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1.
BMC Infect Dis ; 21(1): 187, 2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33602152

RESUMO

BACKGROUND: Thresholds for SARS-CoV-2 antibody assays have typically been determined using samples from symptomatic, often hospitalised, patients. In this setting the sensitivity and specificity of the best performing assays can both exceed 98%. However, antibody assay performance following mild infection is less clear. METHODS: We assessed quantitative IgG responses in a cohort of healthcare workers in Oxford, UK, with a high pre-test probability of Covid-19, in particular the 991/11,475(8.6%) who reported loss of smell/taste. We use anosmia/ageusia and other risk factors as probes for Covid-19 infection potentially undiagnosed by immunoassays by investigating their relationship with antibody readings either side of assay thresholds. RESULTS: The proportion of healthcare workers reporting anosmia/ageusia increased at antibody readings below diagnostic thresholds using an in-house ELISA (n = 9324) and the Abbott Architect chemiluminescent microparticle immunoassay (CMIA; n = 11,324): 426/906 (47%) reported anosmia/ageusia with a positive ELISA, 59/449 (13.1%) with high-negative and 326/7969 (4.1%) with low-negative readings. Similarly, by CMIA, 518/1093 (47.4%) with a positive result reported anosmia/ageusia, 106/686 (15.5%) with a high-negative and 358/9563 (3.7%) with a low-negative result. Adjusting for the proportion of staff reporting anosmia/ageusia suggests the sensitivity of both assays in mild infection is lower than previously reported: Oxford ELISA 89.8% (95%CI 86.6-92.8%) and Abbott CMIA 79.3% (75.9-82.7%). CONCLUSION: Following mild SARS-CoV-2 infection 10-30% of individuals may have negative immunoassay results. While lowered diagnostic thresholds may result in unacceptable specificity, our findings have implications for epidemiological analyses and result interpretation in individuals with a high pre-test probability. Samples from mild PCR-confirmed infections should be included in SARS-CoV-2 immunoassay evaluations.


Assuntos
Anticorpos Antivirais/análise , /diagnóstico , Imunoglobulina G/análise , Adulto , Ageusia/virologia , Infecções Assintomáticas , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Pessoal de Saúde , Humanos , Imunoensaio/normas , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Doenças não Diagnosticadas , Reino Unido
2.
Nat Commun ; 12(1): 113, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397956

RESUMO

The extent of SARS-CoV-2 infection throughout the United States population is currently unknown. High quality serology is key to avoiding medically costly diagnostic errors, as well as to assuring properly informed public health decisions. Here, we present an optimized ELISA-based serology protocol, from antigen production to data analyses, that helps define thresholds for IgG and IgM seropositivity with high specificities. Validation of this protocol is performed using traditionally collected serum as well as dried blood on mail-in blood sampling kits. Archival (pre-2019) samples are used as negative controls, and convalescent, PCR-diagnosed COVID-19 patient samples serve as positive controls. Using this protocol, minimal cross-reactivity is observed for the spike proteins of MERS, SARS1, OC43 and HKU1 viruses, and no cross reactivity is observed with anti-influenza A H1N1 HAI. Our protocol may thus help provide standardized, population-based data on the extent of SARS-CoV-2 seropositivity, immunity and infection.


Assuntos
Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , /imunologia , Anticorpos Antivirais/imunologia , /epidemiologia , /métodos , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pandemias , Padrões de Referência , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/imunologia
3.
Bioanalysis ; 13(1): 13-28, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33319585

RESUMO

Aim: Coronavirus disease 2019 antibody testing often relies on venous blood collection, which is labor-intensive, inconvenient and expensive compared with finger-stick capillary dried blood spot (DBS) collection. The purpose of our work was to determine if two commercially available anti-severe acute respiratory syndrome coronavirus 2 enzyme-linked immunosorbent assays for IgG antibodies against spike S1 subunit and nucleocapsid proteins could be validated for use with DBS. Materials & methods: Kit supplied reagents were used to extract DBS, and in-house DBS calibrators were included on every run. Results: Positive/negative concordance between DBS and serum was 100/99.3% for the spike S1 subunit assay and 100/98% for the nucleocapsid assay. Conclusion: Validation of the DBS Coronavirus disease 2019 IgG antibody assays demonstrated that serum and DBS can produce equivalent results with minimal kit modifications.


Assuntos
/normas , Teste em Amostras de Sangue Seco/normas , Ensaio de Imunoadsorção Enzimática/normas , /imunologia , Anticorpos Antivirais/química , Antígenos Virais/sangue , Antígenos Virais/imunologia , /imunologia , /sangue , Feminino , Humanos , Imunoglobulina G/química , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/sangue , Fosfoproteínas/imunologia , Kit de Reagentes para Diagnóstico/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Glicoproteína da Espícula de Coronavírus/sangue , Glicoproteína da Espícula de Coronavírus/imunologia
5.
PLoS One ; 15(8): e0238196, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32841291

RESUMO

The need for an efficacious vaccine against highly pathogenic filoviruses was reinforced by the devastating 2014-2016 outbreak of Ebola virus (EBOV) disease (EVD) in Guinea, Sierra Leone, and Liberia that resulted in over 28,000 cases and over 11,300 deaths. In addition, the 2018-2020 outbreak in the Democratic Republic of the Congo currently has over 3,400 cases and over 2,200 deaths. A fully licensed vaccine and at least one other investigational vaccine are being deployed to combat this EVD outbreak. To support vaccine development and pre-clinical/clinical testing a Filovirus Animal Nonclinical Group (FANG) human anti-EBOV GP IgG ELISA was developed to measure anti-EBOV GP IgG antibodies. This ELISA is currently being used in multiple laboratories. Reported here is a characterization of an interlaboratory statistical analysis of the human anti-EBOV GP IgG ELISA as part of a collaborative study between five participating laboratories. Each laboratory used similar method protocols and reagents to measure anti-EBOV GP IgG levels in human serum samples from a proficiency panel consisting of ten serum samples created by the differential dilution of a serum sample positive for anti-GP IgG antibodies (BMIZAIRE105) with negative serum (BMI529). The total assay variability (inter- and intra-assay variability) %CVs observed at each laboratory ranged from 12.2 to 30.6. Intermediate precision (inter-assay variability) for the laboratory runs ranged from 8.9 to 21.7%CV and repeatability (intra-assay variability) %CVs ranged from 7.2 to 23.7. The estimated slope for the relationship between log10(Target Concentration) and the log10(Observed Concentration) across all five laboratories was 0.95 with a 90% confidence interval of (0.93, 0.97). Equivalence test results showed that the 90% confidence interval for the ratios for the sample-specific mean concentrations at the five individual labs to the overall laboratory consensus value were within the equivalence bounds of 0.80 to 1.25 for each laboratory and test sample, except for six test samples from Lab D, two samples from Lab B1, and one sample from Lab B2. The mean laboratory concentrations for Lab D were less than those from the other laboratories by 20% on average across the serum samples. The evaluation of the proficiency panel at these laboratories provides a limited assessment of assay precision (intermediate precision, repeatability, and total assay variability), dilutional linearity, and accuracy. This evaluation suggests that the within-laboratory performance of the anti-EBOV GP IgG ELISA as implemented at the five laboratories is consistent with the intended use of the assay based on the acceptance criteria used by laboratories that have validated the assay. However, the assessment of between-laboratory performance revealed lower observed concentrations at Lab D and greater variability in assay results at Lab B1 relative to other laboratories.


Assuntos
Anticorpos Antivirais/sangue , Ebolavirus/imunologia , Ensaio de Imunoadsorção Enzimática/normas , Doença pelo Vírus Ebola/imunologia , Proteínas Virais/imunologia , África Ocidental/epidemiologia , Animais , República Democrática do Congo/epidemiologia , Surtos de Doenças , Vacinas contra Ebola/imunologia , Vacinas contra Ebola/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/estatística & dados numéricos , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Imunoglobulina G/sangue , Laboratórios , Variações Dependentes do Observador
7.
Clin Chim Acta ; 509: 79-82, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32526218

RESUMO

BACKGROUND: Besides SARS-CoV-2 RT-PCR testing, serological testing is emerging as additional option in COVID-19 diagnostics. Aim of this study was to evaluate novel immunoassays for detection of SARS-CoV-2 antibodies in human plasma. METHODS: Using EDITM Novel Coronavirus COVID-19 Enzyme Linked Immunosorbent Assays (ELISAs), we measured SARS-CoV-2 IgM and IgG antibodies in 64 SARS-CoV-2 RT-PCR confirmed COVID-19 patients with serial blood samples (n = 104) collected at different time points from symptom onset. Blood samples from 200 healthy blood donors and 256 intensive care unit (ICU) patients collected before the COVID-19 outbreak were also used. RESULTS: The positivity rates in the COVID-19 patients were 5.9% for IgM and 2.9% for IgG ≤ 5 days after symptom onset; Between day 5 and day 10 the positivity rates were 37.1% for IgM and 37.1% for IgG and rose to 76.4% for IgM and 82.4% for IgG after > 10-15 days. After 15-22 days the "true" positivity rates were 94.4% for IgM and 100% for IgG. The "false" positivity rates were 0.5% for IgM and 1.0% for IgG in the healthy blood donors, 1.6% for IgM and 1.2% for IgG in ICU patients. CONCLUSIONS: This study shows high "true" vs. low "false" positivity rates for the EDITM SARS-CoV-2 IgM and IgG ELISAs.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus , Infecções por Coronavirus/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Pneumonia Viral/sangue , Testes Sorológicos/normas , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/diagnóstico , Testes Sorológicos/métodos
8.
Pol J Microbiol ; 69(2): 217-222, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32548990

RESUMO

Clinical diagnosis of hepatitis E viral (HEV) infection mainly relies on serological assays, and the current status of misdiagnoses regarding HEV infection is uncertain. In this study, patients with acute HEV infection were tested for anti-HEV IgM and IgG, a HEV antigen (Ag), and viral loads (HEV RNA). Serology was performed using four commercial HEV ELISA kits: Wantai, Kehua, Lizhu, and Genelabs IgM and IgG. The HEV RNA was detected using RT-PCR assays. The sensitivities of different kits for anti-HEV IgM ranged from 82.6% to 86%. Each kit for anti-HEV IgM was highly specific (97.8-100%). The sensitivities of all kits to detect anti-HEV IgG with (87.2-91.9%) had a substantial agreement, but the Kehua and Genelabs tests were more specific than the Wantai and Lizhu tests. The Wantai tests for the HEV Ag and HEV RNA were also important for acute HEV infections (Kappa = 0.787). Furthermore, a total of 6.98% of HEV infections were positive for HEV RNA but negative for both the HEV Ag and anti-HEV antibodies of IgM and IgG classes. Our findings demonstrate that the diagnosis of hepatitis E may be missed if only serological assays are used. Thus, a combination of serological and nucleic acid testing provides the optimal sensitivity and specificity to the diagnostic process.Clinical diagnosis of hepatitis E viral (HEV) infection mainly relies on serological assays, and the current status of misdiagnoses regarding HEV infection is uncertain. In this study, patients with acute HEV infection were tested for anti-HEV IgM and IgG, a HEV antigen (Ag), and viral loads (HEV RNA). Serology was performed using four commercial HEV ELISA kits: Wantai, Kehua, Lizhu, and Genelabs IgM and IgG. The HEV RNA was detected using RT-PCR assays. The sensitivities of different kits for anti-HEV IgM ranged from 82.6% to 86%. Each kit for anti-HEV IgM was highly specific (97.8­100%). The sensitivities of all kits to detect anti-HEV IgG with (87.2­91.9%) had a substantial agreement, but the Kehua and Genelabs tests were more specific than the Wantai and Lizhu tests. The Wantai tests for the HEV Ag and HEV RNA were also important for acute HEV infections (Kappa = 0.787). Furthermore, a total of 6.98% of HEV infections were positive for HEV RNA but negative for both the HEV Ag and anti-HEV antibodies of IgM and IgG classes. Our findings demonstrate that the diagnosis of hepatitis E may be missed if only serological assays are used. Thus, a combination of serological and nucleic acid testing provides the optimal sensitivity and specificity to the diagnostic process.


Assuntos
Ensaio de Imunoadsorção Enzimática/normas , Hepatite E/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite E/genética , Humanos , RNA Viral/análise , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
9.
J Neuroimmunol ; 345: 577288, 2020 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-32544754

RESUMO

For the diagnosis of anti-MAG polyneuropathy the commercial ELISA manufacturer currently recommends a cut-off of 1000 Bühlmann Titer Units (BTU). We analyzed sera from 80 anti-MAG neuropathy patients and 383 controls (with other neuropathies or healthy controls) to assess the ELISA sensitivity and specificity at different thresholds. A better combination of sensitivity/specificity was found at a threshold >1500 BTU than at >1000 BTU. The best value of specificity was obtained at threshold >7000 BTU. There was a diagnostic grey area between 1500 and 7000 BTU in which the clinical phenotypes as well as electrophysiological studies need to be carefully assessed particularly to differentiate CIDP and anti-MAG neuropathy.


Assuntos
Autoanticorpos/sangue , Glicoproteína Associada a Mielina/sangue , Polineuropatias/sangue , Estudos de Casos e Controles , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Polineuropatias/diagnóstico , Estudos Retrospectivos
10.
Eur J Clin Microbiol Infect Dis ; 39(10): 1959-1970, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32548683

RESUMO

In recent years, the prevalence of tuberculosis worldwide has increased, and with it, the number of drug-resistant tuberculosis strains. This has brought new challenges towards prevention and control of the disease. Therefore, it is urgent to find reliable and rapid diagnostic methods for tuberculosis in general, and for the drug-resistant forms of the disease. To this aim, we assessed 17 tuberculosis-specific protein candidates for the detection of tuberculosis-specific antibodies. First, we established an indirect ELISA method to detect anti-Mycobacterium tuberculosis IgM and IgG. We tested 453 sera and analyzed the efficacy of the protein candidates for diagnosis of tuberculosis. Next, we screened antigens rich in T cell epitopes for their ability to induce high levels of IFN-γ, in order to define their suitability does develop detection tests based on IFN-γ release assay (IGRAs). The antigens CFP-10, PPE57, 38kDa, and Rv3807 showed higher diagnostic potential for the detection of anti-tuberculosis IgM, whereas PPE57, Ag85B, CFP-10, Rv0220, and 38kDa antigens performed better for anti-tuberculosis IgG detection. Worth noting is that CFP-10, 38kDa, and PPE57 detected efficiently both IgM and IgG. Rv1987, Rv3807, PPE57, Rv0220, and MPT64 proteins alone and combinations of Rv1987 + Rv3807, 16kDa + Rv0220, and MPT64 + Rv1986 tested in IGRAs displayed a good correlation with the positive control constituted by a cocktail of ESAT-6 + CFP-10 + TB7.7 (ECT), known for their stimulating properties (r > 0.5, p < 0.01). Among these antigen candidates, Rv0220 and Rv1987 + Rv3807 were the most potent. We discovered CFP-10, 38kDa, and PPE57 for the detection of anti-M. tuberculosis IgM and IgG, and Rv0220 alone or the combination Rv1987 + Rv3807 as the strongest stimulators in IGRAs. These antigens provide new references for the screening of tuberculosis-specific antibodies and effective stimulation in IGRAs.


Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/metabolismo , Ensaio de Imunoadsorção Enzimática/normas , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose/diagnóstico , Anticorpos Antibacterianos/metabolismo , Humanos , Mycobacterium tuberculosis , Tuberculose/sangue , Tuberculose Resistente a Múltiplos Medicamentos/sangue
11.
Clin Chim Acta ; 509: 18-21, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32485155

RESUMO

BACKGROUND: Here, we report on a head-to-head comparison of the fully-automated Elecsys® Anti-SARS-CoV-2 immunoassay with the EDITM enzyme linked immunosorbent assays (ELISA) for the detection of SARS-CoV-2 antibodies in human plasma. METHODS: SARS-CoV-2 antibodies were measured with the Elecsys® assay and the EDITM ELISAs (IgM and IgG) in 64 SARS-CoV-2 RT-PCR confirmed COVID-19 patients with serial blood samples (n = 104) collected at different time points from symptom onset. Blood samples from 200 healthy blood donors and 256 intensive care unit (ICU) patients collected before the COVID-19 outbreak were also used. RESULTS: In COVID-19 patients, the percentage of positive results rose with time from symptom onset, peaking to positivity rates after 15-22 days of 100% for the Elecsys® assay, of 94% for the EDITM IgM-ELISA and of 100% for the EDITM IgG ELISA. In the 104 blood samples, the agreement between positive/negative classifications of the Elecsys® assay and the EDITM ELISAs (IgM or IgG) was 90%. The false positivity rates in the healthy blood donors and the ICU patients were < 1% for the Elecsys® assay and < 3% for the EDITM ELISAs. CONCLUSIONS: Our results indicate a high sensitivity and specificity for the Elecsys® assay and an acceptable agreement with the EDITM ELISAs.


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus , Técnicas de Laboratório Clínico/métodos , Técnicas de Laboratório Clínico/normas , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Betacoronavirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Imunoensaio/métodos , Imunoensaio/normas , Masculino , Pandemias , Testes Sorológicos/métodos , Testes Sorológicos/normas
12.
Clin Chim Acta ; 509: 1-7, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32485157

RESUMO

BACKGROUND: The evaluation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific antibody (Ab) assay performances is of the utmost importance in establishing and monitoring virus spread in the community. In this study focusing on IgG antibodies, we compare reliability of three chemiluminescent (CLIA) and two enzyme linked immunosorbent (ELISA) assays. METHODS: Sera from a total of 271 subjects, including 64 reverse transcription-polymerase chain reaction (RT-PCR) confirmed SARS-CoV-2 patients were tested for specific Ab using Maglumi (Snibe), Liaison (Diasorin), iFlash (Yhlo), Euroimmun (Medizinische Labordiagnostika AG) and Wantai (Wantai Biological Pharmacy) assays. Diagnostic sensitivity and specificity, positive and negative likelihood ratios were evaluated using manufacturers' and optimized thresholds. RESULTS: Optimized thresholds (Maglumi 2 kAU/L, Liaison 6.2 kAU/L and iFlash 15.0 kAU/L) allowed us to achieve a negative likelihood ratio and an accuracy of: 0.06 and 93.5% for Maglumi; 0.03 and 93.1% for Liaison; 0.03 and 91% for iFlash. Diagnostic sensitivities and specificities were above 93.8% and 85.9%, respectively for all CLIA assays. Overall agreement was 90.3% (Cohen's kappa = 0.805 and SE = 0.041) for CLIA, and 98.4% (Cohen's kappa = 0.962 and SE = 0.126) for ELISA. CONCLUSIONS: The results obtained indicate that, for CLIA assays, it might be possible to define thresholds that improve the negative likelihood ratio. Thus, a negative test result enables the identification of subjects at risk of being infected, who should then be closely monitored over time with a view to preventing further viral spread. Redefined thresholds, in addition, improved the overall inter-assay agreement, paving the way to a better harmonization of serologic tests.


Assuntos
Betacoronavirus , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Testes Sorológicos/normas , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/isolamento & purificação , Criança , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pandemias , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Testes Sorológicos/métodos , Adulto Jovem
13.
J Infect Dis ; 222(3): 362-366, 2020 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-32473021

RESUMO

We comparatively assessed sensitivities and specificities of 4 commercial enzyme-linked immunosorbent assays (ELISAs) and 2 rapid tests in 77 patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 infection, grouped by interval since symptom onset. Although test sensitivities were low (<40%) within the first 5 days after disease onset, immunoglobulin (Ig) M, IgA, and total antibody ELISAs increased in sensitivity to >80% between days 6 and 10 after symptom onset. The evaluated tests (including IgG and rapid tests) provided positive results in all patients at or after the 11th day after onset of disease. The specificities of the ELISAs were 83% (IgA), 98% (IgG), and 97% (IgM and total antibody).


Assuntos
Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática/normas , Imunoensaio/métodos , Pneumonia Viral/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Betacoronavirus/genética , Infecções por Coronavirus/imunologia , Feminino , Humanos , Imunoensaio/normas , Imunoglobulina A/sangue , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Masculino , Pessoa de Meia-Idade , Nasofaringe/virologia , Pandemias , Pneumonia Viral/imunologia , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade , Fatores de Tempo , Adulto Jovem
14.
Int J Gynaecol Obstet ; 150(1): 103-107, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32246772

RESUMO

OBJECTIVE: To evaluate a treponemal enzyme-linked immunosorbent assay (ELISA) as an alternative screening test for syphilis in pregnant women. METHODS: A cross-sectional study of diagnostic test accuracy was carried out in a large volume laboratory from a tertiary care center. A total of 416 serum samples, including 102 archived syphilis Treponema pallidum hemagglutination (TPHA)-positive samples and 314 samples from pregnant women, were used to determine the sensitivity and specificity of ELISA. All the samples were subjected to Venereal Disease Research Laboratory (VDRL), rapid plasma reagin (RPR), ELISA, and TPHA tests. Performance characteristics of VDRL, RPR, and ELISA were calculated with TPHA as a reference standard test. RESULTS: VDRL and RPR exhibited higher false positivity of 10.5% and 9.6%, respectively, compared to 2.5% by ELISA. The sensitivity and specificity of ELISA were 98% and 97.5%, of VDRL were 71.6% and 89.5%, and of RPR were 73.5% and 90.5%, respectively. Moreover, ELISA had an excellent agreement (kappa=0.9) with TPHA compared to VDRL/RPR which had a moderate agreement (kappa=0.6) only. CONCLUSION: ELISA has the potential to replace VDRL/RPR as a screening test for syphilis in centers that can perform ELISA, especially for antenatal screening.


Assuntos
Ensaio de Imunoadsorção Enzimática/normas , Complicações Infecciosas na Gravidez/diagnóstico , Diagnóstico Pré-Natal/métodos , Sorodiagnóstico da Sífilis/métodos , Sífilis/diagnóstico , Adulto , Estudos Transversais , Feminino , Humanos , Gravidez , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Treponema pallidum/isolamento & purificação
15.
J Biosci Bioeng ; 130(2): 217-225, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32284304

RESUMO

A dual monoclonal antibody sandwich enzyme-linked immunosorbent assay (mAb sandwich ELISA) has been developed using rabbit monoclonal antibodies generated by Ecobody technology, which includes the isolation of single B cells binding to a specific antigen, amplification of the heavy and light chains of these immunoglobulins, and expression of the fragment of antigen binding (Fab) by cell-free protein synthesis (CFPS). A rabbit was immunized with swine influenza virus (SIV) vaccine, from which single B cells binding to the antigen were isolated. Then, immunoglobulin mRNA was amplified from single cells by reverse transcription-polymerase chain reaction, followed by the attachment of a T7 promoter, appropriate tags, and a T7 terminator for the expression of the Fab portion by CFPS. By taking advantage of two different peptide tags fused to the same Fab, optimal combinations for coating Fab on assay plates and detecting Fab, both synthesized by CFPS, were investigated for mAb sandwich ELISA. Pairs of Fab detected 0.5 ng SIV in the assay. In summary, this result showed the applicability of Ecobody technology for a variety of immunodetection kits for high throughput analyses.


Assuntos
Anticorpos Monoclonais/metabolismo , Ensaio de Imunoadsorção Enzimática/normas , Testes Imunológicos/métodos , Orthomyxoviridae/isolamento & purificação , Animais , Linfócitos B/imunologia , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Infecções por Orthomyxoviridae/diagnóstico , Biossíntese de Proteínas
16.
Diagn Microbiol Infect Dis ; 97(1): 115011, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32139113

RESUMO

In this study, we described the largest analysis to date conducted with VIDAS® HIV Duo Ultra assay. Additionally, we analyzed the diagnostic performance and cutoff values (TV) of HIV Duo Ultra assay and total cost analysis for HIV testing. Of 11,642 enzyme-linked immunosorbent assay (ELISA)-positive samples referred to our center for confirmation, 2000 were positive with HIV Duo Ultra, and of these, 87% were HIV-1 positive and 0.6% were HIV-1 indeterminate with the confirmatory test. Overall, the false-positivity rate was 1.75% for HIV Duo Ultra assay. The sensitivity and specificity were 100% and 99.1%, respectively, when the TV was set at the recommended cutoff value. Even increasing the cutoff value four times, sensitivity and specificity remained high, pointing out that a TV of 0.99 is highly indicative of HIV positivity. Retesting samples with HIV Duo Ultra assay decreased 80% of the confirmatory tests, revealing a significant decrease of 78% in the total costs and reporting time.


Assuntos
Ensaio de Imunoadsorção Enzimática/normas , Infecções por HIV/diagnóstico , Kit de Reagentes para Diagnóstico/normas , Ensaio de Imunoadsorção Enzimática/métodos , Reações Falso-Positivas , Infecções por HIV/epidemiologia , Infecções por HIV/imunologia , HIV-1 , HIV-2 , Humanos , Programas de Rastreamento , Prevalência , Curva ROC , Sensibilidade e Especificidade , Turquia/epidemiologia
17.
Sci Rep ; 10(1): 2476, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051479

RESUMO

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.


Assuntos
Variação Biológica Individual , Filgrastim/farmacocinética , Polietilenoglicóis/farmacocinética , Animais , Linhagem Celular Tumoral , Proliferação de Células , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Camundongos , Fator de Transcrição STAT3/metabolismo
18.
Diagn Microbiol Infect Dis ; 96(4): 114984, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31954594

RESUMO

Simple measures that can facilitate early recognition of leprosy complications are still lacking. We therefore evaluated a lateral flow-based rapid diagnostic test and fast enzyme-linked immunosorbent assay measuring anti-LID-NDO antibody responses among leprosy cases in Cebu, Philippines. Responses were measured at diagnosis, then during and after the provision of standard multidrug therapy. Our data indicate that both platforms are highly sensitive tools for the primary diagnosis of, in particular, multibacillary leprosy. A gradual, quantifiable decline in both magnitude of response and percent positive responders was observed during and after treatment. As a group, patients that developed erythema nodosum leprosum (ENL) had a significantly higher response at diagnosis than patients that either developed reversal reactions or did not develop reactions. Although higher initial anti-NDO-LID responses were a risk factor for ENL, neither platform, however, could reliably predict the time of emergence of reactional episodes.


Assuntos
Anticorpos Antibacterianos/sangue , Hansenostáticos/uso terapêutico , Hanseníase/diagnóstico , Hanseníase/tratamento farmacológico , Mycobacterium leprae/efeitos dos fármacos , Adulto , Quimioterapia Combinada , Ensaio de Imunoadsorção Enzimática/normas , Eritema Nodoso/diagnóstico , Eritema Nodoso/tratamento farmacológico , Feminino , Humanos , Imunoensaio , Imunoglobulina M/sangue , Hanseníase/complicações , Hanseníase Virchowiana/diagnóstico , Hanseníase Virchowiana/tratamento farmacológico , Hanseníase Multibacilar/diagnóstico , Hanseníase Multibacilar/tratamento farmacológico , Estudos Longitudinais , Masculino , Filipinas , Testes Sorológicos
19.
Crit Care Med ; 48(2): e82-e86, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31939806

RESUMO

OBJECTIVES: Heparin-induced thrombocytopenia is a recognized concern in patients on extracorporeal life support. The purpose of this study was to evaluate the applicability of an enzyme-linked immunosorbent assay optical density threshold less than 1 to rule out heparin-induced thrombocytopenia in patients on extracorporeal membrane oxygenation. DESIGN: Retrospective, single-center study. SETTING: Patients were recruited from a prospectively maintained database of all patients on extracorporeal membrane oxygenation from 2012 to 2018 at a tertiary referral center. PATIENTS: Forty-seven patients on extracorporeal membrane oxygenation support. INTERVENTIONS: The primary objective was to evaluate the application of enzyme-linked immunosorbent assay optical density thresholds and the serotonin release assay in patients on extracorporeal membrane oxygenation. Patients were divided into two cohorts, serotonin release assay negative and serotonin release assay positive. In order to perform a sensitivity and specificity analysis of enzyme-linked immunosorbent assay optical density thresholds, heparin-induced thrombocytopenia negative was defined as an optical density less than 1.0 and heparin-induced thrombocytopenia positive as an optical density greater than or equal to 1.0. MEASUREMENTS AND MAIN RESULTS: Utilizing the prespecified optical density thresholds, a specificity and negative predictive value of 89% and 95% were achieved, respectively. CONCLUSIONS: This assessment has helped to identify optical density thresholds for patients undergoing extracorporeal membrane oxygenation. Our data suggest that an optical density threshold of 1.0 may aid clinicians in objectively ruling out heparin-induced thrombocytopenia without sending a confirmatory serotonin release assay. Increasing the optical density threshold to 1.0 resulted in a high specificity and negative predictive value.


Assuntos
Anticoagulantes/efeitos adversos , Ensaio de Imunoadsorção Enzimática/normas , Oxigenação por Membrana Extracorpórea/efeitos adversos , Heparina/efeitos adversos , Serotonina/sangue , Trombocitopenia/induzido quimicamente , Humanos , Estudos Retrospectivos , Sensibilidade e Especificidade
20.
J Pharm Biomed Anal ; 181: 113090, 2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-31915108

RESUMO

While the role of plasma renin activity (PRA) in heart failure has been widely studied in adults, comprehensive data on pediatric heart failure remain lacking. This drawback is increasingly being addressed by academic research. Nevertheless, such pediatric investigations are commonly conducted only once due to ethical constraints. Therefore, the quality of bioanalytical data must be ensured to acquire meaningful insights into maturing humoral parameters. However, appropriate post-validation assessment of bioanalytical runs is currently underrepresented by regulatory guidance. Thus, for applications in an academic environment, an easy-to-handle six-step bioanalytical quality control system was designed based on regulatory guidelines (e.g. U.S. Food and Drug Administration) combined with international recommendations (e.g. Clinical and Laboratory Standards Institute) and current scientific discussion. Its applicability to an enzyme-linked immunosorbent assay for determination of PRA was investigated within three pediatric trials of the EU-funded "Labeling of Enalapril in Neonates up to Adolescents" project. This quality control system identified 15 % bioanalytical runs as non-compliant to the predefined specifications and ensured the reliable quantification of 940 pharmacodynamic samples. The inter-run assessment of quality controls was able to demonstrate the comparability of the study results. Furthermore, 86 % of incurred sample reanalysis pairs complied with regulatory requirements (>67 %), thus underlining the long-term reproducibility of the utilized ligand-binding assay. Successful participation in interlaboratory testing confirmed the accuracy of the applied method throughout the entire study period. Further investigations showed no notable differences between the five applied lots of the PRA assay. The applicability of this quality control system was proven in an academic environment and ensured reliable results for PRA over the entire 24-month study period.


Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Monitoramento de Medicamentos/métodos , Enalapril/farmacologia , Insuficiência Cardíaca/diagnóstico , Renina/metabolismo , Adolescente , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Criança , Pré-Escolar , Monitoramento de Medicamentos/normas , Enalapril/uso terapêutico , Ensaio de Imunoadsorção Enzimática/normas , Feminino , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/fisiopatologia , Humanos , Lactente , Masculino , Prognóstico , Estudo de Prova de Conceito , Controle de Qualidade , Renina/sangue , Renina/isolamento & purificação , Sistema Renina-Angiotensina/fisiologia , Reprodutibilidade dos Testes
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