CN104448002B - Preparation method of acrylamide monoclonal antibody - Google Patents

Abstract

The invention discloses a preparation method of an acrylamide monoclonal antibody, belonging to the technical field of antibody preparation. The method comprises the preparation of acrylamide antigen and the preparation of acrylamide monoclonal antibody. The antigen comprises hapten, immunogen and coating antigen, and the preparation of the monoclonal antibody comprises the steps of animal immunization, cell fusion and cloning, and ascites preparation and purification of the monoclonal antibody. The preparation method is simple and has good immune effect; can efficiently and stably secrete the anti-acrylamide monoclonal antibody, and the antibody has the characteristics of high titer, strong specificity and low cost.

Description

preparation method of acrylamide monoclonal antibody

Technical Field

the invention relates to a preparation method of an antibody, in particular to a preparation method of a monoclonal antibody with specificity for immunodetection of suspicious carcinogen acrylamide in fried food.

Background

acrylamide is commonly called as 'third poison', is a white crystal, has a molecular formula of CH2CHCONH2, is odorless, has a melting point of 84.5 ℃, is very soluble in water, ethanol, methanol, acetone, dimethyl ether and trichloromethane, is slightly soluble in benzene and toluene, contains amino and double chains in acrylamide molecules, has quite active chemical properties, can generate Hofmann reaction, hydrolysis reaction, Michael type addition reaction, polymerization reaction and the like, and is a raw material for synthesizing polyacrylamide. In 2002, 4 a news agency was called on by the swedish national food administration and published on its official website that higher levels of acrylamide were detected in certain fried or baked farinaceous foods and levels exceeded 500 times the maximum permitted in drinking water. A large number of animal experiments show that acrylamide can cause animal multi-organ tumors including mammary gland, thyroid gland, testis, adrenal gland, central nerve, oral cavity, uterus, pituitary gland, etc. The international center for cancer research evaluated the carcinogenicity of acrylamide, and classified it as a two-class carcinogen, namely, human suspected carcinogen.

Acrylamide has attracted considerable attention from researchers worldwide due to its potential neurotoxicity, genotoxicity and carcinogenicity. And the inspection of acrylamide in food is classified as the subject of 'fifteen' major scientific and technical special item 'food safety key technology' in China, and the food safety plan of the Ministry of health also takes the establishment of an acrylamide detection method in food and the distribution survey of food acrylamide in China as an important part.

At present, the detection methods of acrylamide mainly comprise High Performance Liquid Chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the like. Although the sensitivity of the methods is high, the methods have the defects of complicated sample pretreatment, long detection time, complex operation, high cost and the like, and the requirements of rapid identification and diagnosis are difficult to meet.

In recent years, enzyme-linked immunosorbent assay (ELISA) with the advantages of strong specificity, simplicity, rapidness, high sensitivity and the like is widely regarded, and the ELISA shows unique advantages in rapid detection of food safety. The research reports on the immunoassay of the acrylamide in the fried food are less, and the patent is not available, so that the development of an acrylamide rapid screening immunoassay technology is needed. Since acrylamide is a small molecular substance, has no immunogenicity, belongs to hapten in immunology, cannot stimulate animal organisms to generate antibodies, and can promote the animal organisms to generate immunoreaction only by being coupled with macromolecular substances to form complete antigens, so that the antibodies are obtained. At present, the acrylamide immunodetection technology only stays in the aspects of development of a polyclonal antibody and establishment of a detection method, and the specificity is poor.

disclosure of Invention

Aiming at the defects of the existing acrylamide detection technology, the invention provides a synthesis method of an acrylamide antigen and a preparation method of a monoclonal antibody, so that the acrylamide antigen has the characteristics of high antibody titer, strong specificity and low cost.

the acrylamide monoclonal antibody is realized by the following technical scheme:

1. Preparation of acrylamide antigens

(1) Synthesis of haptens

The specific synthesis method of the hapten is as follows:

acrylamide and m-mercaptobenzoic acid react in water under the catalysis of borax by utilizing Michael addition reaction to obtain an addition product, and the mass spectrum (mass) identification result is correct. The structure of the hapten is:

abbreviated as AM.

(2) Synthesis of immunogens

The specific synthesis method of the immunogen comprises the following steps:

The carboxyl group of hapten is activated by NHS (N-hydroxysuccinimide), EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and condensed with the amino group of KLH (Keyhole Lipnet Hemocyanin) to form amido bond, thereby forming stable artificial antigen-immunogen.

(3) the coating source is synthesized by the following specific synthesis method:

The carboxyl of hapten is activated by NHS and EDC and condensed with the amino of BSA to form amido bond, thus forming stable coating antigen.

2. Preparation of acrylamide monoclonal antibody

(1) animal immunization

The Balb/c mice of 8-10 weeks old are immunized with acrylamide immunogen for three times at intervals, and after the sixth immunization, the boosting immunization is carried out again to activate lymphocytes in vivo and generate acrylamide specific antibody.

(2) Cell fusion and cloning

Spleen cells of Balb/c mice producing acrylamide-specific antibodies were mixed with myeloma cells SP2/0 at a volume of 5:1, culturing in an incubator at 37 ℃ and 5% carbon dioxide volume concentration for 10 days, measuring cell supernatant by direct competitive ELISA, and screening positive wells. And subcloning the positive cells by adopting a limiting dilution method within 2 days after detection until obtaining a stable hybridoma cell strain capable of secreting the acrylamide monoclonal antibody.

(3) Preparation and purification of ascites fluid of monoclonal antibody

Balb/c mice were sensitized with liquid paraffin, and the hybridoma cell lines thus collected were washed with DMEM (Dulbecco's Modified Eagle Medium) and injected into the abdominal cavity of the mice, after one week, the mice were inactive and the abdominal cavity was enlarged. The collected ascites is roughly purified by an octanoic acid-saturated ammonium sulfate method, and is purified by an affinity chromatography method (the ascites is enriched with monoclonal antibodies with acrylamide specificity), and the purified ascites is stored in an environment of 20 ℃ below zero. The ascites is dialyzed at 4 ℃ with 0.01mol/L NaCl before use.

The invention has the advantages that:

(1) according to the invention, the structure of acrylamide is modified to obtain hapten, and then the hapten is coupled with carrier protein to synthesize immunogen, so that the method is simple and has good immune effect;

(2) the hybridoma cell strain is obtained by screening, the anti-acrylamide monoclonal antibody can be efficiently and stably secreted, and the antibody has the characteristics of high titer, strong specificity and low cost.

drawings

FIG. 1 is a mass spectrum of a hapten according to the invention;

FIG. 2 is a UV scanning spectrum of an immunogen of the present invention.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

Example 1:

First step, preparation of acrylamide antigens

(1) synthesis of acrylamide hapten

92.4mg of NaHCO3 and 4ml of water are added into a 10ml flask, after complete dissolution, 38.14mg of borax, 154.19mg of m-mercaptobenzoic acid and 84.1mg of acrylamide are added, and reaction is carried out for 2 hours at 40 ℃; and adjusting the pH value to 5-6 by using 1M hydrochloric acid, separating out a white solid, filtering, washing a filter cake with water, and drying to obtain 200mg of a product, namely the acrylamide hapten. The mass spectrometry mass identifies the product, -M-223.8 (target product MW 225, M-1 signal), and the hapten is correct in structure, as shown in fig. 1.

(2) Synthesis of immunogens

weighing 6.6mg of acrylamide hapten dissolved in 2ml of DMF (N, N-dimethylformamide), adding 10.2mg of NHS (N-hydroxysuccinimide) and 11.4mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), and stirring at room temperature for 2 h; 50mg of KLH (Keyhole Lippet Hemocyanin), 66mg of sodium bicarbonate was dissolved in 6ml of water, the activated drug (NHS, EDC) was added dropwise to the protein in a cold water bath, the mixture was stirred at room temperature for 24 hours, the reaction product was put into 1L of distilled water to wash out a dialysis bag (15cm), 1L of PBS (phosphate buffered saline) having a pH of 7.2 was dialyzed for 3 days, the mixture was stirred at 4 ℃ and dialyzed (100rpm), the solution was changed 3 times a day (once in the morning, at night) for 9 times in total, and the dialyzed product was centrifuged at 4500rpm for 6min to obtain an immunogen (AM-KLH). 1 ml/tube, numbering the immunogen, and storing at-20 ℃ for later use.

Scanning spectrum shows that the light absorption of the hapten-coupled protein at the wavelength of 250nm is increased, which indicates that the hapten containing a benzene ring structure is successfully coupled with the carrier protein. The coupling ratio was 7:1 as measured by UV spectrophotometry, as shown in FIG. 2.

(3) Synthesizing a coating source;

weighing 6.6mg of acrylamide hapten, dissolving in 2ml of DMF, adding 10.2mg of NHS (firstly added) and 11.4mg of EDC, and stirring for 2h at room temperature; 50mg BSA, 66mg sodium bicarbonate dissolved in 6ml water, adding the activated drug dropwise to the protein in a cold water bath, stirring at room temperature for 24h, loading the reaction product into 1 distilled water washing dialysis bag (15cm), dialyzing with 1L PBS (1X, pH7.2) for 3 days, standing at 4 ℃ and stirring (100rpm) for dialysis, changing the solution 3 times a day (once in the morning, noon and evening), changing the solution 9 times in total, centrifuging the dialyzed product at 4500rpm for 6min, subpackaging with 1 ml/tube, and preserving the coating at-20 ℃ for later use.

Second, preparation of acrylamide monoclonal antibody

(1) Animal immunization

The synthesized immunogen (AM-KLH) is dissolved by sterile PBS (phosphate buffered saline) solution with the pH value of 7.0, and then the immunogen and Freund's adjuvant are fully emulsified according to the volume ratio of 1:3 to prepare the oil emulsion vaccine. The first immunization is carried out on Balb/c mice 8-10 weeks old by Freund complete adjuvant, and subcutaneous multi-point injection is carried out on the neck and the back, wherein the immunization dose is 112 mu L/mouse. After 14 days, secondary immunization is carried out, Freund incomplete adjuvant is adopted, and the immunization dose is 56 mu L/mouse; 4 subsequent immunizations with two immunizations; 6, strengthening immunity after immunization, and directly adopting AM-KLH and physiological saline without adding an adjuvant. The immunization protocol is shown in table 1 below:

TABLE 1 Balb/c mice immunization protocol

(2) Cell fusion and culture

mixing splenocytes of Balb/c mice producing acrylamide specific antibody with myeloma cells SP2/0 according to a volume ratio of 5:1, centrifuging the mixed cells (1500rpm, 12min of centrifugation), pouring out the supernatant, making the precipitated cell mass into paste, placing the paste in a 37 ℃ water bath, adding 1mL of fusion agent within 1min, wherein the fusion agent is 0.8mL of polyethylene glycol (PEG), gently mixing the cells uniformly, standing for 90S, adding DMEM (Dulbecco' S Modified Eagle Medium) serum-free culture Medium to terminate the reaction, wherein the adding speed is first 2min 1mL/min, 3min 2mL/min and 4min 5mL/min, then pouring the rest DMEM into a centrifuge tube, the final volume is 25-30 mL, centrifuging at 800rpm for 7min, and discarding the supernatant. Cells were resuspended in 20-50 ml HAT medium and plated in 96-well cell culture plates containing feeder cells (100. mu.l per well) at 150. mu.l per well. The mixture was incubated at 37 ℃ in an incubator containing 5% by volume of carbon dioxide.

(3) Cell selection and subcloning

positive wells were screened by direct competition ELISA when cells grew to 1/2-1/3 of the area of the bottom of the well. And subcloning the positive cells by adopting a limiting dilution method within 2 days after detection until obtaining a stable hybridoma cell strain capable of secreting the acrylamide monoclonal antibody. The hybridoma cell strain is subjected to expanded culture and passage, and acrylamide specific monoclonal antibodies can be produced in an unlimited manner.

(4) Preparation and purification of ascites fluid of monoclonal antibody

A. Mouse sensitization: the Balb/c mice are sensitized by liquid paraffin, and the injection volume is 500 mu l per mouse in 6-8 weeks. Ascites can be prepared 7 days later.

B. cell injection: hybridoma cells were collected and washed twice with DMEM, and 105-106 cells were injected into the abdominal cavity of a mouse, after one week, the mouse was inactive and the abdominal cavity of the mouse was enlarged.

C. Ascites collection: ascites was collected from the abdominal cavity of the mouse with a sterile syringe 5 days after the injection of the cells, and was collected every one to two days, and this was repeated until the mouse died naturally.

D. Ascites purification: the collected ascites is roughly purified by an octanoic acid-saturated ammonium sulfate method and purified by affinity chromatography, the purified ascites is stored in an environment at the temperature of 20 ℃ below zero, the ascites is dialyzed for 48 hours in an environment at the temperature of 4 ℃ by 0.01mol/L NaCl before use, and the fluid is changed every 6 to 8 hours in the middle.

(5) Determination of antibody titer

Coating 100 mu L of diluted coating antigen on each hole of an enzyme label plate, incubating for 2h at 37 ℃, washing the plate for 3 times by using a washing solution, adding 150 mu L of a sealing solution into each hole, sealing for 1h at 37 ℃, washing the plate for 3 times, adding 50ul of PBS (phosphate buffered saline solution) and 50ul of supernatant, and culturing for 30min in a dark place at 37 ℃; washing the plate for 3 times, adding 100 μ l of enzyme-labeled secondary antibody diluted 5000 times with conventional enzyme-labeled secondary antibody diluent, and culturing at 37 deg.C in dark place for 30 min; washing the plate for 3 times, adding 100 μ L of AB solution, reacting for 15min in dark place, and adding 50 μ L of stop solution to stop; the absorbance was measured by a microplate reader, and the absorption wavelength was 450/630 nm. The AB liquid is formed by mixing a color developing liquid A and a color developing liquid B according to the volume ratio of 1:1, and the color developing liquid A and the color developing liquid B can be directly purchased.

Claims (1)

1. A preparation method of acrylamide monoclonal antibody is characterized by comprising the following steps: the monoclonal antibody is used for immunodetection of acrylamide in fried food; the specific preparation method comprises the following steps of,

Firstly, preparing an acrylamide antigen;

(1.1) synthesis of hapten;

92.4mg of NaHCO3 and 4ml of water are added into a 10ml flask, after complete dissolution, 38.14mg of borax, 154.19mg of m-mercaptobenzoic acid and 84.1mg of acrylamide are added, and reaction is carried out for 2 hours at 40 ℃; adjusting the pH value to 5-6 with 1M hydrochloric acid, separating out a white solid, filtering, washing a filter cake with water, and drying to obtain 200mg of a product, namely acrylamide hapten; the specific synthesis method of the hapten is as follows:

(1.2) synthesis of immunogen;

Weighing 6.6mg of acrylamide hapten, dissolving in 2ml of DMF, adding 10.2mg of NHS and 11.4mg of EDC, and stirring for 2h at room temperature; dissolving 50mg of KLH and 66mg of sodium bicarbonate in 6ml of water, dropwise adding an activated drug into protein in a cold water bath, stirring at room temperature for 24 hours, putting a reaction product into 1 distilled water washing dialysis bag, dialyzing for 3 days by 1L of PBS (phosphate buffer solution) with pH7.2, standing at 4 ℃, stirring and dialyzing, changing liquid for 3 times every day, changing liquid for 9 times in total, and centrifuging a dialyzed product at 4500rpm for 6min to obtain an immunogen; carboxyl of the hapten is activated by NHS and EDC and then condensed with amino of KLH to form amido bond, so that stable artificial antigen-immunogen is formed;

(1.3) synthesizing the coating antigen, wherein the specific synthesis method comprises the following steps:

the carboxyl of the hapten is activated by NHS and EDC and condensed with the amino of BSA to form amido bond, thereby forming stable coating antigen, namely weighing 6.6mg of acrylamide hapten, dissolving in 2ml of DMF, adding 10.2mg of NHS and 11.4mg of EDC, and stirring for 2h at room temperature; 50mg BSA, 66mg sodium bicarbonate are dissolved in 6ml water, the activated drug is added into protein drop by drop in a cold water bath, the mixture is stirred for 24 hours at room temperature, the reaction product is put into 1 distilled water to wash out a dialysis bag, 1L PBS is dialyzed for 3 days, the mixture is placed at 4 ℃ to be stirred and dialyzed, the solution is changed for 3 times every day, the total solution is changed for 9 times, and the dialysis product is centrifuged for 6min at 4500rpm to obtain coating antigen;

Secondly, preparing an acrylamide monoclonal antibody;

The method comprises the following steps:

(2.1) immunizing animals;

Dissolving the synthesized immunogen with sterile PBS (phosphate buffer solution) with the pH value of 7.0, and fully emulsifying the immunogen and Freund's adjuvant according to the volume ratio of 1:3 to prepare an oil emulsion vaccine; the first immunization is carried out on Balb/c mice 8-10 weeks old by Freund complete adjuvant, subcutaneous multi-point injection is carried out on the neck and the back, and the immunization dose is 112 mu L/mouse; after 14 days, secondary immunization is carried out, Freund incomplete adjuvant is adopted, and the immunization dose is 56 mu L/mouse; 4 subsequent immunizations with two immunizations; 6, strengthening immunity after immunization, and directly adopting AM-KLH and physiological saline for immunization without adding an adjuvant;

(2.2) fusing and culturing cells;

Mixing splenocytes of Balb/c mice generating acrylamide specific antibody with myeloma cells SP2/0 according to a volume ratio of 5:1, centrifuging the mixed cells, pouring out supernatant, flicking the settled cell blocks into paste, placing the paste in a 37 ℃ water bath, adding 1mL of fusion agent within 1min, wherein the fusion agent is 0.8mL of polyethylene glycol, gently mixing the cells uniformly, standing for 90S, adding serum-free DMEM culture medium to terminate the reaction, wherein the adding speed is first 2min 1mL/min, 3min 2mL/min, 4min 5mL/min, pouring the rest DMEM into a centrifuge tube, the final volume is 25-30 mL, centrifuging at 800rpm for 7min, and removing supernatant; suspending the cells by using 20-50 ml HAT culture medium, and paving 150 mul of HAT culture medium in a 96-well cell culture plate containing feeder cells; culturing at 37 deg.C in an incubator containing 5% carbon dioxide by volume;

(2.3) cell screening and subcloning;

when the cells grow to 1/2-1/3 of the area of the bottom of the hole, directly competing ELISA to measure cell supernatant, and screening positive holes; subcloning the positive cells by adopting a limiting dilution method within 2 days after detection until obtaining a stable hybridoma cell strain capable of secreting an acrylamide monoclonal antibody;

(2.4) preparing and purifying ascites of the monoclonal antibody;

A. mouse sensitization: sensitizing Balb/c mice with liquid paraffin for 6-8 weeks, wherein the injection volume is 500 mul/mouse; preparing ascites 7 days later;

B. cell injection: collecting hybridoma cells, washing the cells twice by using DMEM, taking 105-106 cells, injecting the cells into the abdominal cavity of a mouse, and keeping the mouse inactive and the abdominal cavity of the mouse swollen after one week;

C. ascites collection: ascites is collected in the abdominal cavity of the mouse by using a sterile syringe after injecting the cells for 5 days, and the ascites is collected once every other one to two days, so that the ascites is repeatedly collected for many times until the mouse dies naturally;

D. Ascites purification: the collected ascites is dialyzed by 0.01mol/L NaCl at 4 ℃ before use; and then carrying out coarse purification by using an octanoic acid-saturated ammonium sulfate method and purification by using an affinity chromatography method, placing the purified ascites into an environment at the temperature of-20 ℃ for storage, dialyzing the ascites for 48 hours at the temperature of 4 ℃ by 0.01mol/L NaCl before use, and changing the liquid once every 6-8 hours.