Performance of enzyme-linked immunosorbent assays for detection of antibodies against T. congolense and T. vivax in goats

Indirect ELISAs using denatured antigen preparations of Trypanosoma (T.) congolense (TcAGd) and T. vivax (TvAGd) for detection of anti-trypanosome antibodies in bovine serum (I-TAB ELISAs), were adapted for serodiagnosis in goats. The diagnostic proficiency, the cross-reactivity with sera from heterologous trypanosome infections and the operational performance of the assays were evaluated on experimentally trypanosome-infected goats. The I-TAB ELISA (TcAGd) detected antibodies in all T. congolense infected goats (100% overall sensitivity) from 2 to 4 weeks post-infection (p.i.) until the end of the experiments. Specificity tested on 92 uninfected goats was 96.7%. Extensive cross-reactions of I-TAB ELISA (TcAGd) with sera from T. vivax or T. brucei infected goats were observed. The I-TAB ELISA (TvAGd) detected antibodies in 5 of the 6 T. vivax infected goats, specificity tested on uninfected goats was 100%. Cross-reactivity with sera from T. congolense or T. brucei infected goats remained limited. Infecting species identification based on the highest percent positivity (PP) in both systems, correctly identified all T. congolense infections, but misidentified in 2/19 occasions a T. vivax infection as a T. congolense infection. In the absence of T. brucei specific antigen coated plates, T. brucei infections were identified in, respectively, 7/9 and 2/9 occasions as T. congolense or T. vivax infections. Acceptable inter-plate repeatability was observed. The implications of results and technical requirements for ongoing applied research are discussed

Improved Methods for the Diagnosis of African Trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered

Bioconjugation of protein-repellent zwitterionic polymer brushes grafted from silicon nitride

A new method for attaching antibodies to protein-repellent zwitterionic polymer brushes aimed at recognizing microorganisms while preventing the nonspecific adsorption of proteins is presented. The poly(sulfobetaine methacrylate) (SBMA) brushes were grafted from α-bromo isobutyryl initiator-functionalized silicon nitride (SixN4, x ≥ 3) surfaces via controlled atom-transfer radical polymerization (ATRP). A trifunctional tris(2-aminoethyl)amine linker was reacted with the terminal alkylbromide of polySBMA chains. N-Hydroxysuccinimide (NHS) functionalization was achieved by reacting the resultant amine-terminated polySBMA brush with bifunctional suberic acid bis(N-hydroxysuccinimide ester). Anti-Salmonella antibodies were subsequently immobilized onto polySBMA-grafted SixN4 surfaces through these NHS linkers. The protein-repellent properties of the polySBMA-grafted surface after antibody attachment were evaluated by exposing the surfaces to Alexa Fluor 488-labeled fibrinogen (FIB) solution (0.1 g·L–1) for 1 h at room temperature. Confocal laser scanning microscopy (CLSM) images revealed the minimal adsorption of FIB onto the antibody-coated polySBMA in comparison with that of antibody-coated epoxide monolayers and also bare SixN4 surfaces. Subsequently, the interaction of antibodies immobilized onto polySBMA with SYTO9-stained Salmonella solution without using blocking solution was examined by CLSM. The fluorescent images showed that antibody-coated polySBMA efficiently captured Salmonella with only low background noise as compared to antibody-coated monolayers lacking the polymer brush. Finally, the antibody-coated polySBMA surfaces were exposed to a mixture of Alexa Fluor 647-labeled FIB and Salmonella without the prior use of a blocking solution to evaluate the ability of the surfaces to capture bacteria while simultaneously repelling proteins. The fluorescent images showed the capture of Salmonella with no adsorption of FIB as compared to antibody-coated epoxide surfaces, demonstrating the potential of the zwitterionic layer in preventing the nonspecific adsorption of the proteins during the detection of bacteria in complex matrices.\u