Wuhan Youbo Tetrodotoxin Detection Kit Technology has reached the international first-class level

The tetrodotoxin ELISA test kit developed by Wuhan Youbo Biotechnology Co., Ltd. has a detection limit of 2 ppb and is widely used in food companies and government inspection agencies in the United States, Japan, Russia, Northern Europe, etc. The quality pass rate reaches 100% , error 1 About % , a large number of experimental data indicate that the technology level of tetrodotoxin detection kit is world-class.
Compared with LC-MS instrument, the results were 100% , the difference between batches was less than 0.1% , the kit was stable and high, and the anti-interference ability was strong.

1.   ELISA standard working curve

Competitive ELISA procedure according to the operation, tetrodotoxin different dilutions of the standard solution is measured to logit function of the average OD value of the ratio of the OD values of each well of the control group is the ordinate, with different concentrations of tetrodotoxin logarithm of the concentration standards For the abscissa, draw a standard curve. The results are shown in the figure: the linear regression equation is y = -1.9695x + 3.3899 , R 2 = 0.9902 ; the linear range is 5-500 ng/mL , and the minimum detection limit is 5 ng/mL .
Figure 1 ELISA standard working curve (the ordinate is not the resistance rate logit(B/B0) )

2.   Recovery experiment

When adding 250 ng standards, recovery was 159%, was added 500 ng standards,: add 250 ng and 500 ng puffer fish meat value of 1 g of tetrodotoxin standard, by competitive ELISA assay as shown in Table The recovery rate is 108% . The product recovery rate is high, and our analysis may be due to too many plate washing times, resulting in a small amount of antibody antigen detachment, or due to the long time of the experiment, the antibody titer is weakened. The product recovery rate is high, does not affect the detection sensitivity. Due to the low product detection limit (5 ng/mL), this product can meet the daily testing requirements for puffer fish.
Table 2 simulated sample recovery experiment
sample
0
250ng
500ng
Puffer fish
ND
398ng
542.5ng
Recovery rate %
-
159%
108%
CV%
3.7
4.3
6.2

3. Stability experiment

The standards were tested at concentrations of 10 , 50 , 100 , and 150 ng/mL . The results are shown in the table: 10-150 ng/mL The concentration coefficient of the four standards is 1.93-3.77% , and the average coefficient of variation is 2.76%. .
Coefficient of variation CV %= (standard deviation SD / average Mean ) × 100%
Table 3 stability
Add the theoretical concentration of tetrodotoxin standard (ng/mL)
TT content in solution (ng/mL)
10
50
100
150
1
15.43
60.17
124.80
167.18
2
12.38
57.51
122.02
174.24
3
13.32
54.86
113.49
152.26
4
12.55
56.80
117.83
167.03
5
15.48
57.00
111.84
157.33
6
12.49
60.14
116.74
164.63
average
13.61
57.75
117.79
163.78
CV ( % )
1.93
3.77
2.38
2.97

4. Detection of tetrodotoxin in the sample

Table 4   Distribution of Toxins in Different Parts of Different Varieties of Puffer Fish in Zhejiang and Hainan Provinces         
Sample concentration X ( μg/kg )
Variety
菊1#
male
菊2#
male
菊3#
female
菊4#
female
Double 5#
male
Double 6#
male
Double 7#
female
Double 8#
female
Lunar abdomen
female
heart
ND
ND
ND
ND
60.56
ND
47.40
ND
293.83
liver
ND
ND
ND
ND
1.33
30.09
0.57
ND
623.69
spleen
ND
ND
ND
ND
41.30
89.63
22.46
ND
1034.34
lung
ND
ND
ND
ND
15.89
13.61
89.57
24.34
275.70
kidney
ND
ND
ND
ND
5.72
114.02
102.93
107.4
970.70
Gallbladder
ND
ND
0.875
ND
5.56
43.91
52.83
ND
636.39
eye
ND
ND
ND
ND
5.35
49.37
67.16
20.77
97.75
lip
ND
ND
ND
ND
2.14
6.75
87.55
0.87
312.36
skin
ND
ND
ND
ND
133.93
51.43
ND
20.73
176.74
meat
ND
ND
ND
ND
11.57
81.78
50.19
35.67
254.21
fin
ND
ND
ND
ND
48.30
7.57
54.78
8.25
197.55
blood
ND
ND
ND
ND
2.56
ND
87.73
20.29
N
stomach
ND
ND
ND
ND
ND
113.30
50.89
3
46.7
intestinal
ND
ND
ND
ND
ND
15.86
39.54
ND
45.75
brain
ND
ND
ND
ND
ND
ND
20.09
20.09
59.27
gonad
ND
5.96
ND
ND
ND
ND
32.58
12.5
399.82
Method basis: Determination of tetrodotoxin in fresh puffer fish GBT 5009.206-2007
Calculation formula: X = C × D × V/m
X: Tetrodotoxin content in the sample in micrograms per kilogram ( μg/kg)
C: Tetrodotoxin content measured on the plate, in nanograms per milliliter ( ng/mL )
D: sample dilution factor
V: volumetric volume of the extraction solution in milliliters ( mL )
m: the mass of the test sample, in grams ( g )
About the toxicity of puffer fish , it is generally divided into 4 virulence levels : toxicity is higher than 1000 MU / g is highly toxic ; between 100 MU / g ~ 1000 MU / g is highly toxic ; between 10 MU / Between g and 100 MU/g is attenuated ; below 10 MU/g is non- toxic ( the case of lethality in mice in the non- toxic class can be regarded as micro-toxic ) . According to Zhang Li's definition of the mouse unit (1 MU = 0.22 μg) .
A standard curve is drawn according to the above method, and a regression equation (y = a × xb) is obtained . Where a and b are coefficients, y is the inhibition rate (A0-A /A0%) , and x is Log (100* tetrodotoxin concentration ) . The inhibition rate was calculated from the OD 490 (A) value of the sample , and the concentration of tetrodotoxin was calculated according to the formula. The results are shown in the above table: the distribution of tetrodotoxin in the common puffer fish species in Zhejiang and Hainan Province is significant, and the Phyllostachys pubescens has no detectable toxicity, which is non-toxic; the oriental scorpion scorpion is poisonous but also non-toxic. The spleen and stomach virulence of the male double-spotted oriental scorpion were higher than that of the female; the virulence of the wild scorpion locust was significantly higher than that of the cultured cultivar.

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