Homogeneous liposome immunoassay for insulin using phospholipase C from Clostridium perfringens

A new homogeneous liposome immunoassay system was developed in which analyte-phospholipase C conjugates are used instead of complement or melittin. This system was applied for the determination of insulin. Liposomes incorporated with calcein as a marker were prepared by the reverse-phase evaporation method. The lysis of liposomes was determined by measuring the fluorescence intensity of calcein released from liposomes and it was increased with increasing concentration of phospholipase C. Insulin was conjugated to phospholipase C by a three-step procedure with hetero-bifunctional crosslinking reagents, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester. The lytic activity of phospholipase C was not affected by the reaction for conjugation. Both p-nitrophenylphosphatidylcholine hydrolytic activity and liposome lytic activity of insulin-phospholipase C conjugate were inhibited in the presence of insulin antiserum. However, lower concentration of conjugate and shorter incubation time were required when liposomes were used in the inhibition study. The antibody inhibition of conjugate-induced lysis could be reversed by addition of a competing amount of free insulin. The standard calibration curve was obtained in the range between 4 and 130 microIU/ml (r = 0.994). The detection limit (8 microIU/ml) was comparable with those of conventional heterogeneous enzyme immunoassays. This new assay approach offers a simple, sensitive, and inexpensive testing procedure for determining ultratrace amounts of bioactive substances.