Evaluation of an Enzyme-Linked Immunosorbent Assay with Recombinant Rhoptry-Associated Protein 1 Antigen against Babesia bovis for the Detection of Specific Antibodies in Cattle

Abstract

The gene encoding Babesia bovis rhoptry-associated protein 1 (RAP-1) was used to develop an enzyme-linked immunosorbent assay (ELISA) to measure specific antibodies against B. bovis. The B. bovis RAP-1 gene was subcloned into a baculovirus transfer vector, and the RAP-1 protein was expressed in insect cells infected with a recombinant baculovirus. The recombinant B. bovis RAP-1 of 65 kDa was detected with anti-RAP-1 mouse serum by Western blotting, and this recombinant RAP-1 was used as an antigen in the ELISA. The ELISA was able to differentiate between B. bovis-infected sera and B. bigemina-infected sera or noninfected normal bovine sera. The results demonstrate that the recombinant RAP-1 expressed in insect cells might be a useful antigen for the detection of antibodies to B. bovis.

Improved Enzyme-Linked Immunosorbent Assay Using C-Terminal Truncated Recombinant Antigens of Babesia bovis Rhoptry-Associated Protein-1 for Detection of Specific Antibodies

“…Each of the 3Ј-terminal RAP-1 gene fragments was subcloned into the BamHI and XhoI sites of a baculovirus transfer vector, pFastBac Ht (Life Technologies, Grand Island, N.Y.), and the vectors were designated pFB/RAP-1 CT1, pFB/RAP-1 CT2, and pFB/RAP-1 CT3. By using these transfer vectors, three kinds of recombinant viruses, AcRAP-1 CT1, AcRAP-1 CT2, and Ac-RAP-1 CT3, were produced, respectively, according to the previously described method (1).…”

“…B. bovis genomic DNA was isolated from the culture as described previously (1). Oligonucleotide primers were synthesized with restriction enzyme-compatible ends for the subsequent cloning to find a specific immunogenic region in C-terminal RAP-1 (Table 1).…”

“…Each of the amplified DNA samples was digested with the corresponding restriction enzymes, BamHI and XhoI, and then inserted into a pBluescript SK(ϩ) cloning vector. The PCR amplification and confirmative nucleotide sequencing of the target DNA fragments were performed as described previously (1).…”

“…Previously, we demonstrated that the entire rhoptry-associated protein-1 (RAP-1) of B. bovis synthesized by a baculovirus expression system could be used as a diagnostic enzyme-linked immunosorbent assay (ELISA) antigen for the detection of antibodies to B. bovis, but there were some cross-reactions with another pathogen, B. bigemina (1). The cross-reactions may be due to the high degree of sequence identity in the first 300 amino acids (aa) located at the N-terminal region between the RAP-1 derivates of both parasites (11,12).…”

Identification and Expression of Babesia ovis Secreted Antigen 1 and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay

“…ovis antibodies in some research (15)(16)(17). However, immunoreactive recombinant proteins are widely used in serological assays for the diagnosis of equine, bovine, and canine babesiosis (18)(19)(20)(21). There has been no report of the production of immunoreactive recombinant proteins of B. ovis.…”