Development of a magnetisable solid-phase fluoroimmunoassay for primaquine and carboxyprimaquine
al-Abdulla, I.H.; Sidki, A.M.; Landon, J.; Rowell, F.J.
Southeast Asian Journal of Tropical Medicine and Public Health 20(3): 361-369
1989
ISSN/ISBN: 0125-1562 PMID: 2633347 Document Number: 333528
Primaquine coupled to keyhole limpet hemocyanin was used as an immunogen to produced 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 .mu.l of standard or sample (urine or serum) or 100 .mu.l tracer (150 nmol/l) followed by 100 .mu.l 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 carboxy-primaquine 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. The drug and its metabolites appear in urine within one hour with peak levels after three hours. The highest primaquine level was 1,000 .mu.g/mmol creatinine and dropped quickly to 55 .mu.g/mmol creatinine after 14 hours, being undetectable by 34 hours. The highest level of the metabolites measured was 2,600 .mu.g/mmol creatinine and values fall progressively to 150 .mu.g/mmol creatinine after 24 hours then in increased again to 1,500 .mu.g/l at 36 hours. The second peak may be attributed to the biotransformation of the carboxyprimaquine which seems to be slow and was still detectable after 120 hours. The amount of primaquine recovered in urine during the first 72 hours accounts for 3.0% of the administered dose while the total recovery, including detectable metabolites, accounts for 29.0%.