定量ELISA竞争抑制法测定屋尘螨变应原点刺液生物活性

Determination of biological activity of mite allergen prick solution of Dermatophagoide pteronyssinus by quantitative ELISA

目的建立一种稳定的屋尘螨点刺液生物活性测定方法,以替代现用的放射性吸附抑制测定方法(radio allergosorbent test,RAST)。方法建立定量ELISA竞争抑制法,对屋尘螨点刺液进行生物活性测定,并对该法进行精密性、准确性、适用性验证,确定线性测定范围。应用RAST和定量ELISA竞争抑制法平行测定3批屋尘螨点刺液生物活性,对结果进行比较。结果定量ELISA竞争抑制法测定屋尘螨点刺液制品生物活性,其最佳测定范围为0.052~0.125 HEP/m L;高、中、低3个浓度的日内精密性≤10.3%,日间精密性≤11%,回收率为87%~106%,表明该方法具有较好的精密性;低、中、高3个加标样本的回收率均在73.18%~99.02%之间,表明该方法具有较好的准确性;3批屋尘螨点刺液生物活性最佳测定范围的稀释度为1/48~1/192,所有测定样本的相对活性在80%~110%范围内,表明该方法适用于屋尘螨点刺液生物活性检测。两种方法测定屋尘螨点刺液生物活性,结果差异无统计学意义(P>0.05),且均符合质量标准规定。结论定量ELISA竞争抑制法测定屋尘螨点刺液的生物学活性,重复性好,准确性高,操作简便,可作为现用生物活性测定RAST法的替代方法。

Objective To establish a stable method for determination of biological activity of mite allergen skin prick solution instead of the current radio allergosorbent test（RAST）. Methods A quantitative competitive ELISA method was established to determine the biological activity of house dust mite skin prick solution,and verified for precision,accuracy,suitability,and linear range. Radio allergosorbent test（RAST） and ELISA were used to determine the biological activities of three batches of house dust mite skin prick solution simultaneously,and the results were compared. Results The optimal detection range of quantitative competitive ELISA for biological activity of house mite allergen skin prick solution was 0. 052 ~ 0. 125 HEP/m L. The precision of this method for samples at high,moderate and low concentrations were not more than 10. 3% in intra-assay and not more than 11% in inter-assay,while the recovery rate was 87% ~106%,indicating a high precision. In spike recovery test,the recovery rate of biological activity of house mite allergen skin prick solution was 73. 18% ~ 99. 02%,indicating a high accuracy. The dilutions of three batches of house mite allergen skin prick solution within the optimal detection range of biological activity were 1/48 ~ 1/192,and all the relative activities of the samples were 80% ~ 110%,indicating that the method was suitable for the biological activity of house mite allergen skin prick solution. However,the determination result by quantitative ELISA showed no significant difference with that by RAST（P〉0. 05）,both of which met the quality standard. Conclusion The quantitative ELISA competitive inhibition method showed good reproducibility and accuracy and was easy to handle,which might be used for the determination of biological activity of house dust mite skin solution instead of RAST.