Polyclonal and monoclonal antibodies to PbTx-2-type brevetoxins using minute amount of hapten-protein conjugates obtained in a reversed micellar medium.

Minute amount of Brevetoxin PbTx-3 (400 microg; 0.446 micromol) was converted into an hemisuccinate derivative (PbTx-3 HS) then covalently linked to bovine serum albumin (BSA) and ovalbumin (OVA) in a reversed micellar medium. According to the efficient cyclic synthetic procedure described, the epitope density of the conjugates was around 10 and 20 for OVA and BSA carriers, respectively. The kinetics of antibody production in sequential sera harvested from a single BALB/c mouse immunised by multiple intraperitoneal (i.p.) injections of PbTx-3-BSA conjugate was performed by enzyme-linked immunosorbent assay (ELISA). Two monoclonal antibodies (MAbs) against PbTx-3 were selected from fusion of the mouse immune splenocytes with the P3-X63-Ag 8.653 myeloma cells. In competitive inhibition ELISA experiments, both polyclonal antibodies and MAbs exhibited strong cross-reactivity (> or = 100%) to other PbTx-2-type toxins (PbTx-2 and -9) but low or moderate cross-reactivity (6-15%) to a PbTx-1-type toxin (PbTx-1). Moreover, using these two MAbs, a low cross-reactivity with okadaic acid (3%) was noticed but no significant cross-reactivity was observed with two ciguatoxins (CTX-1B and CTX-3C) over the concentration range studied. The apparent dissociation constant (K(D)) for the interaction of these MAbs with free PbTx-2-type toxins was in the 10(-6)-10(-7)M range. The performance of this MAb-based assay (limit of detection approximately 5ng/well; working range=8-150ng/well) coupled with adequate extraction methods would provide an alternative assay to the mouse i.p. bioassay for routine shellfish monitoring. This production and characterisation of MAbs using small amount of polyether toxins in a reversed micellar medium appear most valuable for the development of immunoassays to other highly potent but poorly available marine polyether toxins like ciguatoxins (CTXs).

Characterization of mice antisera elicited with a ciguatoxin tetracyclic synthetic ring fragment (JKLM) conjugated to carrier proteins.

As a good alternative to the lack of pure ciguatoxin (CTX), conjugates of JKLM ring fragment, a carboxylic derivative of the right-hand tetracyclic terminus portion of CTX-1B (the most potent CTX) with two carrier proteins have been synthesized. Two procedures using different amount of hapten were evaluated: (i) a bulk technique (3-5 mg) via the N-hydroxysuccinimide ester of the carboxylic fragment in the presence of a water-soluble carbodiimide according to the standard method in aqueous buffer, or (ii) a micro-scale technique (300 microg) via the mixed anhydride method performed in a reversed micellar medium. In both cases, bovine serum albumin and ovalbumin were respectively used for immunization of BALB/c mice and antibody screening by a solid phase enzyme-linked immunosorbent assay (ELISA). Using the conjugates obtained through the micro-scale procedure, a long-term immunization schedule appeared to be more efficient to specifically trigger the mice immune system. These antisera titers determined in an end-point titration standard ELISA format were found around 1/128,000 as compared to 1/16,000 obtained in the short-term protocol (immunogen prepared via the bulk procedure). In competitive inhibition ELISA experiments, both types of antisera did not significantly cross-react with a brevetoxin congener (PbTx-3), okadaic acid (OA), monensin or other polyether compounds, but only sera from the short-term protocol did show high cross-reactivity to CTX-1B (133%). With sera from the long-term protocol, a lower detection limit for JKLM (1.23 x 10(-9) M) was achieved by implementation of a biotin-avidin amplification system rather than by miniaturization of the assay in Terasaki plates. This study confirms the feasibility of the immunological approach for CTXs assay in fish tissues, but also emphasizes the importance of (i) the choice of the hapten to construct a relevant well-defined immunogen, (ii) the immunization schedule to obtain hapten-specific Abs still exhibiting high cross-reactivity to CTXs.

Preparation and characterization of domoic acid-protein conjugates using small amount of toxin in a reversed micellar medium: application in a competitive enzyme-linked immunosorbent assay.

With the aim of producing novel antibodies to domoic acid (DA), an original, rapid, and simple procedure for preparing minute amount of hapten-protein conjugates was developed. The amide-bond-generating mixed anhydride method of Erlanger was performed using 0.32-0.64 micromol of DA in a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement. Bovine serum albumin (BSA) and ovalbumin (OVA) conjugates were, respectively, used for immunization of BALB/c mice and antibody screening by enzyme-linked immunosorbent assay (ELISA). Specific polyclonal antibodies were produced upon multiple injections of (DA)(17)-BSA conjugate administered by three different routes: (i) intraperitoneal (i.p.), (ii) intraperitoneal + subcutaneous (i.p. + s.c.), (iii) footpad (f.p.). The i.p. route induced antisera of higher titer (1:350000) than did the other protocols (approximately 1:72900) and was selected throughout further experiments. Using a competitive ELISA format with a peroxidase immunoconjugate and a chromogenic substrate, no significant cross-reactivity was observed with glutamic acid, aspartic acid and kainic acid (KA), a structural analogue of DA. The sensitivity of this assay could be enhanced by 1 order of magnitude by using a beta-galactosidase immunoconjugate with a fluorogenic substrate while preserving DA specificity. The calculated dissociation constant (K(D)) for the interaction of the antibodies with free DA was 5 x 10(-)(7) M (chromogenic assay) and 5 x 10(-)(8) M (fluorogenic assay). Using the optimized assay the limit of detection (LOD) and the limit of quantitation (LOQ) in the ELISA buffer were 1.4 and 3 ng/mL, respectively. Moreover this assay was found applicable for measuring DA levels in spiked mussel extracts pre-cleaned through a solid-phase extraction column, as a very good correlation (r(2) = 0.96) was observed between the actual amounts of DA added and amounts detected by ELISA. Thus, accurate determinations of DA in clean extracts could be achieved between 2 and 180 ng/mL in spiked samples which corresponds to 0.02-1.8 microg/g of original mussel tissue. Owing to the regulation limits of 20 microg DA/g of shellfish tissue, these extraction and assay procedures should provide a useful complement to the standard HPLC analytical technique currently employed in monitoring DA in shellfish tissue.

An improved method for the microscale preparation and characterization of hapten-protein conjugates: the use of cholesterol as a model for nonchromophore hydroxylated haptens.

A minute amount (0.446 micromol) of cholesterol (Chol) was converted into an hemisuccinate derivative (Chol HS) using an excess of succinic anhydride. The optimal conditions for synthesis of Chol HS were explored by checkerboard experiments in which various succinic anhydride/Chol molar ratios ranging from 5:1 to 30:1 were assayed over a wide temperature range (50-85 degrees C) and for various incubation times (3-8 h). Total conversion was obtained at the higher reagent ratios, temperatures, and incubation times. Subsequently, this carboxylic derivative was first covalently linked to bovine serum albumin (BSA) then to various proteins (casein, ovalbumin, and hemocyanins) or to a synthetic homopolymer (poly-DL-Lysine) via a modified version of the mixed anhydride method of Erlanger, performed in a reversed micellar medium. The assessment of the number of haptenic groups per mole of BSA (epitope density) was achieved chromatographically by two methods according to a Chol standard curve established at 207 nm with linearity in the range 0-50 microg. These procedures involving an alkaline hydrolysis of a sample of either the conjugate (direct method) or the unreacted Chol HS (indirect method) yielded an acceptable level of agreement and concordant results in all cases. The influence of the activated hapten/BSA molar ratio on the coupling efficiency was investigated by the direct method within the range 10:1 to 250:1. Using the optimal conditions determined for Chol HS synthesis (a molar reagent ratio of 30:1 with incubation at 65 degrees C for 6 h) and for BSA haptenation (a 100-fold molar excess of activated hapten, with a carrier stock concentration of 5 mg/mL), epitope density of the conjugates lied between 23 and 27. By reacting the same amount of activated hapten ( approximately 216 microg) with identical amounts of various carriers (300 microg), conjugation efficiency was found similar on a microgram of Chol bound per milligram of carrier basis. This simple and reproducible conjugation and analysis procedures should provide a general method applicable to poorly available and weakly immunogenic haptens bearing hydroxyl groups such as polyether-type marine toxins.

An improved method for the production of antibodies to lipophilic carboxylic hapten using small amount of hapten-carrier conjugate.

A rapid, simple and low cost procedure for preparing minute amount of hapten-protein conjugates was developed using 4-acetyl benzoic acid (ABA) and two other closely related small chromophoric haptens. The amide bond-generating mixed anhydride method of Erlanger was modified to promote conjugation to various proteins (bovine serum albumin, ovalbumin, casein and hemocyanin) or to a synthetic homopolymer (Poly-DL-lysine). The key process in this synthesis is the use of a reversed micellar medium allowing strong carrier haptenization as determined by spectrophotometric measurement at characteristic hapten absorption peaks. This coupling procedure is applicable to as little hapten material as 0.2 micromol and is disclosed to be most valuable for other rare lipid haptens which pose analytical problem in biological fluids and matrices. Specific mice polyclonal antibodies were produced following multiple intraperitoneal injections of (ABA)23-BSA conjugate as revealed by indirect and competitive ELISA. Calculated KD for the interaction of the antibodies with free ABA was found to be 5 X 10(-5)M.