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A new virus-like particle based vaccine covering 14 types of high-risk and disease-inducing human papillomavirus (HPV) can offer better coverage against HPV-induced diseases, particularly cervical cancers. However, the assessment of immunogenicity of the vaccine is an important task, representing not only its significant clinical characteristics, but also a major challenge, in terms of both the suitability of methods and the clinical sample testing throughput supporting clinical development. This work covers the development and evaluation of a method based on Luminex technology (a coded-bead and flow-cytometric approach) to assess the HPV-type specific total immunoglobulin G (IgG). This method can evaluate the antibodies in sera post immunization against multiple types of HPV simultaneously (i.e., with multiplexing capability), save time and cost, and improve test throughput with higher sensitivity and precision than the classical, plate-based enzyme-linked immunoassay and competitive Luminex-based immunoassays. Using cynomolgus monkeys as model, we demonstrated the good correlation between the results from the pseudovirion-based neutralization assay (PBNA), and the Luminex-based total IgG assay, supporting that the latter method can be considered as a viable, dependable replacement method for the PBNA supporting immunogenicity evaluation of HPV vaccine in preclinical development and clinical investigation.
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Alphapapillomavirus , Infecções por Papillomavirus , Vacinas contra Papillomavirus , Animais , Anticorpos Antivirais , Humanos , Imunoensaio/métodos , Imunogenicidade da Vacina , Imunoglobulina G , Macaca fascicularis , Papillomaviridae , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/prevenção & controleRESUMO
INTRODUCTION: Dried blood spot (DBS) sampling is a minimally invasive method for specimen collection with potential multifaceted uses, particularly for serosurveillance of previous SARS-CoV-2 infection. In this study, we assessed DBS as a potential specimen type for assessing IgG and total (including IgG and IgM) antibodies to SARS-CoV-2 in vaccinated and naturally infected patients. METHODS: Six candidate buffers were assessed for eluting blood from DBS cards. The study utilized one hundred and five paired plasma specimens and DBS specimens from prospectively collected SARS-CoV-2 vaccinated individuals, remnants from those with PCR confirmed SARS-CoV-2 infections, or remnants from those without history of infection or vaccination. All specimens were tested with the Siemens SARS-CoV-2 total assay (COV2T) or IgG assay (sCOVG). RESULTS: The lowest backgrounds were observed with water and PBS, and water was used for elution. Relative to plasma samples, DBS samples had a positive percent agreement (PPA) of 94.4% (95% CI: 94.9-100%) for COV2T and 79.2 (68.4-87.0) for sCOVG using the manufacturer's cutoff. The NPA was 100 % (87.1-100.0 and 85.13-100) for both assays. Dilution studies revealed 100% (95% CI: 90.8-100%) qualitative agreement between specimen types on the COV2T assay and 98.0% (88.0-99.9%) with the sCOVG using study defined cutoffs. CONCLUSION: DBS specimens demonstrated high PPA and NPA relative to plasma for SARS-CoV-2 serological testing. Our data support feasibility of DBS sampling for SARS-CoV-2 serological testing.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Teste para COVID-19 , Manejo de Espécimes/métodos , Anticorpos Antivirais , Imunoglobulina M , Imunoglobulina G , Teste em Amostras de Sangue SecoRESUMO
People can become infected with cutaneous larva migrans (CLM) through skin penetration by the infective zoonotic larvae of hookworms. Few studies have investigated CLM's immunodiagnosis, and the existing studies were limited to crude somatic or excretory/secretory antigens (Ags) from adult worms. Here, we aimed to develop an indirect enzyme-linked immunosorbent assay (ELISA) to differentiate and diagnose hwCLM by detecting immunoglobulin (Ig)E, IgG, and IgG subclasses 1-4 (IgG1-4) against the somatic Ag of adult Ancylostoma caninum checkerboard titrations of adult A. caninum worm extract. Pooled serum controls were immunocharacterized using an indirect ELISA. The IgG1-4 and IgE results were unsatisfactory; however, the use of total IgG achieved results comparable to those of immunoblotting. Thus, we continued to analyze the IgG-ELISA using serum samples from patients with hwCLM and heterologous infections as well as from healthy controls. The sensitivity and excellent specificity of the total IgG-ELISA were 93.75% and 98.37%, respectively, and its positive and negative predictive values were 75% and 99.67%, respectively. Antibodies from five cases of angiostrongyliasis, gnathostomiasis, and dirofilariasis cross-reacted with the somatic Ag of adult A. caninum. This new assay can adequately serodiagnose hwCLM when combined with clinical features and/or histological examination.
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Background: Together with impaired mucociliary clearance, lung disease in cystic fibrosis (CF) is driven by dysregulation of innate and adaptive immunity caused by dysfunctional CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), leading to airway infection and hyperinflamma-tion. The highly effective CFTR modulator therapy (HEMT) elexacaftor/tezacaftor/ivacaftor (ETI) generates substantial improvements in clinical outcomes of people with CF (pwCF) by restoration of CFTR activity. Aberrant immune responses of lymphocytes due to CFTR dysfunction has been described in the past, but not the effects of CFTR restoration by HEMT on these cells. We aimed to examine the effect of ETI on the proliferative activity of antigen-specific CD154 (+) T cells against bacterial and fungal species relevant in CF and on total IgG and IgE as markers of B cell adaptive immunity. Methods: We performed ex vivo analyses of Ki-67 expression in antigen-specific CD154 (+) T cells against Pseudomonas aeruginosa, Staphylococcus aureus, Aspergillus fumigatus, Scedosporium apiospermum and Candida albicans from 21 pwCF by cytometric assay based on antigen-reactive T cell enrichment (ARTE), and analysis of total serum IgE and IgG before and after initiation of ETI. Results: Mean Ki-67 expression in antigen-specific CD154 (+) T cells against P. aeruginosa, A. fumigatus, S. apiospermum and C. albicans, but not S. aureus, mean total serum IgG and mean total serum IgE decreased significantly after initiation of ETI. No correlation was found to change in sputum microbiology of the examined pathogens. Mean BMI and FEV1 increased significantly. Conclusion: HEMT is associated with decreased antigen-specific CD154 (+) T cell proliferation activity in our cohort, independent of findings in sputum microbiology of the examined pathogens. Together with the observed clinical improvement and the decrease in total IgE and IgG, this indicates effects due to CFTR restoration on CD154 (+) T cells by ETI and a reduction of B cell activation with subsequent lower immunoglobulin synthesis under HEMT therapy. These results endorse earlier evidence of CFTR dysfunction in T and B cells leading directly to aberrant immune responses with hyperinflammation.
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Immunoglobulin G (IgG) antibodies play an important role in the immune response against viruses such as SARS-CoV-2. As the effector functions of IgG are modulated by N-glycosylation of the Fc region, the structure and possible function of the IgG N-glycome has been under investigation in relation to divergent COVID-19 disease courses. Through LC-MS analysis we studied both total IgG1 and spike protein-specific IgG1 Fc glycosylation of 129 German and 163 Brazilian COVID-19 patients representing diverse patient populations. We found that hospitalized COVID-19 patients displayed decreased levels of total IgG1 bisection and galactosylation and lowered anti-S IgG1 fucosylation and bisection as compared to mild outpatients. Anti-S IgG1 glycosylation was dynamic over the disease course and both anti-S and total IgG1 glycosylation were correlated to inflammatory markers. Further research is needed to dissect the possible role of altered IgG glycosylation profiles in (dys)regulating the immune response in COVID-19.
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COVID-19 , Imunoglobulina G , Humanos , SARS-CoV-2 , Glicosilação , BiomarcadoresRESUMO
OBJECTIVES: To examine whether anti-tetanus toxoid (anti-TT) immunoglobulin G (IgG) levels measured in oral fluid and adjusted for collection difficulties and specimen quality are associated with total IgG and anti-TTIgG in oral fluid and assess if statistical adjustment for them improves prediction of anti-TT IgG in serum. METHODS: 267 children, ages 12 to 15 months, enrolled in the M-SIMU randomized controlled trial participated in this nested cross-sectional analysis. Venous blood and oral fluid (OF) specimens were collected, and OF collection difficulties such as crying or gagging were recorded. OF volume was documented and total IgG was measured in OF specimens and anti-TT IgG was measured in OF and serum by enzyme immunoassay (EIA). Collection difficulties, volume and sociodemographic characteristics were assessed in relation to total IgG and anti-TT IgG in OF via multivariate regression. These models were extended to evaluate the association between anti-TT IgG in OF and in serum. A prediction model was developed to adjust anti-TT IgG in OF estimates as proxy for serum. RESULTS: Blood in the specimen, sores in the mouth and crying were positively associated with total IgG concentration while high oral fluid volume and sucking on the swab were inversely associated. None were significant predictors of anti-TT IgG in OF after adjusting for total IgG (geometric mean [GM] ratio: 1.99; 95% confidence interval: 1.78-2.24) and vaccination history (GM ratio: 2.44; 95% CI: 1.98-3.01). When predicting anti-TT IgG levels in serum with OF, total IgG modified the effect of anti-TT IgG in OF. CONCLUSIONS: Anti-TT IgG in OF is a good proxy for levels in serum, after controlling for total IgG in the specimen and other variables. Post hoc adjustments for OF volume and total IgG concentration are an important consideration when conducting serosurveys with oral fluid.
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Antitoxina Tetânica , Toxoide Tetânico , Adolescente , Anticorpos Antibacterianos , Criança , Estudos Transversais , Humanos , Imunoglobulina G , BocaRESUMO
Rubella IgG levels for 157,763 pregnant women residing in Alberta between 2009 and 2012 were analyzed. As there have been no reported cases of indigenous rubella infection in Canada since 2005, there has been a lack of naturally acquired immunity, and the current prenatal population depends almost entirely on vaccine induced immunity for protection. Rubella antibody levels are significantly lower in younger maternal cohorts with 16.8% of those born prior to universal vaccination programs (1971-1980), and 33.8% of those born after (1981-1990) having IgG levels that are not considered protective (<15 IU/mL). Analysis across pregnancies showed only 35.0% of women responded with a 4-fold increase in antibody levels following post-natal vaccination. Additionally, 41.2% of women with antibody levels <15 IU/mL had previously received 2 doses of rubella containing vaccine. These discordant interpretations generate a great deal of confusion for laboratorians and physicians alike, and result in significant patient follow-up by Public Health teams. To assess the current antibody levels in the prenatal population, latent class modeling was employed to generate a two class fit model representing women with an antibody response to rubella, and women without an antibody response. The declining level of vaccine-induced antibodies in our population is disconcerting, and a combined approach from the laboratory and Public Health may be required to provide appropriate follow up for women who are truly susceptible to rubella infection.
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Anticorpos Antivirais/sangue , Rubéola (Sarampo Alemão)/imunologia , Adulto , Alberta/epidemiologia , Feminino , Humanos , Imunoglobulina G/sangue , Gravidez , Complicações Infecciosas na Gravidez/epidemiologia , Complicações Infecciosas na Gravidez/imunologia , Complicações Infecciosas na Gravidez/prevenção & controle , Cuidado Pré-Natal , Diagnóstico Pré-Natal , Rubéola (Sarampo Alemão)/epidemiologia , Rubéola (Sarampo Alemão)/prevenção & controle , Adulto JovemRESUMO
We assessed HPV 16 and 18 antibody responses of female subjects enrolled in a 2- vs. 3-dose quadrivalent HPV (Q-HPV) vaccine trial (ClinicalTrials.gov NCT00501137) using the Merck competitive Luminex (cLIA) and total IgG Luminex (TIgG) immunoassays, and a pseudovirus neutralizing antibody (PsV NAb) assay. Subjects were enrolled in one of three groups: (1) 9-13yr, 2 doses of Q-HPV at 0, 6 months (n=259); (2) 9-13yr, 3 doses at 0, 2, 6 months (n=260); and (3) 16-26yr, 3 doses at 0, 2, 6 months (n=305). Sera were collected from all subjects at baseline, months 7 and 24, and from half the subjects at months 18 and 36. High correlation was observed between all three assays. At month 36, HPV 16 antibodies remained detectable in all subjects by all assays, whereas 86.4%, 99.6% and 100% of subjects respectively were HPV 18 cLIA, TIgG and PsV NAb (partial neutralization endpoint) seropositive. The proportion seropositive for HPV 18 by cLIA at 36 months was not significantly different for 2-dose girls vs. 3-dose adults (85.9% vs. 79.4%; p=0.51), whereas the proportion for 3-dose girls was significantly higher than for 3-dose adults (95.3% vs. 79.4%; p<0.01). The HPV 18 seropositive proportions by the TIgG and PsV NAb (partial neutralization endpoint) assays were the same for all subjects. High baseline HPV 16 and HPV 18 seropositivity was observed for the TIgG assay and it is unclear if all the detected TIgG antibodies are type-specific and/or neutralizing. For the PsV NAb assay, 90% and partial neutralization geometric mean titres were consistently 2-8-fold higher than for 100% neutralization, which enabled detection of HPV 18 NAb in subjects who lost detectable cLIA antibodies over time. We conclude that the PsV NAb assay is more sensitive than the cLIA, and likely more specific than the TIgG assay.
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Anticorpos Antivirais/sangue , Imunoensaio/métodos , Vacinas contra Papillomavirus/administração & dosagem , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Criança , Feminino , Papillomavirus Humano 16 , Papillomavirus Humano 18 , Humanos , Esquemas de Imunização , Testes de Neutralização , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/prevenção & controle , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: Conventional IgG assays require costly equipment and skilled experts. Semiquantitative measurement of total IgG using point-of-care testing devices may be the solution for these limitations. This study evaluated the reproducibility of the ImmuneCheck™ IgG assay (ProteomeTech Inc., Korea) and the correlation of its results with conventional laboratory IgG results in the serum and whole blood. METHODS: Both the serum and whole blood samples from 120 patients were used. To evaluate the intra-test reproducibility and inter-test correlation, intraclass correlation coefficient (ICC) analysis was used. RESULTS: The concentration of serum total IgG measured by cobas® 6000 (Roche Diagnostics, Switzland) ranged from 690.4 to 2,756.4 mg/dL. The intra-test reproducibility of ImmuneCheck™ IgG was high (Serum ICC=0.724, P < 0.001; Whole blood ICC=0.843, P < 0.001). The inter-test correlation between the ImmuneCheck™ IgG and cobas® 6000 results was very good (Serum ICC=0.805, P < 0.001; Whole blood ICC=0.842, P < 0.001). Because there were no samples with a total IgG level lower than 600 mg/dL, the pre-existing serum samples were diluted and then the linearity tests were conducted. The intra-test reproducibility for the diluted serum samples was almost perfect (ICC=0.995, P < 0.001), and the inter-test correlation between the ImmuneCheck™ IgG and cobas® 6000 results was also strong (ICC=0.992, P < 0.001). CONCLUSIONS: The ImmuneCheck™ IgG assay is reproducible and highly correlated with the conventional IgG assay for the serum and whole blood. It could be applied for the rapid detection of total IgG.
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Humanos , Imunoglobulina G , Testes ImediatosRESUMO
Introdução: A tuberculose (TB), doença crônica causada por Mycobacterium tuberculosis (Mtb), é uma das doenças infecciosas que mais acomete a população brasileira, com 2.832 óbitos em 2010. Na infecção causada pelo Mtb, a interação das células T com os macrófagos (MØs) infectados é fundamental na imunidade protetora contra o bacilo. A Mce1A é uma proteína da parede celular do Mtb que confere grande capacidade de aderência, invasão e sobrevivência em MØs. Contudo, a caracterização da proteína Mce1A pode fornecer um biomarcador para diagnóstico e monitoramento do tratamento. Nosso objetivo é avaliar a produção de IgG total anti-Mce1A em pacientes com TB e seus comunicantes domiciliares (CDTB). Material e métodos: Indivíduos diagnosticados com TB pulmonar e CDTB foram submetidos a coleta de sangue por punção venosa. O diagnóstico da TB foi baseado em quadro clínico e/ou radiografia sugestiva e/ou baciloscopia do escarro positiva. Nos CDTB, a infecção foi determinada a partir da reação do teste tuberculínico (TT) e avaliação radiográfica. Soro dos três grupos foi coletado e armazenado a -20ºC, até a determinação dos níveis de IgG total anti-Mce1A por meio de um ensaio imunoenzimático (ELISA). Resultados: Entre janeiro de 2012 e outubro de 2013 foram identificados 50 pacientes com TB pulmonar e 50 CDTB, dentre os quais 23 foram TT positivo e 27 foram TT negativo. A média de idade da população estudada foi de 37,8 anos (DP ± 20,4). O gênero masculino prevaleceu entre os pacientes com TB (68%), porém nos CDTB, o gênero feminino prevaleu (62%)...
Introduction: Tuberculosis (TB), a chronic disease caused by Mycobacterium tuberculosis (Mtb), is an infectious disease that affects the Brazilian population, with 2,832 deaths in 2010. In Mtb infection, the interaction of T cells with infected macrophages (MØs) is critical in protective immunity against the bacillus. The Mce1A is a cell wall protein of Mtb which gives great adhesion characteristics, invasion and survival in MØs. However, the characterization of protein Mce1A can provide a biomarker for diagnosis and monitoring of treatment. Our goal is to evaluate the production of total IgG anti-Mce1A TB patients and their household contacts (HHC). Material and Methods: Individuals diagnosed with pulmonary TB and HHC were subjected to blood collection by venipuncture. The diagnosis of TB was based on clinical and/or suggestive radiography and/or positive sputum smear. In HHC, infection was determined from the reaction of the tuberculin skin test (TST) and radiographic evaluation. Three groups of serum was collected and stored at -20 ° C until determination of the levels of anti-Mce1A total IgG by an enzyme-linked immunosorbent assay (ELISA). Results: Between January 2012 and October 2013, 50 patients were identified with pulmonary TB and 50 HHC, of which 23 were positive TST and 27 were negative TST. The average age of the study population was 37.8 years (SD ± 20.4). The males predominated among patients with TB (68%), but the HHC, the prevailed females (62%)...
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Humanos , Tuberculose/diagnóstico , Tuberculose/epidemiologia , Tuberculose/patologia , Tuberculose/prevenção & controle , Tuberculose/transmissãoRESUMO
AIM: To investigate the changes on immunological function in antigen-sensitized mice injected by dermatophagoides farinaeliposomes (DF) subcutaneously. METHODS: BALB/c mice were sensitized with DF antigen injected subcutaneously. After injected by DF liposomes with 1, 3, and 9 ?g?g -1 dosage respectively, the total IgE, total IgG, DF specific IgE(sIgE), DF specific IgG (sIgG), IL-4, and IFN-? were detected by ELISA. RESULTS: The total IgE, sIgE, total IgG, and sIgG in the serum of sensitized mice increased, the level of IFN-? reduced and IL-4 increased. After injected subcutaneously by DF liposomes, the total IgE, sIgE and IL-4 in the serum of mice of all the treatment groups reduced, and IFN-? raised, and the total IgG, sIgG raised in the serum of mice administrated with small and medium dosage, but the low dosage was more obvious. CONCLUSION: DF liposomes have some effects on the immunological function in mite-sensitized mice in the low dosage.