Improvement of antibody production to PbTx-2 type brevetoxins and development of a new radioimmunoassay.

Publication Type  Book Chapter
Year of Publication  1998
Authors  Naar, J.; Pauillac, S.; Branaa, P.; Dechraoui Bottein, M. Y.; Chinain, M.; Legrand, A-M.
Book Title  Harmful Algae
City  Xunta de Galicia (Spain)
Publisher  Reguera B, Blanco J, Fernandez ML, Wyatt T (Eds). Intergovernmental Oceanographic Commission of UNESCO
Pages  567-70
Language  eng

PbTx3 was converted into an hemisuccinate derivative (PbTx3 HS) and convalently linked to bovine serum albumin (BSA) and ovalbumin (OVA) via the mixed anhydride method. Compared to other studies, five major improvements have been achieved in the process of antigen preparation : 1) PbTx3 HS was produced with a 99% yield with a little as 400 µg starting material ; 2) quantitation of PbTx3 and PbTx3 HS was performed using an original high performance liquid chromatography (HPLC) protocol ; 3) the mixed anhydride method of Erlanger was successfully adapted to minute hapten quantities with a moderate initial hapten/carrier molar ratio ; 4) PbTx3 HS in excess was purified and determined either by HPLC or by its ability to inhibit [3H] PbTx3 binding to rat brain synaptosomes ; 5) radioactive probe was not required to monitor antigen preparation. Multiple intraperitoneal immunization of a BALB/c mouse with a PbTx3-BSA conjugate elicited the production of toxin specific polyclonal antibodies (PAbs) as revealed by ELISA. Moreover the specific reaction of these PAbs with free [3H]- PbTx3 was used to develop a new, rapid and simple competitive radioimmunoassay (RIA) format for the detection of PbTx3. RIA experiments revealed a dissociation constant (KD) of 4. 10-8 for PbTx3 and a detection limit of 0.33 picomoles with a sensitivity range between 0.01 and 1,100 picomoles. The fusion of mouse splenocytes with an histocompatible myeloma cell line yielded 67 antibody-producing hybridomas which are currently analyzed for PbTx3 specificity. It was concluded that analyzed for PbTx3 specificity. It was concluded that this entire procedure could be applicable to other poorly available haptens.

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