Development and Evaluation of a Monoclonal-Antibody-Based Enzyme-Linked Immunosorbent Assay for the Diagnosis of<i>Renibacterium salmoninarum</i>Infection

Hsu, Hui‐Min; Bowser, Paul R.; Schachte, John H.

doi:10.1577/1548-8667(1991)003<0168:daeoam>2.3.co;2

Cited by 28 publications

(11 citation statements)

References 13 publications

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“…However, other investigators have shown a similar lack of agreement of FAT results among laboratories (Armstrong et al 1989b), and FAT results also disagreed with ELISA results , Hsu et al 1991, Pascho et al 1991 and with results of other detection methods more sensitive than the FAT, but less so than the ELISA (Lee & Gordon 1987, Griffiths et al 1991, when 50 to 100 fields were examined. Consequently, examination of more than 30 fields by the FAT did not appear worthwhile in this study.…”

Section: Discussionmentioning

confidence: 88%

“…Due to the ease of application, the most common method has been examination of kidney smears by the fluorescent antibody test (FAT) which replaced the less sensitive Gram stain (Bullock & Stuckey 1975, Bullock et al 1980). More recently, the enzyme-linked immunosorbent assay (ELISA) has been shown to be the most sensitive, accurate, quantitative, and easily automated test for large-scale detection of Rs soluble antigens in salmonids , Turaga et al 1987, Sakai et al 1989, Hsu et al 1991, Pascho et al 1991.…”

Section: Introductionmentioning

confidence: 99%

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Comparison of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for measuring the prevalences and levels of Renibacterium salmoninarum in wild and hatchery stocks of salmonid fishes in Alaska. USA

Meyers¹,

Short²,

Farrington³

et al. 1993

Dis. Aquat. Org.

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ABSTRACT. The enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) were compared for their sensitivity in detection of Renibacterjum salmoninarum (Rs) in kidney tissues of Alaskan salmonids. The ELISA appeared to be more sensitive in detecting Rs infections. The FAT did not detect Rs in 80 % of the ELISA-positive samples but was positive for Rs in 28 'K, of the samples that were ELISA-negative. This contradiction may have been due to low-level washover of Rs cells from smears containing large numbers of Rs cells when slldes containing nlultiple samples were rinsed in d common vessel during the FAT procedures. The FAT routinely did not detect infections in Rs-positive fish the tissues of which produced a mean ELISA optical density value 50.173, and inconsistently detected infections in fish with ELISA values > 0.173 but < 0.978. The 0.978 optical density was the mean ELISA value at which the FAT routinely detected Rs-positive fish. Based on the ELlSA results, Rs occurred in only 9 % of the Alaskan Pacific salmon tested in both wild (85 "/U) and hatchery (81 %) stocks. The very high stock prevalences and levels of Rs antigen detected in wild trout Oncorhynch~~s spp., char Salvelinus spp., and grayling Thymallus arcticus having no clinical signs of bacterial kidney disease suggest these species may be somewhat resistant hosts and important freshwater reservoirs of Rs.

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Section: Discussionmentioning

confidence: 88%

Section: Introductionmentioning

confidence: 99%

Comparison of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for measuring the prevalences and levels of Renibacterium salmoninarum in wild and hatchery stocks of salmonid fishes in Alaska. USA

Meyers¹,

Short²,

Farrington³

et al. 1993

Dis. Aquat. Org.

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“…Similarly, false-positive R. salmoninarum reactions have been reported with the ELISA (Dixon 1985, Turaga et al 1987. In attempts to avoid false-positive reactions, some workers have used monoclonal antibodies directed against selected epitopes of p57 (Wiens & Kaattari 1989, Hsu et al 1991, Rockey et al 1991. Others have used Western blot analysis to make the diagnosis less equivocal (Olivier et al 1992).…”

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confidence: 99%

Bacterial species other than Renibacterium salmoninarum cross-react with antisera against R. salmoninarum but are negative for the p57 gene of R. salmoninarum as detected by the polymerase chain reaction (PCR)

Brown¹,

Evelyn²,

Iwama³

et al. 1995

Dis. Aquat. Org.

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NOTEBacterial species other than Renibacterium salmoninarum cross-react with antisera against R. salmoninarum but are negative for the p57 gene of R. salmoninarum as detected by the polymerase chain reaction (PCR) ABSTRACT: Genomc DNA was extracted from 4 strains of Carnobacterium p~scicola and 2 strains of Corynebacterium aquaticum that had previously been reported to produce a 57 kDa protein that reacted with polyclonal antiserum against Renibacterium salmoninarum. Genomic DNA was also extracted from a Gram-negative bacterium isolated from the ludney tissue of a mature female coho salmon Oncorhynchus kisutch. The bacterium, tentatively identified as Pseudomonas maltophila, cross-reacts with 2 polyclonal antisera, one of which is used in an enzyme-linked immunosorbent assay and the other in a fluorescent antibody test to identlfy R. sal~noninarum The Isolate of P. maltophila, and the Carnobacterium piscicola and Corynebacterium aquahcum strains, were negative by a polymerase chain reaction (PCR) that was designed to amplify a segment of the gene encoding p57, a major protein of R. salmon~narum. These results suggest that although antibodies directed against R. salmoninarum crossreact with antigens of bacterial species other than R. salmoninarum, the cross-reacting antigen(s) is clearly not the same protein, as the non-R. salmoninarum bacteria lacked the gene encoding p57. These findings highlight some of the shortcomings of immunodiagnostic tests for detecting R. salmoninarum and indicate the high degree of specificity associated with a PCR-based diagnostic technique.

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“…For example, localized infections can occur in the head and skin tissues, which are sites not usually assayed in screening programs (29). Several ELISAs have been described utilizing either polyclonal antibodies (21,24,27,30), monoclonal antibodies (33), or a combination of both types of antibodies (23). Assays relying on polyclonal antisera are limited by reagent availability, lot consistency, and cross-reactivity (29).…”

Section: Discussionmentioning

confidence: 99%

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

Wiens

Owen

2005

Appl Environ Microbiol

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Renibacterium salmoninarum is a gram-positive bacterium that causes bacterial kidney disease in salmonid fish. The virulence mechanisms of R. salmoninarum are not well understood. Production of a 57-kDa protein (p57) has been associated with isolate virulence and is a diagnostic marker for R. salmoninarum infection. Biological activities of p57 include binding to eukaryotic cells and immunosuppression. We previously isolated three monoclonal antibodies (4D3, 4C11, and 4H8) that neutralize p57 activity. These monoclonal antibodies (MAbs) bind to the amino-terminal region of p57 between amino acids 32 though 243; however, the precise locations of the neutralizing epitopes were not determined. Here, we use transposon mutagenesis to map the 4D3, 4C11, and 4H8 epitopes. Forty-five transposon mutants were generated and overexpressed in Escherichia coli BL21(DE3). The ability of MAbs 4D3, 4H8, and 4C11 to bind each mutant protein was assessed by immunoblotting. Transposons inserting between amino acids 51 and 112 disrupted the 4H8 epitope. Insertions between residues 78 and 210 disrupted the 4C11 epitope, while insertions between amino acids 158 and 234 disrupted the 4D3 epitope. The three MAbs failed to bind overlapping, 15-mer peptides spanning these regions, suggesting that the epitopes are discontinuous in conformation. We conclude that recognition of secondary structure on the amino terminus of p57 is important for neutralization. The epitope mapping studies suggest directions for improvement of MAb-based immunoassays for detection of R. salmoninarum-infected fish.

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Development and Evaluation of a Monoclonal-Antibody-Based Enzyme-Linked Immunosorbent Assay for the Diagnosis ofRenibacterium salmoninarumInfection

Cited by 28 publications

References 13 publications

Comparison of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for measuring the prevalences and levels of Renibacterium salmoninarum in wild and hatchery stocks of salmonid fishes in Alaska. USA

Comparison of the enzyme-linked immunosorbent assay (ELISA) and the fluorescent antibody test (FAT) for measuring the prevalences and levels of Renibacterium salmoninarum in wild and hatchery stocks of salmonid fishes in Alaska. USA

Bacterial species other than Renibacterium salmoninarum cross-react with antisera against R. salmoninarum but are negative for the p57 gene of R. salmoninarum as detected by the polymerase chain reaction (PCR)

Mapping of Neutralizing Epitopes on Renibacterium salmoninarum p57 by Use of Transposon Mutagenesis and Synthetic Peptides

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