Clinical Evaluation of a Peptide‐ELISA based upon N‐terminal B‐cell Epitope of the VapA Protein for Diagnosis ofRhodococcus equiPneumonia in Foals

Abstract

A total of 227 field samples from naturally exposed foals aged between 3 weeks and 6 months were used in an evaluation of a peptide-based enzyme-linked immunosorbent assay (ELISA) for diagnosis of Rhodococcus equi infection. A biotinylated peptide derived from the virulence-associated protein A (VapA) of R. equi, a horse pathogen, was synthesized and designated as PN11-14. The peptide corresponds to the N-terminal B-cell epitope TSLNLQKDEPNGRASDTAGQ of the VapA protein. Based upon a serum immunoglobulin (Ig)G titre of 512 as a positive cut-off value for the R. equi infection, the ELISA provided the overall sensitivity of 47.62%, specificity of 69.67% and an accuracy of 59.47% with a positive predictive value of 57.47% for true R. equi pneumonia. The assay was improved by detecting VapA-specific IgGb antibodies against N-terminal B-cell epitope of the VapA protein rather than IgG antibodies. The VapA-IgGb ELISA showed the overall sensitivity of 70.47%, specificity of 72.13% and accuracy of 71.36% with a positive predictive value of 68.52%. Diagnosis of R. equi disease in 6-week-old foals showed that the VapA-IgGb ELISA provided an increasing trend (P=0.0572) in sensitivity of 82.4% in comparison with the VapA-IgG ELISA which showed the sensitivity of 58.8%. However, differences in specificity of both tests were statistically insignificant (P=0.357) as analysed by the McNemar test. These results indicated that detection of VapA-specific IgGb antibodies may be a better predictor of R. equi disease in foals.

A role for human endogenous retrovirus-K (HML-2) in rheumatoid arthritis: investigating mechanisms of pathogenesis

“…When biotinylated peptide was used, levels of anti-KPR antibodies detected increased (significantly in RA patients). Results presented here support those of previous investigations in concluding that the biotinylation of antigen improves assay sensitivity significantly [20,21]. This is due probably to significant modifications to the structural and conformational modelling of antigens leading to the masking of potentially important epitopes during the process of adsorbing the peptide to the solid phase [22].…”

Diagnosis, Treatment, Control, and Prevention of Infections Caused byRhodococcus equiin Foals

“…Independent studies evaluating the performance of serological tests available for diagnosis of infection caused by R. equi at endemic farms have demonstrated these tests either have low sensitivity, low specificity, or both . Improving either sensitivity or specificity of ELISA assays by changing the cut‐off value of the tests could only be done to the detriment of the other.…”

“…Independent studies evaluating the performance of serological tests available for diagnosis of infection caused by R. equi at endemic farms have demonstrated these tests either have low sensitivity, low specificity, or both. [3][4][5][6] Improving either sensitivity or specificity of ELISA assays by changing the cut-off value of the tests could only be done to the detriment of the other. The presence of antibodies indicates exposure, subclinical infection, or maternal transfer of antibodies, but it does not necessarily indicate infection leading to clinical disease.…”

“…As a result of the various advantages and limitations associated with this approach to screening, it has both proponents and opponents. Advantages of thoracic ultrasonography include (1) the procedure can be performed relatively quickly for an individual foal; (2) competence with the procedure can be rapidly developed; (3) results are immediately available; (4) the procedure may be more sensitive than radiography for detecting lesions in their early stages of development or in certain regions where soft tissue structures are superimposed over regions of the lung; 63 (5) results are specific for the presence of pulmonary pathology (as contrasted with, for example, results of WBC); and (6) evidence from uncontrolled studies that mortality attributable to R. equi was eradicated using ultrasonographic screening. 53 Disadvantages of ultrasonographic screening include the costs to the farm for repetitive sonographic examinations, the increased labor needed to handle foals repetitively, and an increased number of foals treated for presumptive R. equi pneumonia as a result of the aforementioned apparent increase in cumulative incidence.…”

Epitopic Peptides Identified by LC–ELISA and LC–MS

“…To overcome the disadvantage of past technologies, hyphenated technique of ELISA was considered to develop due to peptide ELISA has also been used in the clinical diagnosis of severe diseases, as evidenced by numerous reports [32][33][34]. Many companies, such as Sigma, Promega, and Perkin Elmer Life Science, have developed ELISA kits for peptides.…”