Indirect Enzyme-Linked Immunosorbent Assay Test Kit Development for Specific Antibody Detection Against Brucella abortus in Cattle

Abstract

The smooth lipopolysaccharide (SLPS) antigen, prepared from Brucella abortus strain 99 was used in an indirect Enzyme-Linked Immunosorbent Assay (iELISA). The data were expressed as optical density and generated using computer software. The positive results were accepted at the cut-off values of ≥ 40 percent positivity (PP). The strong positive serum control (C++), weak positive serum control (C+), negative serum control (C-), and conjugate control (Cc) were used to optimize the test kit. The iELISA was optimization and standardization for antigen concentration, test material dilution and detection system dilution by checkerboard titration. The statistic evaluation using data from sera from 317 infected and 5,300 non-infected cattle revealed that the sensitivity (Se) is 99.369 (98.497-100.0)%, specificity (Sp) is 99.887 (99.796-99.977)%, accuracy (Ac) is 99.858% and Kappa value is 0.987. The iELISA test kit was subsequently validated by testing 9,877 field serum samples submitted to laboratory. The complement fixation test was used as gold standard. The results revealed that the relative Se is 99.026 (97.929-100.0)%, relative Sp is 98.547 (98.308-98.787)%, relative Ac is 98.562% and Kappa value is 0.804 when cattle sera were tested. The components of this iELISA test kit consisted of antigen, control sera, and essential diluents for 800 tests. This test showed high sensitivity, specificity, and accuracy and could be performed in mass. Therefore, this iELISA test kit might be an appropriate tool for brucellosis control in Thailand.