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

Abstract

A non-enzymatic rapid immunofiltration assay (NERIFA) was developed as an alternative field test for rapid detection of anti-Brucella antibody in bovine and ovine sera. The assay was based on Brucella abortus lipopolysaccharide as diagnostic antigen and colloidal gold particle-protein G conjugate as detection reagent. Its diagnostic performance was evaluated using undiluted well-defined positive and negative serum samples in comparison with Rose Bengal test (RBT), complement fixation test (CFT) and a commercial and an in-house indirect enzyme-linked immunosorbent assay (ELISA). A perfect test agreement was found between NERIFA and ELISAs by kappa statistics. In addition, McNemar's analysis of the results showed that the RBT for bovine sera and the CFT for ovine sera were found significantly less performant than indirect ELISAs and NERIFA. The results of the present study indicated that the NERIFA could be considered as a simple, rapid, and accurate field test for screening of ovine and bovine brucellosis. Therefore, this test constitutes a high potential to be used as an alternative model particularly in brucellosis prevalent tropical and subtropical geographical areas.

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

“…There is not a serological test appropriated for all the epidemiological situations; therefore, all the factors influencing in the relevance of the analytical method and the test results for a particular diagnostic interpretation or application must be taken into account. In the case of the rapid lateral flow immunochromatographic tests, even when they are simple and fast running systems, reliable and accurate results are reported (Smits et al, 2003;Genç et al, 2012;Díaz et al, 2015), which, together with the possibility of carrying them to the fields where the herds are (as field tests), make them an alternative to the conventional serological diagnosis of brucellosis (Smits et al, 2003;Nielsen et al, 2004). In the development of the LFIA-PG system, the appropriate concentration of the protein G conjugate was obtained for printing on the pads, and the migration buffer and drying time at 37 °C were effective.…”

“…With the application of this conjugate, IgG antibodies were detected, since protein G had the ability to bind specifically and with high affinity to the Fc fragment of IgG (Saegerman et al, 2004;Díaz et al, 2015). Genç et al (2012) also used a G-gold colloidal conjugate for diagnosis of brucellosis in cattle and sheep with reliable results. .…”

“…Protein A reacts more specifically with the IgG 2 subclass, whereas protein G reacts with both subclasses of bovine IgG (IgG 1 and IgG 2), which allows detecting antibodies at any time of infection (Díaz et al, 2015;Shome et al, 2015). In comparative immunological studies on affinity and specificity of proteins A and G, protein G showed a greater affinity for G-type bovine immunoglobulins (Nielsen et al, 2004;Pajuaba et al, 2010;Genç et al, 2012). 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.…”

A novel, rapid and simple method for detecting brucellosis based on rapid vertical flow technology

“…Then, working dilutions of antigen and complement were added, after which the mixture was incubated at 4°C overnight and the haemolytic system buffer was added to each well. The plates were then incubated at 37°C for 30 min, after which the results were read and expressed as the number of International Complement Fixation Test Units per ml (Genç et al ).…”

“…The rose Bengal test (RBT) and the serum agglutination test (SAT) are widely used to detect serum antibodies for the diagnosis of brucellosis infection. However, these two methods are not effective because they have many limitations, such as low sensitivity and low specificity (Genç et al ). These two methods also require professional and technical personnel to conduct the tests and evaluate the results, and the test results are greatly influenced by subjective factors.…”