Journal of Microbiology

Research Support, Non-U.S. Gov't

Development of a stringent ELISA protocol to evaluate anti-viral hemorrhagic septicemia virus-specific antibodies in olive flounder Paralichthys olivaceus with improved specificity: Hyoung Jun Kim , Jeong Su Park , Se Ryun Kwon; J. Microbiol. 2015;53(7):481-485. Published online June 27, 2015; DOI: https://doi.org/10.1007/s12275-015-5101-9

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Olive flounder were vaccinated with polyinosinic:polycytidylic acid [Poly (I:C)] to prevent viral hemorrhagic septicemia (VHS). Vaccine efficacy was verified by detection of anti- VHS virus (VHSV) antibodies using enzyme-linked immunosorbent assay (ELISA). In the study, ELISA absorbance values of the negative control group [Poly (I:C)-MEM10] were saturated when an ELISA protocol, that includes pretreatment of the fish sera with 5% skim milk, was used. However, the saturated OD values in the negative control did not correlate with a specific immune response against VHSV, because the group showed low survival rate (only 10%) following the VHSV challenge. Also, OD values of Poly (I:C)- VHSV group were high, and the group showed high survival rate (97.5%) against VHSV challenge test. It was suggested that the high OD values were possibly due to the presence of anti-fetal bovine serum (FBS) cross-reactivity. To compensate this, we subtracted the absorbance of infectious hematopoietic necrosis (IHNV)-Ag plates from those of the VHSV-Ag plates. However, the average value for the Poly (I:C)-VHSV group (0.167) was lower than expected even though high survival rate. We used an advanced ELISA system to pre-treat fish sera with 5% skim milk and two novirhabdoviruses as capture antigens as well as 50% FBS. The corrected absorbance values for pre-treated fish sera from the negative control Poly (I:C)-MEM10 and experimental Poly (I:C)-VHSV groups averaged 0.033 and 0.579, respectively. The specific VHSV antibody response of the vaccinated group was assessed using fish sera pretreated with skim milk and FBS and by calculating the corrected absorbance values from ELISA with two novirhabdovirus capture antigens.

Citations

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Development of an indirect ELISA for detection of the adaptive immune response of black carp (Mylopharyngodon piceus)
Tongtong Wang, Shanshan Jin, Ruoxuan Lv, Yuting Meng, Guozhong Li, Yuxing Han, Qiusheng Zhang
Journal of Immunological Methods.2023; 521: 113550. CrossRef
Detection of Salmonid IgM Specific to the Piscine Orthoreovirus Outer Capsid Spike Protein Sigma 1 Using Lipid-Modified Antigens in a Bead-Based Antibody Detection Assay
Lena Hammerlund Teige, Subramani Kumar, Grethe M. Johansen, Øystein Wessel, Niccolò Vendramin, Morten Lund, Espen Rimstad, Preben Boysen, Maria K. Dahle
Frontiers in Immunology.2019;[Epub] CrossRef
Production of a monoclonal antibody against of muskellunge (Esox masquinongy) IgM heavy chain and its use in development of an indirect ELISA for titrating circulating antibodies against VHSV-IVB
Mohamed Faisal, Isaac F. Standish, Mary Ann Vogelbein, Elena V. Millard, Stephen L. Kaattari
Fish & Shellfish Immunology.2019; 88: 464. CrossRef
Phylogenetic analysis and duplex RT-PCR detection of viral hemorrhagic septicemia virus in olive flounder (Paralichthys olivaceus) from Korea
Jee Youn Hwang, Seongdo Lee, Thanthrige Thiunuwan Priyathilaka, Hyerim Yang, Hyukjae Kwon, Mun Gyeong Kwon, Seong Don Hwang, Myoung-Jin Kim, Jehee Lee
Aquaculture.2018; 484: 242. CrossRef
Herpesvirus Infection Induces both Specific and Heterologous Antiviral Antibodies in Carp
Julio M. Coll
Frontiers in Immunology.2018;[Epub] CrossRef
Validation of a serum neutralization test for detection of antibodies specific to cyprinid herpesvirus 3 in infected common and koi carp (Cyprinus carpio)
J Cabon, L Louboutin, J Castric, S Bergmann, G Bovo, M Matras, O Haenen, N J Olesen, T Morin
Journal of Fish Diseases.2017; 40(5): 687. CrossRef
The amino-terminal domain of ORF149 of koi herpesvirus is preferentially targeted by IgM from carp populations surviving infection
F. Torrent, A. Villena, P. A. Lee, W. Fuchs, S. M. Bergmann, J. M. Coll
Archives of Virology.2016; 161(10): 2653. CrossRef

Validation Study

Development and Validation of a Recombinant Nucleocapsid Protein-Based ELISA for Detection of the Antibody to Porcine Reproductive and Respiratory Syndrome Virus: Jia-Qi Chu , Xu-Min Hu , Myung-Cheol Kim , Chang-Sik Park , Moo-Hyung Jun; J. Microbiol. 2009;47(5):582-588. Published online October 24, 2009; DOI: https://doi.org/10.1007/s12275-009-0033-x

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Abstract: Three indirect enzyme-linked immunosorbent assays (iELISA) based on the North American like (NA-like), European like (EU-like) and co-expressed NA- and EU-like recombinant nucleocapsid proteins (N-protein) of porcine reproductive and respiratory syndrome virus (PRRSV) were validated for the detection of the antibodies in porcine sera. A total of 422 serum samples from unvaccinated pigs were tested. The cut-off value was optimized by a two-graph receiver operating characteristics analysis at a 95% confidence level. This assay was validated with Western blot analysis and IDEXX HerdChek™ ELISA. Cross-reactivity results showed that iELISA was PRRSV-specific. Repeatability tests revealed that the coefficients of variation of positive sera within and between runs were less than 10%. The results indicate that iELISA is simpler to produce and perform, time-saving and suitable for large scale surveys of PRRSV infection at low cost, and is potentially useful to evaluate the efficiency of various vaccines against PRRSV.

Journal Article

Performance of the Immunoglobulin G Avidity and Enzyme Immunoassay IgG/IgM Screening Tests for Differentiation of the Clinical Spectrum of Toxoplasmosis: Mehmet Tanyuksel , Cakir Guney , Engin Araz , M.Ali Saracli , Levent Doganci; J. Microbiol. 2004;42(3):211-215.; DOI: https://doi.org/2087 [pii]

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Abstract: Toxoplasmosis has been well known as an important human infection to consider especially in pregnant women. Although many serologic methods are available, the diagnosis of toxoplasmosis can be extremely difficult. The presence of increased levels of Toxoplasma-specific IgG antibodies indicates an infection, but it does not differentiate between a recent and past infection. The purpose of our study was to compare the performance of the ELISA T. gondii IgG/IgM test, a widely used enzyme-linked immunosorbent assay, to the ELISA IgG avidity method. One hundred and four serum samples (from 38 males and 66 females) were tested and evaluated from symptomatic patients (chorioretinitis, lymphadenopathy), and from women in their first trimester of pregnancy who were suspected of having toxoplasmosis. The high IgG avidity and ELISA IgG antibody levels were in agreement for 51 of the specimens (49.0%). Thirty-eight discrepant (borderline) results from the IgG avidity method were positive for IgM (3 specimens) and IgG (37 specimens). Interestingly, out of the eight serum samples that were positive for both IgG and IgM antibodies, two samples were low IgG avidity, and three samples were borderline. There was no statistically significant relation observed between the results of the IgG avidity method and the ELISA IgG test, and the IgG avidity method and ELISA IgM test (c^2=1.987; p=0.370 and c^2=2.152; p=0.341, respectively). The IgG avidity method was considered easy to perform and an acceptable approach for the differentiation of discrepant results (recent/chronic) and for the current detection of T. gondii antibodies. We concluded that the determination of IgG avidity is a helpful tool for the diagnosis of the ocular form of toxoplasmosis and it is a safe method for screening this disease in the first trimester of pregnancy.

Lipoppolysaccharide yields form Rhodobacter capasulatus with indirect ELISA: Yoo, Tae Eun , Lee, Hyun Soon; J. Microbiol. 1996;34(3):255-262.

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Abstract: The lipopolysaccharide (LPS) yields were measured in Rhodobacter capsulatus under several conditions by the ELISA method. The purification of LPS was done by affinity chromatography of IgG coupled CNBr-activated sepharose-4B instead of ultra-centrifugation. The purity of the LPS didn't show much difference between affinity chromatography and ultra-centrifugation method, but affinity chromatography method required much fewer organisms and was more convenient. LPS yield was measured in ng units by the ELISA method. Mannitol was a better single carbon source than other sugars, but mixing two carbon sources resulted in greater LPS yields than any sugar alone. LPS yield was directly proportional to NH₄CI concentration, with optimum yields at 0.05% nitrogen. In contrest to LPS yields, which decreased at 0.005% nitrogen concentration total protein was increased 16 times. Calcium influenced LPS yields. At 0.7 mM CaCI₂, the LPS yield was 16.5 ㎍/mg DW, five times the yield without calcium.

A detection method for vibrio vulnificus using monoclonal antibodies: Chung, Mi Sun , Rim, Bung Moo , Uhm, Tae Boong , Park, Moon Kook; J. Microbiol. 1997;35(2):87-91.

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Abstract: Monoclonal antibodies were prepared in order to an assay method for Vibrio vulnificus. Sixteen mouse ybridoma cell lines were established by immunization of whole cell antigen to BALB/c mice, fusion with SP2/O myeloma cells, and cloning. Most of them secreted IgM.lambda. antibodies. A sandwich enzyme-linked immunosorbent assay was developed with rabbit anti-V. vulnificus polyclonal antibodies as capture antibody, an IgM monoclonal antibody as detector antibody, and goat anti-mouse IgM-alkaline phosphatase conjugate as developer antibody. The range of detection was 10^4 to 10^7 V. vulnificus cells per microplate well. When four related Vibrio species were tested for cross-reactions, V. parahaemolyticus showed 3.5% reactively and V. carchariae, V. fluvialis, and V. furnisii showed negligibal (<1%) cross-reactivity.

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