al-Abdulla I H, Sidki A M, Landon J, Rowell F J
Department of Chemical Pathology, St. Bartholomew's Hospital, London, UK.
Southeast Asian J Trop Med Public Health. 1989 Sep;20(3):361-9.
Primaquine coupled to keyhole limpet hemocyanin was used as an immunogen to produce antiprimaquine antibodies in three sheep. The antisera obtained were characterised by the increase in fluorescence polarisation found upon binding to fluorescein-labelled primaquine prepared via same route. All sheep showed a good antibody response and one antiserum was coupled to magnetisable solid-phase particles to facilitate the separation of the antibody bound from free labelled antigen and the removal of interfering components which may be present in the sample. The fluoroimmunoassay requires addition of 100 microliters of standard or sample (urine or serum) to 100 microliters tracer (150 nmol/l) followed by 100 microliters of magnetisable solid-phase particles (12.5 g/l). After one hour incubation followed by the usual washing and eluting procedures, using a magnetic rack, the fluorescence of the supernatant was measured directly in a fluorimeter. Sodium salicylate was incorporated in the tracer solution to block the non-specific binding of tracer to the protein in serum samples. Cross-reactivity studies showed that the antibodies have high specificity for the 8-aminoquinoline nucleus but not to the 8-N-aminobutyl side chain. Thus carboxyprimaquine cross-reacted equally with primaquine and the assay can be used to measure their combined level. After extraction of primaquine from a basified sample with methylene chloride, the assay may be applied for the quantitation of either primaquine (in the organic phase) or its acidic metabolites including carboxyprimaquine (in the aqueous phase) separately. This approach was applied for the determination of total primaquine (primaquine and its metabolites) and extracted primaquine in urine samples following a single oral dose of 45 mg primaquine.(ABSTRACT TRUNCATED AT 250 WORDS)
将伯氨喹偶联到钥孔戚血蓝蛋白上，以此作为免疫原在三只绵羊体内产生抗伯氨喹抗体。通过相同途径制备的荧光素标记伯氨喹与所获抗血清结合后，荧光偏振度增加，以此对其进行表征。所有绵羊均呈现出良好的抗体反应，并且将一种抗血清偶联到可磁化的固相颗粒上，以利于分离结合抗体的游离标记抗原，并去除样品中可能存在的干扰成分。荧光免疫测定法要求向100微升示踪剂（150纳摩尔/升）中加入100微升标准品或样品（尿液或血清），随后加入100微升可磁化固相颗粒（12.5克/升）。孵育一小时后，按照常规的洗涤和洗脱步骤，使用磁力架，直接在荧光计中测量上清液的荧光。将水杨酸钠加入示踪剂溶液中，以阻断示踪剂与血清样品中蛋白质的非特异性结合。交叉反应研究表明，这些抗体对8-氨基喹啉核具有高特异性，但对8-N-氨基丁基侧链不具有特异性。因此，羧基伯氨喹与伯氨喹具有同等程度的交叉反应，该测定法可用于测量它们的总含量。用二氯甲烷从碱化样品中提取伯氨喹后，该测定法可分别用于定量有机相中的伯氨喹或水相中的酸性代谢物（包括羧基伯氨喹）。在单次口服45毫克伯氨喹后，将该方法应用于测定尿液样品中的总伯氨喹（伯氨喹及其代谢物）和提取的伯氨喹。（摘要截选至250词）
