Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite.

The study compares the effectiveness of serial dilution and single dilution Enzyme Linked Immunosorbent Assays (ELISA) in diagnosing Babesia equi infection in donkeys. The results suggest the single dilution test with a specific antigen, known as the whole merozoite (WM) antigen, is more convenient, economical, sensitive, and specific, and can hence, enhance sero-epidemiological studies on B. equi infections in the field.

Understanding the Research Methods

• The research commenced by standardizing single dilution and serial dilution ELISA for the serodiagnosis of Babesia equi infection.
• The antibody titres from the sera of 24 donkeys were determined separately using three different B. equi antigens – whole merozoite (WM), cell membrane (CM), and high speed supernatant (HSS).
• The measurements involved calculating the ratios of optical density (OD) of known positive and negative sera at distinct dilutions. These ratios are referred to as positive/negative (P/N) ratios.
• The researchers calculated the coefficients of correlation (r) between the P/N ratios at different serum dilutions and the antibody titres ascertained by serial dilution ELISA. The highest ‘r’ value, indicating optimal accuracy, was obtained at 1:200 serum dilution.

Outcome of the Experiment

• Regression equations were formulated from the log10 antibody titre of sera and their P/N ratio at a dilution of 1:200, separately for the three antigens.
• By employing the regression equations, antibody titres of test sera were predicted from the P/N ratio.
• The titres obtained by both ELISAs were compared and no significant difference was observed, suggesting that the single dilution ELISA could effectively replace the conventional serial dilution ELISA.

Statistical Validation of ELISA Methods

• The success of the single dilition ELISA was verified statistically, using 42 B. equi disease-positive and 106 disease-negative sera.
• The whole merozoite antigen (WM) had a higher predictive value for negative sera as compared to the CM or HSS antigens, thus denoting its sensitivity.
• Sera that were known to be positive for other equine infections, including Babesia Caballi, did not cross-react with any of the three B. equi antigens. Hence, the single dilution ELISA method proved to be immunologically specific.

Comparison with Field Data

• The antibody titres of 109 unknown field donkey/horse sera, measured by both ELISA methods using the WM antigen, also showed no significant difference.
• The single dilution ELISA was determined to be more economical, convenient, sensitive, and specific than the serial dilition ELISA.
• Owing to its enhanced characteristics, including its higher predictive value, single dilution ELISA can be used in field sero-epidemiological studies for diagnosing B. equi infections.