Production of Recombinant Protein Pap31 and Its Application for the Diagnosis of Bartonella bacilliformis Infection

Abstract

Tropical bartonellosis is a highly fatal epidemic and endemic infectious disease that occurs throughout the communities of the Andes Mountains in South America. The disease is caused by the facultative intracellular bacteria, Bartonella bacilliformis. The emergence of bartonellosis in new geographic areas and an increase in the number of reported cases suggest the need for a rapid test for epidemiologic study and investigation of the disease burden. The objective of this research is to develop a rapid serologic diagnostic test using recombinant antigens to overcome the limitations of the current standard IFA technique for laboratory diagnosis. Western blot analysis with patient sera of whole cell lysate separated on a 2D gel identified Pap31 as a dominant antigen. PCR primers were designed according to the sequence of ATCC strain 35685 to amplify the gene coding for Pap31 from a local isolate (HOSP 800-09, Peru). The amplicon was subsequently cloned into pET24a, adding the T7 tag, and expressed in E. coli. Patient sera with different IFA titers confirmed the diagnostic band of 31 kDa on a Western blot of SDS-PAGE. The performance of affinity-purified recombinant Pap31 (rPap31) was also evaluated in an ELISA format with 137 patient sera of known IFA titers. The range of ELISA reading from positive sera did not overlap with the range of those from negative sera, suggesting the potential application of rPap31 in both ELISA for high throughput regional hospital settings and in the construction of handheld rapid tests for rural clinical sites.

An Evaluation Study of Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Protein Pap31 for Detection of Antibody against Bartonella bacilliformis Infection among the Peruvian Population

“…The recombinant Pap31 antigen was prepared as described previously. 15 The ELISA was carried out in 96-well ELISA microplates (Polystyrene; Microlon 200, U-bottom, Greiner Bio-one, Monroe, NC). The wells were coated with 0.3 μg/well of recombinant Pap31 antigen prepared in phosphate buffered saline (PBS) and bovine serum albumin (BSA) as the negative control for nonspecific binding of the serum components.…”

“…The protein was found to be highly expressed in cultures of B. bacilliformis and immunologically dominant; thus, it is a good candidate to be used in ELISA. 15 Furthermore, as a homologue of the bacteriophage-associated protein, it was recognized by the host's immune response during Bartonella henselae infections. 16 Recombinant Pap31 (rPap31) can be produced in bulk, is easily purified, and remains antigenic even after several freeze-thaw cycles.…”

“…16 Recombinant Pap31 (rPap31) can be produced in bulk, is easily purified, and remains antigenic even after several freeze-thaw cycles. During the initial assay development by Taye and others, 15 a total of 136 samples from 29 IFA positive and 107 IFA negative sera were tested by ELISA using the rPap31 antigen. The results showed that the 95% confidence interval (CI) of the optical density (OD) values for the IFA negative samples did not overlap with the 95% CI of the OD values for the IFA positive samples.…”

Peptide-Based Antibody Detection for Tuberculosis Diagnosis

“…The need for multiple visits to provide specimens and obtain results before treatment can be initiated also leads to high rates of dropout of infectious patients (30). A rapid and economical diagnostic test that can provide an accurate means for the identification of sputum smear-positive TB cases is a major diagnostic priority of TB control programs in countries where TB is endemic.Antibody detection-based diagnostic assays have been successfully devised for several infectious diseases (7,15,26,30), but efforts to develop a serodiagnostic test for TB have yielded disappointing results for several decades and no currently available commercial immunodiagnostic tests for TB provide high sensitivity and specificity (24). However, interest in developing these assays for the diagnosis of TB continues due to their adaptability to simple, rapid formats, such as the dipstick and lateral-flow cassette formats, which are extremely useful in the field, where a sophisticated laboratory infrastructure and highly trained personnel are not available (8,30).…”

“…Antibody detection-based diagnostic assays have been successfully devised for several infectious diseases (7,15,26,30), but efforts to develop a serodiagnostic test for TB have yielded disappointing results for several decades and no currently available commercial immunodiagnostic tests for TB provide high sensitivity and specificity (24). However, interest in developing these assays for the diagnosis of TB continues due to their adaptability to simple, rapid formats, such as the dipstick and lateral-flow cassette formats, which are extremely useful in the field, where a sophisticated laboratory infrastructure and highly trained personnel are not available (8,30).…”