龙血素A和龙血素B的酶联免疫检测方法的建立

建立了龙血素A和龙血素B间接竞争酶联免疫吸附分析方法,并用于测定龙血素A和龙血素B的含量。分别合成龙血素A和龙血素B人工抗原制备其多克隆抗体,建立二者间接竞争酶联免疫吸附分析方法并评估其分析性能。结果表明:抗龙血素A血清和抗龙血素B血清的效价分别达到8 000和30 000;龙血素A和龙血素B的最低检测限分别为3.9ng·mL-1和26.1ng·mL-1,批内精密度分别为CV≤10.7%和CV≤9.9%,批间精密度分别为CV≤11.5%和CV≤12.3%,回收率分别为87.4%~110.8%和109.8%~113.5%,与各自的6种类似物的交叉反应率最大分别为9.20%和3.64%,在4种龙血竭药物中的最大含量和最小含量分别相差4.05倍和3.57倍。龙血素A和龙血素B间接竞争酶联免疫吸附分析方法的检测精密度、准确度和特异性均较好,可快捷有效地用于药物检测和分析。

Development of Indirect Competitive ELISA for the Detection of Loureirin A and Loureirin B

An indirect competitive enzyme-linked immunosorbent assay（ELISA） was developed in order to determine the content of Loureirin A（LA） and Loureirin B（LB） in the drugs. Artificial antigens of LA and LB were synthesized. Then polyclonal antibodies were both obtained by using animal immunization. The methods of the indirect competitive ELISA for determining LA and LB were established, followed by the evaluation of their analytical performances. Results showed that the highest titer of anti-LA sera and anti-LB sera were 8 000 and 30 000,respectively~the LOD for LA and LB were 3.9 ng·mL-l and 26.1 ng·mL-1 ,respectively;the intra assay precisions were less than 10. 7% and 9.9%,respectively; the inter assay precisions were less than 11.5% and 12. 3% ,respectively;recoveries were 87.4%-110. 8% and 109.8%-113.5%,respectively;and the maximum cross-reactivities of six structural analogues for LA and LB were less than 9.20% and 3.64%, respectively;the contents of maximum and minimum for LA and LB in the four kinds of drugs were differ by 4.05 times and 3.57 times,respectively. Indirect competitive ELISA for the determination of LA and LB has high precision,accuracy and specificity,and can be conveniently applied in drug detection and analysis.