Novel method for preparing gonyatoxin GTX2 and GTX3 artificial antigens
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A technology of genus algae toxin and artificial antigen, which is applied in the field of antigen preparation and achieves the effects of high sensitivity, low cost, easy large-scale production and popularization and application.
Inactive Publication Date: 2012-12-05
GUANGDONG OCEAN UNIVERSITY
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[0005] Related articles have been published about GTX, but there is no detailed report on the preparation of artificial antigens. At the same time, the literature is all GTX-BSA-formaldehyde, but according to the design ideas of the present invention, and experimental results show that only GTX-aldehydes-carrier Proteins can be effectively coupled, which is also the biggest bottleneck problem that the current GTX immunology rapid detection kit lacks
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Embodiment 1
[0035] A new method for the preparation of artificial antigens of gonadotoxin GTX2,3, which uses gonadotoxin GTX 2,3 Coupling with carrier protein BSA and KLH macromolecules to prepare artificial antigens, using glyoxal in the hapten molecule GTX 2,3 Introduce two free amino groups at the imino positions of No. 2 and No. 8, and then use the two-step method to make this intermediate product (H 2 N- GTX2, 3) are respectively coupled to the carrier proteins BSA and KLH, the specific methods are as follows:
[0036] (1) Take 85μL of 2.37mg.mL -1 Add a small amount of 0.1M acetic acid solution to the GTX2,3 toxin stock solution under the dark, and add a certain amount of 0.1M sodium acetate solution, gently flick and mix to make the pH of the solution 4.6;
[0037] (2) Add appropriate amount of freshly prepared glyoxal to the solution in (1) to make the final concentration of aldehydes 0.1%;
[0038] (3) Weigh 0.0023 g BSA and 0.0023 g KLH and dissolve in 160 μL 0.01 mol·L respectively -1...
Embodiment 2
[0050] A new method for the preparation of artificial antigens of gonadotoxin GTX2,3, which uses gongotoxin GTX 2,3 Coupling with carrier protein BSA and KLH macromolecules to prepare artificial antigens, using glyoxal in the hapten molecule GTX 2,3 Introduce two free amino groups at the imino positions of No. 2 and No. 8, and then use the two-step method to make this intermediate product (H 2 N- GTX2, 3) are respectively coupled to the carrier proteins BSA and KLH, the specific methods are as follows:
[0051] (1) Take 85μL of 2.37mg.mL -1 Add a small amount of 0.1M acetic acid solution to the GTX2,3 toxin stock solution under the dark, and add a certain amount of 0.1M sodium acetate solution, gently flick and mix to make the pH of the solution 4.6;
[0052] (2) Add appropriate amount of freshly prepared glyoxal to the solution in (1) to make the final concentration of aldehydes 3%;
[0053] (3) Weigh 0.0023 g BSA and 0.0023 g KLH and dissolve in 160 μL 0.01 mol·L respectively -1 I...
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Abstract
The invention relates to a novel method for preparing gonyatoxin GTX2 and GTX3 artificial antigens. The novel method comprises the following steps: introducing two free amino groups in second imido position and the eighth imido position of hapten molecules GTX2 and GTX3 with glyoxal; respectively coupling an intermediate product (H2N-GTX2,3) with vector proteins BSA (Bovine Serum Albumin) and KLH (Keyhole Limpet Hemocyanin) by adopting a two-step method; and respectively carrying out ultrafiltration and purification on the intermediate product and a coupling product and carrying out full-wave band ultraviolet scanning and HPLC (High Performance Liquid Chromatography) appraisal. The appraisal result shows that gonyaulax-paralytic shellfish poisoning GTX2 and GTX3 artificial antigens are successfully prepared according to the research and also have stronger immunogenicity. According to the novel method, the gonyatoxin GTX2 and GTX3 artificial antigens can be effectively prepared, and thus antibodies of the gonyatoxin GTX2 and GTX3 are produced and have important actual significance for quickly detecting the gonyatoxin GTX2 and GTX3 by adopting an immunoassay technology. The whole preparation process is simple, convenient and feasible without adopting special instrument and equipment; and the cost is low and easiness in scale production, popularization and application is realized. The gonyatoxin GTX2 and GTX3 artificial antigens synthesized by the method have better stability.
Description
Technical field [0001] The invention relates to a preparation method of artificial antigens, in particular to a new method for preparation of artificial antigens of genus genus toxin GTX2,3, and belongs to the technical field of antigen preparation. Background technique [0002] Shellfish eat a lot of toxic algae and accumulate tetrahydropurine toxin in the body. Divided into four categories: carbamate toxins, gynocystins, N-sulfonylcarbamoyl toxins, decarbamoyl toxins, and deoxydecarbamoyl toxins. The toxicity is very strong. Ingestion of 1 mg can cause death. Its chemical properties are very stable. Cooking and heating cannot eliminate its toxicity. Its toxicity has a certain impact on human safety. [0003] At the same time, due to the eutrophication of the water body, the algae proliferate abnormally and release Algae Toxin. These secondary metabolites seriously endanger the safety of human water and the safety of other aquatic organisms. Among them, gonyautox in , GTX )) The...
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