scholarly journals Monoclonal Antibody-Based Antigen Capture Enzyme-Linked Immunosorbent Assay Reveals High Sensitivity of the Nucleocapsid Protein in Acute-Phase Sera of Severe Acute Respiratory Syndrome Patients

2005 ◽  
Vol 12 (1) ◽  
pp. 135-140 ◽  
Author(s):  
Biao Di ◽  
Wei Hao ◽  
Yang Gao ◽  
Ming Wang ◽  
Ya-di Wang ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Acute Phase ◽  
Detection Rate ◽  
Neutralization Test ◽  
Protein Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
N Protein ◽  
Antigen Capture ◽  
Healthy Donors

ABSTRACT Accurate and timely diagnosis of severe acute respiratory syndrome coronavirus (SARS-CoV) infection is a critical step in preventing another global outbreak. In this study, 829 serum specimens were collected from 643 patients initially reported to be infected with SARS-CoV. The sera were tested for the N protein of SARS-CoV by using an antigen capture enzyme-linked immunosorbent assay (ELISA) based on monoclonal antibodies against the N protein of SARS-CoV and compared to 197 control serum samples from healthy donors and non-SARS febrile patients. The results of the N protein detection analysis were directly related to the serological analysis data. From 27 SARS patients who tested positive with the neutralization test, 100% of the 24 sera collected from 1 to 10 days after the onset of symptoms were positive for the N protein. N protein was not detected beyond day 11 in this group. The positive rates of N protein for sera collected at 1 to 5, 6 to 10, 11 to 15, and 16 to 20 days after the onset of symptoms for 414 samples from 298 serologically confirmed patients were 92.9, 69.8, 36.4, and 21.1%, respectively. For 294 sera from 248 serological test-negative patients, the rates were 25.6, 16.7, 9.3, and 0%, respectively. The N protein was not detected in 66 patients with cases of what was initially suspected to be SARS but serologically proven to be negative for SARS and in 197 serum samples from healthy donors and non-SARS febrile patients. The specificity of the assay was 100%. Furthermore, of 16 sera collected from four patients during the SARS recurrence in Guangzhou, 5 sera collected from 7 to 9 days after the onset of symptoms were positive for the N protein. N protein detection exhibited a high positive rate, 96 to 100%, between day 3 and day 5 after the onset of symptoms for 27 neutralization test-positive SARS patients and 298 serologically confirmed patients. The N protein detection rate continually decreased beginning with day 10, and N protein was not detected beyond day 19 after the onset of symptoms. In conclusion, an antigen capture ELISA reveals a high N protein detection rate in acute-phase sera of patients with SARS, which makes it useful for early diagnosis of SARS.

scholarly journals Evaluation of Inapparent Nosocomial Severe Acute Respiratory Syndrome Coronavirus Infection in Vietnam by Use of Highly Specific Recombinant Truncated Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

2005 ◽  
Vol 12 (7) ◽  
pp. 848-854 ◽  
Author(s):  
Fuxun Yu ◽  
Mai Quynh Le ◽  
Shingo Inoue ◽  
Hong Thi Cam Thai ◽  
Futoshi Hasebe ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Large Scale ◽  
Screening Method ◽  
Epidemiological Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Nucleocapsid Proteins ◽  
N Protein ◽  
Care Workers ◽  
Coronavirus Infection

ABSTRACT Severe acute respiratory syndrome (SARS) is a recently emerged human disease associated with pneumonia. Inapparent infection with SARS coronavirus (CoV) is not well characterized. To develop a safe, simple, and reliable screening method for SARS diagnosis and epidemiological study, two recombinant SARS-CoV nucleocapsid proteins (N′ protein and NΔ121 protein) were expressed in Escherichia coli, purified by affinity chromatography, and used as antigens for indirect, immunoglobulin G enzyme-linked immunosorbent assays (ELISA). Serum samples collected from healthy volunteers and SARS patients in Vietnam were used to evaluate the newly developed methods. The N′ protein-based ELISA showed a highly nonspecific reaction. The NΔ121 protein-based ELISA, with a nonspecific reaction drastically reduced compared to that of the nearly-whole-length N′ protein-based ELISA, resulted in higher rates of positive reactions, higher titers, and earlier detection than the SARS-CoV-infected cell lysate-based ELISA. These results indicate that our newly developed SARS-CoV NΔ121 protein-based ELISA is not only safe but also a more specific and more sensitive method to diagnose SARS-CoV infection and hence a useful tool for large-scale epidemiological studies. To identify inapparent SARS-CoV infections, serum samples collected from health care workers (HCWs) in Vietnam were screened by the NΔ121 protein-based ELISA, and positive samples were confirmed by a virus neutralization test. Four out of 149 HCWs were identified to have inapparent SARS-CoV infection in Vietnam, indicating that subclinical SARS-CoV infection in Vietnam is rare but does exist.

scholarly journals False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid-Based Western Blot Assay Were Rectified by the Use of Two Subunits (S1 and S2) of Spike for Detection of Antibody to SARS-CoV

2006 ◽  
Vol 13 (3) ◽  
pp. 409-414 ◽  
Author(s):  
Mimoun Maache ◽  
Florence Komurian-Pradel ◽  
Alain Rajoharison ◽  
Magali Perret ◽  
Jean-Luc Berland ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Western Blot ◽  
False Positive ◽  
High Sensitivity ◽  
Cross Reactivity ◽  
Serum Samples ◽  
Western Blot Assay ◽  
N Protein ◽  
False Positive Diagnosis ◽  
Healthy Donors

ABSTRACT To evaluate the reactivity of the recombinant proteins expressed in Escherichia coli strain BL21(DE3), a Western blot assay was performed by using a panel of 78 serum samples obtained, respectively, from convalescent-phase patients infected with severe acute respiratory syndrome-associated coronavirus (SARS-CoV) (30 samples) and from healthy donors (48 samples). As antigen for detection of SARS-CoV, the nucleocapsid protein (N) showed high sensitivity and strong reactivity with all samples from SARS-CoV patients and cross-reacted with all serum samples from healthy subjects, with either those obtained from China (10 samples) or those obtained from France (38 serum samples), giving then a significant rate of false positives. Specifically, our data indicated that the two subunits, S1 (residues 14 to 760) and S2 (residues 761 to 1190), resulted from the divided spike reacted with all samples from SARS-CoV patients and without any cross-reactivity with any of the healthy serum samples. Consequently, these data revealed the nonspecific nature of N protein in serodiagnosis of SARS-CoV compared with the S1 and S2, where the specificity is of 100%. Moreover, the reported results indicated that the use of one single protein as a detection antigen of SARS-CoV infection may lead to false-positive diagnosis. These may be rectified by using more than one protein for the serodiagnosis of SARS-CoV.

scholarly journals Delayed neutralizing antibody response in the acute phase correlates with severe progression of COVID-19

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hitoshi Kawasuji ◽  
Yoshitomo Morinaga ◽  
Hideki Tani ◽  
Miyuki Kimura ◽  
Hiroshi Yamada ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Neutralization Test ◽  
Neutralizing Antibody ◽  
Pseudotyped Virus ◽  
Serum Samples ◽  
Neutralization Activity ◽  
Neutralizing Activity ◽  
Time Points ◽  
Moderate Disease ◽  
Activity Dynamics

AbstractAdaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) dynamics remain largely unknown. The neutralizing antibody (NAb) levels in patients with coronavirus disease 2019 (COVID-19) are helpful for understanding the pathology. Using SARS-CoV-2 pseudotyped virus, serum sample neutralization values in symptomatic COVID-19 patients were measured using the chemiluminescence reduction neutralization test (CRNT). At least two sequential serum samples collected during hospitalization were analyzed to assess NAbs neutralizing activity dynamics at different time points. Of the 11 patients, four (36.4%), six (54.5%), and one (9.1%) had moderate, severe, and critical disease, respectively. Fifty percent neutralization (N50%-CRNT) was observed upon admission in 90.9% (10/11); all patients acquired neutralizing activity 2–12 days after onset. In patients with moderate disease, neutralization was observed at earliest within two days after symptom onset. In patients with severe-to-critical disease, neutralization activity increased, plateauing 9–16 days after onset. Neutralization activity on admission was significantly higher in patients with moderate disease than in patients with severe-to-critical disease (relative % of infectivity, 6.4% vs. 41.1%; P = .011). Neutralization activity on admission inversely correlated with disease severity. The rapid NAb response may play a crucial role in preventing the progression of COVID-19.

scholarly journals A Rapid Immunochromatography Test Based on Hcp1 Is a Potential Point-of-Care Test for Serological Diagnosis of Melioidosis

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Phornpun Phokrai ◽  
Wisansanee Karoonboonyanan ◽  
Nida Thanapattarapairoj ◽  
Chidchanok Promkong ◽  
Adul Dulsuk ◽  
...  
Keyword(s):  
Point Of Care ◽  
Clinical Manifestations ◽  
Antibody Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Target Antigen ◽  
Northeast Thailand ◽  
Serum Samples ◽  
Serological Method ◽  
Healthy Donors ◽  
Wide Range

ABSTRACTMelioidosis is a fatal infectious disease caused by the environmental bacteriumBurkholderia pseudomallei. It is highly endemic in Asia and northern Australia but neglected in many other tropical countries. Melioidosis patients have a wide range of clinical manifestations, and definitive diagnosis requires bacterial culture, which can be time-consuming. A reliable rapid serological tool is greatly needed for disease surveillance and diagnosis. We previously demonstrated by enzyme-linked immunosorbent assay (ELISA) that a hemolysin-coregulated protein (Hcp1) is a promising target for serodiagnosis of melioidosis. In this study, we developed a rapid immunochromatography test (ICT) using Hcp1 as the target antigen (Hcp1-ICT). We evaluated this test for specific antibody detection using serum samples obtained from 4 groups of human subjects, including the following: (i) 487 culture-confirmed melioidosis patients from four hospitals in northeast Thailand; (ii) 202 healthy donors from northeast Thailand; (iii) 90 U.S. healthy donors; and (iv) 207 patients infected with other organisms. Compared to culture results as a gold standard, the sensitivity of ICT for all hospitals was 88.3%. The specificities for Thai donors and U.S. donors were 86.1% and 100%, respectively, and the specificity for other infections was 91.8%. The results of the Hcp1-ICT demonstrated 92.4% agreement with the Hcp1-ELISA results with a kappa value of 0.829, indicating that the method is much improved compared with the current serological method, the indirect hemagglutination assay (IHA) (69.5% sensitivity and 67.6% specificity for Thais). The Hcp1-ICT represents a potential point-of-care (POC) test and may be used to replace the IHA for screening of melioidosis in hospitals as well as in resource-limited areas.

Chronic neutropenia mediated by Fas ligand

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3219-3222 ◽  
Author(s):  
Jin Hong Liu ◽  
Sheng Wei ◽  
Thierry Lamy ◽  
P. K. Epling-Burnette ◽  
Gordon Starkebaum ◽  
...  
Keyword(s):  
Fas Ligand ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pathogenetic Mechanism ◽  
Serum Samples ◽  
Characteristic Finding ◽  
Healthy Donors ◽  
Fas Pathway ◽  
Neutrophil Survival ◽  
Chronic Neutropenia ◽  
Lgl Leukemia

Abstract Chronic neutropenia, often associated with rheumatoid arthritis, is a characteristic finding in large granular lymphocyte (LGL) leukemia. The mechanism of neutropenia is not known. Normal neutrophil survival is regulated by the Fas–Fas ligand apoptotic system. We hypothesized that neutropenia in LGL leukemia is mediated by dysregulated expression of Fas ligand. Levels of Fas ligand in serum samples from patients with LGL leukemia were measured with a Fas ligand enzyme-linked immunosorbent assay. The effects of serum from patients with LGL leukemia on apoptosis of normal neutrophils were determined by flow cytometry and morphologic assessment. High levels of circulating Fas ligand were detected in 39 of 44 serum samples from patients with LGL leukemia. In contrast, Fas ligand was undetectable in 10 samples from healthy donors. Serum from the patients triggered apoptosis of normal neutrophils that depended partly on the Fas pathway. Resolution of neutropenia was associated with disappearance or marked reduction in Fas ligand levels in 10 of 11 treated patients. These data suggest that high levels of Fas ligand are a pathogenetic mechanism in human disease.

DETECTION OF SPECIFIC ANTIBODIES TO THE NUCLEOCAPSID PROTEIN FRAGMENTS OF SEVERE ACUTE RESPIRATORY SYNDROME-CORONAVIRUS AND SCOTOPHILUS BAT CORONAVIRUS-512 IN THREE INSECTIVOROUS BAT SPECIES

2018 ◽  
Vol 44 (04) ◽  
pp. 179-188 ◽  
Author(s):  
Yi-Ning Chen ◽  
Bo-Gang Su ◽  
Hung-Chang Chen ◽  
Cheng-Han Chou ◽  
Hsi-Chi Cheng
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Nucleocapsid Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Protein Fragments ◽  
Specific Antibodies ◽  
History Of ◽  
Polymerase Chain ◽  
Carboxyl Terminal ◽  
Bat Coronavirus

Bats are the natural reservoirs of severe acute respiratory syndrome-coronavirus (SARS-CoV). Six Alphacoronavirus and five Betacoronavirus have been detected in many bat species, including SARS-related CoV and Middle East respiratory syndrome (MERS)-related CoV. In Taiwan, SARS-related CoV, belonging to Betacoronavirus, has been detected in Rhinolophus monoceros. Scotophilus bat CoV-512, belonging to Alphacoronavirus, has been detected in Scotophilus kuhlii, Miniopterus fuliginosus, and Rhinolophus monoceros by using reverse transcription polymerase chain reaction (RT-PCR). To understand the infection history of CoV in these three insectivorous bat populations, CoV-specific antibodies were surveyed by using western blot (WB) analysis and indirect enzyme-linked immunosorbent assay (ELISA). The carboxyl terminal fragment of nucleocapsid protein (N3) of SARS-CoV and Scotophilus bat CoV-512 were used as the antigen in the assays. Of the 52 serum samples obtained from Scotophilus kuhlii, 29 samples (56%) were tested positive for Scotophilus bat CoV-512-specific antibodies through ELISA. Of the 63 serum samples obtained from Rhinolophus monoceros, 9 samples were tested positive for only SARS-CoV-specific antibodies, 7 samples were tested positive for only Scotophilus bat CoV-512-specific antibodies, and 16 samples (25.4%) were tested positive for both antibodies through WB analysis. Only 1 of 18 Miniopterus bat serum samples tested positive for Scotophilus bat CoV-512-specific antibodies through ELISA. Lactating female bats had higher positive rates of CoV-specific antibodies than non-lactating female and male bats did. Our findings were crucial for understanding CoV infection history in three insectivorous bat species and important for the control of bat-borne zoonosis diseases.

scholarly journals Comparison of the Serum Neutralization Test and a Competitive Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies to Vesicular Stomatitis Virus New Jersey and Vesicular Stomatitis Virus Indiana

2002 ◽  
Vol 14 (3) ◽  
pp. 240-242 ◽  
Author(s):  
Juan Francisco Alvarado ◽  
Gaby Dolz ◽  
Marco V. Herrero ◽  
Brian McCluskey ◽  
Mo Salman
Keyword(s):  
New Jersey ◽  
Confidence Interval ◽  
Vesicular Stomatitis Virus ◽  
Immunosorbent Assay ◽  
Neutralization Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Serum Neutralization ◽  
Serum Neutralization Test ◽  
Vesicular Stomatitis

A competitive enzyme-linked immunosorbent assay (C-ELISA) for the detection of antibodies against vesicular stomatitis virus New Jersey (VSV-NJ) and vesicular stomatitis virus Indiana (VSV-IN) was compared with the serum neutralization test (SNT) using 1,106 serum samples obtained from dairy cattle on sentinel study farms in the Poás region of Costa Rica. Kappa coefficients between the C-ELISA and the SNT were 0.8871 (95% confidence interval [CI]: 0.8587–0.9155) and 0.6912 (95% CI: 0.6246–0.7577) for the VSV-NJ and VSV-IN tests, respectively. These results indicate good to excellent agreement between the 2 tests under these conditions.

scholarly journals Monoclonal antibody-based capture ELISA in the diagnosis of previous dengue infection

2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Chukiat Sirivichayakul ◽  
Kriengsak Limkittikul ◽  
Pornthep Chanthavanich ◽  
Sutee Yoksan ◽  
Anuttarasakdi Ratchatatat ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Neutralization Test ◽  
Dengue Infection ◽  
Severe Dengue ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dengue Vaccine ◽  
Serum Samples ◽  
Specific Immunoglobulin ◽  
Study Population ◽  
Dengue Prevention

Abstract Background Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand. Methods The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3–11 years in Ratchaburi Province, Thailand. Results The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects. Conclusions This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

scholarly journals West Nile Virus Seroprevalence among Qatari and Immigrant Populations within Qatar

2020 ◽  
Author(s):  
Hasna Kunhipurayil ◽  
Muna Ahmed ◽  
Gheyath Nasrallah
Keyword(s):  
West Nile Virus ◽  
Blood Donation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Large Sample Size ◽  
Serum Samples ◽  
West Nile ◽  
Viral Neutralization ◽  
Healthy Donors ◽  
Confirmatory Tests ◽  
Sub Saharan

Background: West Nile virus (WNV) is one of the most widely spread arboviruses worldwide and a highly significant pathogen in humans and animals. Despite frequent outbreaks and endemic transmission being reported in the Middle East and North Africa (MENA), seroprevalence studies of WNV in Qatar are highly lacking. Aim: This study aims to investigate the actual prevalence of WNV among local and expatriate communities in the Qatar using a large sample size of seemingly healthy donors. Method: A total of 1992 serum samples were collected from donors of age 18 or older and were tested for the presence of WNV antibodies. Serion enzyme-linked immunosorbent assay (ELISA) commercial microplate kits were used to detect the presence of the WNV IgM and IgG. The seropositivity was statistically analyzed using SPSS software with a confidence interval of 95%. Results: The seroprevalence of anti-WNV IgG and IgM in Qatar was 10.3% and 3.4%, respectively. The country-specific seroprevalence according to nationality for WNV IgG and IgM, respectively, were Sudan (37.0%, 10.0%), Egypt (31.6%, 4.4%), India (13.4%, 3.2%), Yemen(10.2%, 7.0%), Pakistan (8.6%, 2.7%), Iran (10.6%, 0.0%), Philippines (5.4%, 0.0%), Jordan(6.8%, 1.1%), Syria (2.6%, 9.6%), Palestine (2.6%, 0.6%), Qatar (1.6%, 1.7%), and Lebanon (0.9%, 0.0%). The prevalence of both IgM and IgG was significantly correlated with the nationality (p≤0.001). Conclusion: Among these tested nationalities, Qatar national has a relatively low burden of WNV disease. The highest prevalence of WNV was found in the Sub Saharan African nationalities like Sudan and Egypt. The seroprevalence of WNV is different from the previously reported arboviruses such as CHIKV and DENV, which was highest among Asian countries (India and Philippines). Further confirmatory tests such as viral neutralization assays are needed to confirm the IgM seropositivity in these samples since these samples could be a source of viral transmission through blood donation.

An evaluation of antigen capture assays for detecting active filarial antigens

2014 ◽  
Vol 89 (3) ◽  
pp. 352-358 ◽  
Author(s):  
R. Ravishankaran ◽  
N.S. Radhika ◽  
L. Ansel Vishal ◽  
S. Meenakshisundaram ◽  
A.A. Karande ◽  
...  
Keyword(s):  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Capture Assay ◽  
Antigen Capture ◽  
Circulating Filarial Antigens ◽  
Detection Assay ◽  
Normal Individuals ◽  
Circulating Antigen ◽  
Endemic Normal

AbstractLymphatic filariasis is a parasitic disease of tropical countries. This is a disfiguring and painful disease contracted in childhood, but the symptoms become apparent only in later years. Diagnosis of filarial infection is very crucial for the management of the disease. The main objective of this study was to develop a filarial antigen-based immunological assay for the diagnosis and surveillance of the disease. Monoclonal and polyclonal antibodies were raised to the recombinant protein Brugia malayi vespid allergen homologue (VAH). Capture enzyme-linked immunosorbent assay (ELISA) was standardized utilizing various combinations of antibodies and evaluated with serum samples of endemic normal (EN, n= 110), microfilaraemic (MF, n= 65), chronic pathology (CP, n= 45) and non-endemic normal (NEN, n= 10) individuals. Of the 230 samples tested, VAH capture assay detected circulating antigen in 97.91% of bancroftian and 100% of brugian microfilaraemic individuals, and 5% of endemic normal individuals, comparable to the earlier reported SXP-1 antigen detection assay. However, the combination of VAH and SXP-1 (VS) capture ELISA was found to be more robust, detecting 100% of microfilaraemic individuals and with higher binding values. Thus an antigen capture immunoassay has been developed, which can differentiate active infection from chronic infection by detecting circulating filarial antigens in clinical groups of endemic areas.

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