Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin.

A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX. The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX.

Development and validation of a maleimide-based enzyme-linked immunosorbent assay for the detection of tetrodotoxin in oysters and mussels.

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at -20°C and 4°C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health.

Development of ELISA and colloidal gold immunoassay for tetrodotoxin detetcion based on monoclonal antibody.

A monoclonal hybridoma cell named 5B9 against tetrodotoxin (TTX) was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The 5B9 monoclonal antibody (McAb) with high affinity (about 2.55 × 10(9)) is specific to TTX, and this McAb belongs to the immunoglobulin G (IgG) isotype. Finally, an enzyme-linked immunosorbent assay (ELISA) and colloidal gold immunoassay were established based on this McAb. The linear range of ELISA to detect TTX was 5-500 ng/mL, and the limit of detection (LOD) was 4.44 ng/mL. The average CV of intra- and inter-assay was less than 8%, with the samples recovery range of 70.93-99.99%. A competitive format colloidal gold strip was developed for detection of TTX in real samples, and the LOD for TTX is 20 ng/mL, and the assay time of the qualitative test can be finished in less than 10 min without any equipment. The result from test strip revealed that the test strip has a good agreement with those obtained from ELISA.

Construction of a single chain variable fragment antibody (scFv) against tetrodotoxin (TTX) and its interaction with TTX.

Tetrodotoxin (TTX) is a small molecular weight neurotoxin that occludes voltage-gated sodium channels in nerve and muscle tissue, resulting in respiratory paralysis and death. A high affinity antibody that can neutralize the toxicity of TTX is still lacking, so it is very important to prepare an antibody for TTX therapy and detection. In the present study, a chemical method was used to prepare the tetrodotoxin complete antigen, and a small amount, repeatedly immunity way was carried to immunize 4 mice. The amplified genes encoding monoclonal antibodies against TTX were used to construct the phage display library. After six rounds of biopanning, an antibody named scFv-T53 was characterized from clones showing high affinity and specific to TTX, and its affinity constant was 1.1 × 10(6) L/mol. Three dimensional structure of the scFv-T53 was constructed by computer modeling, and TTX was docked to the scFv-T53 model to obtain the structure of the binding complex. Two predicted essential amino acids, K183 and I189, were mutated to verify the theoretical model. Both mutants lost binding activity significantly against TTX as predicted by the theoretical model. Hence, the above results will be useful for screening the high affinity anti-TTX scFv mutants.

Larval pufferfish protected by maternal tetrodotoxin.

Marine pufferfish contain tetrodotoxin (TTX), an extremely potent neurotoxin. All species of the genus Takifugu accumulate TTX in the liver and ovaries, although the tissue(s) in which it is localized can differ among species. TTX is the major defense strategy the pufferfish appears to use against predators. TTX is also used as a male-attracting pheromone during spawning. Here we demonstrate an additional (and unexpected) use of maternal TTX in the early larval stages of the Takifugu pufferfish. Predation experiments demonstrated that juveniles of all the species of fish used as predators ingested pufferfish larvae, but spat them out promptly. Liquid Chromatography-Tandem Mass Spectrometry (LC-MSMS) analysis revealed that the pufferfish larvae contain a small quantity of TTX, which is not enough to be lethal to the predators. Immunohistochemical analysis with anti-TTX monoclonal antibody revealed that the TTX is primarily localized in the body surface of the larvae as a layer of protection. Our study showed the female parent of the Takifugu pufferfish vertically transfers TTX to the larvae through its accumulation in the ovaries, and subsequent localization on the body surface of the larvae.

Development of competitive indirect ELISA for the detection of tetrodotoxin and a survey of the distribution of tetrodotoxin in the tissues of wild puffer fish in the waters of south-east China.

A monoclonal antibody against tetrodotoxin (TTX) was produced from the hybridoma cell line T6D9, which was established by the fusion of Sp2/0 myeloma cells with spleen cells isolated from a Balb/c mouse immunized with the TTX-keyhole limpet hemocyanin (KLH) conjugate. This monoclonal antibody belongs to the IgG1 subclass; the affinity constant of the antibody is 2.4 × 10(-8) mol l(-1). The relative cross-reactivity of the antibody with TTX was 100%, but with saxitoxin, KLH and bovine serum albumin (BSA) it was less than 1%, respectively. The titre of the antibody in ascites was 6.4 × 10(6); the reference working concentration was 1:1.2 × 10(5). By using this monoclonal antibody, a competitive indirect enzyme-linked immunoabsorbant assay (ELISA) for the analysis of TTX was developed. The linear portion of the dose-response curve of TTX concentration was in range 5-500 ng ml(-1). The limit of detection was 5 ng ml(-1) according 10% inhibition with TTX to anti-TTX monoclonal antibody. The concentration of TTX inhibiting 50% of antibody binding was about 50 ng ml(-1). The recoveries from TTX spiked samples were 79.5-109.5%. In addition, the toxicity of some wild puffer fish specimens captured from south-east China and the Yangzi River in Jiangsu province was determined. The results indicate that the toxicity and toxin tissue distribution vary in different species of wild puffer fish.

[Selecting the phage displaying tetrodotoxin mimic epitope by phage random peptide library].

OBJECTIVE: To screen the positive phage displaying the mimic epitope of tetrodotoxin (TTX) by using phage random peptide display library technology and to establish immunoassay for the detection of tetrodotoxin. METHODS: Monoclonal antibody against TTX was used as a ligand to screen the binding peptide from the Ph. D.-7 peptide library. The library is displayed as a fusion protein with the coat protein III of filamentous phage M13. The positive clones were identified by ELISA. Results After four rounds of panning, 7 positive phages binding to the anti-TTX monoclone antibody were obtained, and through indirect competitive ELISA, 3 positive clones inhibiting TTX were screened. A competitive ELISA was established with phage P4, the linear range of the inhibition is 1-20 ng/ml, R2 = 0.9947, the detecting limit is 1 ng/ml. CONCLUSION: The phage display technique can be successfully applied to screen the mimic epitope of tetrodotoxin. The acquired phages may be used as the surrogate of the toxin to establish immunoassay.

[Study on the antibody against tetrodotoxin made by the immunogen with different carrier proteins].

OBJECTIVE: To compare the immunogenity of the TTX artificial antigen with two different carrier proteins, bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH), and antiserum against each immunogen were well investigated. METHODS: The hapten Tetrodotoxins (TTX) were linked to carrier protein BSA or KLH as immunogen, and linked ovalbumin (OVA) as coating antigen by the Mannich method. Then the 6-8 weeks Babl/c mice were immunized with TTX-BSA or TTX-KLH. And the TTX-BSA also was another coating antigen in the detection of TTX-KLH antiserum. The antiserum was detected by indirect enzyme-linked immunosorbent assay (iELISA) and indirect competitive enzyme-linked immunosorbent assay (icELISA). RESULTS: The high titer of antiserum was 1:64000 after immunizing mouse. In the detection of TTX-KLH antiserum, the A450 value with the TTX-OVA coating antigens was a little higher than the TTX-BSA coating antigen, and IC50 was 10-20 ng/ml by icELISA. CONCLUSION: The artificial antigen was successfully synthesized, and the conjugate of TTX-KLH was testified to have a better immunogenity in comparison with TTX-BSA, this investigation will benefit development of TTX immunoassay.

Influence of carrier proteins on the immunologic response to haptenic antitetrodotoxin vaccine.

Tetrodotoxin (TTX) is a haptenic, highly toxic neurotoxin with no specific antidote available yet. Anti-TTX vaccine is being studied for antitoxin development. The effectiveness of the carrier protein in eliciting TTX-specific antibody response was comparatively studied. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH), Limulus polyphemus hemocyanin (LPH), tetanus toxoid (TT), diphtheria toxoid (DT), and bovine serum albumin (BSA) chemically to form artificial antigens TTH-TTX, LPH-TTX, TT-TTX, DT-TTX, and BSA-TTX, respectively, with which BALB/c mice were immunized, and the antibody response and antitoxic efficacy were detected. The serum anti-TTX antibody response and antitoxic efficacy varied markedly with adopted carrier protein. TTH-TTX elicited the best and BSA-TTX the worst TTX-specific antibody response. The proportion of the immunized mice surviving a 3x lethal dose (LD) dose of TTX challenge was 92%, 75%, 42%, 8%, and 0% for TTH-, TT-, LPH-, DT-, and BSA-TTX conjugates, respectively. The rank order of total efficacy of carrier protein for both anti-TTX antibody response and antitoxic effect was TTH > TT > LPH > DT > BSA. As a result of formaldehyde treatment in coupling of TTX carriers, the relative immunogenicity of TTX vs carrier, that is, the ratio of TTX- to carrier-specific antibody response, evidently varied with respective carrier adopted, in a rank order of TT > BSA > TTH > DT > LPH. The results suggest that the carrier protein used in haptenic TTX vaccine is greatly important in eliciting potent anti-TTX antibody, and both TTH and TT are the preferred carriers for development of excellent experimental TTX vaccine.

Study of a toxin-alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination.

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4-15 ng mL(-1) with a limit of detection (LOD) of 2 ng mL(-1) (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2-50 ng mL(-1) and the LOD was 1 ng mL(-1).

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

Tetrodotoxin (TTX) is a high toxic small molecular neurotoxin. Haptenic vaccine for TTX was investigated and the carrier proteins were compared. TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH) and tetanus toxoid (TT) via formaldehyde to form the artificial antigen TTX-TTH and TTX-TT. BALB/c mice were immunized with the artificial antigen, the TTX-specific antibody response were detected. The immunized animals were intragastrically challenged with increasing doses of TTX repeatedly. The mice which exposed to TTX in doses of 600, 630, 800, 1200, 1500, 2000 and 2400 microg/kg survived at rates of 100, 100, 90, 90, 80, 50 and 20%, with a LD(50) value of 2020 microg/kg for TTH-TTX vaccine, and of 100%, 90.9%, 90.9%, 90.9%, 63.6%, 27.3% and 0%, with a LD(50) value of 1410 microg/kg for TT-TTX vaccine, respectively. All control mice inoculated with carrier protein TTH or TT uniformly died of a dose of 600 microg/kg TTX i.g. challenge. Animals immunized with vaccines could antagonize repeated TTX challenge, half of them surviving about 6 mg/kg, and a few being able to bear a maximal accumulative dose as high as approximate 9 mg/kg of TTX challenges within eight months. The TTH-TTX vaccine was of the more excellent in protective effect from TTX oral intoxication, mainly resulted from the higher antibody affinity than that from TT-TTX vaccine. The present study for the first time demonstrated that the anti-TTX experimental vaccines would high effectively protect animal from multiple, oral TTX intoxication. Immunoprophylaxis would be the hopeful means against TTX poisoning.

Toxin-neutralizing effect and activity-quality relationship for mice tetrodotoxin-specific polyclonal antibodies.

The polyclonal antibodies specific for tetrodotoxin (TTX) were prepared from mice and their capacity of neutralizing TTX was investigated so as to explore the possibility of developing TTX antitoxin. Haptenic TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH) chemically to form artificial antigen TTX-TTH. BALB/c mice were immunized with TTX-TTH and ascites were induced by intraperitoneal administration of Freund's adjuvant. Twenty strains of TTX-specific ascites antibody with apparent affinity varying from 10(-4) to 10(-7)M were obtained. KM mice were challenged with lethal doses (1LD = 14.0 microg/kg, i.p.) of TTX neutralized by antibodies to evaluate the power of antitoxin. The potential of TTX-neutralizing of the antibodies was approved by the increase in survival animal challenged by lethal doses of TTX pre-incubated in vitro or neutralized in vivo with TTX specific antibodies. The highest protection was observed with all animals survived challenge of 1.5 x LD TTX neutralized in vitro, and antibody administration 4 days prior to 1.3 x LD TTX challenge in vivo neutralization. The protective efficiency was antibody quality factor dependent and with the highest detoxifying immunological equivalent as high as 1 300 microg (TTX)/L(ascites) approximately, while the antibody apparent affinity being at the order of 10(-6) to 10(-7)M. These results suggested that chemical vaccine for haptenic TTX could successfully raise high humoral immune response and the antibodies could neutralize TTX effectively both in vitro and in vivo, antibody therapy would be the hopeful means for detoxification of TTX.

[Influence of immunization dose schemes on the immune response to anti-tetrodotoxin vaccine].

OBJECTIVE: To study the relationship between the immune response of anti-tetrodotoxin vaccine, including its dose-response, and to select optimal immunization dose so as to enhance antitoxic effect of the anti-tetrodotoxin vaccine. METHODS: Tetrodotoxin (TTX) was coupled to Tachypleus tridentatus hemocyanin (TTH) chemically to form artificial antigen (TTX-TTH), and with which Balb/c mice were immunized. Influence of different immunization doses [100 microg as the higher (H) and 25 microg as the lower (L) dose group] on the protective effects of TTX vaccine was compared. The quality of antisera and effects of vaccine in anti-TTX poisoning were observed. RESULTS: The sera antibody quality increased more quickly in group L than that in group H after immunization. The dose at which the half of immunized mice survived when challenged once with TTX were 16 x LD (1 LD = 13.5 microg/kg, i.p.) in group L and 11 x LD in group H. When TTX was used time and again, the half of immunized mice could tolerate as high as 40 x LD and 22 x LD of accumulated dose, and the maximum tolerable cumulated dose was 104 x LD and 90 x LD for group L and H respectively. The scheme L was better both in antibody quality and effect of protecting against TTX toxicity than that in scheme H. CONCLUSIONS: The experimental vaccine of TTX could effectively protect animal from TTX intoxication. The lower immunization dose in this study is selected as the optimal immunization scheme.

An amiloride-sensitive and voltage-dependent Na+ channel in an HLA-DR-restricted human T cell clone.

We investigated changes in voltage-gated Na+ currents and effects of extracellular Na+ on proliferation in HLA-DR-restricted human CD4+ alphabeta T cells after stimulation with a non-self antigenic peptide, M12p54-68. In the absence of antigenic peptide, neither single (n = 80) nor APC-contacted (n = 71) T cells showed voltage-gated inward currents recording with whole-cell patch-clamp techniques, even with Ca2+ and Na+ ions present in the perfusion solution. However, with the same recording conditions, 31% (26 of 84) of APC-contacted T cells stimulated with the antigenic peptide showed voltage-dependent inward currents that were elicited from -60 mV. The inward currents were not inhibited in extracellular Ca2+-free conditions or in the presence of 1 mM NiCl2. However, they were completely inhibited in extracellular Na+-free conditions, which were made by replacing Na+ with iso-osmotic N-methyl-d -glucamine or choline. The Na+ currents were insensitive to tetrodotoxin, a classical blocker of Na+ channels, but were dose-dependently inhibited by amiloride, a potassium-sparing pyrazine diuretic. Furthermore, the Ag-specific proliferative response of T cells was completely inhibited in Na+-free Tyrode's solution and was suppressed by amiloride in a dose-dependent manner. Our findings suggest that activation of amiloride-sensitive and voltage-gated Na+ channels would be an important step to allow an adequate influx of Na+ and maintain a sustained high Ca2+ level during T cell activation.

Reexamination of tetrodotoxin production by bacteria.

Vibrio alginolyticus has been reported as a good producer of tetrodotoxin (TTX), but the toxin extracted from this bacterium did not react to the monoclonal antibody against TTX. Surprisingly, chromatographic analyses detected high TTX peaks for polypeptone and yeast extracts used as medium materials, which were, as expected, all negative by the mouse bioassay. These results may require us to revise the bacterial production of TTX.

Prophylaxis and treatment with a monoclonal antibody of tetrodotoxin poisoning in mice.

The ability of a tetrodotoxin (TTX)-specific monoclonal antibody to confer passive protection against lethal TTX challenge was investigated. The monoclonal antibody, T20G10, has an estimated affinity for TTX of approximately 10(-9) M and is about 50-fold less reactive with anhydrotetrodotoxin and unreactive with tetrodonic acid by competitive immunoassay. T20G10 specifically inhibited TTX binding in an in vitro radioligand receptor binding assay, but had no effect on the binding of saxitoxin to the sodium channel on rat brain membranes. In prophylaxis studies, mice were administered T20G10 via the tail vein 30 min prior to i.p. TTX challenge (10 micrograms/kg). Under these conditions, 100 micrograms T20G10 protected 6/6 mice, whereas 3/6 mice were protected with 50 micrograms T20G10. Non-specific control monoclonal antibody did not protect against lethality. Therapy studies simulating oral intoxication were performed with mice given a lethal dose of TTX by gavage in a suspension of non-fat dry milk in phosphate-buffered saline. Death occurred within 25-35 min in 6/6 mice not treated with T20G10. However, 500 micrograms T20G10 administered via the tail vein 10-15 min after oral TTX exposure prevented death in 6/6 mice. Lower doses of mAb conferred less protection.

In vivo neutralization of tetrodotoxin by a monoclonal antibody.

This study examined the ability of monoclonal antibody against TTX to neutralize tetrodotoxin (TTX) in vivo. The mice were injected i.p. with 1.5 mouse units of TTX, followed 3 min later by administering graded doses of antibody immunoglobulin G (IgG). Animals injected with 100 micrograms IgG through the tail vein showed 100% survival. The 50% protective dose was c. 2 mg/kg. The present results indicate that monoclonal antibody can neutralize TTX in vivo.

A monoclonal antibody against tetrodotoxin that reacts to the active group for the toxicity.

A monoclonal antibody against tetrodotoxin was produced. Tetrodotoxin coupled with keyhole limpet hemocyanin was used as an immunogen to BALB/c mice. These mice had no clinical signs for the toxicity of tetrodotoxin during the immunization. The reason may be that the guanidyl group of tetrodotoxin which is an important group for the toxicity was hidden by coupling with keyhole limpet hemocyanin. The monoclonal antibody was highly specific for tetrodotoxin and had no cross-reaction to tetrodotoxin derivatives, paralytic shellfish toxins, keyhole limpet hemocyanin and crude proteins from various organs of puffer fish. Also, tetrodotoxin was neutralized in vitro by this antibody. From the fact that the structural difference between tetrodotoxin and anhydro-tetrodotoxin is recognized by this antibody, it was suggested that this antibody reacted with the OH-groups on C-4 and/or C-9 of tetrodotoxin. In addition, the results from immunization and neutralization tests demonstrated that tetrodotoxin became non-toxic even when one of the active groups of tetrodotoxin was coupled by a molecule.

[Depression of TTX-blocking effect on Na+ current by 8A5, an anti-TTX monoclonal antibody].

Anti-TTX effect of 8A5 on Na+ current was observed by whole-cell voltage-clamp technique in NG108-15 cells. The results show that 8A5 inhibits TTX action efficiently: (1) When 1 mumol/L TTX is added to the normal perfusion solution INa disappears immediately, and can be restored to the control level with futher addition of 8A5. (2) When the cell is pre-incubated with 1 mumol/L 8A5, 1 mumol/L TTX can only cause a partial inhibition of INa.

A tetrodotoxin neutralizing system in the haemolymph of the horseshoe crab, Carcinoscorpius rotundicauda.

The horseshoe crab, Carcinoscorpius rotundicauda, when injected intracardially with tetrodotoxin (TTX) at a dosage of 10 x LD50 of mice showed no mortality. The resulting haemolymph extracted 24 hr later from these crabs caused no fatalities when injected into mice. TLC analysis revealed that cell free haemolymph (CFH) had converted a single yellow fluorescent spot of TTX into two blue fluorescent components when the reaction was carried out at 25 degrees C. However, no fluorescence was observed when TTX was reacted with CFH at 37 degrees C. There was also an increase in the intensity of protein bands on SDS-PAGE gel corresponding to 26,000 and 28,000 mol. wts. This may be the result of the TTX-neutralization reaction in CFH. The pretreatment of TTX with CFH reduced the toxicity of TTX in mice by two-fold. The anti-TTX activity is temperature and time dependent as well as heat stable. Protein denaturation of the haemolymph suggests that proteinaceous factor(s) may also play a role in TTX-neutralizing activity of CFH. It is therefore possible that the detoxification system is attributed to concerted actions of both proteinaceous and non-proteinaceous factors in the haemolymph.