Kumar Sanjay, Kumar Yogesh, Malhotra Dharam V, Dhar Shruti, Nichani Anil K
Department of Veterinary Epidemiology and Preventive Medicine, College of Veterinary Sciences, CCS Haryana Agricultural University, Hisar 125 004, Haryana, India.
Vet Res. 2003 Jan-Feb;34(1):71-83. doi: 10.1051/vetres:2002055.
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 log10 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 log10 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.
对系列稀释和单稀释酶联免疫吸附测定（ELISA）进行了标准化，并比较了它们在马巴贝斯虫感染血清学诊断中的敏感性和特异性。使用三种不同的马巴贝斯虫抗原，即全裂殖子（WM）、细胞膜（CM）和高速上清液（HSS），通过系列稀释ELISA分别测定了24份已知身份的驴血清的抗体滴度。计算了不同血清稀释度下已知阳性和已知阴性血清的光密度（OD）比值，并将其称为阳性/阴性（P/N）比值。计算了不同血清稀释度下P/N比值之间的相关系数（r），并通过系列稀释ELISA确定了相同血清的log10抗体滴度。在血清稀释度为1:200时获得了“r”的最高值。根据血清的log10抗体滴度（y）及其在1:200稀释度下的P/N比值（x），分别计算了三种抗原的回归方程（y = a + bx）。将测试血清稀释至1:200，在重复孔中读取其OD值，并转换为P/N比值。使用三种抗原各自的回归方程，根据P/N比值预测抗体滴度。两种ELISA获得的滴度彼此无显著差异，从而证实单稀释ELISA可成功用于替代传统的系列稀释ELISA。使用42份马巴贝斯虫病阳性血清和106份马巴贝斯虫病阴性血清对单稀释ELISA的敏感性、特异性和预测值进行了统计学验证。发现WM抗原最敏感，与CM或HSS抗原相比，对阴性测试血清具有更高的预测值。包括马巴贝斯虫在内的其他马属动物感染阳性的血清在ELISA中与三种马巴贝斯虫抗原无交叉反应，因此该测试具有免疫特异性。使用WM抗原通过系列稀释和单稀释ELISA获得的109份未知现场驴/马血清的抗体滴度无显著差异。由于发现单稀释ELISA比系列稀释ELISA更经济、方便、敏感、特异且具有较高的预测值，因此适用于现场马巴贝斯虫感染的血清流行病学研究。
