Development of an Individual Rapid Test Based on Enzymatic Immunofiltration Assay for Detection of Anti–Brucella Abortus Antibody in Bovine Sera

Abstract

To detect bovine antibody directed to smooth lipopolysaccharide (LPS), cell lysate (LYS), O-polysaccharide (OPS), and LPS-deprived chromatographic fractions (ChF) of Brucella abortus, 2 bi-antigenic diagnostic models based on the enzymatic rapid immunofiltration assay (ERIFA), ERIFA(LPS/LYS) and ERIFA(OPS/ChF), were developed. Their diagnostic performance was compared with complement fixation test (CFT), Rose Bengal test (RBT), indirect in-house and commercial enzyme-linked immunosorbent assays (iELISA and com-ELISA, respectively), based on the smooth LPS antigen, by using a total of 420 cattle sera collected from aborted-unvaccinated, aborted-unvaccinated and culture-positive, healthy-unvaccinated, and healthy-vaccinated cattle. The results demonstrated excellent agreement and no statistical difference between iELISAs and LPS-, LYS-, OPS-based ERIFA models. However, diagnostic performance of CFT, RBT, and ChF-based ERIFA was less significant than that of LPS-, LYS-, and OPS-based ERIFA models, and iELISAs. The results demonstrated a successful adaptation of the multi-antigenic ERIFA model to anti-B. abortus antibody in bovine sera and suggest that the ERIFA model can be considered as an "individual rapid ELISA" due to its similarity with ELISA, individual applicability, and rapidity in determining reactor animals within 5 minutes. In conclusion, the potential of multi-antigenic applications can make the rapid ERIFA model not only an alternative screening method but also a confirmatory test for bovine brucellosis diagnosis.

Evaluation of two rapid immunochromatographic tests for diagnosis of brucellosis infection in cattle

“…The present study showed the greater affinity of protein G for bovine immunoglobulins compared with protein A, observed in the results of sensitivity and specificity obtained by the LFIA-PG system. Sensitivity and specificity are essential parameters that allow making inferences from the results of an assay (Genç et al, 2011;Ochoa, 2013). Both parameters define the effectiveness of a system since they ensure that the proportion of the infected animals will give positive results to the test; however, the animals still infected give a negative result.…”

Evaluation of Up-Converting Phosphor Technology-Based Lateral Flow Strips for Rapid Detection of Bacillus anthracis Spore, Brucella spp., and Yersinia pestis

“…In the LF assay, some specific common antigens, which are surface exposed and important for virulence, are used in the preparation of antibodies that can recognize as many species as possible, such as F1 antigen [22] and plasminogen activator protein [16] of Y. pestis , anthrose tetrasaccharide of B. anthacis [19], outer membrane proteins and O-polysaccharide of Brucella [20], [21], [38]. However, most surface antigens are difficult to isolate and purify because they are amphipathic molecules.…”

“…However, traditional LF assays that use colloidal gold as label have the following limitations under potential bioterrorism scenarios with mixed bacteria and complex samples: (i) absence of reactions with different strains of the same pathogen species, (ii) low sensitivities caused by macroscopic observations, (iii) results cannot be analyzed because of serious interferences by colored samples such as whole blood [16], and (iv) inapplicability in extreme conditions (e.g., acid, basic, saline, viscose, and protein-rich solutions) because of the interference in conjugation between antibodies and gold particles that depend on physical adsorption. To simultaneously detect different strains of the same pathogen species, specific antigens are used to prepare antibodies, and two or more antibodies are mixed in LF assays to efficiently recognize species of B. anthracis [19], Brucella [20], [21], and Y. pestis [16], [22] under field conditions. However, only a few works have focused on the rapid detection of pathogens from complex samples using LF assay to meet the requirements of POCT diagnostic and system assessments using extreme reagents and on-site samples.…”

Rapid immunofiltration assay based on colloidal gold–protein G conjugate as an alternative screening test for bovine and ovine brucellosis

“…The optimal cutoff value of the iELISA was determined by ROC analysis as 0.600 (OD 405 ) and 0.500 (OD 405 ) for bovine and ovine sera, respectively. The NERIFA is a version of the ERIFA described previously by Genç et al (2011). Briefly, the NERIFA is essentially based on binding of the conjugate, protein G/colloidal gold particles (G/CG conjugate; Arista Biologicals, USA) to the specific antibodies reacting with Brucella LPS dotted onto a large porous nitro-cellulose filter membrane.…”

“…Currently used laboratory-based testing such as complement fixation test (CFT), SAT, enzyme-linked immunosorbent assays (ELISAs), FPA, and recently developed TR-FRET assay (McGiven et al 2009) is inapplicable in field conditions for brucellosis surveillance. In this regard, the lipopolysaccharide (LPS)-based lateral flow (LFA; Abdoel et al 2008) and enzymatic rapid immunofiltration assays (ERIFA; Genç et al 2011) have been recently developed and proposed as field tests for brucellosis diagnosis. Although their usefulness as field tests has been demonstrated, these methods require the use of the species-specific antibodies conjugated with colloidal gold or enzymes as detection reagents.…”