Camp. Biochem. Physiol. Vol. 102C, No. 3, pp. 483487, 1992 Printed in Great Britain 0306~4492/92 $5.00 + 0.00 0 1992 Pergamon Press Ltd ELISA FOR OXYTOCIN. HIGHLY SENSITIVE TESTS AND APPLICATION FOR THE TITRATION OF AN OXYTOCIN-LIKE SUBSTANCE IN THE LEECH ERPOBDELLA OCTOCULATA MICHEL SALZET,* CHRISTIAN WATTEZ,MARIE-CHRISTINE SLOMIANNY, BERNADETTE LEU and KARL J. SIEGERT Laboratoire d’Endocrinologie des InverttbrCs, URA CNRS 148, UniversitC des Sciences et Techniques de Lille 59655 Villeneuve d’Ascq Cedex, France. Tel.: 20434054; Fax: 2043-4995 (Received 18 October 1991) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP Abstract-l. Three different types of ELISA for oxytocin (OT) were developed: direct, inhibiting and competitive. The amounts of OT which could be detected varied with the type of test: 10 to 100 pmol in direct and inhibiting ELISA, 50 fmol to 10 pmol in competitive ELISA. 2. A comparison between the values of percentage of binding obtained with radioimmunoassay (RIA) and in competitive ELISA showed that this latter assay is as sensitive as the RIA but it offers the advantages of being cheaper and more reproducible. 3. The development of these sensitive ELISA provided a means to quantify OT-like molecules. Their usefulness has been first applied to the central nervous system of the leech, Erpobdellu octoculafu. INTRODUCTION Since their description in 1971 by Engvall and Perlmann, ELISA techniques have improved in sensi- tivity, both at the quantitative and the qualitative level. A great deal of research has been related to the quantification of peptides. These efforts have been aided by polystyrene treatment of microwells and amplification of the signal through chromogenic sub- strates such as streptavidin-biotin (Guesdon et al., 1979) and peroxidase-anti-peroxidase (Hsu et al., 1981) or fluorogenic substrates such as FITC-anti- FITC systems (Harmer and Samuel, 1989; Crowther et al., 1990). One way of modifying polystyrene microwells is to pre-coat the surface with polymers which have reactive groups, e.g. poly+lysine, a co-polymer of poly-L-lysine-arginine or poly-glutaraldehyde (Hobbs, 1989). Biomolecules can then be immobilized with linkers such as succinimidyl-esters or condensing agents like carbodiimide. These surface modifi- cations, however, tend to cause increased background and a risk of peptide desorption (Rasmussen, 1990). To resolve these problems, a new generation of microtitre plates (e.g. CovaLink from NUNC) has been introduced. It is now possible to detect small amounts of peptides e.g. the titration window for angiotensin II can be as low as l-100pmol (Sondergard-Andersen et al., 1990). The aim of the present work was to set up three types of ELISA for oxytocin (OT). The direct ELISA (1) allows the detection of the entire (specific and non-specific) immunoreactivity present in a biological sample. The inhibiting ELISA (2), which can be considered as a negative control, measures only the non-specific immunoreactivity. The difference be- *Please address all correspondence to Michel Salzet. tween the two values obtained respectively in direct and inhibiting ELISA gives an estimate for the amount of specific antigen. The competitive ELISA (3) determines directly only the specific amount of antigen. Highly sensitive and specific ELISA for OT are required for developing studies on the hormonal regulation of water balance in leeches where an OT-like substance present in the central nervous system has an anti-diuretic effect (Malecha et al., 1986, 1989; Verger-Bocquet et nl., in press). In a first test, these techniques have been used for the quantifi- cation of OT-like material in the segmental ganglia of the nerve cord of the leech, Erpobdella octoculata. MATERIALS AND METHODS Animals For quantification of OT-like material in sex ganglia and non-sex ganglia, nerve cords were taken on leeches of the species Erpobdelia octoculata collected in Harchies (Belgium) and maintained in aquaria. Antiserum An antiserum against OT generated in our laboratory was used both in ELISA and RIA. This serum has been well characterized, it is specific of the C-terminal part of OT re;sger-Bocquet et al., in press). Its production was in two Coipling of OT. OT was coupled to thyroglobulin with glutaraldehyde by a one-step method: 4mg OT (INTER- CHIM) were added to a solution of 1Omg bovine thyro- globulin (SIGMA) in 2 ml phosphate-buffered saline pH 7.4. Under constant stirring at PC, an equal volume of 0.5% aqueous glutaraldehyde solution was added dropwise. After 3 hr, the reaction was stopped by the addition of sodium metabisulfite (final concentration 2 mg/ml). Immunization. A rabbit was immunized with OT- glutaraldehyde-thyroglobulin at forty different sites. Simul- taneously, vaccines against tuberculosis and whooping 483