CN115073465A - Meropenem hapten derivative, meropenem artificial antigen, hybridoma cell strain, meropenem monoclonal antibody and application - Google Patents
Meropenem hapten derivative, meropenem artificial antigen, hybridoma cell strain, meropenem monoclonal antibody and application Download PDFInfo
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Abstract
The invention belongs to the field of therapeutic drug monitoring, and provides meropenem hapten derivatives, meropenem artificial antigens, hybridoma cell strains, meropenem monoclonal antibodies and application. The invention provides a meropenem hapten derivative which has a structure shown in a formula I. The derivative site (tetrahydropyrrole) of the meropenem hapten derivative is positioned in a common characteristic structure of meropenem and an inactive metabolite (ICI 213,689), and a medium-length linking arm (N-3 bromopropionyl) is adopted, so that the prepared antibody can specifically recognize meropenem.
Formula I.
Description
Technical Field
The invention relates to the technical field of therapeutic drug monitoring, in particular to meropenem hapten derivatives, meropenem artificial antigens, hybridoma cell strains, meropenem monoclonal antibodies and application.
Background
Meropenem is a carbapenem antibiotic, has the advantages of wide antibacterial spectrum, strong antibacterial activity, low drug resistance rate and the like, and has become a leading force for treating severe infection. However, empirical dosing regimens do not ensure that effective blood levels are achieved, resulting in high mortality rates in critically ill patients. Therefore, it is necessary to provide an individual anti-infection treatment scheme for critically ill patients, establish a simple, fast, stable and reliable in-vivo meropenem drug analysis method, monitor the blood concentration of critically ill patients in real time, adjust the administration dosage in time, and ensure the safety and effectiveness of administration.
However, few clinical practices for monitoring the blood concentration of meropenem are developed at present, and the methods are related to tedious detection methods of the blood concentration of meropenem, extremely unstable meropenem molecules, lack of consciousness of blood concentration monitoring of people, interference measurement of metabolites of meropenem and the like. The detection and analysis of meropenem mainly comprises a high performance liquid chromatography-ultraviolet (HPLC-UV) detection method or a high performance liquid chromatography-mass spectrometry (HPLC-MS) detection method, but the method cannot meet the clinical requirements of high-throughput, rapidness and accuracy.
Chinese patent CN112378891A discloses a fluorescence measurement method of meropenem, but the method is not suitable for the measurement of meropenem in complex clinical samples, has poor anti-interference performance, and cannot distinguish structural analogues.
In conclusion, an anti-meropenem antibody and a detection reagent which are high in sensitivity and excellent in specificity are lacked at present.
Disclosure of Invention
In view of the above, the present invention aims to provide meropenem hapten derivatives, meropenem artificial antigens, hybridoma cell strains, meropenem monoclonal antibodies and applications thereof. The meropenem monoclonal antibody generated after an animal is immunized by the meropenem artificial antigen formed by combining the meropenem hapten derivative and the carrier protein can identify meropenem with high sensitivity; meanwhile, the method has specificity superior to that of the existing antibody, and can be used for establishing a method for accurately monitoring the concentration of the meropenem in blood and urine.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a meropenem hapten derivative which has a structure shown in a formula I:
The invention also provides a preparation method of the meropenem hapten derivative in the technical scheme, which comprises the following steps:
mixing meropenem, 3-bromopropionic acid, O-benzotriazole-tetramethylurea hexafluorophosphate and an organic solvent, and carrying out amidation reaction to obtain the meropenem hapten derivative.
Preferably, the molar ratio of meropenem to 3-bromopropionic acid is 1: 1 to 1.1.
Preferably, the temperature of the amidation reaction is 20-25 ℃, and the time is 1-3 h.
The invention also provides a meropenem artificial antigen which comprises a meropenem hapten derivative and a carrier protein;
the meropenem hapten derivative is the meropenem hapten derivative in the technical scheme or the meropenem hapten derivative obtained by the preparation method in the technical scheme.
Preferably, the meropenem artificial antigen has a structure shown in formula II:
in the formula II, n =1~ 100.
The invention also provides a preparation method of the meropenem artificial antigen in the technical scheme, which comprises the following steps:
dissolving the meropenem hapten derivative to obtain a meropenem hapten derivative solution;
dissolving carrier protein to obtain carrier protein solution;
and mixing the meropenem hapten derivative solution and a carrier protein solution, and sequentially carrying out composite reaction and dialysis to obtain the meropenem artificial antigen.
The invention also provides a hybridoma cell strain for producing the meropenem monoclonal antibody, the preservation number is CGMCC number 45164, the hybridoma cell strain is classified and named as hybridoma cell strain, the hybridoma cell strain is preserved in the common microorganism center of China microorganism culture Collection management Committee in 2022, 5 and 19 months, the address is No. 3 of the West Lu No. 1 Hospital of the Chaoyang district in Beijing City, and the postal code is 100101.
The invention also provides a meropenem monoclonal antibody, which is produced by the hybridoma cell strain for producing the meropenem monoclonal antibody in the technical scheme.
The invention also provides application of the meropenem monoclonal antibody in the technical scheme in preparation of a medicament for detecting meropenem.
The invention provides a meropenem hapten derivative which has a structure shown in a formula I. The derivative site (tetrahydropyrrole) of the meropenem hapten derivative is positioned in a common characteristic structure of meropenem and an inactive metabolite (ICI 213,689), and a medium-length linking arm (N-3 bromopropionyl) is adopted, so that the prepared antibody can specifically recognize meropenem.
The invention also provides a preparation method of the meropenem hapten derivative in the technical scheme, and the preparation method provided by the invention is simple to operate.
The invention also provides a meropenem artificial antigen which comprises a meropenem hapten derivative and a carrier protein; the meropenem hapten derivative is the meropenem hapten derivative in the technical scheme or the meropenem hapten derivative obtained by the preparation method in the technical scheme. The meropenem hapten derivative is combined with carrier protein to form a meropenem artificial antigen; the meropenem monoclonal antibody generated by the meropenem artificial antigen immune animal can identify meropenem with high sensitivity; meanwhile, the antibody has better specificity than the existing antibody, namely, the antibody has no cross reaction with active metabolite (ICI 213,689) of meropenem and imipenem.
The invention also provides a preparation method of the meropenem artificial antigen in the technical scheme, and the preparation method provided by the invention is simple to operate.
The invention also provides a hybridoma cell strain for producing the meropenem monoclonal antibody, and the preservation number is CGMCC number 45164. The hybridoma cell strain provided by the invention can generate a meropenem monoclonal antibody, and the generated meropenem monoclonal antibody can identify meropenem with high sensitivity; meanwhile, the antibody has specificity superior to that of the existing antibody, namely, the antibody has no cross reaction with active metabolite (ICI 213,689) of meropenem and imipenem.
The invention also provides a meropenem monoclonal antibody, which is produced by the hybridoma cell strain for producing the meropenem monoclonal antibody in the technical scheme. The meropenem monoclonal antibody can identify meropenem with high sensitivity; meanwhile, the antibody has better specificity than the existing antibody, namely, the antibody has no cross reaction with active metabolite (ICI 213,689) of meropenem and imipenem.
The invention also provides application of the meropenem monoclonal antibody in the technical scheme in preparation of a medicament for detecting meropenem. The meropenem monoclonal antibody has high sensitivity and the capability of specifically recognizing meropenem, so that the meropenem monoclonal antibody can be applied to preparation of a meropenem detection medicament for detecting the blood concentration or urine concentration of meropenem.
Drawings
FIG. 1 is a four parameter fit plot of the magnetic particle luminescence method of example 4;
FIG. 2 is a graph showing the correlation between the results of meropenem by the homogeneous enzymatic method of example 5 and the results of meropenem by the LC-MS method.
Detailed Description
The invention provides a meropenem hapten derivative which has a structure shown in a formula I:
In the present invention, the chemical name of the meropenem hapten derivative is N- (3-bromopropionyl) meropenem.
The invention also provides a preparation method of the meropenem hapten derivative in the technical scheme, which comprises the following steps:
mixing meropenem, 3-bromopropionic acid, O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) and an organic solvent, and carrying out amidation reaction to obtain the meropenem hapten derivative.
In the present invention, unless otherwise specified, the starting materials used in the present invention are preferably commercially available products.
In the present invention, the molar ratio of meropenem and 3-bromopropionic acid is preferably 1: 1 to 1.1.
In the present invention, the molar ratio of meropenem to O-benzotriazole-tetramethylurea hexafluorophosphate is preferably 1: 1.1.
in the present invention, the organic solvent is preferably a mixed solution of acetonitrile and triethylamine, and the ratio of the amount of acetonitrile to triethylamine in the mixed solution is preferably 20 mL: 2.0 mmol.
In the specific embodiment of the invention, preferably, after the meropenem and the acetonitrile are stirred and dissolved, triethylamine is added to prepare a solution 1 for standby; stirring and dissolving the 3-bromopropionic acid, the acetonitrile and the triethylamine, adding HBTU (heterojunction bipolar transistor) and continuously stirring, and then dropwise adding the solution 1, wherein the dropwise adding temperature is preferably 20-25 ℃, and the time is preferably 15 min.
In the invention, the temperature of the amidation reaction is preferably 20-25 ℃, and the time is preferably 1-3 h.
After the amidation reaction, the method preferably further comprises the steps of sequentially carrying out reduced pressure concentration and recrystallization to obtain the meropenem hapten derivative.
In the invention, the pressure for the reduced pressure concentration is preferably 0.5 to 4MPa, more preferably 1 to 3MPa, and even more preferably 2 MPa; the temperature of the reduced pressure concentration is preferably less than or equal to 40 ℃, and the further preferred temperature is 10-25 ℃; in the present invention, the time for the reduced-pressure concentration is not particularly limited as long as the dissolved solvent can be removed cleanly.
In the present invention, the recrystallization preferably includes the steps of: mixing the solid obtained by the reduced pressure concentration, water and ethyl acetate, adjusting the pH value to 2.5, standing for layering, evaporating and crystallizing the obtained organic layer, and then drying in vacuum.
In the invention, hydrochloric acid is preferably used for adjusting the pH value, and the concentration of the hydrochloric acid is preferably 0.1 mol/L.
The present invention is not limited to the specific parameters of the evaporative crystallization and the drying under vacuum, and the methods known to those skilled in the art can be adopted.
The principle of the preparation of the meropenem hapten derivative is shown as the following formula:
the invention also provides a meropenem artificial antigen which comprises a meropenem hapten derivative and a carrier protein;
the meropenem hapten derivative is the meropenem hapten derivative in the technical scheme or the meropenem hapten derivative obtained by the preparation method in the technical scheme.
The meropenem artificial antigen provided by the invention comprises carrier protein; the carrier protein preferably comprises Bovine Serum Albumin (BSA), chicken ovalbumin, bovine thyroglobulin, human serum albumin or rabbit serum albumin, and more preferably Bovine Serum Albumin (BSA).
In the invention, the meropenem artificial antigen has a structure shown in formula II:
in the formula II, n =1~ 100.
In the present invention, n is preferably 10 to 30.
In the invention, the meropenem artificial antigen shown in formula II is a carrier protein connected with n meropenem hapten derivatives. In the invention, an amido bond is formed between the amino group in the carrier protein and the carboxyl group of the meropenem hapten derivative, and the carrier protein and the meropenem hapten derivative are connected.
The invention also provides a preparation method of the meropenem artificial antigen in the technical scheme, which comprises the following steps:
dissolving the meropenem hapten derivative to obtain a meropenem hapten derivative solution;
dissolving carrier protein to obtain carrier protein solution;
and mixing the meropenem hapten derivative solution and a carrier protein solution, and sequentially carrying out composite reaction and dialysis to obtain the meropenem artificial antigen.
The meropenem hapten derivative is dissolved to obtain a meropenem hapten derivative solution.
In the present invention, the reagent for solubilizing the meropenem hapten derivative preferably comprises dimethyl sulfoxide (DMSO).
The invention dissolves carrier protein to obtain carrier protein solution.
In the present invention, the carrier protein-solubilizing reagent preferably includes a PBS solution.
After a meropenem hapten derivative solution and a carrier protein solution are obtained, the meropenem hapten derivative solution and the carrier protein solution are mixed, and then are subjected to composite reaction and dialysis in sequence to obtain the meropenem artificial antigen.
In the invention, the temperature of the complex reaction is preferably room temperature, i.e. neither additional heating nor additional cooling is required; the heat preservation time of the composite reaction is preferably 1-5 h, and more preferably 2 h.
In the present invention, the agent for dialysis preferably comprises a PBS solution. In the invention, the cut-off molecular weight of the dialysis bag is preferably 1000-30000 Da. In the present invention, the number of times of dialysis is preferably 4.
After the meropenem artificial antigen is obtained, the meropenem artificial antigen is preferably frozen and stored at the temperature of-20 ℃.
The invention also provides a hybridoma cell strain for producing the meropenem monoclonal antibody, the preservation number is CGMCC number 45164, the hybridoma cell strain is classified and named as hybridoma cell strain, the hybridoma cell strain is preserved in China general microbiological culture Collection center on 5-19 th 2022, the address is No. 3 of the Kyoho Seisaku No. 1 on the morning and Yangtze district in Beijing, and the postal code is 100101.
In the present invention, the preparation method of the hybridoma cell strain producing meropenem artificial antigen preferably comprises the following steps:
sequentially carrying out first immunization and second immunization on the animal to obtain an immunized animal;
extracting spleen cells of the immune animals and fusing the spleen cells with Sp2/0 cells to obtain fused cells;
and (3) carrying out hybridoma screening on the fused cells.
The invention carries out the first immunity and the second immunity on the animal in sequence to obtain the immune animal.
In the present invention, the animal preferably includes one or more of a mouse, a rabbit, a goat and a sheep, and more preferably a mouse.
In the present invention, the reagent for the first immunization is preferably a mixed emulsified solution of 1mg/mL meropenem artificial antigen-PBS solution and Freund's complete adjuvant in equal volumes. In the present invention, the number of immunizations of the first immunization is preferably 1; the dose of the first immunization is preferably 0.01-0.2 mg/mouse.
In the present invention, the interval between the second immunization and the first immunization is preferably 2 to 6 weeks.
In the invention, the reagent for the second immunization is preferably a mixed emulsified solution of 1mg/mL meropenem artificial antigen-PBS solution and Freund's incomplete adjuvant in equal volume. In the invention, the frequency of the second immunization is preferably 4 times, and the immunization dose of each second immunization is preferably 0.01-0.2 mg/mouse; preferably, two secondary immunizations are separated by 1 week.
After obtaining the immune animal, the invention extracts the spleen cell of the immune animal and fuses with the Sp2/0 cell to obtain the fused cell.
In the present invention, the extraction of spleen cells from an immunized animal is preferably performed one week after the last immunization.
The method of fusion is not particularly limited in the present invention, and means known to those skilled in the art may be used. In a specific embodiment of the present invention, the fusion method is preferably performed with reference to GC Howard, MR Kaser, labeling and using antibodies, a practical handbook, 2013 Book.
After obtaining the fused cells, the invention screens the fused cells for hybridomas.
In the present invention, the hybridoma screening preferably comprises the steps of: coating an ELISA 96-well plate by using 1 mu g/mL of meropenem-BSA (bovine serum albumin), and performing ELISA titer determination on the supernatant of the fusion cells; selecting cell clone supernatant with positive ELISA titer determination to perform competitive ELISA determination; and screening the clone with high competitiveness to subclone to obtain a single cell clone strain.
The invention also provides a meropenem monoclonal antibody, which is produced by the hybridoma cell strain for producing the meropenem monoclonal antibody in the technical scheme.
In the present invention, the method for producing meropenem monoclonal antibody from the hybridoma cell strain producing meropenem monoclonal antibody preferably comprises: ascites culture and Protein A/G purification of mice were performed in this order. The operation of ascites culture and Protein A/G purification of the mouse is not particularly limited in the present invention, and a technical means well known to those skilled in the art may be used.
The invention also provides application of the meropenem monoclonal antibody in the technical scheme in preparation of a medicament for detecting meropenem.
The method for applying the meropenem monoclonal antibody in the preparation of the medicament for detecting meropenem is not particularly limited, and the operation known by the skilled person in the art is adopted.
The meropenem hapten derivative, meropenem artificial antigen, hybridoma cell strain, meropenem monoclonal antibody and application provided by the invention are described in detail below with reference to examples, but the invention is not to be construed as being limited to the scope of the invention.
Example 1
The preparation method of the meropenem hapten derivative from the meropenem comprises the following steps:
stirring and dissolving 2.0mmol of meropenem acetonitrile 20mL, adding 2.0mmol of triethylamine to prepare solution 1 for later use; stirring and dissolving 2.1mmol of 3-bromopropionic acid, 20mL of acetonitrile and 2.0mmol of triethylamine, adding 2.2mmol of HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate), continuously stirring, controlling the temperature to be 20 ℃, dropwise adding the solution 1, keeping the temperature to be 20 ℃ after 15min, and continuously stirring and reacting for 60 min. After the reaction, the solvent was evaporated by concentration under reduced pressure, and 20mL of ethyl acetate (40 mL) was added to the reaction solution, and the pH was adjusted to 2.5 with 0.1mol/L hydrochloric acid. The layers were separated by standing, the organic layer evaporated to crystallize and dried under high vacuum to give an off-white powder (yield 77%).
The structural characterization information of the obtained meropenem hapten derivative is as follows:
1 HNMR (DMSO-d 6 ,400MHz) 12.15 (s, 1H), 6.47-6.44 (m, 1H), 4.47-3.91 (m,5H), 3.87-3.69 (m, 2H), 2.98-2.90 (s, 6H), 2.86-2.69 (m, 6H), 2.59-2.45 (m, 4H), 1.28-1.17 (s, 3H)。
example 2
The preparation method of the meropenem artificial antigen comprises the following steps:
10mg of the meropenem hapten derivative obtained in example 1 was added with 1mL of DMSO to dissolve completely, thereby obtaining a meropenem hapten derivative solution.
Bovine Serum Albumin (BSA) 20mg was weighed and dissolved in 5mL of PBS solution to obtain a BSA solution.
Mixing the meropenem hapten derivative solution and the bovine serum albumin solution, and stirring for 2 hours at room temperature; then dialyzing with PBS (cut-off molecular weight of 7000Da in dialysis bag) for 4 times, and freezing and storing at-20 ℃ to obtain the meropenem artificial antigen.
Example 3
Preparation of meropenem mouse monoclonal antibody
Diluting the meropenem artificial antigen with a PBS solution to obtain a meropenem artificial antigen solution with the concentration of 1 mg/mL; mixing the meropenem artificial antigen solution and Freund's complete adjuvant in equal volume, emulsifying completely, and injecting to mouse; the mice were given a primary immunization at a dose of 0.1 mg/mouse, and the number of primary immunizations was 1.
After 4 weeks, 1mg/mL meropenem antigen and Freund's incomplete adjuvant are mixed and emulsified in equal volume, and then the mice are immunized for the second time; the secondary immunization was performed four times in total, and the secondary immunization was performed every other week at an immunization dose of 0.1 mg/mouse, to obtain immunized mice.
After 7 days of the last immunization, spleen cells of the immunized mice are taken to be fused with Sp2/0 cells according to the method of GC Howard, MR Kaser, Making and using antibodies, a practical handbook, 2013 Book, so as to obtain fused cells; ELISA titer determination was performed on the supernatant of the fused cells using 1. mu.g/mL coated ELISA 96-well plates with meropenem-BSA (bovine serum albumin): the specific process is as follows: adding 50 mu L of fusion cell supernatant on a 1 mu g/mL coated ELISA 96 pore plate of meropenem-BSA (bovine serum albumin) for reaction at 37 ℃ for 30min, and cleaning; then adding 100 mu L of goat anti-mouse IgG-HRP for reaction for 30min, and cleaning; the developing solution is added, and OD450 is read. Selecting cell clone supernatant with positive ELISA titer determination to perform competitive ELISA determination, wherein the specific process comprises the following steps: adding 50 μ L of fusion cell supernatant with positive ELISA titer determination and 50 μ L of competitor (meropenem) for reaction at 37 deg.C for 30min, and washing; then adding 100 mu L of goat anti-mouse IgG-HRP for reaction for 30min, and cleaning; the developing solution is added, and OD450 is read. The 3 cells (1B 2, 5C9, 8H 5) were obtained by screening, and the results of indirect competition ELISA of cell supernatants are shown in Table 1.
TABLE 1 Indirect competitive ELISA results for cell supernatants
As can be seen from table 1: the monoclonal antibody generated by 8B12 shows the highest sensitivity to meropenem, IC50 is 50ng/mL, and the monoclonal antibody has no cross reaction to (ICI 213,689) and imipenem, and is suitable for establishing a meropenem drug concentration monitoring method. Therefore, the 8B12 hybridoma cell strain is subjected to mouse ascites preparation and Protein A/G purification to obtain the meropenem monoclonal antibody for the establishment of the subsequent meropenem method.
Example 4
Establishment of magnetic particle chemiluminescence method for detecting meropenem blood concentration
1) 50mg of Dynal beads M280 Tosyl magnetic beads were diluted in 2mL of 50mM BB (boric acid-borax buffer solution) pH 8.0 buffer solution, 1mg of meropenem monoclonal antibody 8B12 obtained in example 3 was added thereto, and after mixing, the mixture was shaken at 37 ℃ for 8 hours.
2) The supernatant was removed by magnetic attraction, and TBST (50 mM Tris, 0.9wt% NaCl, 0.1wt% TW20, pH 7.4) was added for reaction at 37 ℃ for 12 hours.
3) And (4) magnetically absorbing to remove supernatant, adding TBST, diluting to 0.4mg/mL, named as magnetic particle working solution, and storing at 2-8 ℃ for later use.
4) 1mg of ALP (alkaline phosphatase) was dissolved in 1mL of PBS solution, 0.5mg of meropenem hapten derivative (dissolved in 100. mu.L of DMSO) was added thereto, and the mixture was mixed well1mg of EDC ∙ HCl solid was added and mixed well for 2h at room temperature. Dialyzed into PBS solution with 50mM MES 0.9% NaCl 5mg/mL BSA 1mM MgCl 2 Diluted to 1 mug/mL with pH 6.7 and named as enzyme labeling working solution.
5) Reaction procedure: and (3) incubating and reacting 10 mu L of sample (automatically diluted by 10 times by using PBS solution) with 40 mu L of magnetic particle solution working solution and 50 mu L of enzyme-labeled working solution for 5min at 37 ℃, cleaning, and adding AMPPD luminescent solution for color development.
A standard curve prepared by adopting calf serum matrix to prepare calibration products (0-2.5-5-10-20-40 mu g/mL) of meropenem with different concentrations is shown in a table 2 and a figure 1.
TABLE 2 Meropenem calibration curve data by magnetic particle luminescence method
As can be seen from fig. 1: the calibration curve obtained by the magnetic particle luminescence method is as follows: y = (A-D)/[1+ (X/C) B]+ D, a = 37233641.3497387; b = 1.07270332363271; c = 0.290532904381278; d =57745.1576336999, correlation coefficient R 2 ：0.9999。
Repeatedly measuring the low value and the high value, controlling the quality for 10 times respectively, and calculating CV according to SD/Mean; LoB A zero value sample was taken 20 times and the lowest detection limit was found to be Mean +2SD, as shown in Table 3.
TABLE 3 precision and sensitivity
As can be seen from table 3: the magnetic particle luminescence method meropenem determination reagent prepared by the meropenem monoclonal antibody has high sensitivity.
To a meropenem sample at a concentration of 1.52. mu.g/mL, various concentrations of cross-over test substances (inactive metabolite (ICI 213,689), imipenem, and faropenem) were added for cross-over reaction, and the cross-over reaction rate was calculated, with the results shown in Table 4.
TABLE 4 Cross-reaction results
As can be seen from table 4: the meropenem by the magnetic particle luminescence method has no obvious cross reaction on a main metabolite (ICI 213,689), imipenem and faropenem, and has good specificity.
Example 5
Preparation of meropenem homogeneous enzyme immunoassay reagent
The meropenem homogeneous enzyme immunoassay reagent comprises two reagents which are separately arranged, and specifically comprises the following steps:
preparation of reagent R1: dissolving 20mM NAD, 30 mM glucose-6-phosphate G6P, and 55mM Tris buffer with pH =8.0 to prepare a homogeneous enzyme substrate; 0.01-0.1 mass percent of meropenem monoclonal antibody 8B12 is added into the homogeneous enzyme substrate, and the specific mass percent in the embodiment is 0.05%.
Preparation of reagent R2: 0.1M Tris buffer solution with pH =8.5 is added with 0.01-1% of meropenem hapten derivative-G6 PDH enzyme-labeled conjugate, and the specific mass percentage in the embodiment is 0.05%.
Wherein the meropenem hapten derivative-G6 PDH enzyme-labeled conjugate is prepared by dissolving 600U G6PDH in 1mL PBS solution, adding 0.5mg meropenem hapten derivative (dissolved in 100 muL DMSO), mixing uniformly, adding 1mg EDC solid, and mixing uniformly at room temperature for 2 h; dialyzed into PBS and diluted to 5U/mL with 100mM Tris 0.9% NaCl 5mg/mL BSA pH8.5.
The test procedure was: after 20. mu.L of the sample + 50. mu.L of the reagent R1 reacted for 5min, 50. mu.L of the reagent R2 was added and the reaction was continued for 5min, and the rise rate of OD340 wavelength was measured in Hitachi 7170.
The method comprises the steps of selecting clinical 40 samples of meropenem urine concentration, comparing results of meropenem determination of the homogeneous enzyme method and the LC-MS method constructed by the invention, and comparing the results with the results shown in figure 2.
As can be seen from fig. 2: the correlation between the content of meropenem measured by a homogeneous enzyme method established based on the antibody prepared by the invention and an LC-MS method is good, and the clinical requirement is met.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. A meropenem hapten derivative has a structure shown in formula I:
2. The process for the preparation of meropenem hapten derivatives of claim 1, comprising the steps of:
mixing meropenem, 3-bromopropionic acid, O-benzotriazole-tetramethylurea hexafluorophosphate and an organic solvent, and carrying out amidation reaction to obtain the meropenem hapten derivative.
3. The process according to claim 2, wherein the molar ratio of meropenem to 3-bromopropionic acid is 1: 1 to 1.1.
4. The method according to claim 2 or 3, wherein the temperature of the amidation reaction is 20 to 25 ℃ and the time is 1 to 3 hours.
5. An artificial meropenem antigen which is characterized by comprising a meropenem hapten derivative and a carrier protein;
the meropenem hapten derivative is the meropenem hapten derivative in claim 1 or the meropenem hapten derivative obtained by the preparation method in any one of claims 2-4.
6. The meropenem artificial antigen of claim 5, wherein the meropenem artificial antigen has the structure of formula II:
in the formula II, n =1~ 100.
7. The method for preparing meropenem artificial antigen of claim 5 or 6, which comprises the steps of:
dissolving the meropenem hapten derivative to obtain a meropenem hapten derivative solution;
dissolving carrier protein to obtain carrier protein solution;
and mixing the meropenem hapten derivative solution and a carrier protein solution, and sequentially carrying out composite reaction and dialysis to obtain the meropenem artificial antigen.
8. A hybridoma cell strain for producing a meropenem monoclonal antibody is characterized in that the preservation number is CGMCC number 45164.
9. A meropenem monoclonal antibody produced from the hybridoma cell line producing the meropenem monoclonal antibody of claim 8.
10. Use of the meropenem monoclonal antibody of claim 9 in the preparation of a medicament for detecting meropenem.
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