Amnesic shellfish toxin immunoaffinity column and preparation method and application thereof

Abstract

The invention belongs to the technical field of food safety detection, and particularly relates to an amnesic shellfish toxin immunoaffinity column and a preparation method and application thereof. The preparation method includes: taking sepharose gel activated by adopting an epoxy chloropropane activation method as a carrier; coupling genetic recombinant protein G onto the carrier to obtain protein G-sepharose; connecting an amnesic shellfish toxin antibody onto the protein G-sepharose to obtain antibody-protein G-sepharose; using a crosslinking agent for crosslinking to obtain an antibody-protein G-spharose filler; packing the antibody-protein G-sepharose filler to form the amnesic shellfish toxin immunoaffinity column having high purity and affinity. The characteristic that the geneticrecombinant protein G is combined with the amnesic shellfish toxin antibody specifically is fully utilized, and Fab segment of the amnesic shellfish toxin antibody is exposed outside, so that antibodycombining capacity of the genetic recombinant protein G, capturing ability of amnesic shellfish toxin and purifying efficiency of the amnesic shellfish toxin are improved substantially.

Description

Technical field: [0001] The invention belongs to the technical field of food safety detection, and in particular relates to an amnesic shellfish toxin immunoaffinity column and a preparation method and application thereof. Background technique: [0002] The affinity column is a column installed after the substance that has a specific binding effect with the purification object is connected to an insoluble carrier to make an affinity adsorbent, and it is mostly used for affinity chromatography. [0003] The main component of Amnesic shellfish poisoning (ASP) is a physiologically active amino acid substance-Domoic acid (DA), which is produced by certain species of Pseudonitz and Nitzschia. Produced by diatoms; DA is an isomer of glutamic acid, a product of phytoplankton metabolism, an excitatory proline derivative and neurotoxin, an agonist of kainate receptors, capable of strongly binding Glutamate receptors act on excitatory amino acid receptors and synaptotransmitters, inc...

AI Technical Summary

Problems solved by technology

Past studies believed that bivalve molluscs often have no adverse symptoms when they accumulate a large amount of DA, but with the deepening of research, it was found that red tide toxins have various sub-lethal or lethal effects on bivalve molluscs; for example, producing Paralytic Shellfish Toxins (PST) of Alexandrium tamari have effects on hatchability, survival rate, motility, filter-feeding rate and growth of bivalve molluscs, and can also inhibit shellfish closure motility, filter-feeding rate and filtration rate; Pseudonitzia P.multiserie containing ASP can cause respiratory acidosis and hypoxia of Pacific oyster Crassostreagigas and changes in blood properties; at the same time, toxic algal blooms may also cause a large number of farmed shellfish to die The main reason

[0004] Researchers have developed a variety of DA detection methods, including mouse biological detection, HPLLC-MS, fluorescence HPLC, GC-MS, TLC (thin layer chromatography), amino acid analysis, CE (capillary electrophoresis), CEC (Capillaryelectrochromatography), ELISA, receptor binding assay (Receptor binding assay), etc., wherein the sensitivity (limits of detection, LOD) of fluorescence HPLC and receptor binding assay can reach 0.001-0.002 μg mL-1, and the lower limit of detection (LOD) of ELISA method The shellfish sample is 3.3μgkg-1, and the seawater sample is 6.8ngL-1; the mouse bioassay was established by Yasumoto et al. Inject the acidic extract solution in the abdominal cavity of the mouse, record the death time of the mouse, and use the "mouse unit" (Mu) to express, the American Association of Public Analytical Chemists (AOAC) defines a mouse unit as the minimum dose of toxin that a 20g white mouse dies within 15 minutes after intraperitoneal injection, and the biological detection of mice cannot be determined due to the long time The composition of the toxin is difficult to quantify, with high false positives (usually caused by fatty acids), poor reproducibility, and insufficient sensitivity (only suitable for detecting DA at concentrations above 40-100 μg g-1), and is no longer suitable for 20 μg g-1 The DA limit standard is up

Images