Protection against nerve toxicity by monoclonal antibodies to the sodium channel blocker tetrodotoxin.

Abstract

The sodium channel blocker, tetrodotoxin (TDT), was conjugated to keyhole limpet hemocyanin (KLH) and used to immunize BALB/c mice. Anti-TDT antibodies were detected in serum by ELISA and reached stable levels 4-5 wk after the first immunization. Spleens from immunized mice were fused with NS-1 mouse myeloma cells and -9,329 resultant hybrids were screened by ELISA for reactivity to TDT. Two stable hybrids were isolated, subcloned, and characterized. These hybrids, termed TD13al and TD2C5, secreted specific anti-TDT antibodies that recognized TDT but not the related sodium channel blocker, saxitoxin (STX), as determined by competition ELISA. Both antibodies were of the IgGlk subclass with K.'s approaching 107 M-'. The inhibitory ability of these antibodies was tested by a competitive displacement assay for [3HJSTX on rat brain membranes. Both antibodies strongly inhibited TDT binding to membranes. A nanomole of TD2C5 was able to bind 1.8 nmol of TDT, whereas a comparable amount of TD13al bound half as much. Furthermore, TD2C5 was able to protect against TDT-induced reduction of peripheral nerve action potentials in rat tibial nerve when administered in situ. These antibodies thus represent potentially useful reagents for neurobiologic research, detection of toxin contamination and diagnosis of poisoning, and may provide protection against the toxicity of TDT in vivo.

Immunologic protection of anti-tetrodotoxin vaccines against lethal activities of oral tetrodotoxin challenge in mice

“…The competition-inhibited enzyme immunoassay (CIEIA) described by previous report was used with modifications [11,25,26], the mixtures of TTX in a series of molar concentration (10 À3 -10 À7 M) and test antibody specimens in definite dilution of 10 À2 -10 À5 at which 50% antibody binding yielded by ELISA were incubated with BSA-TTX coated onto 96-well microtiter plate at 37 8C for 0.5 h. The plate was washed, then the GAM-HRP solution was added to wells and the rest steps were conducted same as above. In this assay system, the free TTX compete with BSA-TTX for specific antibody, and the binding of antibody to BSA-TTX was competitively inhibited to different extent by added varying conc.…”

“…Immunoprophylactic treatment was suggested as a new potential approach to protect animals against TTX poisoning. Early monoclonal antibodies to TTX could protect against TTX-induced nerve toxicity in vitro thereby used as useful regents for neurobiological research [11][12][13]. Fukiya and Matsumura [14] first reported active immunization for TTX.…”

A monoclonal antibody‐based immunoassay for detecting tetrodotoxin in biological samples

“…Keyhole limpet hemocyanin-TTX-formaldehyde conjugate (KLH-TTXF) was prepared using a modification of the methods of Huot et al (12) and of Chu and Fan (14); 300 pl TTX (1 mgiml), 700 pl 1 M sodium acetate buffer, pH 7.4, 12 1 ~1 KLH (34.9 mg/ml), and 60 ~1 3 7 % formaldehyde were added dropwise (in that order), to an amber glas vial while stimng on a vortex mixer. The reaction mixture was then placed on a shaker and incubated at 37°C for 3 days.…”

The Use of Enzyme-linked Immunosorbent Assays to Study the Plasma Disposition of Sheep Polyclonal and Rat Monoclonal Digoxin-specific Fab Fragments in the Rabbit

“…They may also have a use as a diagnostic aid in suspected cardiac glycoside overdose involving dysrhythmias (Bagrov et al 1989) and in the treatment of pathological conditions such as eclampsia (Goodlin 1988(Goodlin , 1989 where 'endogenous digitalis' is postulated to have a role. Furthermore, drug-specific Fab fragments could have a use in the treatment of poisoning by other drugs and toxins such as phencyclidine (Owens & Mayersohn I986), desipramine (Hunting et al 1989) and tetrodotoxin (Huot et al 1989).…”

“…Although digoxin-specific Fab fragments used clinically are derived from polyclonal antibodies raised in sheep, there is an increasing tendency to prepare rodent monoclonal antibodies which can be used in drug toxicity reversal (Mudgett-Hunter et al 1982;Bowles et a1 1988;Huot et al 1989). Indeed, it has been demonstrated that mouse monoclonal digoxin-specific Fab fragments effectively reversed the deleterious effects of a potentially lethal dose of digoxin in guinea-pigs (Lechat et al 1984).…”