| TTX is a kind of widespread marine biotoxin, which exists not only in the puffer fish but also other marine animals. Versatil investigates in all over the world have been done, since Tahara proclaimed that had extracted and purified the TTX from the puffer fish roe and designated. It blocks up the sodium ion channel to inhibit conduction of nerve impulse, which made the sensory nerve paralytic, tetraplegia, dyspnoea, then die of respiratory depression. It is toxicity 1000 times as high as sodium cyanide.There are many methods to detect TTX such as biological detection, tissue culture detection, physical chemistry detection, immunodetection and so on. These methods each have advantage and disadvantage. Although it is simple to the biological detection with mouse, it is short of sensitivity and specificity. The ex vivo tissue culture detection is simple and sensitive but it can't discriminate TTX, STX and their derivates. There is convenient operation of fluorometric method and ultraviolet spectrophotometry, but lower sensitivity and poorer specificity. It is senstitive to the instrument of analysis detection, but there is higher cost and more expensive. There are many advantages containing convenience, shortcut, less complicated technique of operation and expensive cost, lower limit of detection and higher sensitivity and specificity to the immunodetection. It is very difficult to the antibody preparation to limit its development. There is possible to choose them according to practical need and condition. There are many directions of the TTX detection in the future, containing locus in quo detection, accuratissime, quickness, high performance, convenience, and lower cost of detecting the monosample. From the situation of development at present, immunodetection is a method which is the most closest to the requirement mentioned above. The ripening monoclonal antibody technique brings the short period of research development, so it would be promising to the immunodetection product for TTX in the future.The method of ELISE is applied to the trials, depending on high affinity of anti-TTX monoclone antibody with antigen and specific binding, so it can attain the target of detecting antigen in samples. The former researcher had prepared two hybridoma cell strains, which was named 2D6 and 4C6. The thawed hybridoma cell 2D6 was less secreting activity than the former, and its generation never recover to the primer level of cryopreservation. It is indicating that the procedure of cryopreservation and thawing made secreting activity of cells become striking diminishment and it can't be restored after passage. As a result, 4C6 can be only used in latter experiments. After culturing of hybridoma cell for proliferation, we have harvested a quantity of hybridoma cell,and then make one part of them to cryopreservation and let another part continue to culture. The bionomics of hybridoma cell had been tested, such as the rate of corpuscular adherenc, optimization innoculation density, and population doubling time etc. Then the growth curve of corpuscular has been drawn. The method that prepares mice ascites with hybridoma cell which secreting monoclonal antibody separated from mice ascites has been adopted. Some immunoparameters have been tested, which includes protein level of ascites(27.54mg/mL), antibody titer of ascites(>105), affinity constant of antibody(K=0.37). Considering the test requirement and cost price of experiment, the method of caprylic acid-ammonium sulfate should be chosen as the method of purification in this experiment.TTX is a kind of half-antigen. There are no structures in TTX that can bind to the plastic microtiter plates. It is application as coating antigen must be coupled with a carrier protein. This experiment has selected bovine serum albumin (BSA) as the carrier protein, which depends on the former work. And the conjugate was analyzed by the methods of sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and ultraviolet spectrophotometry to qualitation analysis and quantitative analysis respectively.Appling model of linear programming the method of ELISA had been optimized and detected ELISA system parameter after optimization, such as sensitivity(minimum limit for detection: 0.1ng/mL), precision(linear range: 0.2～1000 ng/mL, regression equation: y = -0.1892 x + 2.5, R2=0.939), rate of sample recovery(muscle: 101.0% , roe: 101.5%), constancy(180天) and drawn standard curve. According to the detection method of optimized ELISA for TTX, detection kit of TTX has been constructed. And then the kit's parameters, which include sensitivity (minimum limit for detection: 0.1ng/mL), detection range(0.2～1000 ng/mL), repeatability(average intragroup CV: 3.26%,CV<0.05, average interclass CV: 2.70% ,CV<5%), operatability(P>0.05) and safe etc, correlating with detection have been measured.
|
PDF Full Text Request