[Improvement and validation of a micro-complement fixation test for glanders]

Glanders, a major zoonotic disease declared eradicated in China, still faces the risk of external reintroduction. The complement fixation test (CFT) is a key serological diagnostic method for glanders. To address the limitations of conventional CFT, such as high reagent consumption, cumbersome titration procedures, and frequent occurrence of atypical partial hemolysis leading to ambiguous interpretation, this study developed a more economical, user-friendly, and clearly interpretable micro-complement fixation test (mCFT). According to the WOAH guidelines and Chinese agricultural industry standards, we systematically refined the method by focusing on three key dimensions: miniaturization (establishing a 125-μL total reaction volume), system standardization (optimizing and unifying the workflow for determining working concentrations of key components), and endpoint interpretation optimization (enhancing the distinction between positive and negative results). The improved procedure begins with the standardized determination of working concentrations for key reagents. First, the working titers of hemolysin, complement, and antigen are established through serial dilution and reaction. Subsequently, test sera are diluted 1:5, incubated in a 96-well plate with working antigen and complement at 37 ℃ for 1 h, followed by addition of sensitized red blood cells for further 45-min incubation. Finally, the results are assessed by direct visual comparison with standard colorimetric wells: a hemolysis degree ≤50% is considered positive, 50%-90% suspicious, and ≥90% negative. The measured working titers for hemolysin, complement, and antigen were 1:1 000, 1:25, and 1:25, respectively. The validation tests with 40 serum samples provided by world organisation for animal health (WOAH) showed either ≥90% or ≤50% hemolysis, with no sample falling into the suspicious range. The overall result concordance rates of the established method with the WOAH reference and a commercial ELISA kit reached 85% and 87.5%, respectively. While maintaining satisfactory sensitivity and specificity, the method significantly reduces reagent costs, and its clear operational protocol enhances reproducibility. This mCFT provides a reliable technical reserve for the ongoing surveillance, border quarantine, and emergency response to potential glanders outbreaks in China.