Novel Immunofluorescence Assay Using Recombinant Nucleocapsid-Spike Fusion Protein as Antigen To Detect Antibodies against Severe Acute Respiratory Syndrome Coronavirus

ABSTRACT Severe acute respiratory syndrome (SARS) is caused by a novel and highly infectious virus named SARS coronavirus (SARS-CoV). Among the serological tests currently available for the detection of SARS-CoV, a whole-virus-based immunofluorescence assay (IFA) was considered one of the most sensitive assays and served as a “gold standard” during the SARS epidemic in Singapore in 2003. However, the need to manipulate live SARS-CoV in the traditional IFA limits its wide application due to the requirement for a biosafety level 3 laboratory and the risk of laboratory infection. Previously, we have identified two immunodominant epitopes, named N195 and Sc, in the two major structural proteins, the N and S proteins, of SARS-CoV (Q. He, K. H. Chong, H. H. Chng, B. Leung, A. E. Ling, T. Wei, S. W. Chan, E. E. Ooi, and J. Kwang, Clin. Diagn. Lab. Immunol., 11:417-422, 2004; L. Lu, I. Manopo, B. P. Leung, H. H. Chng, A. E. Ling, L. L. Chee, E. E. Ooi, S. W. Chan, and J. Kwang, J. Clin. Microbiol. 42:1570-1576, 2004). In the present study, the N195-Sc fusion protein was highly expressed in insect (Sf9) cells infected with a recombinant baculovirus bearing the hybrid gene under the control of a polyhedrin promoter. An IFA based on Sf9 cells producing the fusion protein was standardized with 23 serum samples from patients with SARS, 20 serum samples from patients with autoimmune diseases, and 43 serum samples from healthy blood donors. The detection rates were comparable to those obtained with a commercial SARS-CoV IFA kit (EUROIMMUN, Gross Groenau, Germany) and a conventional IFA performed at the Singapore General Hospital. Our data showed that the newly developed IFA could detect SARS-CoV in 22 of the 23 SARS-CoV-positive serum samples and gave no false-positive results when the sera from patients with autoimmune diseases and healthy individuals were tested. The detection rate was identical to those of the two whole-virus-based IFAs. Thus, the novel N-S fusion antigen-based IFA could be an attractive alternative to present whole-virus-based IFAs for the diagnosis of SARS-CoV infection.

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Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation

The Journal of Cell Biology ◽

10.1083/jcb.122.4.961 ◽

1993 ◽

Vol 122 (4) ◽

pp. 961-972 ◽

Cited By ~ 170

Author(s):

SM Brady-Kalnay ◽

AJ Flint ◽

NK Tonks

Keyword(s):

Fusion Protein ◽

Protein Tyrosine Phosphatase ◽

Tyrosine Phosphatase ◽

Recombinant Baculovirus ◽

Sf9 Cells ◽

Homophilic Binding ◽

Protein Tyrosine ◽

Baculovirus Infection ◽

Induced Aggregation ◽

Cell Cell

The receptor-like protein tyrosine phosphatase, PTPmu, displays structural similarity to cell-cell adhesion molecules of the immunoglobulin superfamily. We have investigated the ability of human PTPmu to function in such a capacity. Expression of PTPmu, with or without the PTPase domains, by recombinant baculovirus infection of Sf9 cells induced their aggregation. However, neither a chimeric form of PTPmu, containing the extracellular and transmembrane segments of the EGF receptor and the intracellular segment of PTPmu, nor the intracellular segment of PTPmu expressed as a soluble protein induced aggregation. PTPmu mediates aggregation via a homophilic mechanism, as judged by lack of incorporation of uninfected Sf9 cells into aggregates of PTPmu-expressing cells. Homophilic binding has been demonstrated between PTPmu-coated fluorescent beads (Covaspheres) and endogenously expressed PTPmu on MvLu cells. Additionally the PTPmu-coated beads specifically bound to a bacterially expressed glutathione-S-transferase fusion protein containing the extracellular segment of PTPmu (GST/PTPmu) adsorbed to petri dishes. Covaspheres coated with the GST/PTPmu fusion protein aggregated in vitro and also bound to PTPmu expressed endogenously on MvLu cells. These results suggest that the ligand for this transmembrane PTPase is another PTPmu molecule on an adjacent cell. Thus homophilic binding interactions may be an important component of the function of PTPmu in vivo.

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Immunofluorescence Assay for Detection of Human Metapneumovirus-Specific Antibodies by Use of Baculovirus-Expressed Fusion Protein

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.1.202-205.2005 ◽

2005 ◽

Vol 12 (1) ◽

pp. 202-205 ◽

Cited By ~ 16

Author(s):

Nobuhisa Ishiguro ◽

Takashi Ebihara ◽

Rika Endo ◽

Xiaoming Ma ◽

Ryo Shirotsuki ◽

...

Keyword(s):

Trichoplusia Ni ◽

Respiratory Infections ◽

Recombinant Baculovirus ◽

Immunofluorescence Assay ◽

Human Metapneumovirus ◽

Specific Method ◽

Serum Samples ◽

F Protein ◽

Hmpv Infection ◽

Well Correlation

ABSTRACT Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (κ = 0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.

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Development of a Western Blot Assay for Detection of Bovine Immunodeficiency-Like Virus Using Capsid and Transmembrane Envelope Proteins Expressed from Recombinant Baculovirus

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.6.2.168-172.1999 ◽

1999 ◽

Vol 6 (2) ◽

pp. 168-172 ◽

Cited By ~ 9

Author(s):

Y. Abed ◽

G. St-Laurent ◽

H. Zhang ◽

R. M. Jacobs ◽

D. Archambault

Keyword(s):

Recombinant Protein ◽

Fusion Protein ◽

Western Blot ◽

Fusion Proteins ◽

Transmembrane Protein ◽

Recombinant Baculovirus ◽

Serum Samples ◽

Western Blot Assay ◽

Recombinant Fusion Proteins ◽

Syncytial Virus

ABSTRACT A 120-amino-acid polypeptide selected from the transmembrane protein region (tTM) and the major capsid protein p26 of bovine immunodeficiency-like virus (BIV) were expressed as fusion proteins from recombinant baculoviruses. The antigenic reactivity of both recombinant fusion proteins was confirmed by Western blot with bovine and rabbit antisera to BIV. BIV-negative bovine sera and animal sera positive for bovine syncytial virus and bovine leukemia virus failed to recognize the recombinant fusion proteins, thereby showing the specificity of the BIV Western blot. One hundred and five bovine serum samples were tested for the presence of anti-BIV antibodies by the recombinant protein-based Western blot and a reference Western blot assay using cell culture-derived virions as test antigens. There was a 100% concordance when the p26 fusion protein was used in the Western blot. However, the Western blot using the tTM fusion protein as its test antigen identified four BIV-positive bovine sera which had tested negative in both the p26 recombinant-protein-based and the reference Western blot assays. This resulted in the lower concordance of 96.2% between the tTM-protein-based and reference Western blot assays. The results of this study showed that the recombinant p26 and tTM proteins can be used as test antigens for the serodetection of BIV-infection in animals.

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Comparison of a Multiplex Flow Cytometric Assay with Enzyme-Linked Immunosorbent Assay for Quantitation of Antibodies to Tetanus, Diphtheria, and Haemophilus influenzae Type b

Clinical and Vaccine Immunology ◽

10.1128/cdli.9.4.872-876.2002 ◽

2002 ◽

Vol 9 (4) ◽

pp. 872-876 ◽

Cited By ~ 22

Author(s):

Jerry W. Pickering ◽

Thomas B. Martins ◽

M. Carl Schroder ◽

Harry R. Hill

Keyword(s):

Immunofluorescence Assay ◽

World Health ◽

Haemophilus Influenzae Type ◽

Enzyme Linked Immunosorbent Assay ◽

Attractive Alternative ◽

Type B ◽

Serum Samples ◽

Clostridium Tetani ◽

Protective Antibody ◽

Antibody Levels

ABSTRACT We developed a multiplexed indirect immunofluorescence assay for antibodies to Haemophilus influenza type b (Hib) polysaccharide and the toxoids of Clostridium tetani (Tet) and Corynebacterium diphtheriae (Dip) based on the Luminex multiple-analyte profiling system. A pooled serum standard was calibrated against World Health Organization standards for Dip and Tet and an international standard for Hib. The multiplexed Luminex assay was compared to individual enzyme-linked immunosorbent assays (ELISAs) for the same analytes. By both methods, 75 (92.6%) of 81 of random serum samples had protective levels of antibody to Tet (≥0.1 IU/ml). For Dip, 81.5% of the samples had protective antibody levels (≥0.1 IU/ml) by ELISA and 80.2% had protective antibody levels by Luminex. Protective levels (≥1.0 μg/ml) of antibody to Hib were found in 45.0% of the samples tested by ELISA and in 39.0% of the samples tested by Luminex. The correlations (R 2) between ELISA and Luminex of the 81 samples were 0.96, 0.96, and 0.91 for Tet, Dip, and Hib, respectively. There was also similar agreement between Luminex and ELISA for sera collected before and 1 month after Tet, Dip, and Hib vaccine administration. Both methods detected strong postvaccination responses. The Luminex method is an attractive alternative to ELISA since it reduces labor and reagent costs, as well as assay time.

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

Scientific Reports ◽

10.1038/s41598-021-96143-8 ◽

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.

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A rapid point-of-care assay accurately measures vitamin D

Journal of Endocrinological Investigation ◽

10.1007/s40618-021-01575-8 ◽

2021 ◽

Author(s):

K. Albrecht ◽

J. Lotz ◽

L. Frommer ◽

K. J. Lackner ◽

G. J. Kahaly

Keyword(s):

Vitamin D ◽

Metabolic Pathways ◽

Point Of Care ◽

Immunofluorescence Assay ◽

Laboratory Method ◽

Controlled Study ◽

Mean Deviation ◽

Serum Samples ◽

Assay Validation ◽

Accuracy And Precision

Abstract Purpose Vitamin D (VitD) is a pleiotropic hormone with effects on a multitude of systems and metabolic pathways. Consequently, the relevance of a sufficiently high VitD serum level becomes self-evident. Methods A rapid immunofluorescence assay designed for the point-of-care measurement of serum VitD3 solely was tested. Inter- and intra-assay validation, double testing and result comparison with a standardized laboratory method were performed. Results An overall linear correlation of r = 0.89 (Pearson, 95% CI 0.88–0.92, p < 0.01) between the point of care and the conventional reference assay was registered. Accuracy and precision were of special interest at cut-points (10 ng/ml [mean deviation 1.7 ng/ml, SD 1.98 ng/ml, SE 0.16 ng/ml], 12 ng/ml [MD 0.41, SD 1.89, SE 0.19] and 30 ng/ml [MD − 1.11, SD 3.89, SE 0.35]). Only a slight deviation was detected between the two assays when using fresh (r = 0.91, 95% CI 0.86–0.94, p < 0.01) and frozen serum samples (r = 0.86, 0.82–0.89, p < 0.01). Results remained steady when samples were frozen several times. Inter- and intra-assay validation according to the CLSI protocol as well as multiuser testing showed stable results. Conclusion This novel, innovative, and controlled study indicates that the evaluated rapid point of care VitD assay is reliable, accurate, and suited for clinical practice.

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PEDV Infection Generates Conformation-Specific Antibodies That Can Be Effectively Detected by a Cell-Based ELISA

Viruses ◽

10.3390/v13020303 ◽

2021 ◽

Vol 13 (2) ◽

pp. 303

Author(s):

Wei-Ting Hsu ◽

Chia-Yu Chang ◽

Chih-Hsuan Tsai ◽

Sung-Chan Wei ◽

Huei-Ru Lo ◽

...

Keyword(s):

Recombinant Baculovirus ◽

Immunocytochemical Staining ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Samples ◽

Perfect Agreement ◽

Specific Antibodies ◽

Diarrhea Virus ◽

Serological Studies ◽

A Cell ◽

Infected Insect

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes serious and highly contagious enteric disease in swine worldwide. In this study, we constructed a recombinant baculovirus (S-Bac) expressing full-length spike protein of the virulent epidemic genotype 2b (G2b) PEDV strain for serological studies of infected pigs. We found that most spike-specific antibodies produced upon PEDV infection in pigs are conformation-specific and they could be detected on S-Bac-infected insect cells by immunofluorescent assay, but they were insensitive to Western blot analysis, the typical method for antiserum analysis. These results indicated that spike conformation is crucial for serum recognition. Since it is difficult to purify trimeric spike membrane protein for conventional enzyme-linked immunosorbent assay (ELISA), we used S-Bac to generate a novel cell-based ELISA for convenient PEDV detection. We analyzed 100 pig serum samples, and our cell-based ELISA exhibited a sensitivity of 100%, a specificity of 97%, and almost perfect agreement [Cohen’s kappa coefficient value (κ) = 0.98] with immunocytochemical staining results. Our cell-based ELISA rapidly presented antigen for proper detection of conformation-specific antibodies, making PEDV detection more convenient, and it will be useful for detecting many viral diseases in the future.

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Reactivity of serum samples from patients with a flavivirus infection measured by immunofluorescence assay and ELISA

Microbes and Infection ◽

10.1016/s1286-4579(02)01647-7 ◽

2002 ◽

Vol 4 (12) ◽

pp. 1209-1215 ◽

Cited By ~ 73

Author(s):

Penelopie Koraka ◽

Herve Zeller ◽

Matthias Niedrig ◽

Albert D.M.E Osterhaus ◽

Jan Groen

Keyword(s):

Immunofluorescence Assay ◽

Serum Samples ◽

Flavivirus Infection

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Characterization and purification of human retinoic acid receptor-γ 1 overexpressed in the baculovirus-insect cell system

Biochemical Journal ◽

10.1042/bj2870833 ◽

1992 ◽

Vol 287 (3) ◽

pp. 833-840 ◽

Cited By ~ 11

Author(s):

A P Reddy ◽

J Y Chen ◽

T Zacharewski ◽

H Gronemeyer ◽

J J Voorhees ◽

...

Keyword(s):

Retinoic Acid ◽

Dna Binding ◽

Mammalian Cells ◽

Retinoic Acid Receptor ◽

Expression System ◽

Gel Filtration ◽

Recombinant Baculovirus ◽

Sf9 Cells ◽

Acid Receptor ◽

Gel Retardation

The full-length cDNA for the human retinoic acid receptor-gamma 1 (RAR-gamma 1) has been expressed to high levels in Spodoptera frugiferda (Sf9) cells using the baculovirus expression system. Western blot analysis revealed that RAR-gamma 1 expression increased between 32 and 60 h post-infection. The recombinant receptor was expressed primarily as a nuclear protein and displayed a molecular mass of 50 kDa as determined by SDS/PAGE and gel-filtration chromatography, consistent with its cDNA-deduced size. Based on ligand binding, 2 x 10(6) RAR-gamma 1 molecules were expressed per Sf9 cell, a level approx. 2000 times greater than in mammalian cells. The receptor was partially purified 300-fold by sequential anion-exchange, gel-filtration and DNA affinity chromatographies. The overexpressed receptor specifically bound all-trans-retinoic acid (RA) and the synthetic retinoid CD367 with high affinity (Kd 0.15 nM and 0.23 nM respectively). The RA metabolites 4-hydroxy-RA and 4-oxo-RA were poor competitors for [3H]CD367 binding to recombinant RAR-gamma 1 (K(i) > 1 microM), indicating that 4-oxidation of RA greatly reduces its affinity for RAR-gamma 1. Gel-retardation analysis demonstrated that RAR-gamma 1 specifically bound the RA response element of the mouse RAR-beta gene. RAR-gamma 1 species expressed from recombinant baculovirus (in Sf9 cells) and vaccinia virus (in HeLa cells) exhibited similar affinities for RA and CD367 and had comparable DNA-binding properties in gel-retardation experiments. Moreover, a similar requirement for additional DNA-binding stimulatory factor(s) was observed in both cases. These results provide a basis for the use of baculovirus-expressed RAR-gamma 1 in further functional and structural studies.

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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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.1.135-140.2005 ◽

2005 ◽

Vol 12 (1) ◽

pp. 135-140 ◽

Cited By ~ 22

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.

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