Single dilution ELISAs using soluble piroplasm, cellular schizont and soluble schizont antigens for the detection of antibodies against Theileria annulata

Abstract

Single dilution ELISAs were standardised for the determination of antibody titres against Theileria annulata using three antigens namely soluble piroplasm, cellular schizont or soluble schizont antigens. Antibody titres of 20 cattle serum samples of known identity were determined by multi-dilution ELISA using the three antigens. The ratio of the optical density (OD) of known positive and known negative sera at different serum dilutions were calculated and termed as positive/negative (P/N) ratios. Coefficients of correlation (r) were calculated between the P/N ratios at different dilutions of known sera and their log10 antibody titres by multi-dilution ELISA. The value of "r" was the highest at the dilution of 1:400. From the log10 antibody titres of known sera and their P/N ratios at the dilution of 1:400, regression equations (Y = a + bX, where Y = predicted log10 titre, X = the P/N ratio at 1:400 dilution) were calculated separately for the three antigens. Thus, the equations Y = 1.63 + 1.35X for soluble piroplasm, Y = 2.67 + 0.547X for cellular schizont and Y = 1.817 + 0.663X for soluble schizont antigens were derived. Test sera were diluted to 1:400 and their OD were read in duplicate wells and converted to P/N ratios. The antibody titres were predicted from the P/N ratios using the above mentioned regression equations. Twenty randomly selected sera tested by single and multidilution ELISAs showed non-significant differences (P < 0.01) between antibody titres. Antibody titres of 90 unknown field sera of cattle were determined by single dilution ELISA. The piroplasm antigen detected higher antibody titres followed by cellular schizont and soluble schizont antigens. The study revealed that a single dilution ELISA could be successfully used for field epidemiological studies of tropical theileriosis.

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

“…This was in conformity with earlier studies of Briggs and Skeeles [3]; Manuja et al [24] and Khatri et al [17], who adopted a similar strategy while standardising single dilution ELISA for detecting antibodies to other pathogens.…”

“…The P/N ratio was used instead of the OD values at a particular dilution for standardisation of single dilution ELISA since a great variation in OD values for a particular sample is observed depending upon the assay conditions, time of incubation etc. However, the P/N ratio for a particular serum sample at the same dilution remains relatively constant under varying test conditions, as reported previously for standardisation of similar tests for other pathogens [3,24]. Regression equations as derived for use in single dilution ELISA, express the correlation between the two variables (log 10 end titre and P/N ratio) and are necessary in predicting the value of the dependent variable (log 10 end titre) when the value of the independent variable (P/N ratio) is known.…”

“…The conventional ELISA for sero-diagnosis of B. equi is cumbersome, time consuming and requires large quantities of reagents when the end titre of a sample is to be determined. However, the single dilution ELISA has been found to be cheaper, faster and reliable than conventional serial dilution ELISA as reported for other pathogens [17,24]. Thus, the single dilution ELISA was developed to predict the B. equi antibody titre of a sample.…”

“…Regression analysis, as used by Briggs and Skeeles [3] and Manuja et al [24] to predict the end point of antibody titres to Pasteurella multocida in chicken sera and Theileria annulata infection in bovine sera, respectively, was adopted.…”

“…However the cost per sample of ELISA is high and it is tedious and inconvenient when the titre of a sample is to be determined. Recently, single dilution ELISAs have been standardised and used for the sero-diagnosis of the foot and mouth disease [33], bovine babesiosis [1], Pasteurella multocida infection in buffaloes [17] and tropical theileriosis in cattle [17,24]. In conventional serial dilution ELISA, the end titre of a sample is calculated by serial two fold dilution of the sample until the optical density (OD) becomes negative.…”