| Tetrodotoxin is a kind of widespread marine biotoxin ,which exists not only in the puffer fish but also other marine animals. The molecular formula of TTX is C11H17N3O8,relative molecular mass is 319.28. It is one of the most potent halobios neurotoxins. It can block the channel of Na+ and the conduction of nerve pulse, lead to the paralysis of the nerve and the numbness of the muscle,and finally die of respiratory depression. The fatal dose to human is 6～7μg/kg. Jimpy mice intraperitoneal injection LD50 is 8μg/kg. the incidents of TTX toxicosis often occur. As a potential biological warfare agent ,It also take a large threaten to public security. So development a fast,accurate,convenient method to detect tetrodotoxin is very important. It has an important meaning to guard against public safe and to diagnose tetrodotoxin toxicant.In order to produce antibodies against tetrodotoxin ,TTX hapten must be immunogenic by coupling it on a carrier protein. Complete antigens TTX-BSA and TTX-KLH were obtained through coupling with two kinds of protein BSA and KLH by using formaldehyde. The conjugates TTX-BSA and TTX-KLH were analyzed by nondenaturing gel electrophoresis and UV absorption spectrometry method,which the molecule conjugate ratios of TTX to BSA and KLH were 15:1 and 56～1610:1,respectively. The Balb/C mice were immunized with the antigen TTX-KLH, and were screened it with TTX-BSA. Splenocytes from immunized mice were fused with SP2/0 myeloma cells. Three hybridomas of stable secrecting TTX-McAb were selected and were named 5B7,5B9 and 6B9.The subclassof McAb 5B7 was IgG1,5B9 and 6B9 were IgG2a。The supernatant titers were all above 1:500, ELISA titers of the purified ascites was 1:5.1×105. Relative affinity was 5B7>5B9>6B9. According to competitive principle, an ic-ELISA method was established for the quantitative detection of TTX and its optimizal reaction condition as follow: The TTX-BSA was diluted to microtiter plates at a concentration of 1:200 and was incubated 1h at 37℃;then blocked it with 10% cony blood serum and incubated 1h at 37℃;TTX-McAb and variable concentrations of stadard TTX(50μg/well)were add and incubated 1h at 37℃. Then goat anti-mouse IgG-HRP(1:5000) was added and incubated 40min at 37℃. OPD was chosen to be enzyme reaction substrate. The stopping solution(2M H2SO4) was added after ncubated in the dark at 37℃. Absorbance at 490nm was determined by an enzyme immunoassay reader. The established the ic-ELISA method .A linear dose-response standard curve was prepared by plotting log[TTX] versus percent inhibiting. The regression equation of standard curve was y= -0.2204x+0.9568,the correlation coeffecient R2=0.9721,the lower limit detection was 3.16ng/mL,rang from 3.16～1000ng/mL. The result of TTX-McAb establish a experiment foundation for development TTX immunodetection kit and test paper.
|
PDF Full Text Request