obs管家离线版:请问河豚鱼毒素(TTX)的检测方法是什么?有国标吗?有没有快速检测方法及其相关资料呀??

tetrodotoxin
Tetrodotoxin
Formula C11H17N3O8
LD50 5.0 - 8.0 µg/kg
Molecular mass 319.28 u
Tetrodotoxin (anhydrotetrodotoxin 4-epitetrodotoxin, tetrodonic acid, TTX) is a potent neurotoxin, which blocks action potentials in nerves by binding to the pores of the voltage-gated sodium channels in nerve cell membranes. The binding site of this toxin is located at the pore opening of the voltage-gated Na+ channel. Its name derives from Tetraodontiformes, the name of the order that includes the pufferfish, porcupinefish, ocean sunfish or mola, and triggerfish, several species of which carry the toxin. Although tetrodotoxin was discovered in these fish and found in several other animals, it is actually the product of certain bacteria such Pseudoalteromonas tetraodonis, some species of Pseudomonas and Vibrio, as well as some others.

Fish poisoning by consumption of members of the order Tetraodontiformes is one of the most violent intoxications from marine species. The gonads, liver, intestines, and skin of pufferfish can contain levels of tetrodotoxin sufficient to produce rapid and violent death. Toxicity varies between species and at different seasons and geographic localities, and the flesh of many pufferfish may not usually be dangerously toxic.

Tetrodotoxin has also been isolated from widely differing animal species, including western newts of the genus Taricha (where was termed "tarichatoxin"), parrotfish, toads of the genus Atelopus, several species of blue-ringed octopuses of the genus Hapalochlaena (where it was called "maculotoxin"), several seastars, an angelfish, a polyclad flatworm, several nemerteans (ribbonworms) and several species of xanthid crabs. The toxin is variously used as a defensive biotoxin to ward off predation, or as both a defensive and predatory venom (the octopuses and ribbonworms). Tarichatoxin and maculotoxin were shown to be identical to tetrodotoxin in 1964 and 1978, respectively.

Common causes of tetrodotoxin poisoning include the eating of pufferfishes known as fugu, which is a popular delicacy in Japan and Korea and often contains significant amounts of toxin in its liver and other viscera. Between 100 and 200 people suffer serious tetrodointoxication each year in Japan from eating fugu, and about half of them die. There is no antidote to the toxin, which is heat stable and is not destroyed by cooking. The only treatment is gastric lavage early in the course of the poisoning and life supportive measures, including artificial ventilation. If a victim survives for a day or two, recovery is complete with no sequelae. In severe intoxications, the victim is completely paralyzed and some patients have been pronounced dead by competent medical authorities, only to revive later. However, this potent neurotoxin does not cross the blood-brain barrier and the mind is not affected. Throughout the entire ordeal, the victim remains conscious and aware of what is happening. Blue-ringed octopuses, which inhabit tidepools, also contain tetrodotoxin as the toxic faction in their venom, and several people have died from their bites. The lethal dose in mice and humans is 5 to 8 micrograms (millionths of a gram) per kilogram of body mass, making it one of the most potent toxins known. Less than half a milligram can kill an adult human. Only palytoxin and certain bacterial protein endotoxins are more potent.

Biochemistry
Tetrodotoxin binds to what is now known as site 1 of the voltage-gated sodium channel. Site 1 is located at the extracellular pore opening of the ion channel. The binding of any molecules to this site will temporarily disable the function of the ion channel. Saxitoxin and several of the conotoxins also bind the same site.

The use of this toxin as a biochemical probe has elucidated two distinct types of voltage-gated sodium channels present in humans: the tetrodotoxin-sensitive voltage-gated sodium channel (TTX-s Na+ channel) and the tetrodotoxin-resistant voltage-gated sodium channel (TTX-r Na+ channel). Tetrodotoxin binds to TTX-s Na+ channels with a binding affinity of 5-15 nanomolar, while the TTX-r Na+ channels bind TTX with low micromolar affinity. Nerve cells containing TTX-r Na+ channels are located primarily in cardiac tissue, while nerve cells containing TTX-s Na+ channels dominate the rest of the body.

Total Synthesis
Y. Kishi et al Nagoya University, Nagoya, Japan, (now at Harvard University) reported the first total synthesis of D,L-tetrodotoxin in 1972. Isobe et al at Nagoya University, Japan and J. Du Bois et al at Stanford University, USA, reported Asymmetric Total Synthesis of Tetrodotoxin in 2003.

(a) Kishi, Y.; Aratani, M.; Fukuyama, T.; Nakatsubo, F.; Goto, T.; Inoue, S.; Tanino, H.; Sugiura, S.; Kakoi, H. J. Am. Chem. Soc. 1972, 94, 9217-9219. (b) Kishi, Y.; Fukuyama, T.; Aratani, M.; Nakatsubo, F.; Goto, T.; Inoue, S.; Tanino, H.; Sugiura, S.; Kakoi, H. J. Am. Chem. Soc. 1972, 94, 9219-9221.(c)Ohyabu, N.; Nishikawa, T.; Isobe, M. J. Am. Chem. Soc. 2003, 125, 8798-8805 (d) Hinman, A.; Du Bois, J. J. Am. Chem. Soc. 2003, 125, 11510 -11511.

Tetrodotoxin Poisoning

History
The first recorded cases of tetrodotoxin poisoning were from the logs of Captain James Cook. He recorded his crew eating some local tropic fish (pufferfish), then feeding the remains to the pigs kept on board. The crew experienced numbness and shortness of breath, while the pigs were all found dead the next morning. In hindsight, it is clear that the crew received a mild dose of tetrodotoxin, while the pigs ate the pufferfish body parts that contain most of the toxin, thus killing them.

The toxin was first isolated in 1909.

Symptoms and diagnosis
The diagnosis of pufferfish poisoning is based on the observed symptomology and recent dietary history.

The effects of tetrodotoxin poisoning include shortness of breath, numbness, tingling, lightheadedness, paralysis and irregular heartbeat. Symptoms typically onset quickly, minor ones instantaneously. Death is the usual outcome. Although the toxin unbinds from channels as its concentration around nerves diminishes, its molecules are exceptionally potent and unbind only very slowly. Treatment usually consists of respiratory assistance. Nothing equivalent to an antivenom has been developed--presumably because the toxin acts quickly and binds with an affinity that isn't easily overcome.

Course of disease and complications
The first symptom of intoxication is a slight numbness of the lips and tongue, appearing between 20 minutes to three hours after eating poisonous pufferfish. The next symptom is increasing paresthesia in the face and extremities, which may be followed by sensations of lightness or floating. Headache, epigastric pain, nausea, diarrhea, and/or vomiting may occur. Occasionally, some reeling or difficulty in walking may occur. The second stage of the intoxication is increasing paralysis. Many victims are unable to move; even sitting may be difficult. There is increasing respiratory distress. Speech is affected, and the victim usually exhibits dyspnea, cyanosis, and hypotension. Paralysis increases and convulsions, mental impairment, and cardiac arrhythmia may occur. The victim, although completely paralyzed, may be conscious and in some cases completely lucid until shortly before death. Death usually occurs within 4 to 6 hours, with a known range of about 20 minutes to 8 hours.

Areas where tetrodotoxin poisoning is most common
Poisonings from tetrodotoxin have been almost exclusively associated with the consumption of pufferfish from waters of the Indo-Pacific ocean regions. Several reported cases of poisonings, including fatalities, involved pufferfish from the Atlantic Ocean, Gulf of Mexico, and Gulf of California. There have been no confirmed cases of tetrodotoxicity from the Atlantic pufferfish, Sphoeroides maculatus. However, in one study, extracts from fish of this species were highly toxic in mice. Several recent intoxications from these fishes in Florida were due to saxitoxin, which causes paralytic shellfish poisoning with very similar symptoms and signs. The trumpet shell Charonia sauliae has been implicated in food poisonings, and evidence suggests that it contains a tetrodotoxin derivative. There have been several reported poisonings from mislabelled pufferfish and at least one report of a fatal episode in Oregon when an individual swallowed a Rough-skinned Newt, Taricha granulosa.

Relative frequency of disease
From 1974 through 1983 there were 646 reported cases of pufferfish poisoning in Japan, with 179 fatalities. Estimates as high as 200 cases per year with mortality approaching 50% have been reported. Only a few cases have been reported in the United States, and outbreaks in countries outside the Indo-Pacific area are rare, except in Haiti, where Tetrodotoxin plays a key role in the creation of so called zombie poisons.

Target populations
All humans are susceptible to tetrodotoxin poisoning. This toxicosis may be avoided by not consuming pufferfish or other animal species containing tetrodotoxin. Most other animal species known to contain tetrodotoxin are not usually consumed by humans. Poisoning from tetrodotoxin is of major public health concern primarily in Japan, where "fugu" is a traditional delicacy. It is prepared and sold in special restaurants where trained and licensed individuals carefully remove the viscera to reduce the danger of poisoning. Importation of pufferfish into the United States is not generally permitted, although special exceptions may be granted. There is potential for misidentification and/or mislabelling, particularly of prepared, frozen fish products.

Food analysis
The mouse bioassay developed for paralytic shellfish poisoning (PSP) can be used to monitor tetrodotoxin in pufferfish and is the current method of choice. An HPLC method with post-column reaction with alkali and fluorescence has been developed to determine tetrodotoxin and its associated toxins. The alkali degradation products can be confirmed as their trimethylsilyl derivatives by gas chromatography/mass spectrometry. These chromatographic methods have not yet been validated.

河豚毒素（TTX）定量酶联免疫检测试剂盒说明书
ELISA-kit for TTX

本测试盒用间接竞争免疫分析法定量测定河豚鱼中的河豚毒素（TTX）。该测试盒反应孔可拆卸，可测单份样品，也可测多份样品。定量分析时需有含450nm波长的微孔板酶标仪。

1 概要
河豚毒素（TTX）是一种强神经毒素，其性质稳定，加热、盐腌及高温烹煮均不易使其被破坏。河豚鱼中常含有河豚毒素。我国沿海居民素有食用河豚鱼的习惯。因误食或食用加工不当的河豚鱼而发生人畜中毒的事件每年都有发生。故准确检测河豚鱼中的河豚毒素对预防和控制河豚毒素中毒，具有重要意义。本检测方法具有灵敏、快速、简便、特异性好、取样量小并可同时检测大量样品等优点。

2 测定原理
本试剂盒采用固相酶联免疫吸附原理，利用间接竞争ELISA法进行测定。用抗原包被微孔板，加入河豚毒素标准品或样品、抗河豚毒素单克隆抗体进行孵育后，将未与包被抗原结合的抗体洗去；再加入酶标记的抗鼠IgG抗体孵育，加入显色液，经过终止液终止后，用酶标仪在450nm处测定OD值。

3 提供的物品
微孔板
5个浓度的TTX标准溶液（各0.5mL）
标准1：0ng/mL ……………………………………………………………直接使用
标准2：10.0ng/mL ………………………………………………………直接使用
标准3：20.0ng/mL ………………………………………………………直接使用
标准4：50.0ng/mL ………………………………………………………直接使用
标准5：200.0ng/mL ………………………………………………………直接使用
抗体溶液(6mL) …………………………………………………………………直接使用
酶标物(12mL) …………………………………………………………………直接使用
显色液(12mL) …………………………………………………………………直接使用
终止液(6mL) ………………………………………………………………………直接使用
浓缩洗液(30mL) …………………………………………………………………稀释使用

4 盒中未提供，需自备物品
微孔板酶标仪（含450nm）、离心机、电炉、微量移液器、磁力搅拌器
量筒、漏斗、容量瓶、快速定性滤纸、分液漏斗
乙酸、氢氧化钠、去离子水或蒸馏水、PBS缓冲液

5 操作者注意事项
标准溶液中含有TTX毒素，应特别小心。
终止液为0.5M硫酸，避免接触皮肤。
每次加样后立即将所有试剂放回2~8 ℃。
每步加样时间应控制在5min内。
孵育过程中应避光。
不要使用过期试剂盒。
不要交叉使用不同批号试剂盒中的试剂。

6 储存条件
保存试剂盒于2~8℃。不要冷冻
显色液对光敏感，因此要避免直接暴露在光线下。
将不用的微孔板放进原铝箔袋中，置2~8℃保存。

7 试剂变质的标志
标准1的吸光度值小于0.5个单位（A450nm<0.5）时，表示试剂可能变质。

8 样品处理
8.1 鲜河豚鱼中河豚毒素的检测
----称取5g河豚鱼样品(肌肉、皮或内脏)，剪碎，加入25mL 0.1%的乙酸，用烧杯在电炉上煮沸搅拌10min。此步骤应注意不要让液体沸出容器，应控制加热温度，并不断搅拌。
----待冷却至室温后，上清用快速定性滤纸过滤于50mL离心管中
----沉淀用20mL0.1% 乙酸洗到烧杯中，再煮沸3min
----冷却至室温后，所有的液体及煮沸的样品都加到离心管中，3000rpm离心10min。
----取上清，量体积，置分液漏斗中
----加入等体积乙醚，振摇1分钟，静置分层。放出水层，量体积后至另一分液漏斗中，再加入等体积的乙醚，振摇1分钟，静止分层。
----将水层放入50mL容量瓶中，用1M氢氧化钠调节提取液中的pH值至6.5~7.4，然后加入PBS至50mL，提取液4℃保存备用。
8.2 鱼片、鱼干制品中河豚毒素的检测
-----样品中的TTX用0.1%乙酸在水浴中超声提取，提取液中的TTX用ELISA方法进行检测，若样品中TTX含量低于检出限则报告为<100mg/kg；样品中TTX含量在100~3000mg/kg之间，直接按ELISA实验结果报告；如果样品中TTX含量达到3000mg/kg，则需采用小鼠生物测定法进行验证，确认毒性反应后再按照ELISA实验结果报告。

9 酶免疫分析程序
----浓缩洗液为10倍浓缩液，使用时与去离子水或蒸馏水按体积比1:9稀释后使用。
----取所需数量的板条插入微孔架，用洗液洗涤微孔板3min×2次，记录样品及标准的位置。
----加入50mL标准品或处理好的样品到微孔，每个标准和样品必须使用新的吸头。
----加入50mL抗河豚毒素单克隆抗体溶液到每个微孔（注意移液器管尖不要接触到孔中的液体，避免交叉污染）中，37℃孵育90min。
----甩掉孔中液体，用洗液洗涤微孔板3min×3次，最后一次应在吸水纸上拍打以完全除去孔中液体。
----每孔加入酶标物100mL，37℃孵育60min。
----用洗液洗涤微孔板3min×5次，最后一次应在吸水纸上拍打以完全除去孔中液体。
----每孔加入显色液100mL（2滴），37℃孵育12min。
----每孔加入终止液50mL（1滴），立即用酶标仪在波长450nm处测定吸光度值（OD值）。
注：在定量分析中，显色液和终止液需要用移液器准确量取。

10 样品浓度计算
以标准溶液OD值与标准1的OD值的比值为纵坐标（即标准品OD比），所对应标准溶液浓度（ng/mL）的对数值为横坐标，制作标准曲线。根据样品OD值与标准1的OD的比值（即样品OD比），可从曲线上得到对应点的横坐标，即为TTX浓度的对数值，求得反对数即为测定液中TTX浓度C（ng/mL），按下列公式计算出样品中TTX含量：
TTX含量（mg/kg）= 10×C×K
式中：C：测定液中TTX浓度（ng/mL）
K：提取液的稀释倍数
11 主要技术指标及参数
11.1 最低检出浓度10.0 ng/mL。
11.2 加标回收率在70~120%，条内变异系数＜10%，条件变异系数＜15%。