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.

scholarly journals THE EFFECT OF HOMALANTHUS POPULNEUS (GIESEL.) PAX. EXTRACT IN EXPRESSION OF T-CELL RECEPTOR: INHIBITION STUDY OF HIV INFECTION

2019 ◽  
pp. 134-137
Author(s):  
SINTYA E ◽  
WIJAYANTI N ◽  
NORAENI A
Keyword(s):  
Flow Cytometry ◽  
T Cell Receptor ◽  
Defense Mechanism ◽  
Cell Receptor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Inhibition Study ◽  
Peripheral Blood Mononuclear ◽  
Lymphoblast Cell Line ◽  
Receptor Inhibition

Objective: The aim of this study is to analyze the effect of Homalanthus populneus’s extract toward the expression of CD4 and CD8 which both areimportant in body’s defense mechanism against HIV.Methods: Leaves and barks of H. populneus were extracted with 70% ethanol. Freeze dry method had been used in order to get the final extract.PBMCs were extracted and it has been used for CD4 and CD8 expression test using flow cytometry. In oreder to analyze the effect of the extract to theHIV type 1, gp41 and gp120 expression was tested using talicytometry and using enzyme-linked immunosorbent assay, respectively.Results: This study reported that H. populneus’s extract reduced the expression of CD4 receptor in both peripheral blood mononuclear cell (PBMC)and T-lymphoblast cell line (CEM). In contract, this extract increased CD8 expression in PBMC. It was also able to reduce the percentage of proteingp41 and gp120 in CEM cultures.Conclusion: Those results show that H. populneus’s extract is potentially developed as an HIV drug from Indonesia. However, further study needs tobe done including analyzing the effect of variety of concentrations and also exposure periods.

scholarly journals Use of Highly Encapsulated Streptococcus pneumoniae Strains in a Flow-Cytometric Assay for Assessment of the Phagocytic Capacity of Serotype-Specific Antibodies

1998 ◽  
Vol 5 (5) ◽  
pp. 703-710 ◽  
Author(s):  
W. T. M. Jansen ◽  
J. Gootjes ◽  
M. Zelle ◽  
D. V. Madore ◽  
J. Verhoef ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Streptococcus Pneumoniae ◽  
Surface Proteins ◽  
Heat Inactivation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphonuclear Cells ◽  
Phagocytic Capacity ◽  
Specific Antibodies ◽  
Flow Cytometric ◽  
Positive Results

ABSTRACT A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 μl per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

scholarly journals Anti-HPV16 Antibody Titers Prior to an Incident Cervical HPV16/31 Infection

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1548
Author(s):  
Ana Gradissimo ◽  
Viswanathan Shankar ◽  
Fanua Wiek ◽  
Lauren St. Peter ◽  
Yevgeniy Studentsov ◽  
...  
Keyword(s):  
High Risk ◽  
Hpv Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Incident Detection ◽  
Antibody Titers ◽  
Circulating Antibodies ◽  
Prospective Association ◽  
Nested Case Control ◽  
Incident Cases

The goal of this study was to investigate the serological titers of circulating antibodies against human papillomavirus (HPV) type 16 (anti-HPV16) prior to the detection of an incident HPV16 or HPV31 infection amongst vaccinated participants. Patients were selected from a prospective post-HPV vaccine longitudinal cohort at Mount Sinai Adolescent Health Center in Manhattan, NY. We performed a nested case–control study of 43 cases with incident detection of cervical HPV16 (n = 26) or HPV31 (n = 17) DNA who had completed the full set of immunizations of the quadrivalent HPV vaccine (4vHPV). Two control individuals whom had received three doses of the vaccine (HPV16/31-negative) were selected per case, matched on age at the first dose of vaccination and follow-up time in the study: a random control, and a high-risk control that was in the upper quartile of a sexual risk behavior score. We conducted an enzyme-linked immunosorbent assay (ELISA) for the detection of immunoglobulin G (IgG) antibodies specific to anti-HPV16 virus-like particles (VLPs). The results suggest that the average log antibody titers were higher among high-risk controls than the HPV16/31 incident cases and the randomly selected controls. We show a prospective association between anti-HPV16 VLP titers and the acquisition of an HPV16/31 incident infection post-receiving three doses of 4vHPV vaccine.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

scholarly journals Serological Evidence of Filovirus Infection in Nonhuman Primates in Zambia

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1283
Author(s):  
Katendi Changula ◽  
Edgar Simulundu ◽  
Boniface Pongombo Lombe ◽  
Eri Nakayama ◽  
Hiroko Miyamoto ◽  
...  
Keyword(s):  
Nonhuman Primates ◽  
Hemorrhagic Fever ◽  
Enzyme Linked Immunosorbent Assay ◽  
Vervet Monkeys ◽  
Intermediate Hosts ◽  
Significant Difference ◽  
Positive Rate ◽  
Antibody Titers ◽  
Potential Risks ◽  
Reston Virus

Ebolaviruses and marburgviruses are filoviruses that are known to cause severe hemorrhagic fever in humans and nonhuman primates (NHPs). While some bat species are suspected to be natural reservoirs of these filoviruses, wild NHPs often act as intermediate hosts for viral transmission to humans. Using an enzyme-linked immunosorbent assay, we screened two NHP species, wild baboons and vervet monkeys captured in Zambia, for their serum IgG antibodies specific to the envelope glycoproteins of filoviruses. From 243 samples tested, 39 NHPs (16%) were found to be seropositive either for ebolaviruses or marburgviruses with endpoint antibody titers ranging from 100 to 25,600. Interestingly, antibodies reactive to Reston virus, which is found only in Asia, were detected in both NHP species. There was a significant difference in the seropositivity for the marburgvirus antigen between the two NHP species, with baboons having a higher positive rate. These results suggest that wild NHPs in Zambia might be nonlethally exposed to these filoviruses, and this emphasizes the need for continuous monitoring of filovirus infection in wild animals to better understand the ecology of filoviruses and to assess potential risks of outbreaks in humans in previously nonendemic countries.

scholarly journals Evaluation of Recombinant Proteins of Burkholderia mallei for Serodiagnosis of Glanders

2012 ◽  
Vol 19 (8) ◽  
pp. 1193-1198 ◽  
Author(s):  
Vijai Pal ◽  
Subodh Kumar ◽  
Praveen Malik ◽  
Ganga Prasad Rai
Keyword(s):  
Sensitivity And Specificity ◽  
Recombinant Proteins ◽  
False Negative ◽  
Enzyme Linked Immunosorbent Assay ◽  
Burkholderia Mallei ◽  
Content Type ◽  
Whole Cells ◽  
Negative Results ◽  
Elisa Format ◽  
Low Sensitivity

ABSTRACTGlanders is a contagious disease caused by the Gram-negative bacillusBurkholderia mallei. The number of equine glanders outbreaks has increased steadily during the last decade. The disease must be reported to the Office International des Epizooties, Paris, France. Glanders serodiagnosis is hampered by the considerable number of false positives and negatives of the internationally prescribed tests. The major problem leading to the low sensitivity and specificity of the complement fixation test (CFT) and enzyme-linked immunosorbent assay (ELISA) has been linked to the test antigens currently used, i.e., crude preparations of whole cells. False-positive results obtained from other diagnostic tests utilizing crude antigens lead to financial losses to animal owners, and false-negative results can turn a risk into a possible threat. In this study, we report on the identification of diagnostic targets using bioinformatics tools for serodiagnosis of glanders. The identified gene sequences were cloned and expressed as recombinant proteins. The purified recombinant proteins ofB. malleiwere used in an indirect ELISA format for serodiagnosis of glanders. Two recombinant proteins, 0375H and 0375TH, exhibited 100% sensitivity and specificity for glanders diagnosis. The proteins also did not cross-react with sera from patients with the closely related disease melioidosis. The results of this investigation highlight the potential of recombinant 0375H and 0375TH proteins in specific and sensitive diagnosis of glanders.

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 High-throughput quantitation of SARS-CoV-2 antibodies in a single-dilution homogeneous assay

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Markus H. Kainulainen ◽  
Eric Bergeron ◽  
Payel Chatterjee ◽  
Asheley P. Chapman ◽  
Joo Lee ◽  
...  
Keyword(s):  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Negative Results ◽  
A Value ◽  
Elisa Testing ◽  
The World ◽  
Antibody Titers ◽  
Qualitative Assay ◽  
Homogeneous Assay ◽  
Asymptomatic Infections

AbstractSARS-CoV-2 emerged in late 2019 and has since spread around the world, causing a pandemic of the respiratory disease COVID-19. Detecting antibodies against the virus is an essential tool for tracking infections and developing vaccines. Such tests, primarily utilizing the enzyme-linked immunosorbent assay (ELISA) principle, can be either qualitative (reporting positive/negative results) or quantitative (reporting a value representing the quantity of specific antibodies). Quantitation is vital for determining stability or decline of antibody titers in convalescence, efficacy of different vaccination regimens, and detection of asymptomatic infections. Quantitation typically requires two-step ELISA testing, in which samples are first screened in a qualitative assay and positive samples are subsequently analyzed as a dilution series. To overcome the throughput limitations of this approach, we developed a simpler and faster system that is highly automatable and achieves quantitation in a single-dilution screening format with sensitivity and specificity comparable to those of ELISA.

scholarly journals Serological Evaluation of Specific-Antibody Levels in Patients Treated for Chronic Chagas' Disease

2007 ◽  
Vol 15 (2) ◽  
pp. 297-302 ◽  
Author(s):  
Olga Sánchez Negrette ◽  
Fernando J. Sánchez Valdéz ◽  
Carlos D. Lacunza ◽  
María Fernanda García Bustos ◽  
María Celia Mora ◽  
...  
Keyword(s):  
Chagas Disease ◽  
Treatment Effectiveness ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serological Tests ◽  
Recombinant Antigens ◽  
Antibody Titers ◽  
Laboratory Procedures ◽  
The Individual ◽  
Antibody Levels

ABSTRACT Serological tests are the main laboratory procedures used for diagnosis during the indeterminate and chronic stages of Chagas' disease. A serological regression to negativity is the main criterion used to define parasitological cure in treated patients. The aim of this work was to monitor the individual specificities of antibody levels for 3 years posttreatment in 18 adult patients. Conventional serological techniques (hemagglutination assays and enzyme-linked immunosorbent assay [ELISA]) were modified by using recombinant antigens to detect early markers of treatment effectiveness. For this purpose, serum samples were taken before and during treatment and every 6 months after treatment for at least 3 years. When hemagglutination assays were used, a decrease in antibody levels was observed in only one patient. When ELISA with serum dilutions was used, antibody clearance became much more apparent: in 77.7% (14/18) of the patients, antibody titers became negative with time. This was observed at serum dilutions of 1/320 and occurred between the 6th and the 30th months posttreatment. The immune response and the interval for a serological regression to negativity were different for each patient. For some of the recombinant antigens, only 50% (9/18) of the patients reached the serological regression to negativity. Recombinant antigen 13 might be a good marker of treatment effectiveness, since 66.6% (six of nine) of the patients presented with an early regression to negativity for specific antibodies to this antigen (P = 0.002).

Διαχρονικές μεταβολές βιοενέργειας, μεταβολισμού και ανοσολογικής απόκρισης στην οξεία φάση της σοβαρής σήψης

2018 ◽  
Author(s):  
Άννα-Μαρία Σπανάκη-Μπαρμπουνάκη
Keyword(s):  
Body Mass Index ◽  
Flow Cytometry ◽  
Severe Sepsis ◽  
Heat Shock ◽  
Energy Expenditure ◽  
Systemic Inflammatory Response Syndrome ◽  
Enzyme Linked Immunosorbent Assay ◽  
Z Scores ◽  
Luminescent Assay ◽  
Shock Proteins

Εισαγωγή: Το κυτταρικό στρες από σοβαρή σήψη (severe sepsis, SS) ή σύνδρομο συστηματικής φλεγμονώδους απάντησης (Systemic inflammatory response syndrome, SIRS) εκδηλώνεται με οξείες φλεγμονώδεις, ορμονικές, ανοσολογικές και μεταβολικές διαταραχές. Η συσχέτισή τους με πιθανή δυσλειτουργία μιτοχονδρίων δεν έχει επαρκώς μελετηθεί. Σκοπός: Σκοπός της μελέτης ήταν η εκτίμηση των διαχρονικών μεταβολών φλεγμονώδους-ορμονικής αντίδρασης, ενδογενούς-ανοσίας, βιοενέργειας και μεταβολισμού σε ασθενείς, ενήλικες και παιδιά, με σοβαρή σήψη (SS) και η σύγκριση με αντίστοιχες ομάδες ασθενών με SIRS και υγιών (H), ενηλίκων και παιδιών.Υλικά/Μέθοδοι: Μελετήθηκαν 68 παιδιά (SS/18, SIRS/23, H/27) και 79 ενήλικες (SS/23, SIRS/23, H/33) διαχρονικά, την 1, 3η και 5η ημέρα νοσηλείας. Υπολογίστηκαν ο δείκτης μάζας σώματος (Body mass index (BMI) z-scores) και τα scores βαρύτητας νόσου (PeLOD, APACHE, TISS, SOFA). Μετρήθηκαν η καρδιακή συσταλτικότητα (EF, SF), η τροπονίνη (Tn), το γαλακτικό οξύ, η κατανάλωση ενέργειας (Energy expenditure, EE) με Gas Module E-COVX, το ATP στα λευκά αιμοσφαίρια με δοκιμασία λουσιφεράσης (luciferase luminescent assay), τα επίπεδα γλουταμίνης και NO2/NO3 με υγρή χρωματογραφία υψηλής πίεσης (HPLC), τα προϊόντα υπεροξείδωσης λιπιδίων (TBARS) με χρωματομετρική δοκιμασία, η ρεζιστίνη, η αντιπονεκτίνη ορού και οι εξωκυττάριες Heat Shock Proteins (HSP) με την ποσοτική ανοσοενζυμική μέθοδο ELISA (sandwich enzyme-linked immunosorbent assay), και οι ενδοκυττάριες HSP72, HSP90α με κυτταρομετρία ροής (flow cytometry). Αποτελέσματα: Διαχρονικά τόσο σε ενήλικες (ICU) όσο και σε παιδιά (PICU) οι τιμές ρεζιστίνης, αντιπονεκτίνης, εξωκυττάριας HPS72 και 90α παρουσιάζαν σταθερό πρότυπο διέγερσης σε όλη την οξεία φάση των 5 ημέρων. Στη χρονική αυτή περίοδο, οι παράμετροι μεταβολισμού VO2, VCO2, EE παρουσίασαν σταθερό υπομεταβολικό προφίλ, ίδιο σε ενήλικες και παιδιά. Η αυξημένη έκφραση των NO3, NO2, TBARS και αντιπονεκτίνης στη σήψη παρουσίασαν μια πιο ασταθή εικόνα όσον αφορά τη διαχρονική τους έκφραση ανά ηλικιακή ομάδα.Η βιοενέργεια των μιτοχονδρίων ήταν διαχρονικά μειωμένη σε ενήλικες κα παιδιά που δεν επιβίωσαν σε σχέση με εκείνους που επιβίωσαν, και συνοδεύονταν από σημαντικά μειωμένο μεταβολισμό και υπομεταβολικά πρότυπα την 3η και 5η ημέρα (p<0.05). Οι ασθενείς που επιβίωσαν παρουσίαζαν σημαντική διαφοροποίηση των αρχικών τιμών BVR, γαλακτικού, ΕΕ, VO2, VCO2 και μεταβολικού προφιλ σε σύγκριση με ασθενείς που πέθαναν, οι οποίοι έδειξαν αδυναμία ανάκαμψης του υπομεταβολισμού ή της αντιοξειδωτικής κατάστασης την 5η ημέρα. Συμπέρασμα: Η SS χαρακτηρίζεται διαχρονικά, από ισχυρότερη ενδοκυττάρια καταστολή μεταβολισμού, μείωση κατανάλωσης ενέργειας, μείωση των ATP, HPS72, HSP90α, αλβουμίνης, γλουταμίνης και εξωκυττάρια αύξηση φλεγμονωδών ορμονών, ρεζιστίνης και αντιπονεκτίνης και πρωτεϊνών έμφυτης ανοσίας (HSP72). Μια πρώιμη κατάσταση υπομεταβολισμού, με καταστολή βιοενέργειας και ενδογενούς ανοσίας, η οποία και παραμένει διαχρονικά μαζί με συνεχιζόμενη κατάσταση φλεγμονής, με διαχρονικά αυξημένες μεταβολικές ορμόνες και πρωτείνες eHSP72/HSP90α, φαίνεται να ξεχωρίζει τη σήψη από το SIRS, και συνδέεται με αυξημένο κίνδυνο θανάτου.Λέξεις κλειδιά: σήψη, SIRS, βιοενέργεια, HSP, μιτοχόνδρια, μεταβολισμός, θερμιδομετρία, αμινοξέα, οξείδιο του αζώτου, ATP, ρεζιστίνη, αντιπονεκτίνη, τραύμα

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