ABSTRACT
Congenital Zika virus (ZIKV) infection results in neurodevelopmental deficits in up to 14% of infants born to ZIKV-infected mothers. Neutralizing antibodies are a critical component of protective immunity. Here, we demonstrate that plasma IgM contributes to ZIKV immunity in pregnancy, mediating neutralization up to 3 months post-symptoms. From a ZIKV-infected pregnant woman, we isolated a pentameric ZIKV-specific IgM (DH1017.IgM) that exhibited ultrapotent ZIKV neutralization dependent on the IgM isotype. DH1017.IgM targets an envelope dimer epitope within domain II. The epitope arrangement on the virion is compatible with concurrent engagement of all ten antigen-binding sites of DH1017.IgM, a solution not available to IgG. DH1017.IgM protected mice against viremia upon lethal ZIKV challenge more efficiently than when expressed as an IgG. Our findings identify a role for antibodies of the IgM isotype in protection against ZIKV and posit DH1017.IgM as a safe and effective candidate immunotherapeutic, particularly during pregnancy.
Subject(s)
Immunoglobulin M , Pregnancy , Zika Virus Infection , Zika Virus , Animals , Female , Mice , Pregnancy/immunology , Antibodies, Neutralizing , Antibodies, Viral , Epitopes , Neutralization Tests , Zika Virus Infection/immunology , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purificationABSTRACT
BACKGROUND: The potential risk and association of bovine leukemia virus (BLV) with human remains controversial as it has been reported to be both positive and negative in human breast cancer and blood samples. Therefore, establishing the presence of BLV in comprehensive human clinical samples in different geographical locations is essential. RESULT: In this study, we examined the presence of BLV proviral DNA in human blood and breast cancer tissue specimens from Japan. PCR analysis of BLV provirus in 97 Japanese human blood samples and 23 breast cancer tissues showed negative result for all samples tested using long-fragment PCR and highly-sensitive short-fragment PCR amplification. No IgG and IgM antibodies were detected in any of the 97 human serum samples using BLV gp51 and p24 indirect ELISA test. Western blot analysis also showed negative result for IgG and IgM antibodies in all tested human serum samples. CONCLUSION: Our results indicate that Japanese human specimens including 97 human blood, 23 breast cancer tissues, and 97 serum samples were negative for BLV.
Subject(s)
Antibodies, Viral , DNA, Viral , Leukemia Virus, Bovine , Proviruses , Antibodies, Viral/isolation & purification , Blood/virology , Breast Neoplasms/virology , DNA, Viral/isolation & purification , Female , Humans , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Japan , Leukemia Virus, Bovine/genetics , Leukemia Virus, Bovine/immunology , Proviruses/geneticsABSTRACT
The COVID-19 pandemic has resulted in a worldwide health crisis. Rapid diagnosis, new therapeutics and effective vaccines will all be required to stop the spread of COVID-19. Quantitative evaluation of serum antibody levels against the SARS-CoV-2 virus provides a means of monitoring a patient's immune response to a natural viral infection or vaccination, as well as evidence of a prior infection. In this paper, a portable and low-cost electrochemical immunosensor is developed for the rapid and accurate quantification of SARS-CoV-2 serum antibodies. The immunosensor is capable of quantifying the concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against the SARS-CoV-2 spike protein in human serum. For IgG and IgM, it provides measurements in the range of 10.1 ng/mL - 60 µg/mL and 1.64 ng/mL - 50 µg/mL, respectively, both with an assay time of 13 min. We also developed device stabilization and storage strategies to achieve stable performance of the immunosensor over 24-week storage at room temperature. We evaluated the performance of the immunosensor using COVID-19 patient serum samples collected at different time points after symptom onset. The rapid and sensitive detection of IgG and IgM provided by our immunosensor fulfills the need of rapid COVID-19 serological testing for both point-of-care diagnosis and population immunity screening.
Subject(s)
Antibodies, Viral/isolation & purification , Biosensing Techniques , COVID-19 , COVID-19/diagnosis , COVID-19 Serological Testing , Humans , Immunoassay , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Pandemics , SARS-CoV-2 , Sensitivity and Specificity , Spike Glycoprotein, CoronavirusABSTRACT
Serology or antibody tests for COVID-19 are designed to detect antibodies (mainly Immunoglobulin M (IgM) and Immunoglobulin G (IgG) produced in response to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS CoV-2) infection. In this study, 30 lateral flow immunoassays were tested using serum or plasma from patients with confirmed SARS CoV-2 infection. Negative serological controls were accessed from a well-characterised bank of sera which were stored prior to February 2020. Operational characteristics and ease of use of the assays are reported. 4/30 (13%) of kits (Zheihang Orient Gene COVID-19 IgG/IgM, Genrui Novel Coronavirus (2019-nCoV) IgG/IgM, Biosynex COVID-19 BSS IgG/IgM, Boson Biotech 2019-nCoV IgG/IgM) were recommended for SAHPRA approval based on kit sensitivity. Of these, only the Orientgene was recommended by SAHPRA in August 2020 for use within the approved national testing algorithm while the remaining three received limited authorization for evaluation. All kits evaluated work on the same basic principle of immunochromatography with minor differences noted in the shape and colour of cartridges, the amount of specimen volume required and the test duration. Performance of the lateral flow tests were similar to sensitivities and specificities reported in other studies. The cassettes of the majority of kits evaluated (90%) detected both IgG and IgM. Only 23% of kits evaluated contained all consumables required for point-of-care testing. The study highlights the need for thorough investigation of kits prior to implementation.
Subject(s)
Antibodies, Viral/isolation & purification , COVID-19 Serological Testing/instrumentation , COVID-19/diagnosis , Immunoassay/instrumentation , Reagent Kits, Diagnostic/statistics & numerical data , Antibodies, Viral/blood , Antibodies, Viral/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Nucleic Acid Testing/statistics & numerical data , COVID-19 Serological Testing/statistics & numerical data , Humans , Immunoassay/statistics & numerical data , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Immunoglobulin M/blood , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purification , Point-of-Care Testing/statistics & numerical data , RNA, Viral/blood , RNA, Viral/isolation & purification , SARS-CoV-2/genetics , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , Sensitivity and SpecificityABSTRACT
Understanding how human complement proteins interact with human antibodies is important for the development of antibody therapies and understanding autoimmune diseases. At present, many groups use baby rabbit serum as a source of complement because, in contrast to human serum, it lacks preexisting antibodies. However, for characterization of human (monoclonal) antibodies, human serum would be a preferred source of complement. To prevent complement activation via naturally occurring antibodies, this human serum ideally lacks IgG and IgM. Here we describe how to deplete human serum of naturally occurring IgG and IgM using fast protein liquid affinity chromatography (FPLC) while minimizing the loss of serum complement activity. We also describe assays that can be used to validate depletion of IgG and IgM (IgG, IgM, and C1q sandwich ELISAs) and functionally assess remaining serum complement activity (hemolytic assays CH50 and AH50). Finally, we demonstrate how captured IgG and IgM can be purified.
Subject(s)
Blood Component Removal/methods , Complement System Proteins/isolation & purification , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Animals , Chromatography, Liquid/methods , Complement Hemolytic Activity Assay/methods , Complement System Proteins/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Mice , Rabbits , SheepABSTRACT
OBJECTIVES: In Italy, the pandemic of COVID-19 resulted in congestion of hospitals and laboratories and probably determined an underestimation of the number of infected subjects, as the molecular diagnosis of SARS-CoV-2 infection was mainly performed on hospitalised patients. Therefore, limited data are available about the number of asymptomatic/paucisymptomatic subjects in the general population across time. To understand SARS-CoV-2 infection in the general population, we have developed a cross-sectional study (the 'UNIversity against CORoNavirus study') to investigate infection trends in asymptomatic/paucisymptomatic subjects in Milan (Italy), between March and June 2020. PARTICIPANTS: The study population included 2023 subjects asymptomatic at the enrolment. PRIMARY OUTCOME MEASURES: A nasal mid-turbinate swab for the detection of SARS-CoV-2 RNA and blood specimen for testing serum antibodies (immunoglobulin M (IgM) and IgG) were collected. RESULTS: Subjects showing positivity for the SARS-CoV-2 RNA and/or for anti-SARS-CoV-2 Ig is 237 (11.7%). Only 1.2% (n=25) of the total population had a positive nasal swab for SARS-CoV-2 and the large majority (21/25) of them were observed in March. A total of 226 subjects (11%) had IgM (n=19; 0.9%), IgG (n=155; 7.7%) or both (n=52; 2.6%) against SARS-CoV-2. Subjects with a present or past SARS-CoV-2 infection did not differ from other subjects as regards the number of cohabiting family members, travels, fever and upper and lower respiratory infection episodes. CONCLUSIONS: Results from the present study support the hypothesis that the actual spread of the virus in Lombardy was underestimated in the official records. However, as it is not known how long Ig persist, numbers should be taken cautiously.
Subject(s)
Antibodies, Viral/isolation & purification , COVID-19 Serological Testing , COVID-19/diagnosis , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , RNA, Viral/isolation & purification , Adult , COVID-19/blood , Cross-Sectional Studies , Female , Humans , Italy/epidemiology , Male , Middle Aged , SARS-CoV-2ABSTRACT
Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays. This study was a Danish national collaboration and evaluated 15 commercial and one in-house anti-SARS-CoV-2 assays in 16 laboratories. Sensitivity was evaluated using 150 samples from individuals with asymptomatic, mild, or moderate COVID-19, nonhospitalized or hospitalized, confirmed by nucleic acid amplification tests (NAAT); samples were collected 13 to 73 days either from symptom onset or from positive NAAT (patients without symptoms). Specificity and cross-reactivity were evaluated in samples collected prior to the SARS-CoV-2 epidemic from >586 blood donors and patients with autoimmune diseases, cytomegalovirus or Epstein-Barr virus infections, and acute viral infections. A specificity of ≥99% was achieved by all total-Ab and IgG assays except one, DiaSorin Liaison XL IgG (97.2%). Sensitivities in descending order were Wantai ELISA total Ab (96.7%), CUH-NOVO in-house ELISA total Ab (96.0%), Ortho Vitros total Ab (95.3%), YHLO iFlash IgG (94.0%), Ortho Vitros IgG (93.3%), Siemens Atellica total Ab (93.2%), Roche Elecsys total Ab (92.7%), Abbott Architect IgG (90.0%), Abbott Alinity IgG (median 88.0%), DiaSorin Liaison XL IgG (median 84.6%), Siemens Vista total Ab (81.0%), Euroimmun/ELISA IgG (78.0%), and Snibe Maglumi IgG (median 78.0%). However, confidence intervals overlapped for several assays. The IgM results were variable, with the Wantai IgM ELISA showing the highest sensitivity (82.7%) and specificity (99%). The rate of seropositivity increased with time from symptom onset and symptom severity.
Subject(s)
Antibodies, Viral/isolation & purification , COVID-19 Serological Testing/methods , COVID-19/diagnosis , Immunoassay , Cytomegalovirus Infections , Enzyme-Linked Immunosorbent Assay , Epstein-Barr Virus Infections , Herpesvirus 4, Human , Humans , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Laboratories , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
OBJECTIVE: The coronavirus disease (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is now rapidly spreading globally. Serological tests are an important method to assist in the diagnosis of COVID-19, used for epidemiological investigations. In this study, we aimed to investigate the impact of different types of vacuum collection tubes on the detection of SARS-CoV-2 IgM and IgG antibodies, using the colloidal gold immunochromatographic assay (GICA). PATIENTS AND METHODS: A total of 112 patients with COVID-19 and 200 healthy control subjects with no infection were enrolled in this study. Their serum and plasma were collected into four different types of vacuum blood collection tubes. SARS-CoV-2 IgM and IgG specific antibodies in the plasma and serum were then detected by GICA and chemiluminescence assay (CA), respectively. In addition, the particle sizes of different colloidal gold solutions in the presence of different anticoagulants and coagulants were evaluated by both laser diffraction (Malvern) and confocal laser microscope, respectively. RESULTS: Our results revealed that anticoagulated plasma with EDTA-K2 improved the positive detection rate of SARS-CoV-2 IgM antibodies. Furthermore, our results shown that the detection results by GICA and CA were highly consistent, especially, the results of EDTA-K2 anticoagulated plasma detected by GICA was more consistent with CA results. We confirmed that EDTA-K2 could improve the detection sensitivity of SARS-CoV-2 IgG antibodies by chelating excessive colloidal gold compared with sodium citrate or lithium heparin, these methodologies did not appear to cause false positives. Colloidal gold particles could be chelated and aggregated by EDTA-K2, but not by sodium citrate, lithium heparin and coagulants. CONCLUSION: GICA is widely used to detect antibodies for the advantages of convenient, fast, low cost, suitable for screening large sample and require minimal equipment. In this study, we found that EDTA-K2 amplified the positive antibody signal by chelating colloidal gold and improved the detection sensitivity of SARS-CoV-2 IgM and IgG antibodies when using the GICA. Therefore, we suggested that EDTA-K2 anticoagulated plasma was more suitable for the detection of SARS-CoV-2 antibodies.
Subject(s)
Antibodies, Viral/isolation & purification , Chelating Agents/chemistry , Edetic Acid/chemistry , Gold Colloid/chemistry , Immunoassay/methods , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , SARS-CoV-2/immunology , Adult , Antibodies, Viral/blood , Antibody Specificity/immunology , COVID-19/blood , COVID-19/immunology , COVID-19/virology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Male , Middle Aged , Molecular Weight , Particle Size , Polymers/chemistry , Sensitivity and SpecificityABSTRACT
INTRODUCTION: Coronavirus disease 2019 (COVID-19) is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Fast, accurate, and simple blood-based assays for quantification of anti-SARS-CoV-2 antibodies are urgently needed to identify infected individuals and keep track of the spread of disease. METHODS: The study included 33 plasma samples from 20 individuals with confirmed COVID-19 by real-time reverse-transcriptase polymerase chain reaction and 40 non-COVID-19 plasma samples. Anti-SARS-CoV-2 immunoglobulin M (IgM)/immunoglobulin A (IgA) or immunoglobulin G (IgG) antibodies were detected by a microfluidic quantitative immunomagnetic assay (IMA) (ViroTrack Sero COVID IgM + IgA/IgG Ab, Blusense Diagnostics) and compared to an enzyme-linked immunosorbent assay (ELISA) (EuroImmun Medizinische Labordiagnostika). RESULTS: Of the 33 plasma samples from the COVID-19 patients, 28 were positive for IgA/IgM or IgG by IMA and 29 samples were positive by ELISA. Sensitivity for only one sample per patient was 68% for IgA + IgM and 75% IgG by IMA and 80% by ELISA. For samples collected 14 days after symptom onset, the sensitivity of both IMA and ELISA was around 91%. The specificity of the IMA reached 100% compared to 95% for ELISA IgA and 97.5% for ELISA IgG. CONCLUSION: IMA for COVID-19 is a rapid simple-to-use point-of-care test with sensitivity and specificity similar to a commercial ELISA.
Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Immunomagnetic Separation/methods , Point-of-Care Testing , SARS-CoV-2 , Aged , Female , Humans , Immunoglobulin A/blood , Immunoglobulin A/isolation & purification , Immunoglobulin G/blood , Immunoglobulin G/isolation & purification , Immunoglobulin M/blood , Immunoglobulin M/isolation & purification , Male , Middle Aged , RNA, Viral , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
PURPOSE: To report a case of cat scratch disease-associated retinitis diagnosed with an indirect fluorescent antibody (IFA) assay for immunoglobulin M (IgM) specific for a strain (YH-01) of Bartonella henselae recently identified in Japan. METHODS: Case report of a 24-year-old pregnant woman presented with general fever, fatigue, as well as blurred vision, and a central visual field deficiency in her right eye and was suspected as cat scratch disease because she had started to feed a feral dog a month ago. RESULTS: The patient's serum tested negative, however, with an IFA assay for IgG or IgM specific for the Houston-1, common strain of B. henselae. Further testing with an IFA assay for IgM specific for the YH-01 strain yielded a positive result. On the basis of the clinical findings and the IFA results, we were thus able to make a definitive diagnosis of cat scratch disease. CONCLUSION: An IFA assay based on the YH-01 or combination of both YH-01 and Houston-1 strains of B. henselae may show increased sensitivity for the diagnosis of cat scratch disease in Japan.
Subject(s)
Cat-Scratch Disease , Bartonella henselae/immunology , Cat-Scratch Disease/complications , Cat-Scratch Disease/diagnosis , Female , Fluorescent Antibody Technique , Humans , Immunoglobulin M/isolation & purification , Japan , Pregnancy , Pregnancy Complications, Infectious/diagnosis , Retinitis/etiology , Young AdultABSTRACT
The sphingosine 1-phosphate receptor type 1 (S1PR1) has several important functions, including stabilizing endothelial barrier and maintaining lymphocyte circulation. These functions are critically dependent on the regulation of S1PR1 cell surface expression. Currently available antibodies against human S1PR1 are not able to pick up cell surface expression on living cells by flow cytometry due to intracellular epitopes or unspecific binding. Here we describe the generation of a mouse monoclonal antibody specific for the N-terminal region of human S1PR1. It has an immunoglobulin M (IgM) kappa isotype and detects cell surface expression of recombinant human S1PR1 on overexpressing cells. Due to unspecific intracellular cell staining, it cannot be used for staining of dead cells and tissue slides or in microscopic analyses. It is also not suitable for Western blot analysis and immunoprecipitation. However, the antibody can stain for endogenous S1PR1 on human endothelial cell lines and primary human umbilical vein endothelial cells (HUVEC). Incubation of these cells with various S1PR1 agonists revealed potent S1PR1 internalization, which was not the case with the specific antagonist W146. Surprisingly, human T and B cells isolated from blood and palatine tonsils did not show specific staining, demonstrating significantly lower endogenous S1PR1 surface expression on lymphocytes than on endothelial cells.
Subject(s)
Antibodies, Monoclonal/isolation & purification , B-Lymphocytes/metabolism , Burkitt Lymphoma/metabolism , Endothelial Cells/metabolism , Immunoglobulin M/isolation & purification , Sphingosine-1-Phosphate Receptors/metabolism , T-Lymphocytes/metabolism , Anilides/pharmacology , Animals , Burkitt Lymphoma/pathology , Gene Expression Regulation , Human Umbilical Vein Endothelial Cells , Humans , Mice , Organ Specificity , Organophosphonates/pharmacology , Palatine Tonsil/cytology , Sphingosine-1-Phosphate Receptors/immunologyABSTRACT
Understanding and mitigating SARS-CoV-2 transmission hinges on antibody and viral RNA data that inform exposure and shedding, but extensive variation in assays, study group demographics and laboratory protocols across published studies confounds inference of true biological patterns. Our meta-analysis leverages 3214 datapoints from 516 individuals in 21 studies to reveal that seroconversion of both IgG and IgM occurs around 12 days post-symptom onset (range 1-40), with extensive individual variation that is not significantly associated with disease severity. IgG and IgM detection probabilities increase from roughly 10% at symptom onset to 98-100% by day 22, after which IgM wanes while IgG remains reliably detectable. RNA detection probability decreases from roughly 90% to zero by day 30, and is highest in feces and lower respiratory tract samples. Our findings provide a coherent evidence base for interpreting clinical diagnostics, and for the mathematical models and serological surveys that underpin public health policies.
Subject(s)
Betacoronavirus/genetics , Betacoronavirus/immunology , Coronavirus Infections/immunology , Coronavirus Infections/virology , Immunoglobulin G/blood , Immunoglobulin M/blood , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , RNA, Viral/analysis , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Coronavirus Infections/blood , Coronavirus Infections/diagnosis , Enzyme-Linked Immunosorbent Assay , Humans , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Pandemics , Pneumonia, Viral/blood , Pneumonia, Viral/diagnosis , RNA, Viral/isolation & purification , SARS-CoV-2Subject(s)
COVID-19/diagnosis , Coinfection/diagnosis , Mycoplasma pneumoniae/isolation & purification , Pneumonia, Mycoplasma/diagnosis , SARS-CoV-2/isolation & purification , Urticaria/diagnosis , Antibodies, Bacterial/immunology , Antibodies, Bacterial/isolation & purification , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , COVID-19/blood , COVID-19/immunology , COVID-19/virology , COVID-19 Serological Testing , Child , Coinfection/blood , Coinfection/immunology , Coinfection/microbiology , Enzyme-Linked Immunosorbent Assay , False Positive Reactions , Humans , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purification , Male , Middle Aged , Mycoplasma pneumoniae/immunology , Pneumonia, Mycoplasma/blood , Pneumonia, Mycoplasma/immunology , Pneumonia, Mycoplasma/microbiology , SARS-CoV-2/immunology , Skin/blood supply , Skin/microbiology , Urticaria/blood , Urticaria/immunology , Urticaria/microbiologyABSTRACT
OBJECTIVES: Nucleic acid testing is the gold standard method for the diagnosis of coronavirus disease 2019 (COVID-19); however, large numbers of false-negative results have been reported. In this study, nucleic acid detection and antibody detection (IgG and IgM) were combined to improve the testing accuracy of patients with suspected COVID-19. STUDY DESIGN: The positive rate of nucleic acid detection and antibody detection (IgG and IgM) were compared in suspected COVID-19 patients. METHODS: A total of 71 patients with suspected COVID-19 were selected to participate in this study, which included a retrospective analysis of clinical features, imaging examination, laboratory biochemical examination and nucleic acid detection and specific antibody (IgM and IgG) detection. RESULTS: The majority of participants with suspected COVID-19 presented with fever (67.61%) and cough (54.93%), and the imaging results showed multiple small patches and ground-glass opacity in both lungs, with less common infiltration and consolidation opacity (23.94%). Routine blood tests were mostly normal (69.01%), although only a few patients had lymphopenia (4.23%) or leucopenia (12.68%). There was no statistical difference in the double-positive rate between nucleic acid detection (46.48%) and specific antibody (IgG and IgM) detection (42.25%) (P = 0.612), both of which were also poorly consistent with each other (kappa = 0.231). The positive rate of combined nucleic acid detection and antibody detection (63.38%) was significantly increased, compared with that of nucleic acid detection (46.48%) and that of specific antibody (IgG and IgM) detection (42.25%), and the differences were statistically significant (P = 0.043 and P = 0.012, respectively). CONCLUSIONS: Nucleic acid detection and specific antibody (IgG and IgM) detection had similar positive rates, and their combination could improve the positive rate of COVID-19 detection, which is of great significance for diagnosis and epidemic control.
Subject(s)
Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Pneumonia, Viral/diagnosis , Adolescent , Adult , Aged , Antibodies, Viral/isolation & purification , Betacoronavirus/genetics , Betacoronavirus/immunology , COVID-19 , COVID-19 Testing , Child , Child, Preschool , Coronavirus Infections/epidemiology , Female , Humans , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Male , Middle Aged , Nucleic Acids/isolation & purification , Pandemics , Pneumonia, Viral/epidemiology , Reproducibility of Results , Retrospective Studies , SARS-CoV-2 , Young AdultABSTRACT
BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a novel beta-coronavirus that has recently emerged as the cause of the 2019 coronavirus pandemic (COVID-19). Polymerase chain reaction (PCR) based tests are optimal and recommended for the diagnosis of an acute SARS-CoV-2 infection. Serology tests for viral antibodies provide an important tool to diagnose previous exposure to the virus. Here we evaluate the analytical performance parameters of the Diazyme SARS-CoV-2 IgM/IgG serology assays and describe the kinetics of IgM and IgG seroconversion observed in patients with PCR-confirmed COVID-19 who were admitted to our hospital. METHODS: We validated the performance of the Diazyme assay in 235 presumed SARS-CoV-2 negative subjects to determine specificity. Subsequently, we evaluated the SARS-CoV-2 IgM and IgG seroconversion of 54 PCR-confirmed COVID-19 patients and determined sensitivity of the assay at three different timeframes. RESULT: Sensitivity and specificity for detecting seropositivity at ≥15 days following a positive SARS-CoV-2 PCR result, was 100.0% and 98.7% when assaying for the panel of IgM and IgG. The median time to seropositivity observed for a reactive IgM and IgG result from the date of a positive PCR was 5 days (IQR: 2.75-9 days) and 4 days (IQR: 2.75-6.75 days), respectively. CONCLUSIONS: Our data demonstrate that the Diazyme IgM/IgG assays are suited for the purpose of detecting SARS-CoV-2 IgG and IgM in patients with suspected SARS-CoV-2 infections. For the first time, we report longitudinal data showing the evolution of seroconversion for both IgG and IgM in a cohort of acutely ill patients in the United States. We also demonstrate a low false positive rate in patients who were presumed to be disease free.
Subject(s)
Betacoronavirus , Coronavirus Infections , Immunoglobulin G , Immunoglobulin M , Pandemics , Pneumonia, Viral , Seroconversion , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , Betacoronavirus/immunology , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Clinical Laboratory Techniques/methods , Coronavirus Infections/diagnosis , Coronavirus Infections/immunology , Coronavirus Infections/virology , Female , Humans , Immunoglobulin G/blood , Immunoglobulin G/isolation & purification , Immunoglobulin M/blood , Immunoglobulin M/isolation & purification , Male , Middle Aged , Monitoring, Immunologic/methods , Pneumonia, Viral/diagnosis , Pneumonia, Viral/immunology , Pneumonia, Viral/virology , SARS-CoV-2 , Sensitivity and SpecificityABSTRACT
Heterobothrium okamotoi, a monogenean gill parasite, exhibits high host specificity for the tiger puffer, Takifugu rubripes, and it has been experimentally verified that the parasite cannot colonize either closely related species such as the grass puffer Takifugu niphobles or distantly related fish such as the red seabream Pagrus major. Previously, we demonstrated in T. rubripes that immunoglobulin M (IgM) with d-mannose affinity induced deciliation of the oncomiracidia, the first step of parasitism, indicating that the parasite utilizes the molecule as a receptor for infection. In the present study, we purified mannose-specific IgM from 2 nonhost species, T. niphobles and P. major, by affinity and gel-filtration chromatography techniques and compared their deciliation-inducing activity against H. okamotoi oncomiracidia. The IgM of the former showed activity, whereas the latter had no effect, suggesting that in addition to d-mannose-binding ability, the crystallizable fragment domain of IgM, which is not part of the antigen-binding domain, plays an important role in host recognition by the oncomiracidia, such as direct binding to the parasites. It also suggests that the host specificity of H. okamotoi is relatively low upon initial recognition, and the specificity is established by exclusion in nonhosts during a later stage.
Subject(s)
Ectoparasitic Infestations/veterinary , Fish Diseases/parasitology , Immunoglobulin M/physiology , Mannose/immunology , Platyhelminths/immunology , Takifugu/parasitology , Amino Acid Sequence , Animals , Blotting, Western , Cilia/immunology , Cloning, Molecular , DNA, Complementary/genetics , DNA, Complementary/immunology , Ectoparasitic Infestations/immunology , Ectoparasitic Infestations/parasitology , Electrophoresis, Polyacrylamide Gel , Fish Diseases/immunology , Gene Expression , Gills/parasitology , Host Specificity , Hydrogen-Ion Concentration , Immunoglobulin M/blood , Immunoglobulin M/genetics , Immunoglobulin M/isolation & purification , Mucous Membrane/chemistry , Mucous Membrane/immunology , Mucous Membrane/parasitology , Platyhelminths/pathogenicity , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Takifugu/immunology , Trematode Infections/immunology , Trematode Infections/parasitology , Trematode Infections/veterinaryABSTRACT
BACKGROUND: Rapid tests detecting both dengue virus (DENV) NS1 antigen and anti-DENV IgM and IgG antibodies facilitate diagnosis of dengue fever (DF) in resource-poor settings. METHODOLOGY/PRINCIPAL FINDINGS: 92 acute phase serum samples from patients with a PCR-confirmed DENV infection collected in Lao People's Democratic Republic (Lao PDR) in 2013 and 2015 were analyzed with the SD Bioline Dengue Duo test. A subset of 74 samples was additionally tested with the Platelia NS1 antigen test, the Panbio DENV µ-capture ELISA and the Panbio DENV IgG ELISA. IgM seroconversion was assayed using follow-up samples of 35 patients collected in the convalescent phase. 57.6%, 22.8% and 44.6% of acute phase serum samples tested positive in the SD Bioline Dengue Duo NS1, IgM, and IgG test, respectively. Diagnostic sensitivity of the SD Bioline Dengue Duo NS1 test strongly correlated with viral load, decreased rapidly over the acute phase of the disease, and was significantly reduced in presence of high anti-DENV IgG antibody titers resulting from secondary DENV infection. While a good concordance (Cohen's kappa 0.78) was found between the SD Bioline Dengue Duo NS1 test and the Platelia NS1 antigen ELISA, both the SD Bioline Dengue Duo IgM and IgG test displayed a significantly lower sensitivity than the corresponding ELISA tests. CONCLUSIONS/SIGNIFICANCE: The SD Bioline Dengue Duo test is a valuable tool for diagnosis of DENV infections especially when analyzing early acute phase samples with high viral load. Nevertheless, in endemic areas, where secondary flavivirus infections are common, diagnostic sensitivity of the NS1 and IgM test components may be compromised.
Subject(s)
Coinfection/diagnosis , Dengue Virus/isolation & purification , Dengue/diagnosis , Point-of-Care Systems , Reagent Kits, Diagnostic , Adolescent , Adult , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibodies, Viral/isolation & purification , Coinfection/blood , Coinfection/immunology , Coinfection/virology , Dengue/blood , Dengue/immunology , Dengue/virology , Dengue Virus/immunology , Enzyme-Linked Immunosorbent Assay , Female , Follow-Up Studies , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Immunoglobulin G/isolation & purification , Immunoglobulin M/blood , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purification , Laos , Male , Middle Aged , Sensitivity and Specificity , Seroconversion , Viral Load , Viral Nonstructural Proteins/blood , Viral Nonstructural Proteins/immunology , Viral Nonstructural Proteins/isolation & purification , Young AdultABSTRACT
Evolution of antibody repertoire against the Ebola virus (EBOV) proteome was characterized in an acutely infected patient receiving supportive care alone to elucidate virus-host interactions over time. Differential kinetics are observed for IgM-IgG-IgA epitope diversity, antibody binding, and affinity maturation to EBOV proteins. During acute illness, antibodies predominate to VP40 and glycoprotein (GP). At day 13 of clinical illness, a marked increase in antibody titers to most EBOV proteins and affinity maturation to GP is associated with rapid decline in viral replication and illness severity. At one year, despite undetectable virus, a diverse IgM repertoire against VP40 and GP epitopes is observed suggesting occult viral persistence. Rabbit immunization experiments identify key immunodominant sites of GP, while challenge studies in mice found these epitopes induce EBOV-neutralizing antibodies and protect against lethal EBOV challenge. This study reveals markers of viral persistence and provides promising approaches for development and evaluation of vaccines and therapeutics.
Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Ebolavirus/immunology , Hemorrhagic Fever, Ebola/immunology , Animals , Antibodies, Neutralizing/biosynthesis , Antibodies, Viral/biosynthesis , Epitopes/immunology , Hemorrhagic Fever, Ebola/prevention & control , Hemorrhagic Fever, Ebola/therapy , Humans , Immunoglobulin A/isolation & purification , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Mice , Proteome/immunology , Rabbits , Survivors , Viral Envelope Proteins/immunology , Viral Matrix Proteins/immunology , Viral Proteins/immunology , Viral VaccinesABSTRACT
To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAcα-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAcα-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAcα and GalNAcα1-3Galß1-3(4)GlcNAcß. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAcα was quite unexpected. Given that GalNAcα is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAcα-OSer and do not bind a wide variety of GalNAcα-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAcα as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAcα do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAcα residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.
Subject(s)
Antigens, Tumor-Associated, Carbohydrate/immunology , Amino Acid Sequence , Antibody Affinity , Antibody Specificity , Antigen-Antibody Reactions/immunology , Antigens, Tumor-Associated, Carbohydrate/chemistry , Carbohydrate Sequence , Epitopes/chemistry , Epitopes/immunology , Epitopes/isolation & purification , Humans , Immunity, Innate , Immunoglobulin M/immunology , Immunoglobulin M/isolation & purification , Jurkat Cells , Neoplasms/immunologyABSTRACT
Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies' "Human IgG" and "Human IgM", were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.