| In recent years,food safety has become a common concern all over the world.Food poisoning events caused by biological toxins emerge one after another,which not only endanger human health,but also cause huge economic losses to society.Biotoxin is a kind of toxic substances produced by the secretion and metabolism of organisms.According to their different sources,they can be divided into four categories:animal toxins,plant toxins,marine toxins and microbial toxins,of which microbial toxins are the most common in China.According to different chemical structures,biological toxins can be divided into macromolecular toxins and small molecule compound toxins.The former can be subdivided into protein toxins,polysaccharide toxins and polypeptide toxins.Biological toxins act on specific target molecules such as cell membrane,receptor and ion channel through high specific selection to form different degrees of toxic or lethal effects.Biological toxins have the characteristics of complex structure,strong toxicity,wide distribution and difficult detoxification.Due to the inactivation of protein toxins,it is difficult to construct corresponding detection methods,resulting in the imperfection of existing detection methods.Therefore,it is very important to establish a fast and efficient detection method for protein toxins.In this study,the common protein toxins in clinical poisoning were selected,such as Thermostable direct hemolysin（TDH）secreted by Vibrio parahaemolyticus,Botulinum neurotoxin serotype A（Bo NT/A）and Botulinum neurotoxin B（Bo NT/B）secreted by Clostridium botulinum,and the most representative ricin toxin（RT）in plant toxins.Using the characteristics of lanthanide Stokes displacement,long fluorescence lifetime and narrow emission spectrum band,an immunochromatographic test strip based on lanthanide fluorescent microspheres was established for the detection of TDH,Bo NT/A and Bo NT/B;In addition,a homogeneous time resolved fluorescence based on lanthanide cryptate was established for the detection of RT.the main contents are as follows:Lanthanide immunochromatographic test paper and ELISA method for TDH detection were established respectively.Firstly,high purity TDH recombinant protein was prepared by molecular cloning technology,and high affinity polyclonal antibody was obtained by immunizing New Zealand white rabbits with the recombinant protein.It was found that the affinity of the polyclonal antibody prepared in this experiment was up to 1×10-8 M-1.Using the high affinity antibody,the lanthanide immunochromatographic strip detection method and ELISA method were established at the same time.The sensitivity of the former was 50 ng/m L and the quantitative range was 50～5000 ng/m L;The sensitivity of the latter is 78 ng/m L and the quantitative range is 78～312 ng/m L.The specificity and repeatability of the two detection methods are good,the sensitivity has not changed in the detection of seafood samples,and has the ability to detect natural toxins.The results show that both methods are superior to the immunological methods reported in China for TDH detection.Lanthanide immunochromatographic test paper for the detection of Bo NT/A and Bo NT/B was prepared.Firstly,botulinum toxin type A and B were purified by gel column and ion exchange column,and high purity natural toxins were obtained.The purity of Bo NT/A and Bo NT/B were 88.11%and 91.98%respectively.The antibodies prepared in our laboratory were screened by using the toxins,and a high affinity polyclonal antibody was obtained for later method establishment.The lanthanide immunochromatographic test paper for the detection of Bo NT/A was prepared with the antibody,and a good sensitivity was obtained,up to1ng/m L.In order to improve the specificity of the detection method and realize typing,the lanthanide immunochromatographic test paper for Bo NT/A and Bo NT/B was prepared with the nano antibody developed in our laboratory,and the sensitivity was 5 ng/m L respectively.The prepared immunochromatographic test strip was used to detect 52 clinical samples.The clinical coincidence rate was 84.60%,of which the clinical coincidence rate of nano antibody was 79.50%and multi antibody was 100%.The test paper prepared based on nano antibody can be classified and has good specificity,while the test paper based on multi antibody has high coincidence rate of clinical samples.The two methods have their own advantages,and the combined use can improve the accuracy of test results.This method is simple and time-consuming.It provides a good auxiliary diagnostic tool for clinical work and is very suitable for popularization and use in grass-roots hospitals.A homogeneous time resolved fluorescence method for RT detection was designed and established.By combining homogeneous immunoassay with time-resolved fluorescence energy resonance transfer,using lanthanide cryptate as energy donors and allophycocyanin as energy receptors,a homogeneous immunoassay method based on lanthanide cryptate was successfully constructed for the detection of RT.the sensitivity of this method is 1 ng/m L,and its linear range is wide,up to 1～10000 ng/m L.The lanthanide immunochromatographic method and ELISA method for RT detection have been established in our laboratory in the early stage.Compared with them,this method has higher sensitivity and significantly wider linear range.Compared with the sample volume of 80～100μL required by immunochromatography or ELISA,the sample volume of this method is only 10μL,which is 1/8～1/10 of the original,and the whole detection system is only 30μL without cleaning,which provides a new idea for the establishment of a new high-throughput field detection method.In conclusion,the detection method of foodborne protein toxin was established by lanthanide fluorescence.Firstly,an immunochromatographic test paper based on lanthanide fluorescent microspheres was established for the detection of TDH,Bo NT/A and Bo NT/B.This method has the advantages of high sensitivity,strong specificity,simple operation,short time-consuming,no need for professional detection personnel and large detection instruments,and has high application value.It provides a method for early clinical diagnosis and food safety detection.In addition,a homogeneous time resolved fluorescence method for RT detection was established based on lanthanide cryptate.This method does not need cleaning,has simple operation,wide linear range,and can realize the integration of reaction process and detection process.It has a very broad application prospect in the field of high-throughput field detection. |
PDF Full Text Request