scholarly journals Detection of Asymptomatic Antigenemia in Pigs Infected by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) by a Novel Capture Immunoassay with Monoclonal Antibodies against the Nucleocapsid Protein of PRRSV

2009 ◽  
Vol 16 (12) ◽  
pp. 1822-1828 ◽  
Author(s):  
Jian-Piao Cai ◽  
Ya-Di Wang ◽  
Herman Tse ◽  
Hua Xiang ◽  
Kwok-Yung Yuen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Foot And Mouth Disease ◽  
Rapid Screening ◽  
Respiratory Syndrome Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
N Protein ◽  
Specificity And Sensitivity ◽  
Antigen Assay ◽  
Disease Antibody

ABSTRACT Routine surveillance for porcine reproductive and respiratory syndrome virus (PRRSV) infections is crucial for the epidemiological control of this disease. Antibody tests are widely used but cannot differentiate between vaccination and reinfection. We developed a PRRSV antigen capture enzyme-linked immunosorbent assay (ELISA) using well-characterized monoclonal antibodies (MAbs) raised against the nucleocapsid (N) protein of North American and European PRRSV. This antigen assay detected purified N protein from both genotypes at levels as low as 0.4 and 0.8 ng, respectively. The specificity and sensitivity of the N antigen assay were evaluated with ground lung tissues from 8 PRRSV-infected and 16 healthy swine, and culture supernatants from six PRRSV isolates as well as other swine viruses were confirmed by reverse transcriptase PCR (RT-PCR). Antigen assays were positive in all eight infected tissues and with six different PRRSV isolates, with no false positives among healthy tissues and other swine viruses (i.e., pseudorabies and foot and mouth disease viruses). A number of sera, field collected from 466 vaccinated and asymptomatic pigs in Guangdong, China, between 2008 and 2009, tested positive by the N antigen assay (12.45%), RT-PCR (15.02%), and a commercial test for antibodies against PRRSV (78.97%). Of the 466 sera, 47 were positive by both antigen and RT-PCR tests, 11 by antigen test only, and 23 by RT-PCR only; the two assays had an overall agreement of 92.7%, indicating a significant percentage of active PRRSV in asymptomatic pigs despite previous immunization. These findings suggest that the antigen assay is a valuable field tool for the epidemiological control of PRRSV that can be used for rapid screening, particularly in asymptomatic animals.

scholarly journals Evaluation of Sandwich Enzyme-Linked Immunosorbent Assay and Reverse Transcription Polymerase Chain Reaction for the Diagnosis of Foot-and-Mouth Disease

Intervirology ◽  
2021 ◽  
pp. 1-6
Author(s):  
Salman Khan ◽  
Syed Asad Ali Shah ◽  
Syed Muhammad Jamal
Keyword(s):  
Polymerase Chain Reaction ◽  
Viral Genome ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Kappa Value ◽  
Polymerase Chain ◽  
Pcr Assays ◽  
Level Of Agreement

<b><i>Background:</i></b> Foot-and-mouth disease (FMD) is an infectious and highly contagious disease of cloven-hoofed domestic and wild animals, causing heavy economic losses to the livestock industry. Rapid and reliable diagnosis of the disease is essential for the implementation of effective control measures. This study compared sandwich enzyme-linked immunosorbent assay (S-ELISA) and conventional reverse transcription polymerase chain reaction (RT-PCR) for the diagnosis of FMD. <b><i>Methods:</i></b> A total of 60 epithelial samples from suspected cases of FMD were tested using both S-ELISA and RT-PCR assays. The level of agreement between the assays was assessed by calculating the Kappa value. <b><i>Results:</i></b> S-ELISA detected 38 (63%) samples positive for FMD virus (FMDV). Being predominant, serotype O was detected in 22 (57.9%) of the total samples tested positive, whereas 9 (23.7%) and 7 (18.4%) samples were found positive for serotypes A and Asia-1, respectively. RT-PCR detected viral genome in 51 (85%) of the samples using pan-FMDV primers set, 1F/1R. Thirty-six samples were found positive and 7 negative by both the tests. The level of agreement between the tests was assessed by calculating the Kappa value, which was found to be fair (Kappa value = 0.303 and 95% CI = 0.089; 0.517) and significant (<i>p</i> = 0.009). However, 2 samples, which were found positive on S-ELISA tested negative on RT-PCR. This may be attributed to the presence of nucleotide mismatch(es) in the primer-binding sites that may have resulted in failure of amplification of the viral genome. The serotype-specific RT-PCR assays not only confirmed serotyping results of S-ELISA but were also able to establish serotype in 9 S-ELISA-negative but pan-FMDV RT-PCR-positive samples. <b><i>Conclusions:</i></b> The RT-PCR assay contributes significantly to establishing a quick, sensitive, and definitive diagnosis of FMD in resource-constrained countries. Samples giving negative results in S-ELISA should be tested in RT-PCR for the disease detection and virus typing.

scholarly journals Generation and diagnostic application of monoclonal antibodies against Seneca Valley virus

2011 ◽  
Vol 24 (1) ◽  
pp. 42-50 ◽  
Author(s):  
Ming Yang ◽  
Rebekah van Bruggen ◽  
Wanhong Xu
Keyword(s):  
Monoclonal Antibodies ◽  
Indirect Elisa ◽  
Foot And Mouth Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot ◽  
Specific Antibody Response ◽  
Dot Blot Assay ◽  
Infected Cells ◽  
Seneca Valley Virus ◽  
Vesicular Disease

Seneca Valley virus (SVV), a member of the Picornaviridae family, was implicated in a suspicious vesicular disease discovered in pigs from Canada in 2007. Because any outbreak of vesicular disease in pigs is assumed to be foot-and-mouth disease (FMD) until confirmed otherwise, a test for diagnosing the presence of SVV would be a very useful tool. To develop the diagnostic tests for SVV infection, 5 monoclonal antibodies (mAbs) were produced from mice immunized with binary ethylenimine (BEI)-inactivated SVV. Using a dot blot assay, the reactivity of the mAbs was confirmed to be specific for SVV, not reacting with any of the other vesicular disease viruses tested. The mAbs demonstrated reactivity with SVV antigen in infected cells by an immunohistochemistry assay. An SVV-specific competitive enzyme-linked immunosorbent assay (cELISA) was developed using BEI-inactivated SVV antigen and a mAb for serodiagnosis. The cELISA results were compared to the indirect isotype (immunoglobulin [Ig]M and IgG) ELISA and the virus neutralization test. All SVV experimentally inoculated pigs exhibited a positive SVV-specific antibody response at 6 days postinoculation, and the sera remained positive until the end of the experiment on day 57 (>40% inhibition) using the cELISA. The cELISA reflected the profile of the indirect ELISA for both IgM and IgG. This panel of SVV-specific mAbs is valuable for the identification of SVV antigen and the serological detection of SVV-specific antibodies.

scholarly journals Comparative analysis of antigen-specific anti-SARS-CoV-2 antibody isotypes in COVID-19 patients

2020 ◽  
Author(s):  
Hidetsugu Fujigaki ◽  
Masato Inaba ◽  
Michiko Osawa ◽  
Saya Moriyama ◽  
Yoshimasa Takahashi ◽  
...  
Keyword(s):  
Specific Antibody ◽  
Serological Test ◽  
Polymerase Chain Reaction Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serological Tests ◽  
S Protein ◽  
Neutralizing Activity ◽  
N Protein ◽  
Specificity And Sensitivity ◽  
Antibody Isotypes

AbstractSerological tests for detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in blood are expected to identify individuals who have acquired immunity against SARS-CoV-2 and indication of seroprevalence of SARS-CoV-2 infection. Many serological tests have been developed to detect antibodies against SARS-CoV-2. However, these tests have considerable variations in their specificity and sensitivity, and whether they can predict levels of neutralizing activity is yet to be determined. This study aimed to investigate the kinetics and neutralizing activity of various antigen-specific antibody isotypes against SARS-CoV-2 in serum of coronavirus disease 2019 (COVID-19) patients confirmed via polymerase chain reaction test. We developed IgG, IgM and IgA measurement assays for each antigen, including receptor-binding domain (RBD) of spike (S) protein, S1 domain, full length S protein, S trimer and nucleocapsid (N) domain, based on enzyme-linked immunosorbent assay. The assays of the S protein for all isotypes showed high specificity, while the assays for all isotypes against N protein showed lower specificity. The sensitivity of all antigen-specific antibody isotypes depended on the timing of the serum collection and all of them, except for IgM against N protein, reached more than 90% at 15-21 days post-symptom onset. The best correlation with virus neutralizing activity was found for IgG against RBD (RBD-IgG), and levels of RBD-IgG in sera from four severe COVID-19 patients increased concordantly with neutralizing activity. Our results provide valuable information regarding the selection of serological test for seroprevalence and vaccine evaluation studies.

scholarly journals Development and Evaluation of Quantitative Immunoglobulin G Enzyme-Linked Immunosorbent Assay for the Diagnosis of Coronavirus Disease 2019 Using Truncated Recombinant Nucleocapsid Protein as Assay Antigen

2021 ◽  
Vol 18 (18) ◽  
pp. 9630
Author(s):  
Pierre Nsele Mutantu ◽  
Mya Myat Ngwe Tun ◽  
Takeshi Nabeshima ◽  
Fuxun Yu ◽  
Patrick Kakoni Mukadi ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Expression System ◽  
Negative Control ◽  
Spike Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
N Protein ◽  
E Coli ◽  
System A ◽  
Recombinant Nucleocapsid Protein

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the assay’s performance, with the plaque reduction neutralization test and the commercial SARS-CoV-2 spike protein IgG ELISA as gold standards. The SARS-CoV-2 N truncated protein-based ELISA showed similar sensitivity (91.1% vs. 91.9%) and specificity (93.8% vs. 93.8%) between the PRNT and spike IgG ELISA, as well as also higher specificity compared to the full-length N protein (93.8% vs. 89.9%). Our ELISA can be used for the diagnosis and surveillance of COVID-19.

scholarly journals Natural Infection of Cichorium intybus (Asteraceae) by Groundnut Ringspot Virus (Genus Orthotospovirus) Isolates in Brazil

Plant Disease ◽  
2021 ◽  
Author(s):  
Tiago Silva Jorge ◽  
Maria Geane Fontes ◽  
Mirtes Freitas Lima ◽  
Leonardo Silva Boiteux ◽  
Maria Esther N. Fonseca ◽  
...  
Keyword(s):  
Natural Infection ◽  
Cichorium Intybus ◽  
Ringspot Virus ◽  
N Gene ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
N Protein ◽  
Individual Leaf ◽  
Leaf Deformation ◽  
Pcr Assays

Leaf chicory (Cichorium intybus L.) is a nutritionally rich vegetable used in regional cuisine in Brazil. Plants of C. intybus displaying symptoms (viz. chlorotic and necrotic ringspots, mosaic, and leaf deformation) similar to that induced by orthotospoviruses (genus Orthotospovirus, family Tospoviridae) were observed in three fields (≈ 0.2 ha each) in Gama County, in the Federal District, Brazil, from September 2016 to January 2020 in plants of the cultivars ‘Folha-Larga’ and ‘Spadona’ (Fig. 1). Incidence of symptomatic plants was nearly 10% in each field. Transmission electron microscopic examination of thin sections from symptomatic leaf samples showed typical membrane-bounded orthotospovirus particles within cisternae of spongy parenchymal cells (Fig 2). Two individual leaf samples per field were collected and submitted to dot enzyme-linked immunosorbent assay with polyclonal antisera against N protein of tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato chlorotic spot virus (TCSV). Symptomatic samples strongly reacted only against GRSV antibodies. Total RNA was extracted (Trizol®, Sigma) from all six samples and used as template in RT-PCR assays. The primer J13 (5’-CCCGGATCCAGAGCAAT-3’) was employed for cDNA synthesis using M-MLV reverse transcriptase. PCR assays were done with the primer pair BR60/BR65 (Eiras et al., 2001) to obtain ≈ 500 bp fragment of untranslated region and partial N gene in the S RNA segment from each sample. Purified RT-PCR products of two randomly selected individual samples were directly sequenced (GenBank MW467981 and MZ126602) and their BLASTn analyses displayed 99 to 100% nucleotide identity to GRSV isolates previously reported infecting C. endivia L. in Brazil (Jorge et al., 2021). Our analyses combining N protein serology and N-gene sequencing (both directed to the S RNA segment) allowed us to confirm the GRSV infection of C. intybus, but the potential reassortant nature of these isolates (Webster et al., 2015; Silva et al., 2019) are unknown since their M RNA segments were not characterized. Individual leaf extracts (in phosphate buffer, pH 7.0) of the sequenced isolates were mechanically inoculated onto ten seedlings of two C. intybus cultivars (‘Folha Larga’ and ‘Pão-de-Açúcar’) and three plants each of the indicator hosts Capsicum chinense PI 159236, Nicandra physalodes; Nicotiana rustica; Datura stramonium; and tomato cv. Santa Clara. Systemic chlorotic and necrotic ringspots, mosaic, and leaf deformation developed in the indicator hosts and infection by GRSV was confirmed via serological assays 20 days after inoculation. However, no symptoms and no serological reaction to GRSV antibodies were observed on the C. intybus cultivars even after two successive mechanical inoculations. This transmission failure might be due to factors such as the requirement of the thrips vector(s), physicochemical barriers in the foliage or the presence of non-mechanically transmissible helper agent(s) necessary to ensure GRSV infection of C. intybus. The natural infection of C. intybus by a not fully characterized orthotospovirus (mostly likely TSWV) has been observed since 1938 in Brazil (Kitajima, 2020). Our report of GRSV infecting C. intybus is thus confirming previous speculations that similar symptoms in this vegetable crop were induced by orthotospovirus infection in Brazil. References: Eiras, M. et al. 2001. Fitopatol. Bras. 26: 170. Jorge, T. S. et al. 2021. Plant Dis. 105: 714. Kitajima, E.W. 2020. Biota Neotrop. 20: e2019932. Silva, J. M. F. et al. 2019. Viruses 11: 187. Webster, C.G. et al. 2015. Phytopathology 105: 388.

A Double Antibody Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Salmonella Using Biotinylated Monoclonal Antibodies

2001 ◽  
Vol 64 (8) ◽  
pp. 1166-1171 ◽  
Author(s):  
ALFONSO VALDIVIESO-GARCIA ◽  
EDWARD RICHE ◽  
OMAR ABUBAKAR ◽  
THOMAS E. WADDELL ◽  
BRIAN W. BROOKS
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Immunosorbent Assay ◽  
Control Point ◽  
Brain Heart Infusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Culture Methods ◽  
Critical Control Point ◽  
Double Antibody ◽  
Specificity And Sensitivity

A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed using monoclonal antibodies (MAbs) as a rapid, economical alternative to culture isolation procedures for detection of Salmonella. Four MAbs previously shown to react with Salmonella strains representing 18 different serogroups were evaluated as capture antibodies and, after biotinylation, as detection antibodies. One MAb (M183) was selected for use in the ELISA to capture and detect Salmonella antigens. The detection limit of the ELISA was evaluated using Salmonella enterica subspecies enterica serovar Typhimurium and various selective and nonselective Salmonella enrichment media. The highest detection limit (ca. 104 CFU/ml) was achieved using an enrichment broth containing brain heart infusion, yeast extract, sodium hydrogen selenite, and sodium cholate (BYSC) after preenrichment in buffered peptone water. The ELISA detected all Salmonella serovars tested, which included representative serovars of serogroups B, C, D, and E and gave negative results for all non-Salmonella species tested. Samples (106) from various sources, including fecal samples from humans and pigeons, chicken carcass rinses, chicken parts, feed, and the environment, were used to evaluate the performance of the ELISA. The ELISA had a specificity and sensitivity of 100 and 91%, respectively, and a kappa value of 0.93 relative to the culture methods. Such an ELISA has the potential to be used in the implementation of the pathogen reduction and hazard analysis critical control point systems as well as in clinical laboratories.

Screening of durum wheats for pasta-making quality with monoclonal antibodies for gliadin 45

Genome ◽  
1989 ◽  
Vol 32 (6) ◽  
pp. 1096-1099 ◽  
Author(s):  
N. K. Howes ◽  
M. I. Kovacs ◽  
D. Leisle ◽  
M. R. Dawood ◽  
W. Bushuk
Keyword(s):  
Monoclonal Antibodies ◽  
Durum Wheat ◽  
Immunosorbent Assay ◽  
Sodium Dodecyl ◽  
Wheat Breeding ◽  
Rapid Screening ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gluten Strength ◽  
High Sodium ◽  
Sedimentation Volume

Monoclonal antibodies specific for gliadin band 45 (gli 45) of common wheat cultivar 'Marquis' were treated against durum wheat gliadins using an enzyme-linked immunosorbent assay (ELISA). In a test of 15 durum cultivars, high ELISA values were associated with gli 45, high gluten strength, and high sodium dodecyl sulfate (SDS) sedimentation volume; low ELISA values were associated with gli 42, low gluten strength, and low SDS-sedimentation volume. Analysis of 120 F5 lines from the backcrosses Ward/Vic//Vic and Ward/Vic//Ward confirmed that a high reaction to ELISA was statistically correlated with the presence of gli 45, with high gluten strength, and with high SDS-sedimentation volume. It was concluded that monoclonal antibodies specific for gli 45 have potential as a test for rapid screening of durum wheat breeding populations for desirable pasta-making quality.Key words: monoclonal antibodies, gliadin, enzyme-linked immunosorbent assay, durum wheat.

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