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Development of a blocking ELISA based on a monoclonal antibody against a predominant epitope in non-structural protein 3B2 of foot-and-mouth disease virus for differentiating infected from vaccinated animals.

Fu Y, Lu Z, Li P, Cao Y, Sun P, Tian M, Wang N, Bao H, Bai X, Li D, Chen Y, Liu Z - PLoS ONE (2014)

Bottom Line: The parameters for this SPB-ELISA were established by screening panels of sera of different origins.Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal.This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Science, Lanzhou, Gansu, China.

ABSTRACT
A monoclonal antibody (McAb) against non-structural protein (NSP) 3B of foot-mouth-disease virus (FMDV) (3B4B1) was generated and shown to recognize a conserved epitope spanning amino acids 24-32 of 3B (GPYAGPMER) by peptide screening ELISA. This epitope was further shown to be a unique and predominant B cell epitope in 3B2, as sera from animals infected with different serotypes of FMDV blocked the ability of McAb 3B4B1 to bind to NSP 2C3AB. Also, a polyclonal antibody against NSP 2C was produced in a rabbit vaccinated with 2C epitope regions expressed in E. coli. Using McAb 3B4B1 and the 2C polyclonal antibody, a solid-phase blocking ELISA (SPB-ELISA) was developed for the detection of antibodies against NSP 2C3AB to distinguish FMDV-infected from vaccinated animals (DIVA test). The parameters for this SPB-ELISA were established by screening panels of sera of different origins. Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal. This test showed a similar performance as the commercially available PrioCHECK NS ELISA. This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.

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Characteristics of the monoclonal antibodies.(A) The reactivity of the McAbs with various recombinant non-structural proteins. (B) Identification of the epitopes recognized by the McAbs against NSP 3B by peptide ELISA. (C) The N/P values of the McAbs at different dilutions of serum. (D) Conservation of the motifs in FMDV NSP 3B.
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pone-0111737-g001: Characteristics of the monoclonal antibodies.(A) The reactivity of the McAbs with various recombinant non-structural proteins. (B) Identification of the epitopes recognized by the McAbs against NSP 3B by peptide ELISA. (C) The N/P values of the McAbs at different dilutions of serum. (D) Conservation of the motifs in FMDV NSP 3B.

Mentions: Three McAbs, 3B4B1, 3B4E11, and 3B10A10, were screened. McAbs 3B4B1 and 3B4E11 were classified as IgG1, and 3B10A10 was classified as IgG2b using a commercially available isotype classification kit. All three McAbs specifically bind to recombinant proteins His-3B, His-3ABC, and His-2C3AB, but not His-3A or His-3D, as determined by an indirect ELISA (Fig. 1A), indicating that the three McAbs recognize epitopes located within protein 3B. A peptide screening ELISA showed that 3B4B1 and 3B4E11 only react with peptide 3, not with other peptides containing the previously reported QKPLK core motif [7], indicating that 3B4B1 and 3B4E11 specifically bind to the 24GPYAGPMER32 epitope. McAb 3B10A10 reacted with peptide 3 and with peptide 1 (Fig. 1B), indicating that it might recognize a space epitope.


Development of a blocking ELISA based on a monoclonal antibody against a predominant epitope in non-structural protein 3B2 of foot-and-mouth disease virus for differentiating infected from vaccinated animals.

Fu Y, Lu Z, Li P, Cao Y, Sun P, Tian M, Wang N, Bao H, Bai X, Li D, Chen Y, Liu Z - PLoS ONE (2014)

Characteristics of the monoclonal antibodies.(A) The reactivity of the McAbs with various recombinant non-structural proteins. (B) Identification of the epitopes recognized by the McAbs against NSP 3B by peptide ELISA. (C) The N/P values of the McAbs at different dilutions of serum. (D) Conservation of the motifs in FMDV NSP 3B.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219772&req=5

pone-0111737-g001: Characteristics of the monoclonal antibodies.(A) The reactivity of the McAbs with various recombinant non-structural proteins. (B) Identification of the epitopes recognized by the McAbs against NSP 3B by peptide ELISA. (C) The N/P values of the McAbs at different dilutions of serum. (D) Conservation of the motifs in FMDV NSP 3B.
Mentions: Three McAbs, 3B4B1, 3B4E11, and 3B10A10, were screened. McAbs 3B4B1 and 3B4E11 were classified as IgG1, and 3B10A10 was classified as IgG2b using a commercially available isotype classification kit. All three McAbs specifically bind to recombinant proteins His-3B, His-3ABC, and His-2C3AB, but not His-3A or His-3D, as determined by an indirect ELISA (Fig. 1A), indicating that the three McAbs recognize epitopes located within protein 3B. A peptide screening ELISA showed that 3B4B1 and 3B4E11 only react with peptide 3, not with other peptides containing the previously reported QKPLK core motif [7], indicating that 3B4B1 and 3B4E11 specifically bind to the 24GPYAGPMER32 epitope. McAb 3B10A10 reacted with peptide 3 and with peptide 1 (Fig. 1B), indicating that it might recognize a space epitope.

Bottom Line: The parameters for this SPB-ELISA were established by screening panels of sera of different origins.Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal.This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Veterinary Etiological Biology, National Foot-and-Mouth Disease Reference Laboratory, Lanzhou Veterinary Research Institute, Chinese Academy of Agriculture Science, Lanzhou, Gansu, China.

ABSTRACT
A monoclonal antibody (McAb) against non-structural protein (NSP) 3B of foot-mouth-disease virus (FMDV) (3B4B1) was generated and shown to recognize a conserved epitope spanning amino acids 24-32 of 3B (GPYAGPMER) by peptide screening ELISA. This epitope was further shown to be a unique and predominant B cell epitope in 3B2, as sera from animals infected with different serotypes of FMDV blocked the ability of McAb 3B4B1 to bind to NSP 2C3AB. Also, a polyclonal antibody against NSP 2C was produced in a rabbit vaccinated with 2C epitope regions expressed in E. coli. Using McAb 3B4B1 and the 2C polyclonal antibody, a solid-phase blocking ELISA (SPB-ELISA) was developed for the detection of antibodies against NSP 2C3AB to distinguish FMDV-infected from vaccinated animals (DIVA test). The parameters for this SPB-ELISA were established by screening panels of sera of different origins. Serum samples with a percent inhibition (PI) greater than or equal to 46% were considered to be from infected animals, and a PI lower than 46% was considered to indicate a non-infected animal. This test showed a similar performance as the commercially available PrioCHECK NS ELISA. This is the first description of the conserved and predominant GPYAGPMER epitope of 3B and also the first report of a DIVA test for FMDV NSP 3B based on a McAb against this epitope.

Show MeSH
Related in: MedlinePlus