Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona

Surujballi, Om; Elmgren, C.

doi:10.1016/s0378-1135(99)00177-7

Cited by 9 publications

(10 citation statements)

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“…Since MAbs reacted with different serovars from serogroup Sejroe, the absence of reaction with serovar sejroe could be either due to expression of a different protein or lack of gene expression. An attempt to Production of Mabs specific for different serovars of pathogenic Leptospira have been reported by other authors (10,11). Although these MAbs can be used in the development of more specific diagnostic assays, their use is limited to the diagnosis of cases in which that particular serovar is involved.…”

Section: Resultsmentioning

confidence: 99%

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Ludtke¹,

Coutinho²,

Jouglard³

et al. 2003

Braz. J. Microbiol.

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Two hybridomas secreting monoclonal antibodies (MAbs) that react with a lipoprotein (LipL32) of the outer membrane of pathogenic Leptospira were obtained. For hybridoma production, spleen cells from BALB/c mice imunized with recombinant LipL32 (rLipL32) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened in an indirect ELISA. One MAb produced was of the IgG2b isotype and the other was an IgM. MAbs specificity was confirmed by indirect ELISA and immunoblotting using purified rLipL32 and whole-cell antigen preparations from Escherichia coli (E. coli) expressing LipL32 and from pathogenic and nonpathogenic serovars. Both Mabs reacted with most of the pathogenic serovars tested and none reacted with non-pathogenic Leptospira. The MAbs described have potential for use in diagnostic tests for leptospirosis.

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Section: Resultsmentioning

confidence: 99%

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Ludtke¹,

Coutinho²,

Jouglard³

et al. 2003

Braz. J. Microbiol.

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“…The monoclonal antibody (M898) that has been incorporated into this ELISA is very specific for serovar pomona. This monoclonal antibody does not cross-react with any of 13 other pathogenic serovars, including those known to occur in North America (1,3,6,7,8,9,12,13,14,15,16,21,22) and thus the most likely cause of crossreaction in the local cattle population, nor does it cross-react with L. biflexa serovar patoc, an ubiquitous nonpathogen (20). The ELISA data were subjected to ROC curve analysis.…”

Section: Discussionmentioning

confidence: 99%

“…The monoclonal antibody (M898) was produced as described (20). The antigen was prepared from serovar pomona cells as described (20) and then sonicated for 2 min with a 375-W cell disruptor (Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.). The assay was performed as described (20) except for the following modifications.…”

Section: Methodsmentioning

confidence: 99%

“…The antigen was prepared from serovar pomona cells as described (20) and then sonicated for 2 min with a 375-W cell disruptor (Heat Systems-Ultrasonics Inc., Farmingdale, N.Y.). The assay was performed as described (20) except for the following modifications. Batches of microtiter plates were coated with the antigen, incubated overnight at room temperature, and then frozen at Ϫ20°C.…”

Section: Methodsmentioning

confidence: 99%

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Competitive Enzyme-Linked Immunosorbent Assay for Detection of Leptospira interrogans Serovar pomona Antibodies in Bovine Sera

Surujballi

Mallory

2001

Clin Diagn Lab Immunol

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A competitive enzyme-linked immunosorbent assay (ELISA) using a specific monoclonal antibody (M898) was developed for detection of bovine antibodies to Leptospira interrogans serovar pomona. This assay was evaluated using field sera (n ‫؍‬ 190) with serovar pomona microscopic agglutination test (MAT) titers of >100 as the positive population (group A); field sera (n ‫؍‬ 1,445) which were negative in the MAT (1:100 dilution) for serovar pomona (group B); and sera (from a specific-pathogen-free cattle herd [n ‫؍‬ 210]) which were negative in the MAT (1:100 dilution) for serovars canicola, copenhageni, grippotyphosa, hardjo, pomona, and sejroe (group C). At the cutoff point recommended by receiver operating characteristic (ROC) curve analysis of the combined ELISA results of serum groups A, B, and C, the sensitivity and specificity values were 93.7 and 96.3%, respectively. The value for the area under this ROC curve was 0.977, indicating a high level of accuracy for the ELISA. Similar results were obtained from the analysis of the combined results of serum groups A and B and from the analysis of the combined results of serum groups A and C.In the Canadian cattle population, leptospirosis is predominantly caused by serovar hardjo (now generally accepted as being Leptospira borgpetersenii serovar hardjo type hardjobovis) and serovar pomona (1,6,7,8,9,12,13,14,15). Other serovars such as grippotyphosa and icterohaemorrhagiae have also been detected but at relatively lower levels (6,7,13). Direct detection of these organisms by microscopic examination or culture is impractical due to the low success rate and the amount of time and labor required. Instead, leptospirosis is most often diagnosed serologically with the microscopic agglutination test (MAT) (2). The MAT however, despite its widespread usage and international recognition, is encumbered with a number of limitations. These include the need to use hazardous live bacteria and the amount of time and labor required to test each serum sample against multiple serovars of this organism. In addition, the lack of standard operating procedures and source strains among laboratories and the subjective scoring of results may cause quality assurance difficulties. Due to the drawbacks of the MAT we are developing alternative diagnostic tests for the detection of Leptospira serovars which are of economic importance to Canada.In a previous publication (20), we described two monoclonal antibodies (M897 and M898) that are suitable for incorporation into competitive enzyme-linked immunosorbent assays (ELISAs) for the specific detection of serum antibodies to serovar pomona. In this communication, we report the results of a validation study of a competitive ELISA that was developed with monoclonal antibody M898 for the detection of bovine serovar pomona antibodies. MATERIALS AND METHODSBacterial culture and MAT. The Leptospira organisms were cultured and the MAT was performed as previously described (20).Bovine sera. Field serum samples submitted to Canadian Food Inspection Agency ...

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“…Após atingir a densidade de 1 a 2 x 10 8 leptospiras/mL, os cultivos foram centrifugados a 13.000rpm durante 30 minutos, o pellet contendo as leptospiras (14 sorovares de L.interrogans: Australis, Autumnalis, Bataviae, Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Pomona, Pyrogenes, Tarassovi e Wolffi) foi diluído em PBS (NaCl 137mM, KCl 2,7mM, Na 2 HPO 4 12mM, K 2 HPO 4 1,2mM, pH 7,4) estéril, e comparada com o valor 0,5 da escala de Macfarland para que fosse atingida a densidade final aproximada de 1x10 8 leptospiras/mL, e em seguida as bactérias foram inativadas a 56°C por 16-18 horas (Surujballi & Elmgren 2000).…”

Section: Methodsunclassified

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp.

et al. 2013

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Objetivou-se verificar se galinhas imunizadas com uma solução de Leptospira interrogans inativadas e proteínas de membrana externa do sorovar Hardjo, poderiam produzir anticorpos policlonais específicos anti-leptospiras, detectáveis em testes ELISA. Foram imunizados oito galinhas com 25 semanas de idade, da raça White Leghorn, sendo três imunizadas com uma suspensão de leptospiras inativadas, três com uma solução de proteínas de membrana externa extraída do sorovar Hardjo e duas controle. Coletas de sangue foram realizadas quinzenalmente e de ovos diariamente. A IgY foi purificada a partir da gema dos ovos utilizando para a delipidação o método de diluição em água ácida e a precipitação com sulfato de amônio. Nos testes ELISA realizados para verificar a especificidade da IgY, foi demonstrada a produção de anticorpos anti-Leptospira, tanto no soro quanto nas gemas purificadas. O pico de produção de anticorpos específicos ocorreu na 5º semana após a primeira imunização. Ficou demonstrada a possibilidade da indução da produção de anticorpos específicos em galinhas imunizadas com leptospiras do sorovar Hardjo inativadas, bem como, com proteínas de membrana externa (PME) extraidas desse sorovar. As galinhas imunizadas com uma suspensão de leptospiras inativadas ou com PME de Leptospira interrogans do sorovar Hardjo produziram anticorpos reativos a PME Hardjo detectáves por teste ELISA.

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Monoclonal antibodies suitable for incorporation into a competitive enzyme-linked immunosorbent assay (ELISA) for detection of specific antibodies to Leptospira interrogans serovar pomona

Cited by 9 publications

References 14 publications

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Monoclonal antibodies against an outer membrane protein from pathogenic Leptospira

Competitive Enzyme-Linked Immunosorbent Assay for Detection of Leptospira interrogans Serovar pomona Antibodies in Bovine Sera

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp.

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