Optimization and validation of recombinant serological tests for African Swine Fever diagnosis based on detection of the p30 protein produced in Trichoplusia ni larvae

Abstract

We describe the validation of an enzyme-linked immunosorbent assay (ELISA) and confirmatory immunoblotting assays based on a recombinant p30 protein (p30r) produced in insect larvae using a baculovirus vector. Such validation included the following: (i) the scaling up and standardization of p30r production and the associated immunoassays, (ii) a broad immunological analysis using a large number of samples (a total of 672) from Spain and different African locations, and (iii) the detection of the ASF virus (ASFV)-antibody responses at different times after experimental infection. Yields of p30r reached up to 15% of the total protein recovered from the infected larvae at 3 days postinfection. Serological analysis of samples collected in Spain revealed that the p30r-based ELISA presented similar sensitivity to and higher specificity than the conventional Office International des Epizooties-approved ASFV ELISA. Moreover, the p30r ELISA was more sensitive than the conventional ELISA test in detecting ASFV-specific antibodies in experimentally infected animals at early times postinfection. Both the recombinant and conventional ELISAs presented variable rates of sensitivity and specificity with African samples, apparently related to their geographical origin. Comparative analyses performed on the sequences, predicted structures, and antigenicities of p30 proteins from different Spanish and African isolates suggested that variability among isolates might correlate with changes in antigenicity, thus affecting detection by the p30r ELISA. Our estimations indicate that more than 40,000 ELISA determinations and 2,000 confirmatory immunoblotting tests can be performed with the p30r protein obtained from a single infected larva, making this a feasible and inexpensive strategy for production of serological tests with application in developing countries.

FIG. 1.

Production of the recombinant p30 produced in T. ni larvae. Fourth-instar larvae were inoculated with different doses of recombinant p30BAC, and infections were allowed to progress for 48, 72, or 96 h postinfection. Total protein extracts from each group were analyzed by (a) capillary electrophoresis: gray bars represent the percentage of the p30r protein produced out of the total extracted protein, and black bars indicate the mortality rate associated with each group; (b) Coomassie brilliant blue staining of SDS-page gels; and (c) Western blotting using a pool of swine sera from ASFV-seropositive animals as a probe. Arrows indicate the position of the recombinant p30.

FIG. 2.

Reactivity of porcine sera in ELISA assays based on insect-derived antigens. Samples from ASFV-seropositive (n = 165) or seronegative (n = 303) animals, previously characterized by the OIE-approved tests, were tested by ELISA using crude extracts from larvae infected with p30BAC [Ag (+)] or wtBAC [Ag(−)] as detector antigens. Bars represent the mean OD obtained for each group, and standard deviations are indicated in each case by transversal lines.

FIG. 3.

Detection of ASFV-seropositive and -seronegative samples using the p30r-based ELISA and Western blotting. (a) Ratio ODs obtained by ELISA from seropositive and seronegative samples. Values refer to the media of duplicate analyses. Cutoff values are shown as dotted lines on each panel, and assays were carried out as described in Methods. (b) Western blots performed with all the seropositive and seronegative samples that failed to be detected by p30r ELISA.

FIG. 4.

Detection of ASFV-specific antibodies from experimentally infected pigs using the p30r-based ELISA. (a) Pigs 1 and 2 were inoculated with the attenuated strain E75 CV1-4, and serum samples were taken at 10, 14, and 21 dpi and analyzed by the OIE-approved and p30r-based ELISAs. Pig 1 was sacrificed at 21 dpi, and pig 2 was reinoculated (arrow) with the E75L8 virulent strain; (b) ratio ODs obtained using the p30r-based ELISA at 30 dpi for six additional animals (no. 3 to no. 8) infected once with the attenuated E75 CV1-4 strain.

FIG. 5.

Analysis of ASFV African serum samples using the p30r-based ELISA. Samples from Eastern (Uganda) and Western (Nigeria) African countries, previously characterized by the OIE-approved immunoblotting, were analyzed by the p30r-based ELISA. Charts show ratio ODs obtained by p30r ELISA from seropositive and seronegative samples from these two countries. Values refer to the media of duplicate analyses. Cutoff values are shown as dotted lines on each panel, and assays were carried out as described in Materials and Methods.

FIG. 6.

Sequence analysis of p30 proteins from different ASFV isolates. (a) Alignment of p30 sequences obtained from different African locations, including the Spanish E70 isolate. The lower row represents the degree of identity between the Spanish and the different African isolates, and boxed fragments indicate the most variable positions within these sequences. Additional information, including the identity score regarding the E70 virus, country, and year of isolation and hosts from which they were obtained, is included at the end of each of the sequences. *, identical residues in all sequences; :, highly conserved column; ., weakly conserved column. (b) Correlation between antigenicity, structure, and variability in the p30 protein from different isolates. Charts display the variation of the antigenic index as a function of amino acid position for the Spanish E70 and African isolates: Malawi Lil 20/1 1983, Mkuzi 1979, and Kenya 1950. Horizontal black bars indicate regions of predicted intrinsic disorder, and hatched bars mark those fragments presenting greater variability among the isolates as determined in the sequence alignments.