Comparison of a Radioimmunoprecipitation Assay to Immunoblotting and ELISA for Detection of Antibody to African Swine Fever Virus

Alcaraz, C.; Diego, M. De; Pastor, M J; Escribano, J. M.

doi:10.1177/104063879000200307

Cited by 59 publications

(43 citation statements)

References 10 publications

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“…The main consequence of this fact is the increase of false-positive results, making the confirmation of the diagnostic results by IB essential (2,6,20). In order to evaluate the ability of the new ELISAs to analyze properly samples of poor quality, 253 field sera were tested, before and after incubation for 1 month at 37°C, using the prescribed test and recombinant assays.…”

Section: Discussionmentioning

confidence: 99%

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Antigenic Properties and Diagnostic Potential of African Swine Fever Virus Protein pp62 Expressed in Insect Cells

Gallardo¹,

Blanco²,

Rodrı́guez

et al. 2006

J Clin Microbiol

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African swine fever (ASF) is an infectious and economically important disease of domestic pigs. The absence of vaccine renders the diagnostic test the only tool that can be used for the control of new outbreaks of the disease. At present, the enzyme-linked immunosorbent assay (ELISA) test is the most useful method for large-scale ASF serological studies, although false positives have been detected, mainly on poorly preserved sera. In order to improve the current diagnostic test available for ASF, we have studied the antigenic properties of the ASF virus polyprotein pp62 and its suitability for use in a novel ELISA. Two well-known antigenic proteins of ASF virus, p32 and p54, were also included in this study. These proteins were expressed in the baculovirus expression system and used as antigens in ASF serological tests. Our results indicate that the use of these recombinant proteins as antigens in the ELISAs improves the sensitivity and specificity obtained with the conventional diagnosis test used to detect antibodies against ASF virus. Furthermore, the use of polyprotein pp62 in an ELISA for testing poorly preserved sera allows performance of the diagnosis of ASF without the need to confirm the results by the immunoblot test. These features make pp62 one of the most interesting viral proteins to be used for serological ASF diagnosis.

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Section: Discussionmentioning

confidence: 99%

“…The results obtained by the analysis of sera from infected pigs were compared with the results obtained with the ELISA prescribed for international trade (18) and with the results of ELISAs using recombinant proteins (rP-ELISAs) p32 (p32-ELISA) and p54 (p54-ELISA) (p32 and p54 are two of the most antigenic ASFV proteins [2]). …”

mentioning

confidence: 99%

Antigenic Properties and Diagnostic Potential of African Swine Fever Virus Protein pp62 Expressed in Insect Cells

Gallardo¹,

Blanco²,

Rodrı́guez

et al. 2006

J Clin Microbiol

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“…Virus infection of MS cells was carried out with Dulbecco's modified Eagle's medium (DMEM) containing 2% swine serum. Virus infection of CV 1 and Vero cells was carried out with DMEM containing 2% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

“…These OIE-approved tests are based on the use of live virus as the antigen and involve the requirement of level 3 biosafety facilities for the production and handling of the pathogen (16,20,21). The risk associated with the handling of live virus, together with the lack of reliability of the OIE-ELISA for the analysis of poorly preserved samples so often encountered in sera of African origin (1,3), provides the stimulus for the development of alternative and more robust systems for the detection of anti-ASFV antibodies. Indeed, previous studies have demonstrated that recombinant viral proteins can give improved specificity and sensitivity when they are applied to the analysis of European field sera (9,19,22).…”

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confidence: 99%

Recombinant Antigen Targets for Serodiagnosis of African Swine Fever

Gallardo

Reis

Kalema‐Zikusoka

et al. 2009

Clin Vaccine Immunol

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African swine fever (ASF) is an infectious and economically important disease of domestic pigs. There is no vaccine, and so reliable diagnosis is essential for control strategies. The performance of four recombinant ASF virus (ASFV) protein (pK205R, pB602L, p104R, and p54)-based enzyme-linked immunosorbent assays (ELISAs) was evaluated with European porcine field sera that had been established by Office International des Epizooties (OIE)-approved tests to be ASFV negative (n ‫؍‬ 119) and ASFV positive (n ‫؍‬ 80). The values showed that there was almost perfect agreement between the results of the "gold standard" test (immunoblotting) and the results obtained by the p54-specific ELISA ( ‫؍‬ 0.95; 95% confidence interval [CI], 0.90 to 0.99) and the pK205R-specific ELISA or the pB602L-specific ELISA ( ‫؍‬ 0.92; 95% CI, 0.86 to 0.97). For the pA104R-specific ELISA, there was substantial to almost perfect agreement ( ‫؍‬ 0.81; 95% CI, 0.72 to 0.89). Similar results were observed by the OIE-approved ELISA ( ‫؍‬ 0.89; 95% CI, 0.82 to 0.95). Importantly, antibodies against these proteins were detectable early after infection of domestic pigs. Preliminary testing of 9 positive and 17 negative serum samples from pigs from West Africa showed identical results by the recombinant protein-based ELISA and the OIE-approved tests. In contrast, there was a high degree of specificity but a surprisingly a low level of sensitivity with 7 positive and 342 negative serum samples from pigs from East Africa. With poorly preserved sera, only the p104R-specific ELISA showed a significant reduction in sensitivity compared to that of the OIE-approved ELISA. Finally, these recombinant proteins also detected antibodies in the sera of the majority of infected warthogs. Thus, recombinant ASFV proteins p54, pB602L, and pK205R provide sensitive and specific targets for the detection of antibodies in European and West African domestic pigs and warthogs.

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“…The radioimmunoprecipitation assay was performed as described (Alcaraz et al, 1990) except that Streptococcus Protein G (Calbiochem) was used instead of Staphylococcus Protein A to obtain binding to horse Igs.…”

Section: Methodsmentioning

confidence: 99%

Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting

Laviada¹,

Arias²,

Sánchez-Vizcaíno³

1993

Journal of General Virology

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The structural and non-structural proteins induced by African horsesickness virus serotype 4 (AHSV-4) in infected Vero cells were analysed by SDS-PAGE. Twenty-two virus-induced polypeptides were detected in infected cells by comparison with the polypeptides of mock-infected cells, of which four major (VP2, VP3, VP5 and VP7) and three minor (VP1, VP4 and VP6) structural proteins and four non-structural proteins (P58, P48, P21 and P20) were shown to be virus-coded, as deduced from electrophoretic and antigenic studies of purified virions and infected cells. The proteins that elicit the major antibody responses both in vaccinated and naturally or experimentally infected horses were shown to be three structural proteins, VP2, VP5 and VP7, and the four major non-structural proteins, P58, P48, P21 and P20, as deduced by radioimmunoprecipitation and immunoblotting assays. The crossreactivity between AHSV-4 and sera obtained from horses experimentally infected with seven other serotypes was also determined. The results showed that VP5, VP7, P48, P21 and P20 are conserved and can be used to diagnose the infection of any of these eight serotypes.

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Comparison of a Radioimmunoprecipitation Assay to Immunoblotting and ELISA for Detection of Antibody to African Swine Fever Virus

Cited by 59 publications

References 10 publications

Antigenic Properties and Diagnostic Potential of African Swine Fever Virus Protein pp62 Expressed in Insect Cells

Antigenic Properties and Diagnostic Potential of African Swine Fever Virus Protein pp62 Expressed in Insect Cells

Recombinant Antigen Targets for Serodiagnosis of African Swine Fever

Characterization of African horsesickness virus serotype 4-induced polypeptides in Vero cells and their reactivity in Western immunoblotting

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