Issue |
Vet. Res.
Volume 34, Number 1, January-February 2003
|
|
---|---|---|
Page(s) | 71 - 83 | |
DOI | https://doi.org/10.1051/vetres:2002055 | |
How to cite this article | Vet. Res. (2003) 71-83 |
DOI: 10.1051/vetres:2002055
Standardisation and comparison of serial dilution and single dilution enzyme linked immunosorbent assay (ELISA) using different antigenic preparations of the Babesia (Theileria) equi parasite
Sanjay Kumara, Yogesh Kumara, Dharam V. Malhotraa, Shruti Dharb and Anil K. Nichaniba Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar 125 004, Haryana, India
b All India Coordinated Research Project on Blood Protista, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar 125 004, Haryana, India
(Received 26 December 2001; accepted 16 July 2002)
Abstract
Serial dilution and single dilution enzyme linked immunosorbent assays (ELISA)
were standardised and their sensitivity and specificity were compared for
serodiagnosis of Babesia equi infection. The antibody titres of 24 donkey sera
of known identity were determined separately by serial dilution ELISA using
three different B. equi antigens namely whole merozoite (WM), cell membrane
(CM) and high speed supernatant (HSS). The ratios of the optical density
(OD) of known positive and known negative sera at different serum dilutions
were calculated and termed as the positive/negative (P/N) ratio.
The coefficients of correlation (r) were calculated between the P/N ratios
at different dilutions of sera and the log
10 antibody titres of the same
sera were ascertained by serial dilution ELISA. The highest value
of `r' was obtained at a serum dilution of 1:200. From log
10 antibody
titre of sera (y) and their P/N ratio at a dilution of 1:200 (x), regression
equations (y = a + bx) were calculated separately for the three antigens.
Test sera were diluted to 1:200, their OD were read in duplicate wells
and were converted to the P/N ratio. Antibody titres were predicted from
the P/N ratio using a regression equation separately for the three antigens.
Titres obtained by both ELISAs were not significantly different from each
other, thus confirming that single dilution ELISA could be successfully
used to replace conventional serial dilution ELISA. The sensitivity,
specificity and predictive value of single dilution ELISA was validated
statistically using 42 B. equi disease-positive sera and 106
B. equi disease-negative sera. The WM antigen was found to be the most
sensitive with a higher predictive value for negative test sera as
compared to the CM or HSS antigens. Sera positive for other equine
infections including Babesia caballi showed no cross-reaction with the
three B. equi antigens in ELISA, thus the test was immunologically
specific. Antibody titres of 109 unknown field donkey/horse sera obtained
by serial and single dilution ELISA using the WM antigen did not show any
significant difference. Since the single dilution ELISA was found to be
more economical, convenient, sensitive, specific than the serial dilution
ELISA and has a high predictive value, it is suitable for use in
sero-epidemiological studies on B. equi infections in the field.
Key words: Babesia equi / Theileria equi / protozoan / sero-diagnosis / ELISA
Correspondence and reprints: Sanjay Kumar fax: (81) 155 49 5643;
e-mail: sanjayk2002@hotmail.com
© INRA, EDP Sciences 2003