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Novel method for preparing gonyatoxin GTX2 and GTX3 artificial antigens
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A technology of gonyal toxin and artificial antigen, which is applied in the field of antigen preparation to achieve good effect, good stability and easy operation
Inactive Publication Date: 2014-07-30
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 method for preparing the gonylin GTX2,3 artificial antigen, the method is to use the gonylin GTX 2,3 A method for preparing artificial antigen by coupling with carrier protein BSA and KLH macromolecule, using glyoxal in hapten molecule GTX 2,3 The 2nd and 8th imino positions introduce 2 free amino groups, and then the intermediate product (H 2 N-GTX2,3) were coupled with carrier proteins BSA and KLH respectively, the specific method is as follows:
[0036] (1) Take 85μL of 2.37mg.mL -1 Add a small amount of 0.1M acetic acid solution and a certain amount of 0.1M sodium acetate solution to the GTX2,3 toxin stock solution in the dark, flick and mix to make the pH of the solution 4.6;
[0037] (2) Add an appropriate amount of freshly prepared glyoxal to the solution of (1) respectively, so that the final concentration of aldehydes is 0.1%;
[0038] (3) Weigh 0.0023 g BSA and 0.0023 g KLH and dissolve them in 160 μL 0.01 mol·L -1 PBS with pH 7.6 was added to the abov...
Embodiment 2
[0050] A method for preparing the gonylin GTX2,3 artificial antigen, the method is to use the gonylin GTX 2,3 A method for preparing artificial antigen by coupling with carrier protein BSA and KLH macromolecule, using glyoxal in hapten molecule GTX 2,3 The 2nd and 8th imino positions introduce 2 free amino groups, and then the intermediate product (H 2 N-GTX2,3) were coupled with carrier proteins BSA and KLH respectively, the specific method is as follows:
[0051] (1) Take 85μL of 2.37mg.mL -1 Add a small amount of 0.1M acetic acid solution and a certain amount of 0.1M sodium acetate solution to the GTX2,3 toxin stock solution in the dark, flick and mix to make the pH of the solution 4.6;
[0052] (2) Add an appropriate amount of freshly prepared glyoxal to the solution of (1), so that the final concentration of aldehydes is 3%;
[0053] (3) Weigh 0.0023 g BSA and 0.0023 g KLH and dissolve them in 160 μL 0.01 mol·L -1 In the PBS of pH 7.6, add to the above solution resp...
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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 method for preparing an artificial antigen, in particular to a method for preparing an artificial antigen of gonylin GTX2,3, and belongs to the technical field of antigen preparation. Background technique [0002] Tetrahydropurine toxins accumulated in the body of shellfish from ingesting large quantities of toxic algae. Divided into four classes: carbamate toxoids, gonylins, N-sulfonylcarbamoyl toxoids, decarbamoyl toxoids, deoxydecarbamoyl toxoids. 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, and 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 toxins (Algae Toxin). These secondary metabolites seriously endanger the safety of human water and the safety of other aquatic organisms. in , GTX...
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