CN112920163B - Hapten, antigen and antibody of imatinib and N-demethyl imatinib and application thereof - Google Patents

Hapten, antigen and antibody of imatinib and N-demethyl imatinib and application thereof Download PDF

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CN112920163B
CN112920163B CN202110103999.2A CN202110103999A CN112920163B CN 112920163 B CN112920163 B CN 112920163B CN 202110103999 A CN202110103999 A CN 202110103999A CN 112920163 B CN112920163 B CN 112920163B
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imatinib
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张玉
韩勇
陶凯雄
史琛
张鹏
呂永宁
周红
赵晓亚
黄潇
张望
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Tongji Medical College of Huazhong University of Science and Technology
Beijing Diagreat Biotechnology Co Ltd
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Abstract

The application relates to the technical field of immunological detection, and particularly discloses hapten, antigen and antibody of imatinib and N-demethyl imatinib and application thereof, wherein the hapten has a structure shown in formula 10:the antigen is a conjugate formed by coupling the hapten and a carrier protein; the antibodies are generated in response to the antigen. The antigen and the antibody prepared by adopting the hapten can simultaneously identify the imatinib and the N-demethyl imatinib, and have strong specificity and high sensitivity.

Description

Hapten, antigen and antibody of imatinib and N-demethyl imatinib and application thereof
Technical Field
The application relates to the technical field of immunological detection, in particular to hapten, antigen and antibody of imatinib and N-demethyl imatinib and application thereof.
Background
Imatinib mesylate (imatinib mesylate) is a small-molecule tyrosine kinase inhibitor, takes KIT and platelet-derived growth factor receptor (PDGFRA) as targets to exert anti-tumor activity, and is mainly used for treating Philadelphia chromosome (Bcr-Abl) -positive chronic granulocytic leukemia (CML for short) and malignant gastrointestinal stromal tumor (GIST) patients incapable of surgical excision or metastasis.
Imatinib is metabolized in humans mainly by the CYP3A enzyme system to N-desmethyl piperazine derivatives (N-desmethyl imatinib), and the in vitro efficacy of the metabolite is similar to that of the original drug. There is therefore a need for a detection method that is capable of simultaneously determining imatinib and N-desmethyl imatinib. In recent years, studies have shown that imatinib mesylate steady-state trough blood concentration (Cmin) is closely related to clinical benefit and disease progression in CML and GIST patients; in addition, the imatinib blood concentration has larger inter-individual variation and intra-individual variation, which suggests that the blood concentration monitoring can play an important role in judging the imatinib curative effect, evaluating the curative effect, avoiding side effects, adjusting the personalized medication scheme and the like.
The detection and analysis of imatinib mainly comprises HPLC-UV, LC-MS-MS, MESED-LC-MS-MS, electrochemical analysis methods and the like, but the methods can not meet the requirements of clinical high-throughput, rapid and accurate detection.
For example, patent documents with the authority of 2016, 03 and 30, and the authority of 102625702B disclose an imatinib immunoassay method, and propose an imatinib molecule derivative, and an antibody prepared by using the derivative specifically recognizes imatinib, but does not have cross reaction with N-demethyl imatinib, and the prepared detection reagent may cause low valley of blood concentration of imatinib and toxicity of imatinib.
For example, patent documents with the authority of publication No. CN104804079B and 2018, publication No. 07 and 13 disclose an imatinib immunogen, a derivative, a synthesis method, a specific antibody, a detection reagent and a preparation method. The patent document does not specifically specify whether or not N-desmethyl imatinib is recognized, but is not recognized from the standpoint of description of specificity.
In view of the above, there is currently a lack of immunological detection methods for simultaneously determining imatinib and N-desmethyl imatinib with high sensitivity.
Disclosure of Invention
In order to establish an immunological detection method which is strong in specificity and high in sensitivity and can simultaneously detect imatinib and N-desmethyl imatinib, the application provides hapten, antigen and antibody of imatinib and N-desmethyl imatinib and application thereof.
In a first aspect, the hapten of imatinib and N-desmethyl imatinib provided by the application adopts the following technical scheme:
hapten of imatinib and N-demethyl imatinib, the structure of which is shown in formula 10:
in a second aspect, the preparation method of hapten of imatinib and N-demethyl imatinib provided by the application adopts the following technical scheme:
a preparation method of hapten of imatinib and N-demethyl imatinib comprises the following steps:
(i) The compound shown in the formula 1 and trimethyl orthoformate are subjected to an aldehyde group protection reaction to obtain a compound shown in the formula 2;
(ii) The compound shown in the formula 2 and the compound shown in the formula 3 are subjected to condensation reaction to obtain a compound shown in the formula 4;
(iii) The compound shown in the formula 4 and the compound shown in the formula 5 are subjected to reductive amination reaction to obtain a compound shown in the formula 6;
(iv) Deprotection reaction of the compound shown in the formula 6 to obtain a compound shown in the formula 7;
(v) The compound shown in the formula 7 and ethyl bromoacetate undergo substitution reaction to obtain a compound shown in the formula 8;
(vi) The compound shown in the formula 8 and m-chloroperoxybenzoic acid undergo oxidation reaction to obtain a compound shown in the formula 9;
(vii) The compound shown in the formula 9 is subjected to hydrolysis reaction to obtain a compound shown in the formula 10;
the specific synthetic route is as follows:
preferably, in the step (i), the molar ratio of the compound shown in the formula 1 to trimethyl orthoformate is 1 (2-3), the aldehyde group protection reaction uses p-toluenesulfonic acid as a catalyst, and the molar ratio of the p-toluenesulfonic acid to the compound shown in the formula 1 is (0.3-0.5): 1.
Preferably, in the step (ii), the molar ratio of the compound shown in the formula 2 to the compound shown in the formula 3 is 1 (1-1.1), the condensation reaction is carried out by taking HATU as a condensing agent and triethylamine as an acid-binding agent, and the molar ratio of the HATU, the triethylamine and the compound shown in the formula 2 is (1-1.2): (2-2.4): 1.
Preferably, the molar ratio of the compound shown in the formula 4 to the compound shown in the formula 5 in the step (iii) is 1 (1-1.2), the reductive amination reaction uses sodium triacetyl borohydride as a reducing agent, and the molar ratio of the sodium triacetyl borohydride to the compound shown in the formula 4 is (1-1.2): 1.
Preferably, the deprotection reaction in step (iv) is carried out by using trifluoroacetic acid as a deprotection agent, and the molar ratio of trifluoroacetic acid to the compound represented by formula 6 is (20-50): 1.
Preferably, the molar ratio of the compound of formula 7 to ethyl bromoacetate in step (v) is 1 (1-1.2), anhydrous potassium carbonate.
Preferably, the molar ratio of the compound of formula 8 to m-chloroperoxybenzoic acid in step (vi) is 1 (1-1.2).
Preferably, in the step (vii), sodium hydroxide is used as a hydrolysing agent, and the molar ratio of sodium hydroxide to the compound represented by formula 9 is (1.5-3): 1.
In a third aspect, the present application provides an antigen of imatinib and N-desmethyl imatinib, which adopts the following technical scheme:
an antigen of imatinib and N-desmethyl imatinib is a conjugate formed by coupling the hapten and carrier protein.
Preferably, the hapten and carrier protein are coupled using a coupling agent selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
Preferably, the carrier protein comprises at least one of bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin and rabbit serum albumin.
In a fourth aspect, the preparation method of the antigen of imatinib and N-desmethyl imatinib provided by the application adopts the following technical scheme:
a method for preparing an antigen of imatinib and N-desmethylimatinib, comprising the steps of:
(i) Dissolving hapten in dimethyl sulfoxide to obtain dimethyl sulfoxide solution of hapten;
(ii) Dissolving a coupling agent in water to obtain an aqueous solution of the coupling agent;
(iii) Mixing a dimethyl sulfoxide solution of hapten and a coupling agent aqueous solution, and reacting for 0.5-2 hours at room temperature to obtain a reaction solution;
(iv) Dissolving carrier protein in PBS buffer solution to obtain carrier protein solution;
(v) Mixing the carrier protein solution with the reaction solution, and stirring at room temperature for 1-3 hours to obtain a reaction mixed solution;
(vi) Dialyzing the reaction mixed solution against PBS buffer solution to obtain the antigen, wherein the volume ratio of the reaction mixed solution to the PBS buffer solution is 1 (400-600);
the weight ratio of hapten to coupling agent to carrier protein is 1 (0.5-1.5) (1-2).
Preferably, the coupling agent is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
Preferably, the carrier protein comprises at least one of bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin and rabbit serum albumin.
In a fifth aspect, the present application provides an antibody of imatinib and N-desmethyl imatinib, which adopts the following technical scheme:
an antibody of imatinib and N-desmethylimatinib is produced in response to the antigen described above.
Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.
Preferably, the antibody is specifically prepared by the following method:
(i) Selecting a host for antibody production;
(ii) Inoculating a host with the antigen;
(iii) Fusing a cell line from the vaccinated host with Sp2/0 cells to obtain hybridoma cells producing said antibody;
(iv) The hybridoma cells are prepared by adopting host ascites, and the antibody is obtained by purification.
Preferably, the host is selected from any one of mice, rabbits, goats, sheep.
In a sixth aspect, the present application provides an antibody of imatinib and N-desmethyl imatinib, which is applied by the following technical scheme:
use of an antibody to imatinib and N-desmethyl imatinib for detecting imatinib and/or N-desmethyl imatinib.
In a sixth aspect, the present application provides a detection kit according to the following technical solution:
a test kit comprising an antibody as described above.
In summary, the application has the following beneficial effects:
the derivative sites of hapten structures of the imatinib and the N-demethyl imatinib provided by the application are far away from the characteristic structure of the imatinib, so that the antibody prepared by the application has no cross reaction with two inactive metabolites of the Pyridine-N-oxide-imatinib and the Hydroxymethyl-phenyl-imatinib; in addition, the derivative site of the hapten structure is positioned at the methyl position of the N-demethyl imatinib and is coupled with carrier protein by adopting an extremely short connecting arm, and the carrier protein can shield the methyl part of the N-demethyl imatinib, so that the antibody prepared by the application can simultaneously identify the imatinib and the N-demethyl imatinib.
Second, the antigen and antibody provided by the application have the capability of simultaneously recognizing imatinib and N-desmethyl imatinib, and have antibody specificity superior to that of the related art, namely, have no cross reaction with Pyridine-N-oxide-imatinib and hydroymethyl-phenyl-imatinib.
Drawings
FIG. 1 is a correlation analysis of the total concentration of imatinib and N-desmethyl imatinib by a magnetic particle luminescence method established by the antibody provided by the application and an HPLC-MS method.
Detailed Description
The present application will be described in further detail with reference to examples.
Preparation examples of hapten of imatinib and N-desmethyl imatinib
The structures of the hapten of imatinib and N-desmethyl imatinib are shown as formula 10, and the specific synthetic route of the compound of formula 10 is as follows:
the hapten of the target products imatinib and N-demethyl imatinib is obtained by taking carboxybenzaldehyde and trimethyl orthoformate as starting materials and sequentially carrying out seven steps of reactions such as aldehyde group protection reaction, condensation reaction, reductive amination reaction, substitution reaction, oxidation reaction, hydrolysis reaction and the like.
Step (i): the compound shown in the formula 1 and trimethyl orthoformate undergo an aldehyde group protection reaction to obtain a compound shown in the formula 2:
to a 500mL single-necked flask were added p-carboxybenzaldehyde (15 g,0.01 mol), 150mL of methanol, p-toluenesulfonic acid monohydrate (0.75 g,0.00394 mol) and trimethyl orthoformate (3 mL), and the reaction was completed by TLC at room temperature for 24 hours. After the system was evaporated to dryness, 200mL of water was added, the aqueous phase was extracted three times with 50mL of methylene chloride each time, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, evaporated to dryness to give a crude product, which was purified by column chromatography to give the compound of formula 2 (7.8 g, yield 40%).
Step (ii): the compound shown in the formula 2 and the compound shown in the formula 3 undergo condensation reaction to obtain a compound shown in the formula 4:
to a 250mL single flask was added the compound of formula 2 (7.8 g,4.00 mmol), HATU (16.6 g,4.40 mmol), dichloromethane 150mL, triethylamine (8.10 g,8.8 mmol), the compound of formula 3 (11.1 g,4.00 mmol), the system was reacted at 25℃for 12 hours, the reaction was completed by TLC detection, and the crude product was obtained by spin-drying and purified by column chromatography to give the compound of formula 4 (13 g, yield 72%).
Step (iii): the compound shown in the formula 4 and the compound shown in the formula 5 are subjected to reductive amination reaction to obtain a compound shown in the formula 6:
to a 250mL single-port flask, the compound (4.55 g,10 mmol), glacial acetic acid 5mL and methanol 150mL were added, the system was stirred for 3 hours, then the compound (1.86 g,10 mmol) of the compound shown in the formula 5 and sodium triacetylborohydride (2.11 g,10 mmol) were added, the system was reacted at room temperature for 16 hours, TLC detection reaction was completed, the system was filtered, the filtrate was evaporated to dryness to obtain a crude product, and the crude product was purified by column chromatography to obtain the compound (1.27 g, yield 22%) of the compound shown in the formula 6.
Step (iv): deprotection of a compound of formula 6 affords a compound of formula 7:
to a 100mL single-port flask, the compound represented by formula 6 (1.16 g,2 mmol), 30mL of methylene chloride and 5mL of trifluoroacetic acid were added, the reaction was carried out at room temperature for 12 hours, TLC detection was carried out, water was added for 20mL, and sodium bicarbonate solution was used for adjusting pH=8, the organic phase was separated, dried, filtered and evaporated to dryness to obtain a crude product, and the crude product was subjected to column chromatography to obtain the compound represented by formula 7 (800 mg, yield 84%).
Step (v): the compound shown in the formula 7 and ethyl bromoacetate undergo substitution reaction to obtain a compound shown in the formula 8:
to a 100mL single-port flask, the compound represented by formula 7 (800 mg,1.67 mmol), tetrahydrofuran (20 mL), anhydrous potassium carbonate (460 mg,3.34 mmol) and ethyl bromoacetate (280 mg,1.67 mmol) were added, the reaction was carried out at room temperature for 16 hours, the reaction was completed by TLC, the system was filtered, the filtrate was evaporated to dryness to give a crude product, and the crude product was purified by column chromatography to give the compound represented by formula 8 (700 mg, yield 74%).
Step (vi): the compound shown in the formula 8 and m-chloroperoxybenzoic acid undergo oxidation reaction to obtain a compound shown in the formula 9:
to a 100mL single-port flask, the compound represented by formula 8 (566 mg,1 mmol), methylene chloride 20mL, and m-chloroperoxybenzoic acid (172 mg,1 mmol) were added, the reaction was carried out at room temperature for 16 hours, the TLC detection reaction was completed, the system was filtered, the filtrate was evaporated to dryness to obtain a crude product, and the crude product was purified by column chromatography to obtain the compound represented by formula 9 (203 mg, yield 34%).
Step (vii): the compound shown in the formula 9 is subjected to hydrolysis reaction to obtain a compound shown in the formula 10:
to a 100mL single-port flask, a compound (200 mg,3.44 mmol) represented by formula 9, sodium hydroxide (28 mg,6.88 mmol) methanol (20 mL) and water (1 mL) were added, the reaction was carried out at room temperature for 2 hours, TLC detection was completed, and the system was evaporated to dryness; then adding 50mL of water, extracting the secondary water phase by adopting ethyl acetate, wherein the dosage of the ethyl acetate is 30mL each time; then the pH value of the water phase is regulated to be 3 by hydrochloric acid, the water phase is extracted by ethyl acetate for three times, and the consumption of the ethyl acetate is 30mL each time; the organic phases were combined, dried, filtered, evaporated to dryness to give a crude product, which was purified by column chromatography to give the compound of formula 10 (110 mg, yield 58%).
Characterization of hapten of imatinib and N-desmethyl imatinib:
1 H-NMR(400MHZ,DMSOd6)：δ2.12-2.49(m,3H),2.80(m,4H),3.34(m,4H),3.66(m，2H),4.18(m,2H),7.20(d,1H),7.39-7.55(m,5H),7.57(d,1H),7.92(d,2H),8.45-8.50(m,1H),8.50(d,
1H),8.68(dd,1H),8.89(s,1H),9.24(d,1H),10.32(d,1H),12.22(brs,1H)；
13 C-NMR(400MHZ,DMSOd6)：δ17.6,50.6,50.6,55.0,55.0,59.0,64.4,103.3,107.9,111.5,124.0,124.6,127.2,127.2,128.9,128.9,130.0,133.0,132.7,133.7,134.0,142.0,142.2,147.5,147.9,157.6,157.9,164.7,168.2,168.6；
MS：m/Z 554(M+H)。
preparation examples of antigens of imatinib and N-desmethyl imatinib
The antigen of imatinib and N-desmethyl imatinib is a conjugate formed by coupling hapten of imatinib and N-desmethyl imatinib and carrier protein.
The specific preparation method of the antigen of the imatinib and the N-demethyl imatinib comprises the following steps:
(i) Dissolving 12mg of imatinib and N-desmethyl imatinib hapten in 1mL of dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten;
(ii) 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was dissolved in 100uL of water to obtain an aqueous solution of the coupling agent;
(iii) Mixing a dimethyl sulfoxide solution of hapten and a coupling agent aqueous solution, and reacting for 1 hour at room temperature to obtain a reaction solution;
(iv) Dissolving 16mg of carrier protein in 5mL of PBS buffer solution to obtain a carrier protein solution;
(v) Mixing the carrier protein solution with the reaction solution, and stirring at room temperature for 2 hours to obtain a reaction mixed solution;
(vi) And dialyzing the reaction mixed solution against PBS buffer solution, wherein the volume ratio of the reaction mixed solution to the PBS buffer solution is 1:500, and repeating the dialyzing step four times to obtain the antigen of the imatinib and the N-demethyl imatinib.
It is worth noting that the conditions for preservation of the antigens of imatinib and N-desmethyl imatinib are cryopreservation, in particular at-20 ℃.
Preparation examples of antibodies to imatinib and N-desmethyl imatinib
Antibodies to imatinib and N-desmethyl imatinib are generated in response to antigens to imatinib and N-desmethyl imatinib.
The specific preparation method of the monoclonal antibody of the imatinib and the N-demethyl imatinib comprises the following steps:
(i) Selecting a host for antibody production: wherein, the host can be mice, rabbits, goats, sheep and the like, and the preparation example adopts the mice as the host;
(ii) The antigens of imatinib and N-desmethyl imatinib were inoculated into the host: diluting the antigen of the imatinib and the antigen of the N-demethyl imatinib to 1mg/mL by adopting PBS buffer solution, adding an equal volume of Freund complete adjuvant, completely emulsifying, and immunizing a mouse for the first time according to the dosage of 0.1 mg/mouse; after four weeks of interval, 1mg of imatinib and N-desmethylimatinib antigen were mixed with 1mg of Freund's incomplete adjuvant and stirred at a stirring speed of 2000rpm/min for 2 hours to complete emulsification, and the mice immunized for the first time were boosted at a dose of 0.1 mg/dose;
(iii) Spleen cells from the inoculated host are fused with Sp2/0 cells, antigen coated ELISA 96-well plates of imatinib and N-desmethyl imatinib are adopted, titer and competition measurement are respectively carried out on the fused cells by adopting an indirect ELISA method and an indirect competition ELISA method, and 3 cells are obtained by screening, wherein the cells are respectively 6A, 11B1 and 21C1.
(iv) The three strains of cells are respectively prepared by adopting mouse ascites and protein A/G affinity purification to obtain the antibody.
The binding capacities of the three antibodies with four small molecule compounds, i.e., imatinib, N-desmethyl imatinib, pyridine-N-oxide-imatinib, hydroxymethyl-phenyl-imatinib, were measured by indirect competition ELISA, and the results are shown in Table 1.
TABLE 1 Imatinib antibody specificity assay
6A 11B1 21C1
Imatinib 100％ 100％ 100％
N-desmethyl imatinib 54％ 62％ 96％
Pyridine-N-oxide-imatinib ＜0.1％ ＜0.1％ ＜0.1％
Hydroxymethyl-phenyl-imatinib ＜0.1％ ＜0.1％ ＜0.1％
As can be seen from Table 1, the antibodies produced by 21C1 cells showed substantially consistent affinity for imatinib and N-desmethyl imatinib and very low cross-reactivity to inactive Pyridine-N-oxide-imatinib and Hydroxymethyl-phenyl-imatinib. Thus, the antibodies raised by 21C1 cells are suitable for simultaneous detection of imatinib and establishment of a method for monitoring N-desmethyl imatinib blood concentration.
This is probably due to the fact that the derivative site of the hapten structure provided by the application is far away from the characteristic structure of imatinib, so that the antibody prepared by the application has no cross reaction with two imatinib inactive metabolites of Pyridine-N-oxide-imatinib and hydroymethyl-phenyl-imatinib; and, the derivative site of hapten structure is positioned at the methyl position of N-demethyl imatinib and is coupled with carrier protein by adopting a very short connecting arm, and the carrier protein can shield the methyl part of N-demethyl imatinib, so that the antibody prepared by the application can simultaneously identify imatinib and N-demethyl imatinib, which is very beneficial to simultaneously determining two active substances of imatinib and N-demethyl imatinib.
In conclusion, the antigen and the antibody provided by the application have the capability of simultaneously recognizing imatinib and N-desmethyl imatinib, and have antibody specificity superior to that of the related art, namely, have no cross reaction with Pyridine-N-oxide-imatinib and hydroymethyl-phenyl-imatinib.
Preparation examples of detection reagents for the detection of imatinib and/or N-desmethyl imatinib are worth noting: "detection of imatinib and/or N-desmethyl imatinib" means: if the sample contains imatinib and does not contain N-demethyl imatinib, the concentration of the imatinib can be independently detected; if the sample does not contain imatinib and contains N-demethyl imatinib, the concentration of N-demethyl imatinib can be independently detected; if the sample contains both imatinib and N-desmethyl imatinib, the total concentration of imatinib and N-desmethyl imatinib can be detected simultaneously.
Preparation examples of detection reagents for the detection of imatinib and/or N-desmethyl imatinib
The detection reagent for detecting the imatinib and/or the N-desmethyl imatinib comprises a magnetic particle working solution, an enzyme-labeled working solution and an indicator solution.
S1, obtaining a magnetic particle working solution:
s1-1: 50mg of Dynal beads M270 COOH magnetic particles were diluted in 2mL of MES buffer (50 mM, pH=6.0) to obtain a magnetic particle solution, and 1mg of an antibody was added to the magnetic particle solution to obtain a magnetic particle-antibody mixture;
s1-2: adding 2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into the magnetic particle-antibody mixed solution, and vibrating at room temperature for 2 hours;
s1-3: removing the supernatant by magnetic attraction, adding TBST buffer solution to enable the concentration of magnetic particles to reach 0.5mg/mL, and reacting for 2 hours at room temperature;
s1-4: removing the supernatant by magnetic attraction, adding TBST buffer solution to obtain magnetic particle working solution, wherein the concentration of the magnetic particles in the magnetic particle working solution is 0.4mg/mL.
S2, obtaining enzyme-labeled working solution:
s2-1: 1mg of alkaline phosphatase is dissolved in 1mL of PBS buffer to obtain alkaline phosphatase solution;
s2-2: dissolving 0.5mg of imatinib and/or hapten of N-desmethyl imatinib in 0.1mL of dimethyl sulfoxide to obtain hapten solution;
s2-3: uniformly mixing an alkaline phosphatase solution and a hapten solution, adding 1mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and uniformly mixing at room temperature for 2 hours;
s2-4: and (3) obtaining the enzyme-labeled working solution by adopting a PBS (phosphate buffer solution) dialysis method, wherein the concentration of the marker in the enzyme-labeled working solution is 1ug/mL.
S3, obtaining an indicator solution: commercial AMPPD luminescent solutions.
The detection reagent for detecting the imatinib and/or the N-desmethylimatinib can be prepared at present or can be stored and taken at the temperature of 2-8 ℃; in a further step, the detection reagent for detecting the imatinib and/or the N-desmethyl imatinib is prepared into a detection kit, and the detection kit needs to be stored at the temperature of 2-8 ℃.
Detection example for immunological detection of imatinib and/or N-desmethyl imatinib in sample
The immunological detection method for detecting the imatinib and/or the N-desmethyl imatinib comprises the following specific operation steps:
uniformly mixing 20uL of blood sample, 40uL of magnetic particle liquid working solution and 50uL of enzyme-labeled working solution, incubating at 37 ℃ for 5min, cleaning, adding an indicator solution for color development, and collecting optical signals of 400-550nm by adopting a photomultiplier. The specific step of cleaning comprises the steps of magnetically removing the supernatant, adding TBST buffer solution for resuspension, and magnetically removing the supernatant again.
The method comprises the steps of selecting 50 clinical patient blood samples taking imatinib, respectively measuring total concentration of imatinib and N-desmethyl imatinib in the blood samples by adopting an immunological detection method and an HPLC method for detecting imatinib and/or N-desmethyl imatinib, and carrying out correlation analysis on the measurement results, wherein the analysis results are shown in figure 1.
As can be seen from FIG. 1, the total concentration of imatinib and N-desmethyl imatinib measured by the magnetic particle luminescence method based on the antibody prepared by the application has good correlation with the HPLC method, and meets the clinical requirements.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims (8)

1. An antigen of imatinib and N-desmethyl imatinib, characterized in that the antigen is a conjugate formed by coupling hapten and carrier protein with a structure shown in a formula 10, and the hapten and the carrier protein are coupled by taking 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride as a coupling agent;。
2. a method of preparing the antigen of imatinib and N-desmethyl imatinib of claim 1, comprising the steps of: (i) Dissolving the hapten in dimethyl sulfoxide to obtain a dimethyl sulfoxide solution of the hapten; (ii) Dissolving a coupling agent in water to obtain an aqueous solution of the coupling agent; (iii) Mixing a dimethyl sulfoxide solution of hapten and a coupling agent aqueous solution, and reacting for 0.5-2 hours at room temperature to obtain a reaction solution; (iv) Dissolving carrier protein in PBS buffer solution to obtain carrier protein solution; (v) Mixing the carrier protein solution with the reaction solution, and stirring at room temperature for 1-3 hours to obtain a reaction mixed solution; (vi) Dialyzing the reaction mixed solution against PBS buffer solution to obtain the antigen, wherein the volume ratio of the reaction mixed solution to the PBS buffer solution is 1 (400-600); the weight ratio of hapten to coupling agent to carrier protein is 1 (0.5-1.5) (1-2).
3. The method of preparing an antigen of imatinib and N-desmethyl imatinib according to claim 2, characterized in that the method of preparing the hapten comprises the steps of: (i) The compound shown in the formula 1 and trimethyl orthoformate are subjected to an aldehyde group protection reaction to obtain a compound shown in the formula 2; (ii) The compound shown in the formula 2 and the compound shown in the formula 3 are subjected to condensation reaction to obtain a compound shown in the formula 4; (iii) The compound shown in the formula 4 and the compound shown in the formula 5 are subjected to reductive amination reaction to obtain a compound shown in the formula 6; (iv) Deprotection reaction of the compound shown in the formula 6 to obtain a compound shown in the formula 7; (v) The compound shown in the formula 7 and ethyl bromoacetate undergo substitution reaction to obtain a compound shown in the formula 8; (vi) The compound shown in the formula 8 and m-chloroperoxybenzoic acid undergo oxidation reaction to obtain a compound shown in the formula 9; (vii) The compound shown in the formula 9 is subjected to hydrolysis reaction to obtain a compound shown in the formula 10; the specific synthetic route is as follows:
。
4. the method for preparing an antigen of imatinib and N-desmethylimatinib according to claim 3, wherein the molar ratio of the compound represented by formula 1 to trimethyl orthoformate in the step (i) for preparing a hapten is 1 (2-3), the aldehyde group protecting reaction is carried out by using p-toluenesulfonic acid as a catalyst, and the molar ratio of the p-toluenesulfonic acid to the compound represented by formula 1 is (0.3-0.5): 1; in the step (ii), the molar ratio of the compound shown in the formula 2 to the compound shown in the formula 3 is 1 (1-1.1), the condensation reaction is that HATU is taken as a condensing agent, triethylamine is taken as an acid-binding agent, and the molar ratio of the HATU, the triethylamine and the compound shown in the formula 2 is (1-1.2): (2-2.4): 1; in the step (iii), the mol ratio of the compound shown in the formula 4 to the compound shown in the formula 5 is 1 (1-1.2), the reductive amination reaction takes sodium triacetyl borohydride as a reducing agent, and the mol ratio of the sodium triacetyl borohydride to the compound shown in the formula 4 is (1-1.2): 1; in the step (iv), trifluoroacetic acid is used as a deprotection agent, and the molar ratio of the trifluoroacetic acid to the compound shown in the formula 6 is (20-50): 1; the molar ratio of the compound shown in the formula 7 to ethyl bromoacetate in the step (v) is 1 (1-1.2); the molar ratio of the compound shown in the formula 8 and the m-chloroperoxybenzoic acid in the step (vi) is 1 (1-1.2); in the step (vii), sodium hydroxide is used as a hydrolyzer, and the molar ratio of the sodium hydroxide to the compound shown in the formula 9 is (1.5-3): 1.
5. An antibody of imatinib and N-desmethylimatinib, produced in response to the antigen of claim 1.
6. The antibody of claim 5, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
7. Use of an antibody according to any one of claims 5-6 for the detection of imatinib and/or N-desmethyl imatinib, which use is not for the diagnosis and treatment of diseases.
8. A test kit comprising the antibody of any one of claims 5-6.
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