters increased while heterologous combination induced greater response. able predict response, however varied since

Language: Английский

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Advances in Surrogate Neutralization Tests for High-Throughput Screening and the Point-of-Care

Analytical Chemistry, Journal Year: 2025, Volume and Issue: unknown

Published: March 4, 2025

InfoMetricsFiguresRef.SI Analytical ChemistryASAPArticle This publication is Open Access under the license indicated. Learn More CiteCitationCitation and abstractCitation referencesMore citation options ShareShare onFacebookXWeChatLinkedInRedditEmailBlueskyJump toExpandCollapse ReviewMarch 4, 2025Advances in Surrogate Neutralization Tests for High-Throughput Screening Point-of-CareClick to copy article linkArticle link copied!Simon StreifSimon StreifInstitute of Chemistry, Chemo- Biosensors, University Regensburg, Universitaetsstrasse 31, 93053 GermanyMore by Simon StreifView Biographyhttps://orcid.org/0000-0001-6081-7571Antje J. Baeumner*Antje BaeumnerInstitute Germany*Email: [email protected]More Antje BaeumnerView Biographyhttps://orcid.org/0000-0001-7148-3423Open PDFAnalytical ChemistryCite this: Anal. Chem. 2025, XXXX, XXX, XXX-XXXClick citationCitation copied!https://pubs.acs.org/doi/10.1021/acs.analchem.5c00666https://doi.org/10.1021/acs.analchem.5c00666Published March 2025 Publication History Received 29 January 2025Accepted 20 February 2025Revised 10 2025Published online 4 2025review-article© The Authors. Published American Chemical Society. licensed CC-BY 4.0 . License Summary*You are free share (copy redistribute) this any medium or format adapt (remix, transform, build upon) material purpose, even commercially within parameters below:Creative Commons (CC): a Creative license.Attribution (BY): Credit must be given creator.View full license*DisclaimerThis summary highlights only some key features terms actual license. It not has no legal value. Carefully review before using these materials. underCC-BY share(copy adapt(remix, below: Attribution *DisclaimerThis creator. View ACS Publications© SocietySubjectswhat subjects Article automatically applied from Subject Taxonomy describe scientific concepts themes article. Assays Biopolymers Immunology Peptides proteins SARS-CoV-2 Special IssuePublished as part Chemistry special issue "Fundamental Applied Reviews 2025".IntroductionClick section linkSection copied!Serological testing long played crucial role disease management clinical diagnostics. Both infection with viruses vaccination mediate humoral immune response, including generation specific antibodies. presence antibodies can therefore used qualitatively detect recent past infections quantitatively determine status patient. Antibody profiles generated different vary show differences versus vaccination, latter often uses one antigen rather than whole virus. Furthermore, while vaccinations result long-term immunity, others require booster shots every few years, yearly. Hence, necessity benefits serological typically tailored respective diseases.Diseases preventable through include, e.g., hepatitis A, influenza, SARS, chickenpox, measles, mumps, rubella, tetanus, poliomyelitis. Low mutation rates due constraints, such limited host range case (1) ensuring immunity memory B cell T persistence. Vaccination lead up 30 years longer (2) influenza viruses, especially rapid antigenic drift, necessitating extensive modeling predict most likely strains targeted annual vaccine. (3) Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also shows high rates, facilitating its escape. COVID-19 pandemic caused virus sparked advancement innovation field regard both binding neutralizing antibody detection. consists four proteins: nucleocapsid (N), envelope (E), membrane (M), spike (S). Interaction mediated S protein receptor domain (RBD) S1 subunit binds human angiotensin converting enzyme (ACE2) receptor. (4) Cellular transmembrane protease serine (TMPRSS2) lysosomal cathepsin proteases cleave S2 subunits, followed fusion initiated subunit. (5) While target protein, majority directed against more specifically RBD. (6,7) Most vaccines make use observation introducing mRNA viral vectors induce expression (8,9) directly protein. (10) Testing antinucleocapsid thus check infections, unless an inactivated vaccine was used, all proteins. (11) Antispike anti-RBD antibodies, which produced after provide means assert might serve correlate protection (CoP). (12−14) For information on definition CoP other reviews Perry et al., (14) Sobhani (15) Plotkin (16) recommended.Two categories quantified, (nAbs). former includes certain antigen, that prevent infection, i.e., blocking virus–host interaction preventing host–cell fusion. tests mimic quantify indirectly via their ability block interaction. gold standard plaque reduction neutralization test (PRNT), live incubated patient serum dilutions prior addition cells expressing Infection results formation plaques, quantified manual automatic counting. PRNT50 value correlates dilution required reduce observed without 50%. makes PRNT conventional (cVNTs), micro (microNT), highly accurate but requires biosafety level (BSL) facility turn-around times 3 days. (17) In mentioned above, BSL-3 would most. To safety requirements at least BSL-2, pseudovirus-neutralization (pVNTs) have been developed, relying lentiviruses vesicular stomatitis pseudotyped responsible (18−20) pVNT development, Rocha (21) Sun (22) Vaidya (23) recommended. SARS-CoV-2, RBD identified main providing opportunity further simplify tests. (sVNTs), focus review, rely competitive relevant ACE2 RBD, respectively. These cell-free assays divided into two categories: high-throughput screening (HTS) point-of-care (POC) assays. They do facilities times, making widely available. COVID showed monitor development titers studies, insights SARS-CoV-2. Still, sVNTs endorsed regulatory agencies status, yet. (24) date, three granted emergency authorization (EUA) Food Drug Administration (FDA). (25) Lacking standardization validation during well difficulties defining titer currently hinders progress take advantage sVNTs. However, they technological answer broad needed situations. following, overview last provided. Advantages disadvantages formats critically analyzed, future potential, applicability toward discussed.Binding TestsClick copied!Quantification assessment minutes hours POC. Such secondary recognize sections molecules. Thus, approaches need account isotypes seroconversion. Specifically, immunoglobulin M (IgM) levels rise quickly drop shortly recovery, IgA IgG increase decrease reliable indicators reinfection. (26,27) Time-resolved combinations IgM detailed about seroconversion vaccination. selected specialized facilities. important note strength lies large parts population. because infectiousness precedes multiple days, unsuitable diagnostic stop spread disease. research improving sensitivity specificity, maintaining ease-of-use lateral flow assay (LFA), intensified over 5 remarkable novelties. example, Hossain al. (28) alkaline phosphatase (AP)-conjugated enable off-the-shelf glucometer strips incorporation maltose phosphatase. Streptavidin magnetic nanoparticles were modified biotinylated Alkaline yellow (pNPP) enzymatically degraded PO43–, phosphorylase stoichiometrically converts glucose, then amperometrically minipotentiostat. A similar approach investigated Peng (29) who AP-conjugated electrochemical detection commercial hand-held potentiostat. Their platform (SPEEDS) consisted streptavidin-coated carbon working electrode counter electrode, Ag/AgCl reference electrode. determined conversion p-aminophenyl phosphate p-aminophenol AP, oxidized p-quinonimine chronoamperometry. Other researchers focused new materials sensor Nunez (30) based zinc oxide nanorods min. positive charge ZnO it interesting option adsorption negatively charged Further optimization technology market ready, system stable 15 days so far. Nanorods involved Shen al.'s work, developed magnetofluid-integrated multicolor immunochip (MMI-chip). (31) eight liquid storage wells MMI-chip connected mineral oil layer reaction washing steps (Figure 1). horseradish peroxidase (HRP)-labeled oxidize substrate 3,3′,5,5′-tetramethylbenzidine (TMB), etches nanorods, decreasing length thereby changing absorption spectra, enabling semiquantitative visual read-out. Although being multistep assay, chip design sealed environment, minimizing external exposure increasing user-friendliness. Chip fabrication many publications. Kim microfluidic fluorescent LFA integrated dry reagents, mixer, vacuum pump. (32) sample first passes DyLight 550-labeled reagent chamber, mixed herringbone spike-labeled polystyrene particle chamber. particles captured zone pillars, fluorescence measurements inverted microscope.Figure 1Figure 1. Schematic illustration MMI-chip. (A) Structural diagram loading (I), solution (II III), (IV), (V–VII), signal (VII). (B) Magnetic bead transportation between mixing magnet. (C) Workflow peripheral blood. Reproduced permission ref (31). Copyright 2022 Society.High Resolution ImageDownload MS PowerPoint SlideMultiplexing offers another intriguing avenue, allowing simultaneous variety antigens, opening possibility screen several variants prevalent simultaneously. growing interest, continuously mutates dominating variant changes quickly. Several multiplexing platforms machine learning, barcode beads, nanopore sensing, more. (33−37) paper-based vertical (xVFA) Eryilmaz able five antigens <20 (33) device 3D-printed mobile-phone-based optical reader. neural network 89.5% accuracy 31 samples tested training. Importantly, addressed limitations choice panel later application. Serum panels consisting local sera small numbers may biased less variations status. Nan naked-eye readable microarray (NRM) thickness sensing nanoplasmonic ruler provided POC friendly multiplex 2). (35) NRM chips 16 (∼2 μL each) simultaneously <30 nanoparticle (AuNP) monolayer, where increased decreased reflectance, which, turn, measurable gray analysis smartphone images. manufacturing tape-based transfer AuNP deemed impractical, however. Exploiting effects, Huang immunosensor nanoporous hollow anti-IgG coated chip. (38) generate min amplification washing. Similarly, dual-affinity ratiometric quenching (DARQ) fast homogeneous format. Kilgour fluorescein-labeled rhodamine-labeled L, obtaining signals just (39) Liang combined Raman spectroscopy. (40) synthesized silver ultrathin shells embedded 4-mercaptobenzoic acid conjugated them HS-PEG-COOH EDC/NHS chemistry. enabled dual-mode qualitative quantitative SERS read-out portable spectrometer 785 nm laser strip. changed allow instead (41) excellent example demonstrating noncompetitive format, technologies universally applicable. fact, discussed next chapters evolved out found written Lee (42) Yari (43) Dong (44)Figure 2Figure 2. Visualization micromosaic (a) photograph illustrating ready-to-use (top) channels incubation (bottom). (b) representation immobilization layout (top). Smartphone image chip, (c) Heatmap box plots (bottom) showing random donors reopening China December (Cohort (35). 2023 SlideCommercial Binding Neutralizing copied!The diseases established, starting enzyme-linked immunosorbent (ELISA) (45−47) and, recently, LFAs. (48) pandemic, companies adapted existing tools Many resulting products gained EUA FDA, indicated distribution, go complete FDA process. Now, beginning outbreak, EUAs revoked, (49) off market. Once discontinued, finding principles behind difficult, datasheets become unavailable. cases, publications Web sites listing best obtain information. At point writing, still lists 75 EUA, issued April 2020 July 2024 traditional marketing authorization. (50) Of these, IgG, IgM, total levels. Only targeting antibodies: SCoV-2 Detect Ab ELISA (InBios International, Inc.), Diazyme CLIA Kit (Diazyme Laboratories, cPass Detection (GenScript). list reveals that, besides classification tests, there major groups tests: ELISAs chemiluminescence immunoassays (CLIAs) LFAs POCT. mainly microtiter plate microarrays labeled HRP, fluorescence, markers. predominant sVNT literature GenScript's Kit, RBD-conjugated HRP ACE2-coated 96-well microplate 3a). (51−54) Two separate 37 °C TMB, overall time ∼1 h. market, varying times; examples Leinco ImmunoRank (Leinco Technologies, Inc.) (53) TECO SARS-CoV-2-AK Neutralisation Test (TECOmedical). (51,53) Fluorescence read-outs shorten fully automated systems. Examples Roche Elecsys Anti-SARS-CoV-2 S, (55) ruthenium–RBD conjugates, streptavidin-conjugated detection, RBD-modified microbeads ACE2-ABEI. 18 34 min, Graninger compared seven robust correlation live-virus test, ACE2-RBD DiaPro displayed highest nAb (51) AuNPs markers reached Healgen Scientific Rapid Cassette (55,56) comprises AuNPs, line absence reflectance spectrum analyzer response. VERI-Q (MiCo BioMed) (57) captures AuNP-RBD bound ACE2-mouse-Fc goat antimouse line, requiring serum. sensitive obtained conjugates ichroma test. (58) capturing ACE2–biotin conjugate streptavidin susceptible interference biotin. Users advised 24 h stopping intake biotin supplements ensure functionality, concentrations 500 ng/mL shown interfere assay. When 1:5 factored in, slightly below threshold 3510 (14367 nM) Clinical Laboratory Standards Institute (CLSI) guideline (EP37), physiological concentration measured dose uptake. (59) Actually, common problem streptavidin–biotin interaction, should kept mind development. pursued PremaLabs Diagnostics NAb kit. According McLean capture neutralized fluorescently (60) ACE2, site antibody. appears problematic, position could theory causing false negative result. An works blood, plasma. LFIA Finecare 2019-nCoV S-RBD advertised short (61)Figure 3Figure 3. Illustration principle kit Ahn biotin-based sVNT. Reprinted (62). Elsevier.High SlideTo compete available products, newly factor characteristics. (I) type, HTS consideration steps, time, equipment, interconnected cost (II) type serum, plasma, preferably (finger prick) blood (III) Desired sensitivity, qualitative, semiquantitative, Besides signaling agent, volume affects sensitivity. (IV) conjugation strategy needs adaptation viruses. intertwined purification strategy.New Concepts sVNTsClick following will discuss advantages sVNTs, taking possible product. better overview, grouped formats, subchapters concerning method cases signal-on sVNTs.High-Throughput copied! HRP-Based DetectionMany HRP-based published, conjugates. Correlations either complicating comparison formats. Table 1 conjugate, substrate, correlations easier comparison. immobilized His6-tag improved 3b) GenS

Language: Английский

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Temporal correlations between RBD-ACE2 blocking and binding antibodies to SARS-CoV-2 variants in CoronaVac-vaccinated individuals and their persistence in COVID-19 patients

Scientific Reports, Journal Year: 2025, Volume and Issue: 15(1)

Published: May 6, 2025

Abstract Antibodies play a crucial role in protection against SARS-CoV-2. Understanding the correlation between binding and functional antibodies is essential to determine whether antibody levels can reliably predict neutralizing activity. We assessed responses 111 individuals vaccinated with inactivated vaccine CoronaVac COVID-19 patients Thailand. Plasma of ACE2-blocking targeting receptor-binding domain (RBD) SARS-Co-V2 variants were measured before vaccination at 14 28 days after second dose using multiplex surrogate virus neutralization test. Anti-spike anti-nucleocapsid quantified by electrochemiluminescence immunoassay, anti-RBD IgG ELISA. After vaccination, blocking, anti-spike, increased but declined rapidly within month, whereas persisted. Blocking anti-spike correlated day post-vaccination not 28. In patients, correlations moderate 14, stronger Correlations weaker for Omicron subvariants than ancestral strain non-Omicron variants. The weak blocking suggests might These findings highlight temporal nature CoronaVac-induced immunity need booster doses variant-adapted vaccine.

Language: Английский

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Anti-SARS-CoV-2 serology based on ancestral RBD antigens does not correlate with the presence of neutralizing antibodies against Omicron variants

Microbiology Spectrum, Journal Year: 2024, Volume and Issue: 13(1)

Published: Nov. 20, 2024

ABSTRACT Neutralizing antibody titers and binding levels are considered correlates of protection against severe SARS-CoV-2 infection. The clinical utility serology should be reevaluated in light the emergence escape variants, as commercial antibody-binding assays have not been adapted to virus’ antigenic evolution. We compared anti-SARS-CoV-2 four quantitative serological tests based on variable ancestral spike antigens (three in-house ELISAs prototype VIDAS IgG QUANT assay) neutralization pseudotyped Wuhan, BA.2, BA.4/5, BQ.1.1, XBB.1.1 viruses a cohort 100 patients infected 2020 or during Omicron waves. Binding correlated well with neutralizing for but association decreased BQ.1.1 XBB.1 (for assay, Spearman’s correlation was 0.82 [95% CI 0.74–0.88] 0.61 [0.46–0.72] BA.2 XBB.1, respectively). In 15% no antibodies assay still yielded ranging from 74 7,652 units/mL. Using an adjusted threshold receiver operating characteristic (ROC) curve analysis, specificity detection increased 0.15 (95% 0.02–0.45) 0.17 (0.04–0.41) 0.92 (0.64–1.00) 0.83 (0.59–0.96) respectively. Serological receptor-binding domain virus fail predict activity latest circulating variants. Adapting may improve their immunocompromised patients. IMPORTANCE Anti-SARS-CoV-2 developed response COVID-19 pandemic diagnose infection monitor individual’s immunity following natural vaccination. Given relationship between infection, many studies evaluated pre-Omicron era. An important potential use serology, which explores antibodies, is estimating level new particularly immunosuppressed individuals those at risk COVID. However, era, evade induced by previous infections vaccination, determined re-examined order determine whether these optimized adapting strains.

Language: Английский

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Comparison of anti‐nucleocapsid antibody assays for the detection of SARS‐CoV‐2 Omicron vaccine breakthroughs after various intervals since the infection

Journal of Medical Virology, Journal Year: 2023, Volume and Issue: 95(11)

Published: Nov. 1, 2023

Abstract Antibody assays with the nucleocapsid (NC) protein as target antigen can identify severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infections when polymerase chain reaction (PCR) analyses are unavailable. Regarding kinetics of NC‐specific antibodies, vaccine breakthroughs Omicron subvariants may differ from ancestral wild‐type virus. Therefore, we evaluated which have highest sensitivity for detecting antibodies after various intervals since breakthrough an subvariant. The study included 279 samples vaccinated subjects who experienced PCR‐confirmed between 21 and 266 days before sampling. were comparatively assessed Elecsys® Anti‐SARS‐CoV‐2 N (Roche), Anti‐SARS‐CoV‐2‐NCP‐ELISA (Euroimmun), recomLine SARS‐CoV‐2 IgG (Mikrogen), ViraChip (Viramed). In whole cohort, assay displayed (93%, p < 0.0001), followed by (70%), (41%) (35%). Although measured antibody levels time‐dependent sensitivities differed, extent decrease was similar among all assays. As demonstrated this study, manufacturer‐dependent differences in should be considered serology is applied to link previous potential post‐COVID sequelae.

Language: Английский

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tANCHOR fast and cost-effective cell-based immunization approach with focus on the receptor-binding domain of SARS-CoV-2

Biology Methods and Protocols, Journal Year: 2023, Volume and Issue: 8(1)

Published: Jan. 1, 2023

Abstract Successful induction of antibodies in model organisms like mice depends strongly on antigen design and delivery. New designs for immunization are helpful developing future therapeutic monoclonal (mAbs). One the gold standards to induce is express purify vaccination. This especially time-consuming when mAbs needed rapidly. We closed this gap used display technology tetraspanin anchor develop a reliable technique without need antigen. able speed up step enormously we have demonstrated that were against different proteins with focus receptor-binding domain SARS-CoV-2 extracellular loop canine cluster differentiation 20 displayed surface human cells.

Language: Английский

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