Novel Purification Method for Recombinant 3AB1 Nonstructural Protein of Foot-and-Mouth Disease Virus for Use in Differentiation between Infected and Vaccinated Animals

Abstract

An indirect enzyme-linked immunosorbent assay (ELISA) was developed for differentiation of animals infected with foot-and-mouth disease virus (FMDV) from vaccinated animals. The test was based on a highly pure and concentrated preparation of recombinant 3AB1 protein obtained by expression in a prokaryotic system, protein separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and electro elution. Experimental- and field-serum samples from naive, vaccinated, and infected cattle were tested for anti3AB1 antibody using the ELISA. A cutoff level was set at 35% of the maximum absorbance obtained with a positive control serum (FMDV-infected animal, 21 days postinfection [dpi]). This assay could detect antibodies from sera of animals experimentally infected by contact (n = 118) with a sensitivity of 97.5%. The specificity was 100%, based on negative test results obtained on 109 sera from naive animals. Remarkably, all sera from animals vaccinated either once (n = 102) or twice (n = 30) were negative. In addition, this 3AB1-ELISA could detect seroconversion at 7 dpi in animals inoculated intradermolingually. This assay constitutes an important tool for the rapid detection of FMDV outbreaks in a vaccinated population. In addition, it presents a reliable, economical, and simple method for testing large numbers of serum samples.

Keywords

3AB1-ELISA foot-and-mouth disease nonstructural protein recombinant protein purification

References

Bergmann

Malirat

Neitzert

: 2000, Improvement of serodiagnostic strategy for foot and mouth disease virus surveillance in cattle under systematic vaccination: enzyme linked immunoelectrotransfer blot assay. Arch Virol 145:473–489.

De Diego

Brocchi

Mackay

: 1997, The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle. Arch Virol 142:2021–2033.

Lubroth

Lopez

Ramalho

: 1998, Cattle response to foot and mouth disease virus non-structural proteins as antigens within vaccines produced using different concentrations. Vet Q 20(suppl. 2):s13–s17.

Mackay

Forsyth

Davies

: 1998, Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA. Vaccine 16(5):446–459.

Malirat

Neitzert

Bergmann

: 1998, Detection of cattle exposed to foot and mouth disease virus by means of an indirect ELISA test using bioengineered non-structural polyprotein 3ABC. Vet Q 20(suppl. 2):s24–s26.

Mezencio

JMS

Babcock

Meyer

: 1998, Differentiating foot and mouth disease virus-infected from vaccinated animals with baculovirus-expressed specific proteins. Vet Q 20(suppl. 2):s11–s13.

Rodriguez

Dopazo

Saiz

Sobrino

: 1994, Immunogenicity of non-structural proteins of foot-and-mouth disease virus: differences between infected and vaccinated swine. Arch Virol 136:123–131.

Sambrook

Fritsch

Maniatis

: 1989, Molecular Cloning. In: A laboratory manual, 2nd ed., pp. 18. 60–18.75. Cold Spring Harbor Laboratory Press, New York.

Shen

Chen

Walfield

: 1999, Differentiation of convalescent animals from those vaccinated against foot-and-mouth disease by a peptide ELISA. Vaccine 17:3039–3049.

10.

Silberstein

Kaplan

Taboga

: 1997, Foot-and-mouth disease virus-infected but not vaccinated cattle develop antibodies against recombinant 3AB1 non-structural protein. Arch Virol 142:795–805.

11.

Smith

Corcoran

: 1990 In: Current protocols in molecular biology, ed. Ausubel

, pp. 16 17. John Wiley and Sons Inc. New York, NY.

12.

Sorensen

Madsen

: 1998, Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus. Arch Virol 143:1461–1476.