CN109490457B

CN109490457B - Application of DA monoclonal antibody immunoaffinity column in determination of chondroacronic acid toxins

Info

Publication number: CN109490457B
Application number: CN201811395731.5A
Authority: CN
Inventors: 张小军; 陈思; 严忠雍
Original assignee: Zhejiang Marine Fisheries Research Institute
Current assignee: Zhejiang Marine Fisheries Research Institute
Priority date: 2018-11-22
Filing date: 2018-11-22
Publication date: 2021-06-22
Anticipated expiration: 2038-11-22
Also published as: CN109490457A

Abstract

The invention relates to an immunoaffinity column, in particular to a preparation method of a DA monoclonal antibody immunoaffinity column, which is characterized in that an antibody with specificity to DA toxin is screened out to prepare the DA monoclonal antibody immunoaffinity column with high performance and strong recognition, the obtained DA monoclonal antibody immunoaffinity column can be applied to analysis and determination of DA toxin in different marine organisms, the problem of unsatisfactory purification of a sample in pretreatment is effectively solved, the interference of a sample matrix is reduced, the analysis time is shortened, the recovery rate of the method is improved, and the blank of the immunoaffinity technology in the field of DA toxin detection is filled. The invention also provides an application of the DA monoclonal antibody immunoaffinity column.

Description

Application of DA monoclonal antibody immunoaffinity column in determination of chondroacronic acid toxins

Technical Field

The invention relates to an immunoaffinity column, in particular to an application of a DA monoclonal antibody immunoaffinity column in determination of chondroacronic acid toxins.

Background

The memory-deficient shellfish poison (ASP) is toxin secreted by diatom in the red tide, and Domoic Acid (DA), a main active ingredient, is a nervous amino acid toxin. The toxin was first discovered in 1987 in the toxic death event caused by eating common mussels on the east coast of the island of the Edward prince, Canada. The intoxication is manifested as abdominal pain, diarrhea, vomiting, severe headache, memory loss, confusion, coma, etc. It was found that DA is mainly produced by the columns of rhomboidium. When the alginic acid content in the shellfish tissue reaches 40mg/kg, the shellfish tissue can cause poisoning of consumers, when 150mg/kg, the shellfish tissue has death risk, the maximum tolerable limit of human beings by eating is 20mg/kg, Canada firstly establishes a safety limit standard of 20 mu g/g shellfish meat, and Europe and Japan also successively list the toxin as a shellfish conventional detection project. The research of China on DA still belongs to the starting stage, and an effective detection means and a monitoring network are not available. Although the report of detecting DA in China is rare at present, a plurality of toxigenic rhombohedral algae strains are detected in China coastal regions. Considering that the toxin has high toxicity, quick response and no antidote, the development of detection research on DA toxin in marine organisms is very important for ensuring the safety of aquatic products and the health of human bodies.

An immunoaffinity column (IAC) is a novel chromatographic column based on antigen-antibody reaction, is manufactured by utilizing the affinity recognition and reversible binding characteristics of a specific antibody to a target toxin, and has high selection specificity and good adsorption and purification performance. At present, no related report of using immunoaffinity column as pretreatment purification means to detect DA toxin exists in China.

Disclosure of Invention

The invention aims to provide a preparation method of a DA monoclonal antibody immunoaffinity column, the prepared immunoaffinity column has the characteristics of high performance and strong recognition by screening out an antibody with specificity to DA toxin, can be applied to analysis and determination of DA toxin in different marine organisms, effectively solves the problem of unsatisfactory purification of a sample in pretreatment, reduces sample matrix interference, shortens analysis time, improves method recovery rate, and fills the blank of the immunoaffinity technology in the field of DA toxin detection.

The invention also provides an application of the DA monoclonal antibody immunoaffinity column.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention relates to a preparation method of a DA monoclonal antibody immunoaffinity column, which comprises the following steps:

(1) synthetic immunogens

10mg of bovine serum albumin, 2mL of 0.05M sodium acetate buffer solution with pH7.4, 1mg of domoic acid and 60 mu L of formaldehyde solution with the mass concentration of 37% are sequentially added into a small flask, mixed uniformly, stirred at 37 ℃ for 48 hours, dialyzed with 0.1M PBS buffer solution with pH7.3 at 4 ℃ for 3 days, and changed every day for 2 times to obtain immunogen DA-BSA.

(2) Synthetic assay antigen

10mg of chicken egg white albumin, 2mL of 0.05M sodium acetate buffer solution with pH7.4, 1mg of domoic acid and 60 mu L of formaldehyde solution with the mass concentration of 37% are sequentially added into a small flask, mixed uniformly, stirred at 37 ℃ for 48 hours, dialyzed by 0.1M PBS buffer solution with pH7.3 at 4 ℃ for 3 days, and changed every day for 2 times to obtain the original DA-OVA for detection.

(3) Monoclonal antibody preparation and purification

Taking 5 female Balb/c mice with the age of 6-8 weeks, injecting DA-BSA (Dada-bovine serum albumin) 20 mu g/mouse subcutaneously at the back, starting to strengthen the immunity after immunizing for 4 weeks, and strengthening the immunity for 1 time every 2 weeks; in the primary immunization, emulsifying immunogen DA-BSA and an isovolume Freund's complete adjuvant, and emulsifying immunogen DA-BSA and an isovolume Freund's incomplete adjuvant in the boosting immunization, wherein the immunization dose and the immunization mode are unchanged;

after 10 days of 2-time boosting immunization, taking tail venous blood of mice for detection, coating a detection source DA-OVA, detecting the titer of serum by using an indirect competitive ELISA method, selecting a BALB/c mouse with the highest titer for preparing hybridoma, boosting the abdominal cavity of the mouse once 3 days before fusion, taking the spleen cell of the BALB/c mouse with the highest titer to fuse with a myeloma cell Sp2/0, and screening to obtain the hybridoma;

BALB/c mice were intraperitoneally injected with 0.5mL liquid paraffin for sensitization, and 8 days later, mice were intraperitoneally injected with 1X 10⁷And (3) extracting ascites from the abdominal cavity of the mouse by using a syringe after 10 days, extracting the ascites for 1 time every 1 day, centrifuging, collecting supernate, purifying the supernate by adopting a saturated ammonium sulfate method, and further purifying by adopting a protein affinity chromatography to obtain the DA monoclonal antibody.

(4) Affinity column preparation

Weighing 0.5g of CNBr activated sepharose 4B dry powder, swelling the sepharose 4B dry powder by using 100mL of 1M HCl solution, and washing; then washing with 100mL of coupling buffer solution to obtain gel;

taking 1.5mL of the swelled gel, adding 1.5mL of 10mg/mL DA monoclonal antibody, carrying out oscillation coupling reaction at room temperature for 2h, carrying out suction filtration, washing the gel with 30mL of coupling buffer solution, carrying out suction filtration, adding 10mL of blocking buffer solution, carrying out oscillation reaction at room temperature for 2h, carrying out suction filtration, washing the gel with 100mL of 0.01M PBS buffer solution with pH7.3, transferring the gel into a 5mL column tube, adding 0.01M PBS solution with the mass concentration of 0.05% of thimerosal and 0.05% of bovine serum albumin respectively and storing the gel at 2-8 ℃.

Preferably, in step (4), the formulation of the coupling buffer is: 0.1 mol. L^-1 NaHCO₃，0.5 mol·L^-1 NaCl，pH8.3。

Preferably, in step (4), the formulation of the blocking buffer is: 0.1 mol. L^-1 Tris-HCl，pH 8.0。

An application of DA monoclonal antibody immunoaffinity column in determination of chondroacronic acid toxin is disclosed.

Therefore, the invention has the following beneficial effects: the DA toxin immunoaffinity column with high performance and strong recognition is prepared by screening out the antibody with specificity to the DA toxin, and the obtained DA toxin immunoaffinity column can be applied to analysis and determination of the DA toxin in different marine organisms, so that the problem of unsatisfactory sample purification in pretreatment is effectively solved, the interference of a sample matrix is reduced, the analysis time is shortened, the recovery rate of the method is improved, and the blank of the immunoaffinity technology in the field of DA toxin detection is filled.

Detailed Description

The invention is further described below by means of specific embodiments.

In the present invention, all percentages are by weight unless otherwise specified, all equipment and materials are commercially available or commonly used in the industry, and the methods in the following examples are conventional in the art unless otherwise specified.

Instrument and reagent

ACQUITY ultra high performance liquid chromatography-tandem Mass spectrometer Xevo TQ-S (Waters, USA); MS2 vortex mixer (IKA, germany); nitrogen blow dryer (organo matio corporation, usa); centrifuge5810 high speed Centrifuge (Eppendorf, germany); 12 channel solid phase extraction device (supelco, usa).

Domoic Acid (DA) standard (purity ≥ 98.0%, canadian national research council); acetonitrile, methanol (chromatographically pure, Merck, germany); ammonium acetate, formic acid (Sigma, usa); disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, and ammonia water (Shanghai national drug group); the experimental water is ultrapure water.

(II) preparation of PBS buffer solution

2.18g of sodium dihydrogen phosphate dihydrate, 12.90g of disodium hydrogen phosphate dodecahydrate and 8.50g of sodium chloride are weighed respectively, dissolved by water and fixed to 1000 mL.

(III) 25. mu.g/mL Domoic Acid (DA) standard solution

Accurately transferring a proper amount of domoic acid standard solution, diluting with methanol to a constant volume of 50mL, and storing at-20 ℃ in the dark for 3 months.

Examples

(a) Synthetic immunogens

Adding 10mg of bovine serum albumin, 2mL of 0.05M sodium acetate buffer solution with pH of 7.4, 1mg of domoic acid and 60 mu L of formaldehyde solution with the mass concentration of 37% into a small flask in sequence, mixing uniformly, stirring at 37 ℃ for 48h, dialyzing with 0.1M PBS buffer solution with pH of 7.3 at 4 ℃ for 3 days, and changing the solution 2 times every day to obtain immunogen DA-BSA;

(b) synthetic assay antigen

Sequentially adding 10mg of chicken egg white albumin, 2mL of 0.05M sodium acetate buffer solution with pH7.4, 1mg of domoic acid and 60 mu L of formaldehyde solution with the mass concentration of 37 percent into a small flask, uniformly mixing, stirring at 37 ℃ for 48h, dialyzing by using 0.1M PBS buffer solution with pH7.3 at 4 ℃ for 3 days, and changing the solution 2 times every day to obtain the original DA-OVA for detection;

(c) monoclonal antibody preparation and purification

BALB/c mice were intraperitoneally injected with 0.5mL liquid paraffin for sensitization, and 8 days later, mice were intraperitoneally injected with 1X 10⁷Extracting ascites from the abdominal cavity of the mouse by using an injector after 10 days, then extracting the ascites for 1 time every 1 day, centrifuging, collecting supernate, purifying the supernate by adopting a saturated ammonium sulfate method, and further purifying by using a protein affinity chromatography to obtain a DA monoclonal antibody;

(d) affinity column preparation

Weighing 0.5g of CNBr activated sepharose 4B dry powder, swelling the sepharose 4B dry powder by using 100mL of 1M HCl solution, and washing; then washing with 100mL of coupling buffer solution to obtain gel, wherein the formula of the coupling buffer solution is as follows: 0.1 mol. L^-1 NaHCO₃，0.5 mol·L^-1NaCl，pH8.3；

Taking 1.5mL of the swelled gel, adding 1.5mL of 10mg/mL DA monoclonal antibody, oscillating at room temperature for coupling reaction for 2h, filtering, and washing the gel with 30mL of coupling buffer solution, wherein the formula of the coupling buffer solution is as follows: 0.1 mol. L^-1 NaHCO₃，0.5 mol·L^-1NaCl, pH8.3, suction filtration, and the addition of 10mL of blocking buffer, the formulation of the blocking buffer is: 0.1 mol. L^-1Tris-HCl, pH8.0, room temperature oscillation reaction for 2h, suction filtration, then 100mL PBS buffer solution of 0.01M, pH7.3 to wash the gel, and transfer to 5mL column tube, add PBS solution of 0.01M, pH7.3 containing the mass concentration of 0.05% thimerosal, 0.05% bovine serum albumin, respectively, store at 2-8 ℃.

The application method of the DA monoclonal antibody immunoaffinity column obtained by the invention in determination of chondroacronic acid toxins comprises the following steps:

(1) sample pretreatment: accurately weighing 2.00g of a fully homogenized sample into a 50mL centrifuge tube with a plug, adding 8mL of 75% methanol aqueous solution in volume concentration, carrying out vortex oscillation for 2min, carrying out ultrasonic extraction for 10min, centrifuging at 7000r/min for 5min, and transferring the supernatant into another 50mL centrifuge tube to obtain a primary extract; adding 8mL of 75% methanol aqueous solution into the residual residue, and repeatedly extracting once to obtain a secondary extracting solution; mixing the primary extract and the secondary extract, and diluting to a constant volume of 20mL to obtain an extract; transferring 1mL of the extracting solution into another 50mL of centrifugal tube, and adding 5mL of PBS buffer solution for dilution to obtain a sample solution to be purified;

(2) purifying the immunoaffinity column: taking a DA monoclonal antibody immunoaffinity column, removing an affinity column plug after the DA monoclonal antibody immunoaffinity column is recovered to room temperature, discharging a preservation solution in the column, and then loading a sample solution; after the sample loading is finished, eluting the affinity column by using 6mL of methanol water solution with volume concentration of 50%, squeezing out residual liquid in the column, discarding all effluent liquid, eluting by using 3mL of methanol solution containing ammonia water with mass concentration of 2%, drying the collected eluent by using nitrogen at 50 ℃, dissolving by using 1mL of initial mobile phase with constant volume, filtering by using a 0.22 mu m filter membrane, and then supplying liquid chromatography-mass spectrometry for analysis;

the liquid chromatography conditions were: a chromatographic column: ACQUITY UPLC BEH C₁₈A column (2.1 mm x 50 mm, 1.7 μm); the sample injection volume is 2 mu L; the temperature of the sample chamber is 10 ℃; the column temperature is 40 ℃; the flow rate is 0.2 mL/min; mobile phase a was acetonitrile and B was a 2mmol/L ammonium acetate solution containing 0.1% formic acid, gradient elution: 0-1.0 min, 90% A; 1.0-3.0 min, 90-10% A; 3.0-4.0 min, 10% A; 4.0-4.1 min, 10% -90% A; 4.1-5.5 min, 90% A;

the mass spectrum conditions are as follows: electrospray ion source, positive ion scanning (ESI-); the detection mode is as follows: multiple Reaction monitoring mode (MRM); capillary voltage: 3.0 kV; ion source temperature: 150 ℃; desolventizing gas temperature: 600 ℃; taper hole gas flow: 150L/h; desolventizing agent gas flow: 1000L/h; taper hole voltage: 12V; collision energy: 16 eV; analyte qualitative ion pair:m/z 312.2 >248.2; analyte quantification ion pair:m/z 312.2 > 266.2。

59 parts of 5 varieties of coastal cities, namely mussels, ark shells, scallops and oysters are analyzed and detected by the method, and the obtained detection results are shown in table 1. The DA detection rate is 10%, the detection rate comprises 5 batches of scallops and 1 batch of oysters, the scallop detection rate is highest, and the DA pollution level is different from 0.9 to 11.8 mug/g; the oyster detection rate is 7%, and the DA content is 0.07 mug/g.

TABLE 1 contamination level of DA in Positive samples

Variety of (IV) C	DA concentration (μ g/g)
		Scallop	11.8
Scallop	14.9
		Scallop	0.7
Scallop	9.3
		Scallop	0.9
Oyster shell	0.07

Therefore, the DA monoclonal antibody immunoaffinity column obtained by the invention has the characteristics of high performance and strong identification, and can completely meet the analysis and determination requirements of DA toxin in marine organisms.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way, and other variations and modifications may be made without departing from the spirit of the invention as set forth in the claims.

Claims

1. The application of the DA monoclonal antibody immunoaffinity column in determination of chondroacronic acid toxins is characterized in that the specific application method is as follows:

(a) sample pretreatment: accurately weighing 2.00g of a fully homogenized mussel tissue sample, arca subcrenata tissue sample, scallop tissue sample or oyster tissue sample in a 50mL centrifuge tube with a plug, adding 8mL of 75% methanol aqueous solution, carrying out vortex oscillation for 2min, carrying out ultrasonic extraction for 10min, centrifuging at 7000r/min for 5min, and transferring the supernatant to another 50mL centrifuge tube to obtain a primary extract; adding 8mL of 75% methanol aqueous solution into the residual residue, and repeatedly extracting once to obtain a secondary extracting solution; mixing the primary extract and the secondary extract, and diluting to a constant volume of 20mL to obtain an extract; transferring 1mL of the extracting solution into another 50mL of centrifugal tube, and adding 5mL of PBS buffer solution for dilution to obtain a sample solution to be purified;

(b) purifying the immunoaffinity column: taking a DA monoclonal antibody immunoaffinity column, removing an affinity column plug after the DA monoclonal antibody immunoaffinity column is recovered to room temperature, discharging a preservation solution in the column, and then loading a sample solution; after the sample loading is finished, eluting the affinity column by 6mL of methanol water solution with volume concentration of 50%, squeezing out residual liquid in the column, discarding all effluent liquid, eluting by 3mL of methanol solution containing ammonia water with mass concentration of 2%, drying the collected eluent by nitrogen at 50 ℃, dissolving by 1mL of initial mobile phase with constant volume, filtering by a filter membrane, and analyzing by liquid chromatography-mass spectrometry; the liquid chromatography conditions were: a chromatographic column: ACQUITY UPLC BEH C₁₈The column specification is 2.1mm multiplied by 50 mm, 1.7 mu m; the sample injection volume is 2 mu L; the temperature of the sample chamber is 10 ℃; the column temperature is 40 ℃; the flow rate is 0.2 mL/min; mobile phase a was acetonitrile and B was a 2mmol/L ammonium acetate solution containing 0.1% formic acid, gradient elution: 0-1.0 min, 90% A; 1.0-3.0 min, 90-10% A; 3.0-4.0 min, 10% A; 4.0-4.1 min, 10% -90% A; 4.1-5.5 min, 90% A; the mass spectrum conditions are as follows: electrospray ion source, positive ion scanning; the detection mode is as follows: a multiple reaction monitoring mode; capillary voltage: 3.0 kV; ion source temperature: 150 ℃; desolventizing gas temperature: 600 ℃; taper hole gas flow: 150L/h; desolventizing agent gas flow: 1000L/h; taper hole voltage: 12V; collision energy: 16 eV; analyte qualitative ion pair:m/z 312.2 >248.2; analyte quantification ion pair:m/z 312.2 > 266.2。