CN112630420A

CN112630420A - Method for realizing directional coupling by using glycosyl of antibody and solid phase carrier material

Info

Publication number: CN112630420A
Application number: CN202011419566.XA
Authority: CN
Inventors: 时国庆; 毛心怡; 于冰儿; 弓爱君; 李旭琴
Original assignee: University of Science and Technology Beijing USTB
Current assignee: University of Science and Technology Beijing USTB
Priority date: 2020-12-07
Filing date: 2020-12-07
Publication date: 2021-04-09
Anticipated expiration: 2040-12-07
Also published as: CN112630420B

Abstract

The invention relates to the field of biochemical synthesis, and provides a method, a directional conjugate and an application for realizing directional coupling by utilizing the sugar group of an antibody and a solid-phase carrier material. The surface modification of the solid phase carrier material has a binding arm with a certain length and rich in amino groups, and the binding arm can extend into the Fc region of the antibody and react with the glycosyl group of the antibody; then the glycosyl group of the antibody is oxidized to an aldehyde group; the aldehyde group and the modified The amino group of the solid support material reacts to form a Schiff base, and the Schiff base is reduced to an amine compound to obtain a directional conjugate. The invention solves the problem that the solid-phase carrier material is difficult to couple with the glycosyl group in the antibody; and solves the problem of random fixation of the antibody and the solid-phase carrier material, and the active site of the antibody and the antigen can be fully exposed; Directed conjugates are used in immunoassays, and more antigens can be recognized with less directed conjugates, thereby improving the sensitivity of immunoassay assays.

Description

Method for realizing directional coupling by using glycosyl of antibody and solid phase carrier material

Technical Field

The invention relates to the field of biochemical synthesis, in particular to a method for realizing directional coupling by utilizing glycosyl of an antibody and a solid-phase carrier material, a directional conjugate and application.

Background

The protein coupling is to connect small molecular substances (hapten and the like) or large molecular substances (enzyme and the like) with protein in a covalent bond mode, and can prepare artificial antigen, enzyme-labeled antibody, antibody targeted drug and the like in a protein coupling mode. Antibodies have found wide application in immunoassay and detection techniques. Antibody conjugation is the use of antibodies covalently or non-covalently bound to solid support materials (e.g., enzymes, fluorescent complexes, and biotin) to optimize specific antibodies by using these methods, to make them easier to track in complex mixtures using specific recognition of antigens and antibodies, and to provide quantifiable and intuitive test results.

There are various methods for labeling antibodies with solid phase carrier materials, such as:

and (4) carrying out physical adsorption. The method is easily influenced by a plurality of factors such as isoelectric points of the antibody, hydrophobic group distribution, temperature, ion concentration and the like by utilizing the combination of hydrophobic interaction and electrostatic force of the antibody and a solid phase carrier material, and reversible adsorption has certain influence on the stability of the conjugate.

Covalent coupling. The antibody is coupled with the solid phase carrier material by forming covalent bonds. The antibody is rich in carboxyl or amino, and the distribution in the tertiary structure is uniform, so that the coupling of the antibody and the solid phase carrier material is non-directional, the amino or carboxyl on the solid phase carrier material can be randomly coupled with the antibody, the conjugated structure of the antibody is changed, and the active part for combining the antibody and the antigen is blocked probably due to steric hindrance, so that the combined antigen activity of the antibody is reduced, and the stability and the sensitivity of an immunoassay method are influenced.

There are other functional groups on the antibody that can be used to perform chemical conjugation, such as glycosyl groups. The functional groups are distributed on the antibody in a specific number and are distributed on the antibody in a discrete mode, and the functional groups at specific positions on the surface of the specific antibody are used for directional coupling, so that the binding sites of the antigen and the antibody can be kept away, and the binding capacity of the antibody to the antigen is reserved. The glycosyl groups are distributed only in the Fc region, away from the active site of antibody binding to antigen. When the glycosyl and the solid phase carrier material are used for combination, the active site for combining the antibody and the antigen can be ensured not to be blocked by the solid phase carrier material, thereby influencing the combination of the antibody and the antigen. The glycosylation sites on the surface of the antibody are known, and thus also the binding ratio of the antibody to the solid support material can be controlled. However, the carbohydrate groups are located within the Fc region of the antibody (as shown in FIG. 1), and if coupled directly to a solid support material, the amount of binding is greatly reduced due to steric hindrance.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, improve the sensitivity of an immunoassay method, and provide a method for realizing directional coupling by using glycosyl of an antibody and a solid-phase carrier material, a directional conjugate and application. The method comprises the steps of modifying a solid phase carrier material, modifying the surface of the solid phase carrier material with a binding arm with a certain length, extending into an Fc region of an antibody, and enabling the other end of the binding arm to be provided with an amino group, reacting with an aldehyde group of the activated antibody to form Schiff base, and reducing the Schiff base into an amine compound, thereby realizing the directional coupling of the antibody and the solid phase carrier material.

The invention adopts the following technical scheme:

a method for realizing directional coupling by utilizing glycosyl of an antibody and a solid phase carrier material, which utilizes a modifying reagent to modify the solid phase carrier material so that the surface of the solid phase carrier material is modified with a binding arm which has a certain length and is rich in amino, wherein the binding arm can extend into an Fc region of the antibody and react with the glycosyl of the antibody; then oxidizing glycosyl of the antibody into aldehyde group; the aldehyde group reacts with the amino group of the modified solid phase carrier material to form Schiff base, and then the Schiff base is reduced into amine compounds, so that the directional coupling of the antibody and the solid phase carrier material is realized.

Further, the method specifically comprises the following steps:

s1, solid phase carrier material modification: modifying a binding arm with a certain length and the other end rich in amino on the surface of the solid phase carrier material by using a modifying reagent;

s2, antibody activation: oxidizing hydroxyl groups on two adjacent carbon atoms of a sugar chain on the glycosyl of the antibody into aldehyde groups by using an oxidizing reagent, and dialyzing and purifying the antibody after quenching reaction;

s3, directional conjugate preparation: after dialysis and purification, aldehyde groups of the activated antibody react with amino groups of the modified solid phase carrier material to form unstable Schiff base, reducing agent is added to reduce the Schiff base to generate stable amine compounds, then sealant is added to seal unreacted aldehyde groups, and redundant small molecules are removed to obtain the directional conjugate.

Further, in step S1, the modifying reagent includes, but is not limited to, bovine serum albumin, polylysine, chitosan, etc.; after incubation with the solid phase carrier material, modifying the surface of the solid phase carrier material with a binding arm with a certain length and carrying a large amount of amino; the solid phase carrier material comprises time-resolved fluorescent microspheres, nano magnetic particles, immune latex particles, quantum dots, up-conversion luminescent particles and the like.

Further, in step S2, the oxidizing agent is sodium periodate.

Further, in step S3, the reducing agent includes, but is not limited to, one of sodium borohydride, sodium cyanoborohydride, amine borane, and ascorbic acid; the blocking agent includes, but is not limited to, ethanolamine, glycine, tris.

Further, when the sodium periodate solution is mixed with the antibody, the sodium periodate solution is gradually added into the antibody solution drop by drop at a uniform dropping speed and a uniform stirring speed, and after the reaction at room temperature, the mixture is stirred on a magnetic stirrer at 4 ℃ in a dark place. Then sodium sulfite reacts with unreacted sodium periodate to quench the reaction of sodium periodate oxidizing glycosyl on the antibody.

Further, the activated antibody was purified using dialysis. Dialysis solutions include, but are not limited to, dialysis with sodium acetate solution (1mmol/L, pH 4.4). Dialyzing with stirring at 4 ℃. After dialysis, the cells were stored at 4 ℃.

Further, when the antibody and the solid phase carrier material are mixed and reacted, the solid phase carrier material is dissolved in carbonate buffer solution (0.2mol/L, pH9.6), and is continuously stirred and the rotating speed is uniform; gradually and slowly adding the antibody dropwise, adjusting the pH to about 9.5, and stirring at 4 ℃ for reaction. After the antibody was added completely, the pH was adjusted to 9.5 and the mixture was stirred at 4 ℃.

Further, the directional conjugates were purified using dialysis, including but not limited to dialysis against phosphate solution (10mol/L, pH 7.2). Dialyzing with stirring at 4 ℃.

Further, the purified directional conjugate is preserved using a preservation solution. Wherein the conjugate storage solution comprises, but is not limited to, 5mg/mL BSA, 0.02% sodium azide and 0.1% Proclin 300.

Further, in step S1, the specific method for modifying the solid phase support material comprises the following steps:

s1.1, taking out the solid phase carrier material, centrifuging for 15min at 4 ℃ and 12000r, removing the protective solution, and centrifuging to obtain a precipitate;

s1.2, adding a certain amount of modification reagent into the centrifuged precipitate, and standing and incubating for 2 hours at 25 ℃; centrifuging at 12000r at 4 deg.C for 15min to remove excessive modifying reagent; washing twice with phosphate buffer solution, PB, 20mol/L, pH7.2, centrifuging at 4 deg.C and 12000r for 15min, and removing liquid phase;

s1.3, adding a certain amount of preservation solution into the centrifuged precipitate, wherein the preservation solution is 0.1% Proclin 300.

Further, the monoclonal antibody is dissolved in one of a phosphate buffer, a carbonate buffer, a borate buffer, a disodium hydrogen phosphate-citrate buffer, and a disodium hydrogen phosphate-potassium dihydrogen phosphate buffer. Buffer solutions in the pH range 7-8, preferably pH 7.2.

Further, in step S2, the specific steps of antibody activation include:

s2.1 antibody lysis: dissolving the antibody in a brown bottle by using 0.01mol/L sodium phosphate and 0.15mol/L NaCl, and obtaining an antibody solution at the pH value of 7.2;

s2.2 antibody and sodium periodate mixed reaction: slowly adding sodium periodate solution into the antibody solution dropwise at a speed of about 20 μ L/s and a stirring speed of about 1r/s, reacting at room temperature for 30min, and stirring on a magnetic stirrer at 4 ℃ in a dark place for 1 h;

s2.3 quenching reaction: quenching the oxidation reaction of the antibody with sodium periodate by adding sodium sulfite;

s2.4 dialysis purification: after the reaction is finished, putting the reactant into a dialysis bag with the specification of D6mm and the cut-off molecular weight of 7000, and dialyzing with sodium acetate solution at 4 ℃;

s2.5 preservation of activated antibodies: and taking out the activated antibody solution in the dialysate, and storing the antibody solution in a centrifuge tube at 4 ℃.

Further, in step S3, the specific steps of preparing the directional conjugate include:

s3.1, mixing and reacting the activated antibody prepared in the step S2 with the modified solid phase carrier material: adding 0.2mol/L carbonate buffer solution with pH of 9.6 into a brown bottle, putting a stirrer into the bottle, continuously stirring at the rotating speed of 1r/s, adding a solid phase carrier material, slowly and gradually adding the partially activated antibody solution in batches, adding a certain amount of carbonate buffer solution, adjusting the pH to about 9.5 by using pH test paper, and stirring at 4 ℃ for reaction; adding the rest part of the antibody every 20min, adding a certain amount of carbonate buffer solution to adjust the pH to about 9.5, and stirring at 4 ℃ for reaction; after the antibody is completely added, adjusting the pH value to 9.5, and stirring at 4 ℃;

s3.2 reduction of unstable Schiff bases: dropwise adding 5mol/L sodium borohydride solution into the reaction solution in the step S3.1 on a magnetic stirrer, uniformly stirring, and standing for 2h at 4 ℃;

s3.3 blocking of Targeted conjugates: 1mol/L ethanolamine was added to block unreacted aldehyde groups while keeping the solution cool on ice. Reacting at room temperature for 30 min; transferring the reactant into a sterile centrifuge tube, and centrifuging for 15min at 4500rpm4 ℃;

s3.4 washing and dialysis purification: repeatedly washing with phosphate buffer solution for three times, discarding supernatant, dissolving precipitate with precooled 10mmol/L phosphate buffer solution, filling into dialysis bag with specification of D6mm and cut-off molecular weight of 7000, dialyzing with precooled 10mmol/L phosphate buffer solution at 4 deg.C for 24 hr, and replacing dialysate for 1 time;

s3.5 preservation of the Targeted conjugates: taking the directional conjugate, diluting with 10mmol/L phosphate buffer solution by 10 times, and ultrafiltering with 100KD ultrafiltering tube at 3500r/min for 15 min; the supernatant was put into a centrifuge tube, 50mg/mL BSA was added to give a final BSA concentration of 5mg/mL, and the tube was stored at-20 ℃.

The invention also provides a directional conjugate which is obtained by the method for realizing the directional conjugate by utilizing the glycosyl of the antibody and the solid phase carrier material.

The invention also provides an application of the directional conjugate in immunoassay determination and immune extraction, which can greatly improve the sensitivity of immunoassay.

The invention has the beneficial effects that: compared with the traditional direct coupling of the antibody and the solid phase carrier material, the invention can directionally fix the antibody on the solid phase carrier material, solves the problem of random fixation of the antibody and the solid phase carrier material, and fully exposes the active site of the antibody combined with the antigen; meanwhile, the solid phase carrier material is modified by the binding arm, so that the binding arm can extend into an Fc region of the antibody, is more easily bound with glycosyl of the antibody, and can improve the binding capacity of the solid phase carrier material and the antibody; when the directional conjugate constructed by the invention is applied to immunoassay, more antigens can be identified only by fewer directional conjugates, so that the sensitivity of immunoassay is improved; the method is applied to immune extraction, and can greatly improve the capture capability and extraction efficiency of the target object.

Drawings

FIG. 1 is a schematic representation of the position of the sugar group on an antibody molecule; wherein the dark rod-like structure is glycosyl.

FIG. 2 is a schematic diagram showing the directional coupling of antibody molecules to a solid support material in the examples; wherein, A is solid phase carrier material, B is antibody, and C is binding arm.

FIG. 3 is a graph showing the comparison of fluorescence intensity between two conjugates prepared by direct coupling and the method of the present invention; wherein, A is a direct coupling method, and B is the method of the embodiment of the invention.

Detailed Description

Specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that technical features or combinations of technical features described in the following embodiments should not be considered as being isolated, and they may be combined with each other to achieve better technical effects.

Example 1

Directional coupling of aflatoxin B1 antibody and time-resolved fluorescent microsphere (solid phase carrier material).

S1, modification of the time-resolved fluorescent microspheres:

1) taking out 0.5mg of time-resolved fluorescent microspheres, centrifuging at 4 ℃ and 12000r for 15min, and removing the protective solution;

2) after centrifugation, 0.2mg of bovine serum albumin was added to the pellet, and the resulting mixture was incubated at 25 ℃ for 2 hours. Excess bovine serum albumin was removed by centrifugation at 12000r for 15min at 4 ℃. Washing twice with phosphate buffer (PB, 20mol/L, pH 7.2), centrifuging at 12000r at 4 deg.C for 15min, and removing the liquid phase;

3) to the pellet after centrifugation, 250. mu.l of a preservative solution (0.1% Proclin300) was added.

S2, antibody activation:

1) preparing 2L of 1mmol/L sodium acetate solution with pH4.4 of dialysate, adding acetic acid to adjust pH to 4.4, and pre-cooling at 4 deg.C;

2) preparing 0.1mol/L sodium periodate solution: 107mg of sodium periodate were added to 5mL of ultrapure water (15mL brown vial);

3) weighing 0.108mg of aflatoxin B1 monoclonal antibody, dissolving in 1mL of 0.01mol/L sodium phosphate, 0.15mol/L NaCl, and pH7.2 (15mL of brown small bottle), and adding into a self-made stirrer;

4) 100uL of sodium periodate solution is gradually and gradually added into 0.108mg of AFB1 monoclonal antibody drop by drop at the speed of about 20 mu L/s and the stirring speed of about 1r/s, the reaction is carried out for 30min, and the mixture is stirred on a magnetic stirrer for 1 hour at 4 ℃ in the dark;

5) immediately by adding 4.33mg sodium sulfite (Na)₂SO₃) The reaction was quenched to provide a 2-fold higher molar excess than the periodate initially added.

6) After the reaction, 1.1mL of reactant is put into a dialysis bag with the specification of D6mm and the cut-off molecular weight of 7000, and dialyzed overnight by sodium acetate solution at 4 ℃ without changing dialysate;

7) the following day, activated AFB1 mab in the dialysate was removed and stored at 4 ℃ in a 2mL centrifuge tube.

Preparing an S3, aflatoxin B1 antibody and time-resolved fluorescent microsphere directional conjugate:

1) putting 500 mu L of carbonate buffer solution (0.2mol/L, pH9.6) into a 15mL brown vial, continuously stirring with a stirrer at the rotation speed of 1r/s, adding 1.2mg MS, then slowly adding 200 mu L of AFB1 monoclonal antibody dropwise, adding a certain amount of carbonate buffer solution (about 50 mu L), adjusting the pH value to about 9.5 with pH test paper, and stirring at 4 ℃ for reaction;

2) adding 200 mu L of aflatoxin B1 monoclonal antibody every 20min, adding a certain amount of carbonate buffer solution to adjust the pH to about 9.5, and stirring at 4 ℃ for reaction;

3) after 0.108mg of aflatoxin B1 monoclonal antibody is completely added, adjusting the pH value to 9.5, and stirring overnight at 4 ℃;

4) dropwise adding 16 mu L of 5mol/L sodium borohydride solution into the reaction solution on a magnetic stirrer, uniformly stirring, and standing for 2 hours at 4 ℃;

5) unreacted aldehyde groups were blocked by adding 50. mu.L of 1mol/L ethanolamine (pH 9.6) per ml of binding solution. By adding 300. mu.L ethanolamine to 5mL deionized water, an approximately 1mol/L ethanolamine solution can be prepared. The pH of the ethanolamine solution was adjusted by the addition of concentrated hydrochloric acid while keeping the solution cool on ice. The reaction was carried out at room temperature for 30 min.

6) The above reaction was transferred to a 50mL sterile centrifuge tube and centrifuged at 4500rpm for 15min at 4 ℃. Washing with PBS for three times, discarding supernatant, dissolving precipitate with 1mL of precooled 1 × PBS, loading into dialysis bag with specification of D6mm and cut-off molecular weight of 7000, dialyzing with precooled 2L 1 × PBS at 4 deg.C for 24 hr, and changing dialysate for 1 time;

7) 1mL of the time-resolved fluorescent conjugate was diluted 10-fold with 1 × PBS, ultrafiltered (3500r/min, 15min) using a 100KD ultrafilter tube, the supernatant (about 500 μ L) was taken out and put into a centrifuge tube, 50mg/mL of BSA was added to give a final BSA concentration of 5mg/mL, and the sample was stored at-20 ℃.

Comparative analysis

The conventional method for covalently coupling the time-resolved fluorescent microspheres and the antibodies is compared with the effect of directionally coupling the time-resolved fluorescent microspheres and the antibodies in example 1.

The conventional covalent coupling method of the time-resolved fluorescent microspheres and the antibody activates the time-resolved fluorescent microspheres through EDC and NHS, and the time-resolved fluorescent microspheres are incubated with the antibody and then are sealed and stored.

Antigen is coated in a micropore plate in advance, conjugates diluted by 100 times by PBST are added respectively after the micropore plate is sealed for incubation, and a time resolution fluorescence module in a micropore plate reading instrument is used for detecting fluorescence intensity of the conjugates prepared by the two methods, so that the coupling effects of the two methods are compared, and the result is shown in figure 3.

The result shows that the effective antibody amount of the conventional antibody coupling method is small, the antibody is randomly coupled with the time-resolved fluorescent microsphere, and the active sites of the antibody combined with the antigen are probably blocked, so that the antibody is prevented from being combined with more antigens; the method utilizes the unique glycosyl of the antibody to perform directional coupling with the time-resolved fluorescent microsphere, greatly increases the coupling amount of the effective antibody, fully displays the Fab region of the antibody for combining with the antigen, avoids the random coupling of the antibody and the time-resolved fluorescent microsphere, and solves the problem of the blocking of the active site for combining the antibody and the antigen.

Example 2

And (3) directionally coupling the antibody of the C peptide with immunomagnetic beads, and extracting the C peptide in purified serum.

S0, magnetic bead pretreatment:

s0.1 washing of magnetic beads

1) Vortex and shake the resuspended carboxyl magnetic bead, absorb 100 microliter magnetic bead to 2mL centrifuge tube, magnetic separation and suck and abandon the supernatant;

2) adding 500 μ L coupling buffer (50mmol/L MES, pH 6.0, 0.01% Triton X-100) into the tube, vortexing for 20s to wash the magnetic beads, placing the centrifuge tube on a magnetic rack for 60s, magnetically separating and discarding the supernatant; repeating the washing step three times;

s0.2 activation of magnetic beads

1) EDC (50mg/mL) and NHS (50mg/mL) were prepared with coupling buffer (now ready for formulation), and 60. mu.L of coupling buffer, 20. mu.L of fresh EDC solution and 20. mu.L of fresh NHS solution were added to the beads and vortexed to mix.

2) After incubation for 15min at room temperature, the centrifuge tubes were placed on a magnetic rack for 60s, magnetically separated and the supernatant was aspirated. The beads were washed with 500 μ L of coupling buffer, vortexed, magnetically separated and the supernatant was aspirated off and washed twice.

S1, magnetic bead modification:

1) adding 5mg of polylysine into the activated magnetic beads, and standing and incubating for 2h at 25 ℃; magnetically separating and absorbing the supernatant to remove redundant modifying reagents; washing twice with phosphate buffer (PB, 20mmol/L, pH 7.2), magnetically separating and discarding the supernatant;

2) to the beads, 250. mu.L of a preservation solution (0.1% Proclin300) was added.

S2, antibody activation:

1) preparing 2L of 1mmol/L dialysate sodium acetate solution with pH4.4, adding acetic acid to adjust pH to 4.4, and pre-cooling at 4 deg.C;

3) weighing 0.1mg of C peptide labeled antibody, dissolving in 1mL of 0.01mol/L sodium phosphate, 0.15mol/L NaCl, pH7.2 (15mL of brown bottle), and placing in a self-made stirrer;

4) slowly adding 100 μ L of sodium periodate solution into 0.1mg of C peptide labeled antibody dropwise at a speed of about 20 μ L/s and a stirring speed of about 1r/s, reacting for 30min, and stirring on a magnetic stirrer at 4 ℃ for 1 hour in a dark place;

7) the following day, the activated C-peptide labeled antibody in the dialysate was removed and stored at 4 ℃ in a 2mL centrifuge tube.

S3, preparing directional conjugates of the antibodies and the magnetic beads:

1) putting 500uL of carbonate buffer solution (0.2mol/L, pH9.6) into a 15mL brown bottle, putting a self-made stirrer into the bottle, continuously stirring the bottle at the rotation speed of 1r/s, adding 2mg of magnetic beads, then dropwise and slowly adding 200 mu L C of peptide labeled antibody, adding a certain amount of carbonate buffer solution (about 50 mu L), adjusting the pH value to about 9.5 by using pH test paper, and stirring the solution at 4 ℃ for reaction;

2) adding 200 mu L C peptide labeled antibody every 20min, adding a certain amount of carbonate buffer solution to adjust the pH to about 9.5, and stirring at 4 ℃ for reaction;

3) after 0.1mg of C peptide labeled antibody is completely added, adjusting the pH value to 9.5, and stirring overnight at 4 ℃;

4) dropwise adding 16 mu L of 5mol/L sodium borohydride solution into the reaction solution on a magnetic stirrer, uniformly stirring, and standing for 2h at 4 ℃;

5) unreacted aldehyde groups were blocked by adding 50. mu.L of 1mol/L ethanolamine (pH 9.6) per ml of binding solution. Approximately 1mol/L ethanolamine solution can be prepared by adding 300uL ethanolamine to 5mL deionized water. The pH of the ethanolamine solution was adjusted by the addition of concentrated hydrochloric acid while keeping the solution cool on ice. The reaction was carried out at room temperature for 30 min.

6) The above reaction was transferred to a 50mL sterile centrifuge tube and centrifuged at 4500rpm for 15min at 4 ℃.

7) Washing with PBS for three times, discarding supernatant, dissolving precipitate with 1mL of precooled 1 × PBS, loading into dialysis bag with specification of D6mm and cut-off molecular weight of 7000, dialyzing with precooled 2L 1 × PBS at 4 deg.C for 24 hr, and changing dialysate for 1 time;

8) 1mL of immunomagnetic bead conjugate was diluted 10-fold with 1 × PBS, ultrafiltered with a 100KD ultrafilter tube (3500r/min, 15min), the supernatant (about 500 μ L) was taken out and put into a centrifuge tube, 50mg/mL of BSA and glycerol were added to make the final concentration of BSA 5mg/mL and the final volume of glycerol 50%, and the mixture was stored at-20 ℃.

S4, C peptide in purified serum:

1) washing: taking 100 mu L of immunomagnetic bead conjugate to a centrifuge tube, adding 1mL of PBST (10mM PB, 0.5% Tween 20) to wash for 2 times, and removing supernatant after magnetic separation;

2) combining: 1mL of the sample solution was added to the immunomagnetic bead conjugate, mixed and incubated at 37 ℃ for 30min, then subjected to magnetic separation, the supernatant was discarded, and gently washed with 1mL of PBST for 2 times.

3) Dissociation: adding 500 μ L dissociation solution (20mmol/L glycine-hydrochloric acid buffer solution, pH 2.4) into immunomagnetic bead-C peptide complex to dissociate C peptide bound on immunomagnetic bead, adding into supernatant, performing magnetic separation, collecting supernatant, placing into centrifuge tube containing 6 μ L1 mol/L NaOH, washing magnetic bead with 1mL PBST for 2 times, adding 100 μ L quenching buffer TBS (25mol/LTris-Cl,130mmol/LNaCl,2.7mmol/LKCl), and storing;

4) desalting: desalting the collected dissociated supernatant, namely C peptide extracted from the sample by using a solid phase extraction column. Adding 200 μ L acetonitrile into the solid phase extraction column for activation, and centrifuging at 2300g for 2 min. Acetonitrile was then washed with 200. mu.L of eluent (0.1% aqueous formic acid) and centrifuged at 2300g for 2 min. Adding a formic acid solution into the collected supernatant to enable the final concentration to be 0.1%, slowly adding the solution into a solid phase extraction column, centrifuging the solution for 2min at 2300g, and adsorbing the C peptide on the solid phase extraction column; adding 200 μ L eluent (0.1% formic acid water solution) to wash off acetonitrile, centrifuging 2300g for 2min, and eluting excessive salts; finally, 200 μ L of 60% acetonitrile water solution is used to elute the C peptide adsorbed on the small column for solid phase extraction, thereby obtaining the purified C peptide.

Example 3

The directed coupling of the zearalenone antibody and the quantum dots is used for detecting the zearalenone in the edible oil.

S1, quantum dot modification:

1) taking out 0.5mg quantum dots, centrifuging at 4 deg.C and 12000r for 15min, and removing protective solution;

2) activating, adding 250 μ L MES buffer solution (50mmol/L, pH 5) containing 5mg EDC and 1.44mg NHS into quantum dot, blowing up and down, shaking at 25 deg.C for 20min, centrifuging at 4 deg.C and 12000r for 15min, and removing liquid phase;

3) washing, centrifuging, adding 250 μ L MES buffer (50mmol/L, pH 5) into the precipitate, blowing up and down, ultrasonic treating for 3min, centrifuging at 4 deg.C and 12000r for 15min, and removing liquid phase;

4) after centrifugation, 0.1mg of bovine serum albumin was added to the pellet, and the resulting mixture was incubated at 25 ℃ for 2 hours. Excess bovine serum albumin was removed by centrifugation at 12000r for 15min at 4 ℃. Washing twice with phosphate buffer (PB, 20mmol L, pH 7.2), centrifuging at 12000r at 4 deg.C for 15min, and removing the liquid phase;

5) to the pellet after centrifugation, 250. mu.L of a preservation solution (0.1% Proclin300) was added.

S2, activation of zearalenone antibody:

3) weighing 0.2mg of zearalenone monoclonal antibody, dissolving in 1mL of 0.01mol/L sodium phosphate, 0.15mol/L NaCl, and pH7.2 (15mL of brown small bottle), and placing in a self-made stirrer;

4) slowly adding 100 μ L of sodium periodate solution into 0.2mg of zearalenone monoclonal antibody dropwise at a speed of about 20 μ L/s and a stirring speed of about 1r/s, reacting for 30min, and stirring on a magnetic stirrer at 4 ℃ for 1 hour in a dark place;

7) the next day, the activated zearalenone monoclonal antibody in the dialysate was removed and stored in a 2mL centrifuge tube at 4 ℃.

S3, preparing a directional conjugate of the zearalenone antibody and the quantum dot:

1) putting 500 mu L of carbonate buffer solution (0.2mol/L, pH9.6) into a 15mL brown bottle, continuously stirring by a self-made stirrer at the rotation speed of 1r/s, adding 1.5mg of quantum dots, then slowly adding 200 mu L of zearalenone monoclonal antibody dropwise, adding a certain amount of carbonate buffer solution (about 50 mu L), adjusting the pH value to about 9.5 by pH paper, and stirring at 4 ℃ for reaction;

2) adding 200 μ L zearalenone monoclonal antibody every 20min, adding a certain amount of carbonate buffer solution to adjust pH to about 9.5, and stirring at 4 deg.C for reaction;

3) after 0.2mg of zearalenone monoclonal antibody is completely added, adjusting the pH value to 9.5, and stirring overnight at 4 ℃;

4) dropwise adding 20 mu L of 5mol/L sodium borohydride solution into the reaction solution on a magnetic stirrer, uniformly stirring, and standing for 2 hours at 4 ℃;

6) Transferring the reactant into a 50mL sterile centrifuge tube, and centrifuging at 4500rpm4 ℃ for 15 min;

8) 1mL of the quantum dot conjugate was diluted 10-fold with 1 × PBS, ultrafiltered with a 100KD ultrafilter tube (3500r/min, 15min), the supernatant (about 500 μ L) was taken out of the centrifuge tube, 50mg/mL BSA and glycerol were added to give a final BSA concentration of 5mg/mL and a final glycerol volume of 50%, and the mixture was stored at-20 ℃.

Detection was performed using the quantum dot conjugates prepared above.

Preparing a zearalenone test strip:

the NC membrane is humidified for 1h at 25 ℃ and 55% humidity in advance, 0.5mg/mL zearalenone antigen is marked on a T line, 1mg/mL goat-anti-mouse secondary antibody is marked on a C line, and the NC membrane is dried for 12h at 37 ℃. Sample pads and absorbent paper were attached, and cut into 4mm pieces for use.

Sample pretreatment:

according to the patent of 'a method for rapidly and immunologically detecting pollutants in vegetable oil and application (ZL 201510477644.4)', zearalenone in edible oil is extracted.

A detection step:

premixing 2 mu L of quantum dot conjugate and 150 mu L of sample solution in a micropore, oscillating for 3min, dropwise adding 100 mu L of the premixed quantum dot conjugate into a prepared zearalenone quantum dot immunochromatography detection card, and detecting by using a quantum dot reading instrument after 15 min. And calculating the concentration of zearalenone in the sample solution according to the standard curve of the detection card.

Detecting the performance parameters of the card:

the detection range of the detection method for detecting the zearalenone in the edible oil is 0.5-10ng/g, and the average recovery rate is 104.2 +/-5.6%.

The invention solves the problem of difficult coupling with glycosyl in an antibody by modifying a solid phase carrier material; and the problem of random fixation of the antibody and the solid phase carrier material is solved, and the active site of the antibody combined with the antigen can be fully exposed. When the directional conjugate constructed by the invention is applied to immunoassay, more antigens can be identified by less directional conjugates, and the sensitivity of immunoassay determination is improved.

While several embodiments of the present invention have been presented herein, it will be appreciated by those skilled in the art that changes may be made to the embodiments herein without departing from the spirit of the invention. The above examples are merely illustrative and should not be taken as limiting the scope of the invention.

Claims

1. A method for realizing directional coupling by utilizing glycosyl of an antibody and a solid phase carrier material is characterized in that the method utilizes a modifying reagent to modify the solid phase carrier material, so that the surface of the solid phase carrier material is modified with a binding arm which has a certain length and is rich in amino, and the binding arm can extend into an Fc region of the antibody and react with the glycosyl of the antibody; then oxidizing glycosyl of the antibody into aldehyde group; the aldehyde group reacts with the amino group of the modified solid phase carrier material to form Schiff base, and then the Schiff base is reduced into amine compounds, so that the directional coupling of the antibody and the solid phase carrier material is realized.

2. The method for achieving directed conjugation using antibody glycosyl and solid support material according to claim 1, wherein the method comprises the following steps:

3. The method of claim 2, wherein in step S1, the modifying reagent comprises bovine serum albumin, polylysine, chitosan; the solid phase carrier material comprises time-resolved fluorescent microspheres, nano magnetic particles, immune latex particles, quantum dots and up-conversion luminescent particles.

4. The method of claim 2, wherein in step S2, the oxidizing agent is sodium periodate.

5. The method of claim 2, wherein in step S3, the reducing agent is one of sodium borohydride, sodium cyanoborohydride, amine borane and ascorbic acid; the blocking agent comprises ethanolamine, glycine and tris (hydroxymethyl) aminomethane.

6. The method for achieving directional coupling of antibody glycosyl and solid phase carrier material according to claim 2 wherein the step S1, the solid phase carrier material is modified by the following steps:

s1.1, taking out a solid phase carrier material, and removing a protective solution through centrifugation to obtain a precipitate;

s1.2, adding a certain amount of modification reagent into the centrifuged sediment, standing and incubating at 25 ℃, centrifuging, and removing redundant modification reagent; washing twice with phosphate buffer;

7. The method for achieving directed coupling of antibody glycosyl and solid phase carrier material as claimed in claim 2 wherein step S2, the specific step of antibody activation comprises:

s2.1 antibody lysis: dissolving the antibody with a buffer solution to obtain an antibody solution;

s2.2 antibody and sodium periodate mixed reaction: slowly adding sodium periodate solution into the antibody solution drop by drop, reacting at 25 ℃ for 30min, and stirring on a magnetic stirrer at 4 ℃ in a dark place for 1 h;

s2.3 quenching reaction: quenching the oxidation reaction of the antibody with sodium periodate by adding a quenching reagent, wherein the quenching reagent is sodium sulfite;

s2.4 dialysis purification: after the reaction is finished, putting the reactant into a dialysis bag, and dialyzing by using a sodium acetate solution at 4 ℃;

8. The method for achieving directional coupling by using glycosyl and solid phase carrier material of antibody as claimed in claim 2, wherein in step S3, the specific steps of directional conjugate preparation comprise:

s3.1, mixing and reacting the activated antibody prepared in the step S2 with the modified solid phase carrier material: adding carbonate buffer solution into a stirrer to be continuously stirred, adding the solid phase carrier material, then dropwise and slowly adding the activated antibody solution in batches, adding a certain amount of carbonate buffer solution to adjust the pH value to about 9.5, and stirring and reacting at 4 ℃;

s3.2 reduction of unstable Schiff bases: dropwise adding a reducing agent into the reaction liquid in the step S3.1 on a magnetic stirrer, uniformly stirring, and standing for 2 hours at 4 ℃;

s3.3 blocking of Targeted conjugates: adding a sealing agent to seal unreacted aldehyde groups, and reacting for 30min at 25 ℃; transferring the reactant into a sterile centrifuge tube, and centrifuging to remove the redundant sealant;

s3.4 washing and dialysis purification: repeatedly washing with phosphate buffer solution, dissolving the precipitate with precooled phosphate buffer solution, filling into a dialysis bag, and dialyzing with precooled phosphate buffer solution at low temperature of 4 ℃ for 24 hours;

s3.5 preservation of the Targeted conjugates: taking the directional conjugate, diluting by 10 times with phosphate buffer solution, ultrafiltering with 100KD ultrafiltration tube, and centrifuging; taking the supernatant liquid into a centrifuge tube, adding a conjugate preservation solution, and preserving at-20 ℃; the conjugate preserving fluid comprises bovine serum albumin, sodium azide and Proclin 300.

9. A directed conjugate obtained by the method of using the glycosyl of an antibody and a solid phase carrier material according to any one of claims 1 to 8 to achieve directed conjugation.

10. Use of a targeted conjugate in immunoassay and immuno-extraction, wherein the targeted conjugate is obtained by the method of any one of claims 1 to 8 using a glycosyl group of an antibody and a solid support material to achieve targeted conjugation.