CN113512107A - Warfarin complete antigen and preparation method thereof - Google Patents

Abstract

The application belongs to the technical field of poison detection, and particularly relates to a warfarin complete antigen and a preparation method thereof. It is prepared by directly coupling warfarin and BSA, or by firstly preparing hapten and then preparing holoantigen. In the process of preparing antibodies by animal immunization, because small molecule compounds lack immunogenicity, proper molecular modification is often needed to enable the small molecule compounds to become complete antigens with immunogenicity, and then animal mice can be further immunized to generate and prepare monoclonal antibodies. In the present application, the inventors prepared WFR-BSA complete antigens coupled with BSA by direct coupling and first hapten and then complete antigen, respectively. And the monoclonal antibodies are prepared by utilizing the prepared complete antigens respectively. Preliminary experiment results show that the in vivo serum titer of the immunized mouse is higher, the immune reaction of the mouse can be effectively activated, and a better technical basis is laid for the preparation of related monoclonal antibodies.

Description

Warfarin complete antigen and preparation method thereof

Technical Field

The application belongs to the technical field of poison detection, and particularly relates to a warfarin complete antigen and a preparation method thereof.

Background

Warfarin, a name of warfarin, is an anticoagulant type rodenticide, is mainly used for killing domestic rats such as mice, rats and brown rats, and can also be used for killing wild rats, and the action mode of the warfarin is that after entering a rat body, the warfarin hinders the synthesis of prothrombin in blood, so that the rat body is hemorrhagic through the anticoagulation effect to kill the rat, and the warfarin is widely used in the world due to good environmental safety.

Although warfarin has better environmental safety, the phenomenon that some poultry, livestock and even children take the warfare by mistake is frequently caused due to strong animal toxicity, easiness in purchasing and wide application of warfarin, so that whether related toxicity characteristics are caused by warfarin or not is timely detected and judged from the perspective of public safety, and the warfarin has very important technical significance for related treatment and prevention.

In the prior art, when the warfarin residue is detected in a sample, a liquid chromatography-mass spectrometry detection method is commonly used, but the method needs to be equipped with professional technicians and expensive experimental equipment, and the detection time is long, so that related treatment is easily delayed. The immunological detection method has the characteristics of high detection speed, low experimental technical requirement, simple requirement on experimental equipment and the like, and has better technical significance for the rapid detection and judgment of the warfarin. However, the implementation of the immunological method requires that the related immunoassay products such as colloidal gold test strips, SPR detection chips, ELISA kits and the like can be prepared based on specific antibodies. However, related research and reports on the warfarin antibody in the prior art are relatively limited, so that the prior art is lack of related immune product development. Therefore, relevant warfarin-related antigen and antibody products are developed, and relevant immune products are developed on the basis, so that the method has very important technical significance for rapid detection and relevant treatment of warfarin.

Disclosure of Invention

The application aims to provide the warfarin complete antigen for preparing the warfarin monoclonal antibody and the monoclonal antibody based on the warfarin complete antigen, thereby laying a certain technical foundation for preparing related immune products for detection.

The technical solution of the present application is detailed as follows.

A Warfarin complete antigen WFR-BSA is prepared by coupling Warfarin (Warfarin) and BSA, and is prepared by the following steps:

(I) Material preparation

Preparing related materials for later use according to the following material proportions and requirements respectively:

weighing Warfarin, and dissolving with methanol to obtain 5mg/ml solution;

dissolving BSA into a binding buffer solution to prepare a solution of 5mg/ml for later use;

EDC (2mM) (carbodiimide, (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) (EDC) (18 mg) and NHS (5mM) (N-hydroxysuccinimide) (27 mg) were added to 30ml of coupling buffer, and were sufficiently dissolved to prepare an activator for use;

the binding buffer (i.e., coupling buffer) was a 0.1M MES solution at pH = 4.5-5.0;

in the preparation process, Sulfo-NHS (N-hydroxy thiosuccinimide) can be used to replace NHS;

(di) radical coupling

When the groups are coupled, a direct coupling mode is adopted, or a mode of firstly preparing hapten and then preparing holoantigen is adopted;

when the complete antigen Warfarin-BSA (WFR-BSA) is prepared by adopting a direct coupling mode, the following steps are referenced:

(1) mixing the BSA solution with an activating agent (in a volume ratio of 1: 1), and reacting at room temperature for 1h (or keeping the temperature at 37 ℃ for 30 min) to ensure that all amino groups are coupled with activated groups;

(2) mixing 1ml of Warfarin methanol solution with 2ml of activated BSA solution in step (1), and freeze-drying at-80 ℃ to ensure freeze-drying;

(3) placing the freeze-dried mixture obtained in the step (2) in a coupling instrument, and performing coupling reaction for 1 hour at 50 ℃ under the nitrogen atmosphere;

(4) adding 5ml of PBS (pH7.4) solution into the coupling reaction end product in the step (3) for dissolution;

then, the lysate is transferred to a 3KD ultrafiltration tube, centrifuged at 6000g for 1h at 4 ℃, the supernatant is discarded, and the precipitate is suspended with 5ml of PBS (pH7.4) and centrifuged at 6000g for 1h at 4 ℃ again to ensure that the uncoupled small molecules are removed; finally obtaining the trapped Warfarin-BSA conjugate;

(5) finally dissolving and recovering the product by using a PBS (pH7.4) solution to prepare the Warfaring-BSA conjugate;

when the complete antigen WFR-BSA is prepared by adopting a mode of firstly preparing hapten and then preparing the complete antigen WFR-BSA, the following steps are referenced:

(1) adding 50mg of warfarin into 10mL of 1M potassium permanganate solution, stirring for 6 hours at the temperature of 25 ℃, and pouring into an ice-water mixture to obtain a solid precipitate;

filtering the solid precipitate and recrystallizing with ethanol to obtain modified hapten WFR-COOH with-COOH groups;

then, dissolving WFR-COOH into an activation buffer solution to prepare a solution with the concentration of 3 mg/ml for later use;

the activation buffer is a 0.1M MES (2- (N-morphine) ethanesulfonic acid) solution containing 0.5M NaCl and having a pH = 6.0;

(2) adding 10ml of the activator prepared in the step (one) into 5ml of hapten WFR-COOH activated solution prepared in the step (1), and reacting at 37 ℃ for 15min to activate groups;

after the reaction was complete, 2-mercaptoethanol was added to a final concentration of 20mM to quench EDC;

(3) adding 4ml of BSA solution in the step (1), and reacting at 37 ℃ for 2 hours to couple BSA with the WFR-COOH activated in the step (2);

after coupling occasionally, hydroxylamine was added to a final concentration of 10mM to form hydroxamic acid with unreacted NHS;

(4) and finally, removing redundant quenching reagent by adopting a desalting column to obtain the WFR-BSA after BSA coupling.

The warfarin complete antigen WFR-BSA is applied to the preparation of monoclonal antibodies and is used for preparing anti-WFR antibodies.

In the process of preparing antibodies by animal immunization, because small molecule compounds lack immunogenicity, proper molecular modification is often needed to enable the small molecule compounds to become complete antigens with immunogenicity, and then animal mice can be further immunized to generate and prepare monoclonal antibodies. In the present application, the inventors prepared WFR-BSA complete antigens coupled with BSA by direct coupling and first hapten and then complete antigen, respectively. And the monoclonal antibodies are prepared by utilizing the prepared complete antigens respectively. Preliminary experiment results show that the in vivo serum titer of the immunized mouse is higher, the immune reaction of the mouse can be effectively activated, and a better technical basis is laid for the preparation of related monoclonal antibodies.

Drawings

FIG. 1 is an enzyme-labeled assay for Warfarin-BSA conjugate;

FIG. 2 shows the results of serum antibody titers obtained after mice were immunized with the relevant antigens.

Detailed Description

The present application is further illustrated by the following examples.

Example 1

In this example, Warfarin (Warfarin) was coupled to BSA protein by direct coupling to prepare the complete antigen, Warfarin-BSA, which is briefly described below.

(1) Weighing Warfarin, and dissolving with methanol to obtain 5mg/ml solution;

at the same time, BSA was activated, specifically:

firstly, dissolving BSA into a binding buffer solution to prepare a solution of 5mg/ml for later use;

subsequently, 18mg of EDC (2mM) (carbodiimide, (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride), 27mg of NHS (5mM) (N-hydroxysuccinimide) were added to 30ml of the coupling buffer, and the activator was prepared after sufficient dissolution;

finally, the above BSA solution was mixed with the activating agent (volume ratio, 1: 1) and allowed to react at room temperature for 1h (or incubated at 37 ℃ for 30 min) to ensure that all amino groups had been coupled to the activated groups.

The binding buffer (coupling buffer) was a 0.1M MES solution at pH = 4.5-5.0.

(2) 1ml (5 mg/ml) of Warfaring methanol solution was mixed with 2ml (5 mg/ml) of activated BSA (all amino groups coupled to activating groups) solution, and lyophilized at-80 ℃ to ensure lyophilization.

(3) And (3) placing the freeze-dried mixture obtained in the step (2) in a coupling instrument, and coupling at 50 ℃ for 1 hour under the nitrogen atmosphere.

(4) Adding 5ml of PBS (pH7.4) solution into the coupling reaction end product in the step (3) for dissolution;

then, the lysate is transferred to a 3KD ultrafiltration tube, centrifuged at 6000g for 1h at 4 ℃, the supernatant is discarded, and the precipitate is suspended with 5ml of PBS (pH7.4) and centrifuged at 6000g for 1h at 4 ℃ again to ensure that the uncoupled small molecules are removed; the final product is the trapped Warfarin-BSA conjugate.

(5) Dissolving and recovering the final product by using 2ml of PBS (pH7.4), and measuring the A280 value, wherein the concentration of the prepared Warfarin-BSA conjugate is 4.41 mg/ml; further sampling, identifying, packaging and storing for application.

Example 2

In view of the problems of preparation efficiency, preparation amount, preparation difficulty and the like of directly preparing the complete antigen in example 1, the inventors adopted a technical idea of first preparing the hapten and further preparing the complete antigen in this example. That is, in this example, the inventors chemically modified warfarin to prepare a hapten having a COOH group and further coupled BSA protein to prepare a complete antigen.

It should be noted that, in the preparation process, based on chemical structure similarity and functional similarity, as a control, the inventors synchronously used BDL (bromoadiolone) and BDF (brodifacoum) to prepare the relevant hapten and complete antigen.

The specific preparation process is briefly described as follows.

Chemical modification of rodenticide

Chemically modifying and preparing the warfarin into hapten with COOH groups, wherein the specific method comprises the following steps:

(1) adding 50mg of warfarin into 10mL of 1M potassium permanganate solution, and stirring for 6 hours at the temperature of 25 ℃;

(2) pouring the mixture obtained in the step (1) into an ice-water mixture to obtain a solid precipitate;

(3) filtering the solid precipitate in the step (2) and recrystallizing with ethanol to obtain the modified hapten: WFR-COOH (Warfaring-COOH), BDL-COOH and BDF-COOH.

(II) chemical coupling with BSA protein

(1) Firstly, respectively dissolving WFR-COOH, BDL-COOH and BDF-COOH into an activation buffer solution to prepare a solution with the concentration of 3 mg/ml for later use;

meanwhile, 18mg of EDC (2mM) (carbodiimide, (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 27mg of NHS (5mM) (N-hydroxysuccinimide) were added to 30ml of the coupling buffer, and were sufficiently dissolved to prepare an activator for use;

synchronously, dissolving BSA into a binding buffer solution to prepare a solution of 5mg/ml for later use;

the activation buffer is a 0.1M MES (2- (N-morphine) ethanesulfonic acid) solution containing 0.5M NaCl and having a pH = 6.0;

the binding buffer (coupling buffer) was a 0.1M MES solution at pH = 4.5-5.0.

(2) Subsequently, 10ml of the above activator is added into 5ml of WFR-COOH, BDL-COOH and BDF-COOH solution respectively, and reacted for 15min at 37 ℃ to activate groups;

after the reaction was completed, 22. mu.l of 2-mercaptoethanol (final concentration: 20mM) was added thereto to quench EDC.

(3) Then, 4ml of the BSA solution obtained in step (1) (i.e., 20mg of BSA was added) was added thereto, and the mixture was reacted at 37 ℃ for 2 hours to couple BSA with WFR-COOH, BDL-COOH and BDF-COOH activated in step (2);

after coupling, hydroxylamine (hydroxylamine hydrochloride solution) was added to a final concentration of 10mM (or to a concentration of 20-50mM in lysine, glycine or ethanolamine Tris buffer) to form hydroxamic acid with unreacted NHS.

(4) And finally, removing redundant quenching reagents (neutralizing reagents) by using a desalting column, and finally obtaining the coupled BSA WFR-BSA, BDL-BSA and BDF-BSA which are used as complete antigens for subsequent immunization.

Blank (unconjugated Warfarin) activated BSA was adjusted to a concentration of 4.41mg/ml in PBS (pH7.4) to serve as a blank control. And (3) identifying the prepared antigen by adopting an enzyme labeling method, wherein the specific operation is as follows:

when enzyme labeling analysis is carried out, respectively taking 200 mu l of blank activated BSA and Warfaring-BSA conjugate, and respectively adding the blank activated BSA and Warfaring-BSA conjugate into 2 holes of a 96-hole plate; performing full-wavelength absorption scanning by using a Spectra max 190 full-wavelength microplate reader, and stepping by 3 nm; baseline zeroing was performed with blank post-activation BSA as blank control.

The results of the scans of the Warfarin-BSA conjugate are shown in fig. 1. As can be seen from the figure, the Warfarin-BSA conjugate has a characteristic absorption peak at 340nm of 240-minus-one, and the result indicates that the small molecule-BSA has been successfully coupled, and the subsequent experiments can be carried out.

Example 3

Based on the complete antigens WFR-BSA, BDL-BSA and BDF-BSA prepared in examples 1 and 2, the inventors further injected them into mice for immunization to obtain B cells in the spleen, fused the B cells with hybridoma cells, and performed titer detection of the antibody in the supernatant of the hybridoma cells. The specific experimental procedures are briefly described as follows.

(I) mouse immunization and serum titers

Respectively dissolving the complete antigens prepared in the examples 1 and 2 in physiological saline to prepare a solution with the concentration of 0.2mg/mL, and then uniformly mixing the complete antigens with Freund's complete adjuvant which is distributed and has the same volume by adopting a double-pushing method (the concentration of the antigen is equivalent to 0.1 mg/mL);

abdominal subcutaneous multipoint injection, immunization of BALB/c mice (injection amount 0.25 mL);

after 2 weeks, the same amount of antigen and incomplete Freund's adjuvant are mixed uniformly by the same method, and then the mixture is injected again for immunization; after 1 week, boosts were given 1 time per week (i.e., equal amount of antigen in incomplete freund's adjuvant) for a total of 3 times;

after 3 days of the last immunization, serum titers were determined by tail-off blood sampling.

The results of the measurement of the serum titer of the mice at different dilution ratios are shown in FIG. 2 (the abscissa represents the dilution ratio of the serum, and the ordinate represents the antibody content in the serum of the mice). Analysis can see that:

the red line (lower curve) represents the antibody titer in the serum of the mice immunized with the complete antigen prepared in example 1, and the green line (upper curve) represents the antibody titer in the serum of the mice immunized with the complete antigen prepared in example 2. by comparison, the method of immunizing the mice with the complete antigen first and then with the hapten in example 2 can promote the mice to produce more antibodies (the results of BDL and BDF are similar, and thus are not shown specifically).

Preparation of monoclonal antibody

Based on the results of the serum titer after the immunization, the inventors further performed the preparation of the relevant monoclonal antibody, and the specific process is briefly described as follows.

(1) Preparation of myeloma cells

Myeloma cells are expanded and cultured 48 hours before fusion;

on the day of fusion, gently blowing down the cells from the bottle wall by using an elbow dropper, collecting the cells in a 50 ml centrifugal tube, centrifuging the cells at 1000r/min for 10 minutes, and discarding the supernatant; adding 30ml of incomplete culture medium, and centrifuging and washing once; the cells were then resuspended in 10ml of incomplete medium and mixed until needed.

(2) Preparation of splenocytes

The immunized BALB/c mouse (the complete antigen WFR-BSA in example 2 was used for immunization) was sacrificed and the spleen was removed, placed in a dish containing 10ml of incomplete medium, ground into a cell suspension with a syringe needle, and then pipetted several times to prepare a single cell suspension.

(3) Cell fusion

Mixing myeloma cells in the step (1) and spleen cells in the step (2) in a 50 ml fusion tube, supplementing incomplete culture medium to 30ml, and fully and uniformly mixing;

adding preheated 50% PEG 1ml in 30s with a 1ml pipette, and stirring gently while adding; then adding preheated incomplete culture solution to stop the action of PEG;

adding 5ml of complete culture medium, slightly blowing and sucking the precipitated cells to suspend and uniformly mix the cells, and then supplementing the complete culture medium to 40 ml;

subpackaging to 96-well cell culture plate with 100ul per well, standing at 37 deg.C with 5% CO₂ Culturing in an incubator.

(6) Cloning of hybridoma cells (by limiting dilution method)

When screening hybridoma cells, limited dilution (to 0.8 cells/well, calculated by Poisson method, 36% of wells should be 1 cell/well) is performed first, and then culture is performed, and during the culture process, fused cells which can survive for more than one week are screened. When the cells are cultured to cover about 20 percent of the bottom of the hole, the culture supernatant is absorbed to detect the content of the antibody by ELISA, and then high-secretion specific cell strains are screened and cloned for expanding culture or frozen storage for later use.

The results of antibody detection of the partially screened and cloned hybridoma cells are shown in the following table. It can be seen that the antibody expression level of part of hybridoma cell strains is more prominent, and a good technical basis can be laid for the preparation of related antibodies.

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Claims (2)

1. The warfarin complete antigen WFR-BSA is characterized in that the complete antigen is prepared by coupling warfarin and BSA, and is prepared by the following steps:

(I) Material preparation

Preparing related materials for later use according to the following material proportions and requirements respectively:

weighing Warfarin, and dissolving with methanol to obtain 5mg/ml solution;

dissolving BSA into a binding buffer solution to prepare a solution of 5mg/ml for later use;

adding 18mg of EDC and 27mg of NHS into 30ml of coupling buffer solution, and preparing an activator for later use after fully dissolving;

the binding buffer, i.e., coupling buffer, was a 0.1M MES solution at pH = 4.5-5.0;

(di) radical coupling

When the groups are coupled, a direct coupling mode is adopted, or a mode of firstly preparing hapten and then preparing holoantigen is adopted;

when the complete antigen WFR-BSA is prepared by adopting a direct coupling mode, the following steps are carried out:

(1) mixing BSA solution with an activating agent, and reacting at room temperature for 1h to ensure that all amino groups are coupled with activated groups;

(2) 1ml of Warfaring methanol solution and 2ml of activated BSA solution are mixed evenly and freeze-dried to obtain a solution;

(3) placing the freeze-dried mixture obtained in the step (2) in a coupling instrument for coupling reaction under the condition of nitrogen atmosphere;

(4) adding a PBS solution into the coupling reaction finished product in the step (3) for dissolving; then, transferring the lysate to a 3KD ultrafiltration tube, centrifuging, discarding the supernatant, and removing uncoupled micromolecules; finally obtaining the trapped Warfarin-BSA conjugate;

when the complete antigen WFR-BSA is prepared by adopting a mode of firstly preparing hapten and then preparing the complete antigen WFR-BSA, the specific steps are as follows:

(1) transforming warfarin into hapten WFR-COOH with-COOH groups, dissolving the warfarin into an activation buffer solution, and preparing into a solution with the concentration of 3 mg/ml;

the activation buffer is a 0.1M MES solution containing 0.5M NaCl with pH = 6.0;

(2) adding 10ml of the activator prepared in the step (one) into 5ml of hapten WFR-COOH activated solution prepared in the step (1), and reacting at 37 ℃ for 15min to activate groups;

after the reaction was complete, 2-mercaptoethanol was added to a final concentration of 20mM to quench EDC;

(3) adding 4ml of BSA solution in the step (1), and reacting at 37 ℃ for 2 hours to couple BSA with the WFR-COOH activated in the step (2);

after coupling occasionally, hydroxylamine was added to a final concentration of 10mM to form hydroxamic acid with unreacted NHS;

(4) and finally, removing redundant quenching reagent by adopting a desalting column to obtain the WFR-BSA after BSA coupling.

2. Use of the warfarin complete antigen WFR-BSA of claim 1 in the preparation of monoclonal antibodies for the preparation of antibodies against WFR.

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