scholarly journals SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors

2020 ◽  
Author(s):  
Ludivine Grzelak ◽  
Sarah Temmam ◽  
Cyril Planchais ◽  
Caroline Demeret ◽  
Christèle Huon ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Blood Donors ◽  
Hospitalized Patients ◽  
High Antibody ◽  
Serological Analysis ◽  
Healthy Donors ◽  
Response Profile ◽  
Antibody Titers ◽  
Antibody Profiling ◽  
Nucleoprotein N

AbstractIt is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their antibody response profile. Here, we performed a pilot study to assess the levels of anti-SARS-CoV-2 antibodies in samples taken from 491 pre-epidemic individuals, 51 patients from Hôpital Bichat (Paris), 209 pauci-symptomatic individuals in the French Oise region and 200 contemporary Oise blood donors. Two in-house ELISA assays, that recognize the full-length nucleoprotein (N) or trimeric Spike (S) ectodomain were implemented. We also developed two novel assays: the S-Flow assay, which is based on the recognition of S at the cell surface by flow-cytometry, and the LIPS assay that recognizes diverse antigens (including S1 or N C-terminal domain) by immunoprecipitation. Overall, the results obtained with the four assays were similar, with differences in sensitivity that can be attributed to the technique and the antigen in use. High antibody titers were associated with neutralisation activity, assessed using infectious SARS-CoV-2 or lentiviral-S pseudotypes. In hospitalized patients, seroconversion and neutralisation occurred on 5-14 days post symptom onset, confirming previous studies. Seropositivity was detected in 29% of pauci-symptomatic individuals within 15 days post-symptoms and 3 % of blood of healthy donors collected in the area of a cluster of COVID cases. Altogether, our assays allow for a broad evaluation of SARS-CoV2 seroprevalence and antibody profiling in different population subsets.

scholarly journals A comparison of four serological assays for detecting anti–SARS-CoV-2 antibodies in human serum samples from different populations

2020 ◽  
Vol 12 (559) ◽  
pp. eabc3103 ◽  
Author(s):  
Ludivine Grzelak ◽  
Sarah Temmam ◽  
Cyril Planchais ◽  
Caroline Demeret ◽  
Laura Tondeur ◽  
...  
Keyword(s):  
Symptom Onset ◽  
Blood Donors ◽  
Serum Samples ◽  
Neutralization Activity ◽  
S Protein ◽  
Antibody Assays ◽  
Antibody Titers ◽  
Different Populations ◽  
Carboxyl Terminal ◽  
Nucleoprotein N

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti–SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti–SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.

Haptoglobin polymorphisms and fucosylation change: possible influence of variation on the identified lung cancer-relevant biomarkers

2020 ◽  
Vol 17 ◽  
Author(s):  
Kongsak Boonyapranai ◽  
Sakaewan Ounjaijean ◽  
Kanokwan Kulprachakarn ◽  
Laddawan Potpromanee ◽  
Miles Chih-Ming Chen ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Healthy Donor ◽  
Blood Donors ◽  
Acute Phase Protein ◽  
Biological Properties ◽  
Diagnostic Biomarker ◽  
Lung Cancer Patients ◽  
Healthy Donors ◽  
Phase Protein ◽  
Blotting Technique

Background: Haptoglobin (Hp), an acute-phase protein, is known as a potential diagnostic biomarker in human diseases. Two alleles of Hp (Hp1 and Hp2) exist in humans allowing three phenotypes (Hp1-1, Hp2-1, and Hp2-2), which influence the biophysical and biological properties of Hp. Objective: This work aimed to investigate the variation of serum level and fucosylation change among Hp phenotypes in pa-tients with lung cancer compared to healthy donors. Method: 44 patients with lung cancer and 26 healthy blood donors who lived in the Northern-Thailand region were investi-gated by glycoproteomic procedure. Results: The phenotypic distribution of the Hp (Hp1-1:Hp2-1:Hp2-2) in healthy donors were 0.04:0.38:0.58, while the pa-tient group were 0.09:0.52:0.39. The Hp1 allele frequency of the patients with lung cancer (0.34) was higher than the healthy donor (0.23). Glycoprotein blotting technique represented that the level of serum Hp and its fucosylation were sig-nificantly higher among lung cancer patients compared to those of the healthy donors. However, a downward trend in the fucosylation level from Hp1-1 to Hp2-1, Hp2-2, was seen in the patient group, but varying in the serum Hp level. An N-linked glycan was enzymatically released from discrete Hp multimers of Hp2-1 and Hp2-2 samples. Analysis of glycan pro-filing by MALDI-TOF-MS showed that reduction of the fucosylated glycan was associated with the size of Hp multimers, resulting in the lower level of fucosylation in Hp2-1 and Hp2-2, respectively. Conclusion: Our finding demonstrates that the Hp phenotype is a dependent risk factor for lung cancer and should be incor-porated into further clinical and biochemical investigations of diseases, including lung cancer.

542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A578-A578
Author(s):  
Andreia Maia ◽  
Joana Lerias ◽  
Markus Maeurer ◽  
Mireia Castillo-Martin
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Nk Cell ◽  
Tumour Cells ◽  
List Type ◽  
Mhc I ◽  
Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

scholarly journals A Multiplex, Droplet Digital PCR Assay for the Detection of T-Cell Receptor Excision Circles and Kappa-Deleting Recombination Excision Circles

2019 ◽  
Vol 66 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Tracie Profaizer ◽  
Patricia Slev
Keyword(s):  
Flow Cytometry ◽  
T Cell ◽  
T Cell Receptor ◽  
Reference Gene ◽  
Cell Receptor ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Blood Samples ◽  
Healthy Donors ◽  
Excision Circles

Abstract BACKGROUND T-cell receptor excision circles (TREC) and κ-deleting recombination receptor excision circles (KREC) concentrations can be used to assess and diagnose immune deficiencies, monitor thymic and bone marrow immune reconstitution, or follow responses to drug therapy. We developed an assay to quantify TREC, KREC, and a reference gene in a single reaction using droplet digital PCR (ddPCR). METHODS PCR was optimized for 3 targets: TREC, KREC, and ribonuclease P/MRP subunit p30 (RPP30) as the reference gene. Multiplexing was accomplished by varying the target's fluorophore and concentration. Correlation with clinical results was evaluated using 47 samples from healthy donors, 59 samples with T-cell and B-cell markers within the reference interval from the flow cytometry laboratory, 20 cord blood samples, and 34 samples submitted for exome sequencing for severe combined immunodeficiency disease (SCID). RESULTS The limit of the blank was 4 positive droplets, limit of detection 9 positive droplets, and limit of quantification 25 positive droplets, or 2.0 copies/μL. TREC and KREC copies/μL were as expected in the healthy donors and cord blood samples and concordant with the healthy flow cytometry results. Of the samples from the SCID Panel, 56.5% had a TREC count &lt;20 copies/μL and 17.7% had a KREC count &lt;20 copies/μL, suggestive of low T- and B-cell numbers, respectively. CONCLUSIONS Our multiplex ddPCR assay is an analytically sensitive and specific method for the absolute quantification of TREC and KREC. To the best of our knowledge, this paper is the first to describe the simultaneous quantification of TREC, KREC, and a reference gene by use of ddPCR.

scholarly journals Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19

2021 ◽  
Vol 22 (5) ◽  
pp. 2723
Author(s):  
Linhua Tian ◽  
Elzafir B. Elsheikh ◽  
Paul N. Patrone ◽  
Anthony J. Kearsley ◽  
Adolfas K. Gaigalas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Vaccine Development ◽  
Neutralizing Antibody ◽  
Epidemiologic Studies ◽  
Cross Reactivity ◽  
Receptor Binding Domain ◽  
Proof Of Concept ◽  
Reference Standard ◽  
Improve Measurement ◽  
Antibody Titers

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here, we report a proof-of-concept approach for the development of quantitative, multiplexed flow cytometry-based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.

scholarly journals Comparison of a Classical Phagocytosis Assay and a Flow Cytometry Assay for Assessment of the Phagocytic Capacity of Sera from Adults Vaccinated with a Pneumococcal Conjugate Vaccine

2001 ◽  
Vol 8 (2) ◽  
pp. 245-250 ◽  
Author(s):  
Wouter T. M. Jansen ◽  
Merja Väkeväinen-Anttila ◽  
Helena Käyhty ◽  
Moon Nahm ◽  
N. Bakker ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Defense Mechanism ◽  
Worker Safety ◽  
Enzyme Linked Immunosorbent Assay ◽  
Phagocytosis Assay ◽  
Phagocytic Capacity ◽  
Killing Assay ◽  
Potential Efficacy ◽  
Antibody Titers ◽  
Low Sensitivity

ABSTRACT Antibody- and complement-mediated phagocytosis is the main defense mechanism against Streptococcus pneumoniae. A standardized, easy to perform phagocytosis assay for pneumococci would be a great asset for the evaluation of the potential efficacy of (experimental) pneumococcal vaccines. Such an assay could replace the laborious phagocytosis assay of viable pneumococci (classical killing assay). Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was compared with the conventional killing assay and enzyme-linked immunosorbent assay (ELISA), using sera obtained from adults pre- and postvaccination with either a bivalent conjugate, a tetravalent conjugate, or the 23-valent polysaccharide vaccine. Highly significant correlations were observed between flow assay phagocytosis titers, killing assay phagocytosis titers, and ELISA antibody titers for serotype 6B and 23F as well. For serotype 19F, strong correlations were only observed between killing assay and ELISA titers. A potential drawback of the flow assay might be the low sensitivity compared with that of the killing assay. The choice of what assay to use, however, will depend on the objectives of the assay. When speed, easy performance, sample throughput, improved worker safety, absence of influence of antibiotics, and absence of false positives are the major criteria, the flow assay is the method of choice. When higher sensitivity is the major requirement, the classical killing assay should be used.

PASSIVE IMMUNIZATION

PEDIATRICS ◽  
1963 ◽  
Vol 32 (4) ◽  
pp. 599-609
Author(s):  
Geoffrey Edsall
Keyword(s):  
Diphtheria Toxin ◽  
Gamma Globulin ◽  
Passive Immunization ◽  
Gas Gangrene ◽  
High Antibody ◽  
Antibody Titers ◽  
The Subject ◽  
Guiding Principles ◽  
Routine Procedures ◽  
The Ideal

Passive immunization has existed for over 70 years, ever since Von Behring and Kitasato demonstrated its effectiveness in neutralizing diphtheria toxin. In fact, at first glance one might think that there was little new to say on this subject. However, the very fact that its concepts and practices have been so long accepted and–in the minds of many–have fallen into the pattern of purely routine procedures, is in itself sufficient justification to re-examine the subject. In addition, moreover, there have been a number of changes in the range of diseases for which passive immunization may be employed, the type of antiserum used, and the guiding principles for use of such preparations. Therefore, it may be timely to deal with some of the present considerations that apply to passive immunization, its prospects, its scope, and its limitations. At the risk of repeating old and familiar cliches it appears desirable to summarize, at first, the guiding principles which apply to the effectiveness (or ineffectiveness) of passive immunization. First of all, it is well established that some techniques of passive immunization are highly effective–e.g., diphtheria prophylaxis with antitoxin; some are very useful but fall short of the ideal of routine success with the purpose intended–e.g., the use of gamma-globulin for the modification of measles; whereas others are of relatively uncertain value so that their usefulness in medical practice still continues to be debated–e.g., gas gangrene antitoxin. The reasons for such great disparity in the efficacy of different antisera cannot easily be put into generalizations, but surely the varied pathogenesis of the diseases in question must be a major factor, as well as the fact that high antibody titers can readily be obtained for some such sera, whereas they are difficult or impossible to achieve with others.

scholarly journals Seroprevalence rates of transfusion-transmitted infections among blood donors in Jordan

2016 ◽  
Vol 10 (04) ◽  
pp. 377-383 ◽  
Author(s):  
Lina Souan ◽  
Faten Tout ◽  
Mahmoud Siag ◽  
Maher A. Sughayer
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Blood Donors ◽  
Hepatitis B Surface Antigen ◽  
Cancer Center ◽  
Type I ◽  
Human T Cell ◽  
Healthy Donors ◽  
Elisa Technique ◽  
Anti Hiv

Introduction: Statistics on the prevalence of donor screening for hepatitis B surface antigen (HBsAg) and anti-hepatitis C virus (anti-HCV) in Jordan are outdated. There are no studies on the prevalence of anti-HIV I/II, anti-human T-cell lymphotropic virus type I and II (anti-HTLV-I/II), or anti-syphilis. Data are also lacking on the prevalence and significance of using anti-HBc screening in Jordan. This study aimed to evaluate the prevalence of transfusion-transmissible infections (TTI) among donors at King Hussein Cancer Center and compare it with neighboring countries and to evaluate the significance of screening for anti-HB core total (anti-HBc) antibodies. Methodology: A retrospective analysis covering the period from 2009 to 2013 was conducted on records of healthy donors. The number of donors was 10,101, 12,694, 13,387, 14,256, and 12,495, respectively. Donors were screened for HBsAg, anti-HBc, anti-HIV I/II, anti-HCV, and anti-HTLV-I/II using ELISA technique, while syphilis antibodies were detected using rapid chromatographic immunoassay. Results: Among 62,933 donors, the prevalence of HBsAg was 0.52%, of anti-HBc was 6.04%, and of anti-HCV was 0.16%. None of the donors were positive for anti-HIV I/II, anti-HTLV I/II, or anti-TP. Conclusions: This study demonstrates that the seroprevalence for HBsAg, anti-HBc, and anti-HCV in Jordan was low compared to neighboring countries. None of the donors were confirmed positive for anti-HIV I/II, anti-HTLV I/II, or anti-TP during the studied period. This study demonstrates the importance of screening for anti-HBc to improve blood and platelet safety and stresses the need to complement it with an algorithm that qualifies reentry of anti-HBc false-positive donors.

scholarly journals Can we predict antibody responses in SARS-CoV-2? A cohort analysis

2021 ◽  
Author(s):  
Mary Gaeddert ◽  
Philip Kitchen ◽  
Tobias Broger ◽  
Stefan Weber ◽  
Ralf Bartenschlager ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Severe Disease ◽  
Cohort Analysis ◽  
Antibody Responses ◽  
High Antibody ◽  
Igg Antibodies ◽  
Rt Pcr ◽  
Median Increase ◽  
Antibody Titers

AbstractBackgroundAfter infection with severe acute respiratory syndrome coronavirus (SARS-CoV-2), Immunoglobulin G (IgG) antibodies and virus-specific neutralizing antibodies (nAbs) develop. This study describes antibody responses in a cohort of recovered COVID-19 patients to identify predictors.MethodsWe recruited patients with confirmed SARS-CoV-2 infection from Heidelberg, Germany. Blood samples were collected three weeks after COVID-19 symptoms ended. Participants with high antibody titers were invited for follow-up visits. IgG titers were measured by the Euroimmun Assay, and nAbs titers in a SARS-CoV-2 infection-based assay.Results281 participants were enrolled between April and August 2020 with IgG testing, 145 (51.6%) had nAbs, and 35 (12.5%) had follow-up. The median IgG optical density (OD) ratio was 3.1 (Interquartile range (IQR) 1.6-5.1), and 24.1% (35/145) had a nAb titer>1:80. Higher IgG titers were associated with increased age and more severe disease, and higher nAbs were associated with male gender and CT-value of 25-30 on RT-PCR at diagnosis. The median IgG OD ratio on follow-up was 3.7 (IQR 2.9-5.9), a median increase of 0.5 (IQR −0.3-1.7). Six participants with follow-up nAbs all had titers ≤ 1:80.ConclusionsWhile age and disease severity were correlated with IgG responses, predictive factors for nAbs in convalescent patients remain unclear.

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