CN112920163A - Hapten, antigen and antibody of imatinib and N-demethyl imatinib and application thereof - Google Patents
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Abstract
The application relates to the technical field of immunological detection, and particularly discloses a hapten, an antigen and an antibody of imatinib and N-demethylation imatinib and application thereof, wherein the hapten has a structure shown as a formula 10:
the antigen is a conjugate formed by coupling the hapten and a carrier protein; the antibody is generated in response to the antigen. The antigen and the antibody prepared by the hapten can simultaneously recognize imatinib and N-demethyl imatinib, and have strong specificity and high sensitivity.
Description
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 micromolecular tyrosine kinase inhibitor, exerts antitumor activity by taking KIT and platelet-derived growth factor receptor (PDGFRA) as targets, and is mainly used for treating Philadelphia chromosome (Bcr-Abl) -positive chronic myelogenous leukemia (CML for short) and patients with malignant gastrointestinal stromal tumors (GIST) which cannot be resected or metastasized by operation.
Imatinib is metabolized into an N-demethylpiperazine derivative (N-demethyl imatinib) in a human body mainly through a CYP3A enzyme system, and the in vitro drug effect of the metabolite is similar to that of a raw drug. There is therefore a need for an assay that is capable of simultaneously determining imatinib and N-desmethyl imatinib. In recent years, studies have shown that steady state blood trough concentrations (Cmin) of imatinib mesylate are closely associated with clinical benefit and disease progression in CML and GIST patients; in addition, the imatinib blood concentration has larger inter-individual and intra-individual variation, which prompts the blood concentration monitoring to play an important role in judging imatinib curative effect, evaluating treatment effect, avoiding side reaction, adjusting individual medication scheme and the like.
However, few clinical practices are developed in the imatinib blood concentration monitoring at present, and the method is related to the tedious imatinib blood concentration detection method, the uncertain imatinib treatment window of Chinese people, the lack of consciousness of blood concentration monitoring of people and the like. The detection and analysis of imatinib mainly comprises HPLC-UV, LC-MS-MS, MESED-LC-MS-MS, electrochemical analysis method and the like, but the methods can not meet the clinical high-flux, rapid and accurate detection requirements.
If the patent document with the grant publication date of 2016, 03, 30 and the grant publication number of CN102625702B discloses an Imatinib immunoassay, an Imatinib molecule derivative is provided, an antibody prepared by using the derivative specifically recognizes Imatinib, but does not have cross reaction with N-demethyl Imatinib, and a prepared detection reagent may cause a low trough to the blood concentration of Imatinib, so that the toxicity of Imatinib is caused.
For example, patent documents with the publication authorization date of 2018, 07, 13 and publication authorization number of CN104804079B disclose imatinib immunogen, derivatives and synthetic method thereof, specific antibody and detection reagent and preparation method thereof. The patent document does not specifically specify whether N-desmethyl imatinib is recognized, but cannot be recognized from the description of specificity.
In summary, there is currently a lack of immunological detection methods that are highly sensitive and that simultaneously measure imatinib and N-desmethyl imatinib.
Disclosure of Invention
In order to establish an immunological detection method which has strong specificity and high sensitivity and can simultaneously detect imatinib and N-demethyl imatinib, the application provides a hapten, an antigen and an antibody of imatinib and N-demethyl imatinib and application thereof.
In a first aspect, the hapten of imatinib and N-demethyl imatinib provided by the present application adopts the following technical scheme:
hapten of imatinib and N-demethylimatinib, which has a structure shown in formula 10:
in a second aspect, the present application provides a method for preparing a hapten of imatinib and N-demethyl imatinib, which adopts the following technical scheme:
a method for preparing haptens of imatinib and N-demethylimatinib, comprising the steps of:
(i) performing aldehyde group protection reaction on the compound shown in the formula 1 and trimethyl orthoformate to obtain a compound shown in a formula 2;
(ii) carrying out condensation reaction on the compound shown in the formula 2 and the compound shown in the formula 3 to obtain a compound shown in a formula 4;
(iii) carrying out reductive amination reaction on the compound shown in the formula 4 and the compound shown in the formula 5 to obtain a compound shown in a formula 6;
(iv) carrying out deprotection reaction on the compound shown in the formula 6 to obtain a compound shown in a formula 7;
(v) carrying out substitution reaction on the compound shown in the formula 7 and ethyl bromoacetate to obtain a compound shown in a formula 8;
(vi) carrying out oxidation reaction on the compound shown in the formula 8 and m-chloroperoxybenzoic acid to obtain a compound shown in a formula 9;
(vii) carrying out hydrolysis reaction on the compound shown in the formula 9 to obtain a compound shown in a 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 is carried out by taking 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 represented by the formula 2 to the compound represented by the formula 3 is 1 (1-1.1), the condensation reaction is carried out by taking HATU as a condensation agent and triethylamine as an acid-binding agent, and the molar ratio of the HATU, the triethylamine and the compound represented by the formula 2 is (1-1.2): 2-2.4): 1.
Preferably, in the step (iii), the molar ratio of the compound represented by the formula 4 to the compound represented by the formula 5 is 1 (1-1.2), the reductive amination reaction is performed by using sodium triacetyl borohydride as a reducing agent, and the molar ratio of the sodium triacetyl borohydride to the compound represented by the formula 4 is (1-1.2): 1.
Preferably, the deprotection reaction in the step (iv) is trifluoroacetic acid as a deprotection agent, and the molar ratio of the trifluoroacetic acid to the compound shown in the formula 6 is (20-50): 1.
Preferably, the molar ratio of the compound shown in the formula 7 to the ethyl bromoacetate in the step (v) is 1 (1-1.2), and anhydrous potassium carbonate is adopted.
Preferably, the molar ratio of the compound shown in the formula 8 in the step (vi) to m-chloroperoxybenzoic acid is 1 (1-1.2).
Preferably, in the step (vii), the hydrolysis reaction is performed by using sodium hydroxide as a hydrolyzing agent, and the molar ratio of the sodium hydroxide to the compound represented by the formula 9 is (1.5-3): 1.
In a third aspect, the present application provides an antigen of imatinib and N-demethyl imatinib, which adopts the following technical scheme:
an antigen of imatinib and N-demethylimatinib is a conjugate formed by coupling the hapten and a carrier protein.
Preferably, the hapten and the carrier protein are coupled by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride as a coupling agent.
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 present application provides a method for preparing an antigen of imatinib and N-demethyl imatinib, which adopts the following technical scheme:
a method for preparing antigens of imatinib and N-desmethyl imatinib comprising the steps of:
(i) dissolving the hapten of claim 1 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 the dimethyl sulfoxide solution of the hapten and the aqueous solution of the coupling agent, and reacting at room temperature for 0.5-2 hours to obtain a reaction solution;
(iv) dissolving carrier protein in PBS buffer solution to obtain carrier protein solution;
(v) mixing the carrier protein solution and the reaction solution, and stirring for 1-3 hours at room temperature 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 the hapten to the coupling agent to the carrier protein is 1 (0.5-1.5) to 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 against imatinib and N-demethyl imatinib, wherein the following technical scheme is adopted:
an antibody to imatinib and N-desmethyl imatinib produced in response to the antigen described above.
Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.
Preferably, the antibody is prepared by the following method:
(i) selecting a host for antibody production;
(ii) inoculating the host with the antigen;
(iii) fusing a cell line from the inoculated host with Sp2/0 cells to obtain hybridoma cells producing the antibody;
(iv) the hybridoma cell is prepared from host ascites and purified to obtain the antibody.
Preferably, the host is selected from any one of mouse, rabbit, goat and sheep.
In a sixth aspect, the application of an antibody against imatinib and N-demethyl imatinib provided by the present application adopts 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, which adopts the following technical scheme:
a detection kit comprises the antibody.
In summary, the present application has the following beneficial effects:
firstly, the hapten structure of imatinib and N-demethyl imatinib is derived from a site far away from the characteristic structure of imatinib, so that the antibody prepared by the method has no cross reaction with two imatinib inactive metabolites, namely Pyridine-N-oxide-imatinib and hydroxymethy-phenyl-imatinib; and the derivative site of the hapten structure is positioned at the methyl position of the N-demethyl imatinib and is coupled with the carrier protein by adopting a very 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 recognize imatinib and the N-demethyl imatinib.
Secondly, the antigen and the antibody provided by the application have the capability of simultaneously recognizing imatinib and N-demethyl imatinib, and simultaneously have the antibody specificity superior to that of the related technology, namely, the antigen and the antibody do not have cross reaction with Pyridine-N-oxide-imatinib and hydroxymethy-phenyl-imatinib.
Drawings
FIG. 1 is a correlation analysis of total concentration of imatinib and N-demethylimatinib determined by magnetic particle luminescence method established by the antibody provided in the present application and HPLC-MS method.
Detailed Description
The present application will be described in further detail with reference to examples.
Preparation examples of haptens of Imatinib and N-demethyl Imatinib
The structure of hapten of imatinib and N-demethyl imatinib is shown in formula 10, and the specific synthetic route of the compound of formula 10 is as follows:
the target products imatinib and N-demethyl imatinib hapten are obtained by using 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 are subjected to aldehyde group protection reaction to obtain a compound shown in a formula 2:
a500 mL single-neck flask was charged with p-carboxybenzaldehyde (15g, 0.01mol), 150mL of methanol, p-toluenesulfonic acid monohydrate (0.75g, 0.00394mol), and trimethyl orthoformate (3mL), and the system was reacted at room temperature for 24 hours to complete the reaction by TLC. After the system is evaporated to dryness, 200mL of water is added, the water phase is extracted with dichloromethane for three times, the dosage of dichloromethane is 50mL each time, the organic phases are combined, dried by anhydrous sodium sulfate, filtered and evaporated to dryness to obtain a crude product, and the crude product is purified by column chromatography to obtain the compound of formula 2 (7.8g, yield 40%).
Step (ii): carrying out condensation reaction on a compound shown in a formula 2 and a compound shown in a formula 3 to obtain a compound shown in a formula 4:
a250 mL single-neck flask is charged with the compound shown in formula 2 (7.8g, 4.00mmol), HATU (16.6g, 4.40mmol), dichloromethane 150mL, triethylamine (8.10g, 8.8mmol) and the compound shown in formula 3 (11.1g, 4.00mmol), the system reacts at 25 ℃ for 12 hours, TLC detection reaction is finished, and the crude product is obtained by spin-drying, and is purified by column chromatography to obtain the compound shown in formula 4 (13g, 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:
adding a compound shown in a formula 4 (4.55g and 10mmol), glacial acetic acid 5mL and methanol 150mL into a 250mL single-neck bottle, stirring the system for 3 hours, adding a compound shown in a formula 5 (1.86g and 10mmol) and sodium triacetyl borohydride (2.11g and 10mmol), reacting the system for 16 hours at room temperature, finishing TLC detection reaction, filtering the system, evaporating filtrate to obtain a crude product, and purifying the crude product by column chromatography to obtain a compound shown in a formula 6 (1.27g and the yield of 22%).
Step (iv): and carrying out deprotection reaction on the compound shown in the formula 6 to obtain a compound shown in a formula 7:
adding a compound shown as a formula 6 (1.16g, 2mmol), dichloromethane 30mL and trifluoroacetic acid 5mL into a 100mL single-neck bottle, reacting the system at room temperature for 12 hours, detecting by TLC, adding water 20mL, adjusting the pH to 8 by using sodium bicarbonate solution, separating an organic phase, drying, filtering, evaporating to obtain a crude product, and performing column chromatography on the crude product to obtain a compound shown as a formula 7 (800mg, yield 84%).
Step (v): and (3) carrying out substitution reaction on the compound shown in the formula 7 and ethyl bromoacetate to obtain a compound shown in a formula 8:
adding the compound shown in the formula 7 (800mg, 1.67mmol), tetrahydrofuran 20mL, anhydrous potassium carbonate (460mg, 3.34mmol) and ethyl bromoacetate (280mg, 1.67mmol) into a 100mL single-neck flask, reacting at room temperature for 16 hours, detecting by TLC to finish the reaction, filtering the system, evaporating the filtrate to obtain a crude product, and purifying the crude product by column chromatography to obtain the compound shown in the formula 8 (700mg, yield 74%).
Step (vi): the compound shown in the formula 8 and m-chloroperoxybenzoic acid undergo oxidation reaction to obtain a compound shown in a formula 9:
adding the compound shown in the formula 8 (566mg, 1mmol), dichloromethane 20mL and m-chloroperoxybenzoic acid (172mg, 1mmol) into a 100mL single-neck flask, reacting for 16 hours at room temperature in a system, finishing TLC detection reaction, filtering the system, evaporating filtrate to obtain a crude product, and purifying the crude product by column chromatography to obtain the compound shown in the formula 9 (203mg, yield 34%).
Step (vii): the compound shown in the formula 9 is subjected to hydrolysis reaction to obtain a compound shown in a formula 10:
adding 20mL of a compound shown in formula 9 (200mg, 3.44mmol), 20mL of sodium hydroxide (28mg, 6.88mmol) methanol and 1mL of water into a 100mL single-neck bottle, reacting the mixture at room temperature for 2 hours, finishing TLC detection reaction, and evaporating the system to dryness; then adding 50mL of water, and extracting a secondary water phase by adopting ethyl acetate, wherein the dosage of the ethyl acetate is 30mL each time; then adjusting the pH value of the water phase to 3 by hydrochloric acid, extracting the water phase for three times by adopting ethyl acetate, wherein the dosage of the ethyl acetate is 30mL each time; the organic phases were combined, dried, filtered and evaporated to dryness to give a crude product, which was purified by column chromatography to give the compound represented by formula 10 (110mg, yield 58%).
Characterization of haptens of imatinib and N-desmethyl imatinib:1H-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)；
13C-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-demethyl Imatinib
The antigen of imatinib and N-demethyl imatinib is a conjugate formed by a hapten of imatinib and N-demethyl imatinib and a carrier protein by conjugation.
The specific preparation method of the antigen of the imatinib and the N-demethyl imatinib comprises the following steps:
(i) dissolving hapten of 12mg of imatinib and N-demethyl imatinib in 1mL of dimethyl sulfoxide to obtain dimethyl sulfoxide solution of the hapten;
(ii) dissolving 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 100uL of water to obtain a coupling agent aqueous solution;
(iii) mixing a dimethyl sulfoxide solution of a hapten with a coupling agent aqueous solution, and reacting at room temperature for 1 hour 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 for 2 hours at room temperature to obtain a reaction mixed solution;
(vi) and (3) 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 dialysis step for four times to obtain the antigens of the imatinib and the N-demethyl imatinib.
It is worth pointing out that the preservation conditions for the antigens of imatinib and N-demethylimatinib are low temperature cryopreservation, in particular cryopreservation 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 of imatinib and N-desmethyl imatinib.
The specific preparation method of the monoclonal antibody of imatinib and N-demethyl imatinib comprises the following steps:
(i) selection of hosts for antibody production: wherein, the host can adopt mice, rabbits, goats, sheep and the like, and the mice are adopted as the host in the preparation embodiment;
(ii) vaccinating a host with antigens of imatinib and N-desmethyl imatinib: diluting antigens of imatinib and N-demethyl imatinib to 1mg/mL by PBS buffer solution, adding equivalent volume of Freund's complete adjuvant, emulsifying completely, and immunizing mouse according to 0.1 mg/mouse for the first time; after four weeks, 1mg of the antigen of imatinib and N-demethyl imatinib and 1mg of Freund's incomplete adjuvant were mixed 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/mouse;
(iii) splenocytes from the inoculated host are fused with Sp2/0 cells, antigen coating ELISA 96-well plates of imatinib and N-demethyl 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 strains of cells are obtained by screening, wherein the cell strains are 6A, 11B1 and 21C1 respectively.
(iv) The three strains of cells are respectively prepared by mouse ascites and affinity purified by Protein A/G to obtain the antibody.
The indirect competition ELISA method is adopted to determine the binding capacity of the three antibodies with four small molecular compounds such as imatinib, N-demethylimatinib, Pyridine-N-oxide-imatinib, hydroxymethy-phenyl-imatinib and the like, and the results are shown in Table 1.
TABLE 1 Imatinib antibody specificity assay
| 6A | 11B1 | 21C1 | |
| Imatinib | 100％ | 100％ | 100％ |
| N-demethyl 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 uniform affinity for imatinib and N-desmethyl imatinib, and very low cross-reactivity to inactive Pyridine-N-oxide-imatinib and hydroxymethy-phenyl-imatinib. Therefore, the antibody produced by the 21C1 cell is suitable for establishing a monitoring method for simultaneously detecting imatinib and N-demethylation imatinib blood concentration.
This is probably because 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, namely Pyridine-N-oxide-imatinib and hydroxymethy-phenyl-imatinib; furthermore, the derivative site of the hapten structure is positioned at the methyl position of N-demethyl imatinib and is coupled with a carrier protein by using a very 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 recognize imatinib and N-demethyl imatinib, which is very favorable for simultaneously measuring 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-demethyl imatinib, and simultaneously have the antibody specificity superior to that of the related art, namely, the antigen and the antibody do not have cross reaction with Pyridine-N-oxide-imatinib and hydroxymethy-phenyl-imatinib.
Preparation examples of detection reagents for detecting imatinib and/or N-desmethyl imatinib deserve to be noted: "detecting imatinib and/or N-desmethyl imatinib" represents: if the sample contains imatinib and does not contain N-demethyl imatinib, the concentration of imatinib can be separately detected; if the sample does not contain imatinib and contains N-demethyl imatinib, the concentration of the N-demethyl imatinib can be separately detected; if the sample contains both imatinib and N-demethyl imatinib, the total concentration of imatinib and N-demethyl imatinib can be detected simultaneously.
The detection reagent for detecting imatinib and/or N-demethylimatinib comprises magnetic particle working solution, enzyme labeling working solution and indicator solution.
S1 obtaining of magnetic particle working solution:
s1-1: diluting 50mg Dynal beads M270 COOH magnetic particles in 2mL MES buffer (50mM, pH 6.0) to obtain a magnetic particle solution, and adding 1mg antibody 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 carrying out oscillation reaction at room temperature for 2 hours;
s1-3: removing supernatant through 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: and (4) magnetically absorbing to remove supernatant, adding TBST buffer solution to obtain magnetic particle working solution, wherein the concentration of the magnetic particles in the magnetic particle working solution is 0.4 mg/mL.
S2 obtaining enzyme-labeled working solution:
s2-1: dissolving 1mg of alkaline phosphatase in 1mL of PBS buffer solution to obtain an alkaline phosphatase solution;
s2-2: dissolving hapten of 0.5mg of imatinib and/or N-demethyl imatinib in 0.1mL of dimethyl sulfoxide to obtain a hapten solution;
s2-3: uniformly mixing the alkaline phosphatase solution and the 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 1 ug/mL.
Acquisition of the indicator solution of S3: commercial AMPPD luminescent liquid.
The detection reagent for detecting imatinib and/or N-demethyl imatinib can be prepared at present, or the detection reagent for detecting imatinib and/or N-demethyl imatinib can be stored at 2-8 ℃ for use; further, the detection reagent for detecting imatinib and/or N-demethyl imatinib is prepared into a detection kit, and the detection kit needs to be stored at the temperature of 2-8 ℃.
Examples of the immunological detection of Imatinib and/or N-desmethyl Imatinib in a sample
An immunological detection method for detecting imatinib and/or N-desmethyl imatinib comprises the following specific operation steps:
uniformly mixing a 20uL blood sample, 40uL magnetic particle liquid working solution and 50uL enzyme-labeled working solution, incubating and reacting for 5min at 37 ℃, cleaning, adding an indicator solution for color development, and collecting a light signal of 400-550nm by using a photomultiplier tube. The specific steps of cleaning comprise magnetic absorption to remove supernatant, adding TBST buffer solution for heavy suspension, and magnetic absorption again to remove supernatant.
Selecting blood samples of 50 clinical patients taking imatinib, respectively measuring the total concentration of imatinib and N-demethyl imatinib in the blood samples by adopting the immunological detection method and the HPLC method for detecting imatinib and/or N-demethyl 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 correlation between the total concentration of imatinib and N-demethyl imatinib measured by a magnetic particle luminescence method established based on the antibody prepared by the invention and an HPLC method is good, and the clinical requirement is met.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (10)
1. Hapten of imatinib and N-desmethyl imatinib, having a structure represented by formula 10:
2. a method for preparing hapten of imatinib and N-demethyl imatinib, which comprises the following steps:
(i) performing aldehyde group protection reaction on the compound shown in the formula 1 and trimethyl orthoformate to obtain a compound shown in a formula 2;
(ii) carrying out condensation reaction on the compound shown in the formula 2 and the compound shown in the formula 3 to obtain a compound shown in a formula 4;
(iii) carrying out reductive amination reaction on the compound shown in the formula 4 and the compound shown in the formula 5 to obtain a compound shown in a formula 6;
(iv) carrying out deprotection reaction on the compound shown in the formula 6 to obtain a compound shown in a formula 7;
(v) carrying out substitution reaction on the compound shown in the formula 7 and ethyl bromoacetate to obtain a compound shown in a formula 8;
(vi) carrying out oxidation reaction on the compound shown in the formula 8 and m-chloroperoxybenzoic acid to obtain a compound shown in a formula 9;
(vii) carrying out hydrolysis reaction on the compound shown in the formula 9 to obtain a compound shown in a formula 10;
the specific synthetic route is as follows:
3. the preparation method according to claim 2, wherein the molar ratio of the compound represented by the formula 1 to trimethyl orthoformate in the step (i) is 1 (2-3), the aldehyde group protection 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 the 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), HATU is used as a condensing agent in the condensation reaction, triethylamine is used as an acid-binding agent in the condensation reaction, and the molar ratio of the HATU to the triethylamine to the compound shown in the formula 2 is (1-1.2) to (2-2.4) 1;
in the step (iii), the molar 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 molar ratio of the sodium triacetyl borohydride to the compound shown in the formula 4 is (1-1.2): 1;
in the deprotection reaction in the step (iv), trifluoroacetic acid is used as a deprotection agent, and the molar ratio of the trifluoroacetic acid to the compound shown as the formula 6 is (20-50): 1;
the molar ratio of the compound shown in the formula 7 to the ethyl bromoacetate in the step (v) is 1 (1-1.2);
the molar ratio of the compound shown in the formula 8 to the m-chloroperoxybenzoic acid in the step (vi) is 1 (1-1.2);
in the hydrolysis reaction in the step (vii), sodium hydroxide is used as a hydrolyzing agent, and the molar ratio of the sodium hydroxide to the compound represented by the formula 9 is (1.5-3): 1.
4. An antigen of imatinib and N-desmethyl imatinib that is a conjugate formed by coupling the hapten of claim 1 to a carrier protein.
5. The antigen of claim 4, wherein the hapten and the carrier protein are coupled using self-1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride as a coupling agent.
6. A method for preparing antigens of imatinib and N-demethylimatinib, comprising the steps of:
(i) dissolving the hapten of claim 1 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 the dimethyl sulfoxide solution of the hapten and the aqueous solution of the coupling agent, and reacting at room temperature for 0.5-2 hours to obtain a reaction solution;
(iv) dissolving carrier protein in PBS buffer solution to obtain carrier protein solution;
(v) mixing the carrier protein solution and the reaction solution, and stirring for 1-3 hours at room temperature 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 the hapten to the coupling agent to the carrier protein is 1 (0.5-1.5) to 1-2.
7. An antibody to imatinib and N-desmethyl imatinib produced in response to the antigen of any one of claims 4-6.
8. The antibody of claim 7, wherein the antibody is a monoclonal antibody or a polyclonal antibody.
9. Use of an antibody according to any one of claims 7 to 8 for the detection of imatinib and/or N-desmethyl imatinib.
10. A test kit comprising the antibody according to any one of claims 7 to 8.
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