CN116023442A - Polymyxin B hapten, artificial antigen, specific antibody and preparation methods and application thereof - Google Patents
Polymyxin B hapten, artificial antigen, specific antibody and preparation methods and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of biochemistry, and particularly relates to a polymyxin B hapten, an artificial antigen, a specific antibody, a preparation method and application thereof. Derivatization in the polymyxin hapten structure of the inventionThe site is-COCH 2 CH 2 COOH, which is remote from the cyclic domain of polymyxin B and exposes the common features of polymyxins B1 and B2, and the prepared antibody is capable of equivalently recognizing polymyxins B1 and B2; meanwhile, as the polymyxin E and the polymyxin B have different structures on the annular structural domain, the prepared antibody and the polymyxin E have no cross reaction, and the sensitivity and the specificity of identifying the polymyxin B1 and B2 are further improved.
Description
Technical Field
The invention belongs to the technical field of biochemistry, and particularly relates to a polymyxin B hapten, an artificial antigen, a specific antibody, a preparation method and application thereof.
Background
Polymyxin (polymyxin) is a class of non-ribosomal alkaline polypeptide antibacterial drugs, and was first isolated from Paenibacillus polymyxa in 1947, including A, B, C, D, E, etc. Among them, polymyxin E (PE or colistin) and Polymyxin B (PB) were approved for clinical use in 1950. Bacterial resistance has become a global medical problem, with the problem of multiple resistance of gram-negative bacteria being particularly pronounced. As the last line of defense of multi-drug resistant gram-negative bacteria, polymyxin is mainly used for severe infections such as multi-drug resistant Acinetobacter baumannii, klebsiella pneumoniae, pseudomonas aeruginosa and the like.
For clinical use, PB is administered intravascularly, intrathecally, by nebulization or topically in the form of polymyxin B sulfate. The PB has narrow therapeutic window and high incidence rate of adverse reactions, and common adverse reactions are dose-dependent nephrotoxicity and neurotoxicity, wherein the nephrotoxicity is most common and mainly appears as Acute Kidney Injury (AKI) and is common in acute tubular necrosis; neurotoxicity is mainly manifested as abnormal sensation, so that blood concentration monitoring of polymyxin is carried out, and adverse reactions can be reduced while curative effects are ensured.
The currently proposed mechanism of PB drug elimination includes the renal pathway (reabsorption through the tubule) and the non-renal pathway. Studies indicate that PB preferentially accumulates in kidney tissue in rodent models, which may explain its elimination via the renal pathway. Furthermore, several studies in animals and humans have shown low urine recovery (< 5%) of PB, indicating its selective uptake and retention processes in kidney cells, which is also responsible for its adverse effects as acute kidney injury. Therefore, detection of polymyxin B content in urine is an important means for studying metabolic pathways.
However, current PB blood concentration monitoring is seldom developed in domestic clinical practice, and is related to complicated PB blood concentration detection methods, lack of consciousness of blood concentration monitoring for people, and need of multiple blood sampling points for PB to calculate AUC. PB is composed of polypeptide and fatty chain, and has no benzene ring or other conjugated rigid structure, so that the PB has low ultraviolet absorptivity and more limited natural fluorescence, and therefore, the effect of directly measuring by adopting an HPLC method is not ideal. At present, the PB detection and analysis mainly comprises an LC-MS method, but the method cannot meet the requirements of clinical high-throughput, rapid and accurate detection.
The premise of establishing high-sensitivity and high-specificity PB immunological detection is to obtain high-quality anti-PB antibodies, and the design and preparation of PB artificial antigens are further relied on. Chinese patent CN111830153a, publication No. 2020, 10, 27, discloses a method for measuring polymyxin B1 and B2 in plasma by high performance liquid chromatography-mass spectrometry, which requires complex pretreatment and specialized operators. Chinese patent CN110923208A, publication No. 2020, no. 03, no. 27, discloses a PB monoclonal antibody hybridoma cell strain OVA17 and its use in food safety. However, the presence of polymyxins B1 and B2 in blood levels and clinical concomitant medication are complex, and thus it is required that antibodies specifically recognize polymyxins B1 and B2 without cross-reaction with other analogues. The antibodies in this invention recognize only PB and do not disclose specificity for PB analogs, and thus do not meet the need for PB Therapeutic Drug Monitoring (TDM). Chinese patent CN113717255a, published as 2021, 11 and 30, discloses a PB and colistin hapten, an artificial antigen, and preparation methods and applications thereof, and the corresponding antibody and colistin E have significant cross-reaction, and cannot meet PB specific blood concentration monitoring.
In summary, there is currently a lack of highly sensitive anti-PB antibodies that simultaneously recognize equally polymyxin B1 and B2 and that are very specific.
Disclosure of Invention
The invention aims to provide a polymyxin B hapten, an artificial antigen, a specific antibody, a preparation method and application thereof, wherein the polymyxin B specific antibody prepared from the polymyxin B hapten can be used for equivalently identifying polymyxin B1 and B2 with high sensitivity, has higher specificity and does not have cross reaction with polymyxin E.
The invention provides a polymyxin B hapten, which has a structure shown in a formula I:
formula I.
The invention also provides a polymyxin B artificial antigen, which is obtained by coupling a carrier protein with the polymyxin B hapten according to the technical scheme by adopting a coupling agent.
Preferably, the carrier protein comprises bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin or rabbit serum albumin;
the coupling agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide or carbonylimidazole.
Preferably, the molar ratio of the polymyxin B hapten to the carrier protein is (2-80): 1.
the invention also provides application of the polymyxin B hapten or the polymyxin B artificial antigen in preparation and/or detection of polymyxin B specific antibodies.
The invention also provides a hybridoma cell strain of the polymyxin B specific antibody, which is obtained by immunizing animals by taking the polymyxin B artificial antigen as an immunogen; the hybridoma cell strain comprises a monoclonal hybridoma cell strain C-PB-2-F.
Preferably, the preservation number of the monoclonal hybridoma cell strain C-PB-2-F is CGMCC No.45341.
The invention provides a polymyxin B monoclonal antibody, which is secreted and generated by a polymyxin B monoclonal antibody hybridoma cell strain C-PB-2-F according to the technical scheme.
The invention also provides application of the polymyxin B monoclonal antibody C-PB-2-F in one or more of the following I-IV:
i: detecting polymyxin B at a non-diagnostic destination;
II: preparing an immunochromatography test strip for detecting polymyxin B;
III: preparing a magnetic particle immunodetection reagent of polymyxin B;
IV: preparing the polymyxin B homogeneous enzyme immunoassay kit.
The invention also provides a magnetic particle immune detection reagent of polymyxin B, which comprises a magnetic particle working solution, an enzyme-labeled working solution and an indicator solution which are independently packaged;
the magnetic particle working solution comprises a combination body obtained by combining magnetic beads with the polymyxin B monoclonal antibody in the technical scheme;
the enzyme-labeled working solution comprises a conjugate obtained by coupling a polymyxin B hapten and a marker;
the indicator solution includes an indicator for quantitatively detecting the concentration of the marker.
The beneficial effects are that:
the invention provides a polymyxin B hapten, which has a structure shown in a formula I:
formula I.
The new derivative site contained in the polymyxin hapten structure is specifically-COCH 2 CH 2 COOH, which is far from the cyclic domain of polymyxin B and exposes the common characteristic structure (cyclic region) of polymyxin B1 and B2, and thus an antibody prepared using the hapten as an immunogen can equivalently recognize polymyxinBacteriocins B1 and B2; meanwhile, as the polymyxin E and the polymyxin B have different structures on the annular structural domain, the prepared antibody has no cross reaction with analogues including polymyxin E, vancomycin, linezolid and teicoplanin, and the sensitivity and the specificity of identifying the polymyxin B1 and B2 are further improved.
Biological material preservation information
The classification of the monoclonal antibody hybridoma cell strain C-PB-2-F is named as hybridoma cell strain, and the hybridoma cell strain is preserved in the China general microbiological culture Collection center with the preservation date of 2022 and 12 months and 26 days. The preservation address is number 1 and number 3 of North Chen Xili of the Chaoyang area of Beijing city, and the preservation number is CGMCC No.45341.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a preparation route diagram of intermediate 1 prepared from polymyxin B in example 1 of the present invention;
FIG. 2 is a preparation route diagram of a polymyxin carboxyl derivative (polymyxin B hapten) prepared from intermediate 1 in example 1 of the present invention;
FIG. 3 is a graph showing the calibration of polymyxin B by the magnetic particle luminescence method in example 5 of the present invention;
FIG. 4 is a correlation analysis chart of the polymyxin B homogeneous enzyme immunoassay kit and HPLC-MS method in example 6 of the present invention.
Detailed Description
The invention provides a polymyxin B hapten, which has a structure shown in a formula I:
formula I.
The derivative site in the polymyxin B hapten structure is-COCH 2 CH 2 COOH, which is remote from the cyclic domain of polymyxin B and exposes the common features of polymyxins B1 and B2The structure is characterized, and the prepared antibody can equivalently recognize polymyxins B1 and B2.
The invention also provides a preparation method of the polymyxin B hapten, which is characterized by comprising the following steps:
performing an amide reaction on polymyxin B and monomethyl succinate acyl chloride in toluene to obtain an amide reaction solution;
mixing and layering the amide reaction liquid and water to respectively obtain a first water phase and a first toluene phase, wherein the first water phase contains an intermediate 1;
and (3) carrying out hydrolysis reaction on the first aqueous phase under alkaline conditions, extracting the obtained hydrolysis reaction solution by toluene to obtain a second toluene phase and a second aqueous phase respectively, wherein the second aqueous phase is an aqueous solution containing the polymyxin B hapten.
In the invention, the toluene solution of polymyxin B is preferably mixed with monomethyl succinate acyl chloride and toluene for amide reaction. In the invention, the concentration of the toluene solution of the polymyxin B is preferably 2-6 mg/mL, more preferably 3mg/mL; the mass ratio of the polymyxin B to the monomethyl succinate acyl chloride is preferably (4-16): 1, more preferably 8:1.
in the present invention, the mixing of the toluene solution of polymyxin B with monomethyl succinate acid chloride and toluene is preferably slow addition of the monomethyl succinate acid chloride and toluene to the toluene solution of polymyxin B. The mass concentration of the monomethyl succinate acyl chloride in toluene is preferably 0.5-2.5 mg/mL, and more preferably 1.5mg/mL.
In the present invention, the temperature of the amide reaction is preferably 10 to 15 ℃, more preferably 12 ℃. The time for the amide reaction is preferably 2-10 h, more preferably 6h.
After the amide reaction, the obtained amide reaction solution is mixed with water for layering, so that a first water phase and a first toluene phase are respectively obtained. In the mixing and layering process, stirring is preferably carried out, and the stirring time is preferably 10-30 min, more preferably 20min. The first aqueous phase contains an intermediate 1; the structure of the intermediate 1 is shown as a formula II.
A formula II;
after the first aqueous phase is obtained, the first aqueous phase is preferably subjected to hydrolysis reaction under alkaline conditions, and the obtained hydrolysis reaction solution is extracted by toluene to obtain a second toluene phase and a second aqueous phase respectively, wherein the second aqueous phase is preferably an aqueous solution containing the polymyxin B hapten. In the invention, the second aqueous phase is preferably cooled to 5-10 ℃ and then subjected to the hydrolysis reaction, and the temperature of the hydrolysis reaction is more preferably 8 ℃. The pH value of the alkaline condition is preferably 10-13, more preferably 12. The invention preferably uses a liquid base to adjust the pH. The mass concentration of the liquid alkali is preferably 10-90%, more preferably 50%. The hydrolysis reaction is preferably 10-50 min, more preferably 30min.
After the extraction, the pH value of the aqueous solution containing the polymyxin B hapten is preferably adjusted to 3-5, and then the aqueous solution is mixed with ethanol and distilled under reduced pressure to obtain the polymyxin B hapten. The present invention preferably adjusts the pH of the aqueous solution containing polymyxin B hapten to 4.0. The present invention preferably uses dilute sulfuric acid to adjust the pH of the aqueous solution containing polymyxin B hapten. The mass concentration of the dilute sulfuric acid is preferably 10-90%, more preferably 50%. The number of times of the reduced pressure distillation in the present invention is preferably 1 to 4 times, more preferably 2 times. The amount of ethanol used in the present invention is not particularly limited, and the water and ethanol in the aqueous solution containing polymyxin B hapten can be removed by distillation under reduced pressure as is conventional in the art.
The invention also provides a polymyxin B artificial antigen, which is obtained by coupling a carrier protein with the polymyxin B hapten according to the technical scheme by adopting a coupling agent.
The carrier protein of the present invention preferably comprises bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin or rabbit serum albumin, more preferably bovine serum albumin or bovine thyroglobulin. The coupling agent of the present invention preferably comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide (DCC) or Carbonylimidazole (CDI), more preferably 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. The amino group on the carrier protein is coupled with the carboxyl group on the polymyxin B hapten, and an amide bond is formed through condensation reaction. The molar ratio of the polymyxin B hapten to the carrier protein is (2-80): 1, more preferably 40:1.
the polymyxin B artificial antigen can be used as an immunogen or a coating antigen.
The invention also provides a preparation method of the polymyxin B artificial antigen, which is characterized by comprising the following steps:
mixing and dissolving the polymyxin B hapten and dimethyl sulfoxide, and mixing and reacting the obtained polymyxin B hapten solution with a coupling agent aqueous solution to obtain a solution A;
mixing carrier protein with PBS buffer solution to obtain carrier protein solution;
mixing the solution A with a carrier protein solution for reaction to obtain a mixed solution containing the polymyxin B artificial antigen;
and dialyzing the mixed solution containing the polymyxin B artificial antigen by using a PBS buffer solution, wherein the obtained retention solution comprises the polymyxin B artificial antigen.
The method is characterized in that the polymyxin B hapten is mixed and dissolved with dimethyl sulfoxide, and the obtained polymyxin B hapten solution is mixed and reacted with a coupling agent aqueous solution to obtain a solution A. The mass concentration of the polymyxin B hapten in the polymyxin B hapten solution is preferably 5-10 mg/mL, and more preferably 7mg/mL. The mass concentration of the coupling agent in the aqueous solution of the coupling agent is preferably 0.05-0.2 mg/mu L, and more preferably 0.1 mg/mu L. The temperature of the reaction is preferably room temperature, and the reaction time is preferably 0.2-2 h, more preferably 1h.
The present invention preferably mixes the carrier protein with PBS buffer to obtain a carrier protein solution. The mass concentration of the carrier protein in the carrier protein solution is preferably 10-30 mg/mL, more preferably 20mg/mL.
After the solution A and the carrier protein solution are obtained, the solution A and the carrier protein solution are preferably mixed and reacted to obtain the mixed solution containing the polymyxin B artificial antigen. Stirring is carried out during the reaction, and the temperature of stirring is preferably room temperature; the stirring time is preferably 0.2 to 2 hours, more preferably 2 hours. The stirring process is not particularly limited, and conventional stirring processes in the art can be adopted.
After the mixed solution containing the polymyxin B artificial antigen is obtained, the present invention preferably uses PBS buffer solution to dialyze the mixed solution containing the polymyxin B artificial antigen, and the obtained retention solution contains the polymyxin B artificial antigen. The molecular weight cut-off of the dialysis is preferably 7000-33000 Da, more preferably 15000Da. The number of times of dialysis in the present invention is preferably 2 to 8 times, more preferably 4 times.
Based on the characteristics of the immunogen and the coating antigen of the polymyxin B artificial antigen, the invention also provides application of the polymyxin B hapten or the polymyxin B artificial antigen in preparation and/or detection of polymyxin B specific antibodies. The invention preferably takes the polymyxin B artificial antigen as an immunogen, and prepares the polymyxin B specific antibody through an immunization means; the human antigen of polymyxin B is used as coating antigen to make organism produce immune reaction, and then combined with polymyxin B specific antibody in vitro so as to attain the goal of detecting polymyxin B specific antibody.
The invention also provides a hybridoma cell strain of the polymyxin B specific antibody, which is obtained by immunizing animals by taking the polymyxin B artificial antigen as an immunogen; the hybridoma cell strain comprises a monoclonal hybridoma cell strain C-PB-2-F.
The hybridoma cell strain of the polymyxin B specific antibody is preferably prepared by adopting a hybridoma cell fusion technology. The animals of the present invention preferably include, but are not limited to, mice, rabbits, goats, and sheep. The preservation number of the monoclonal hybridoma cell strain C-PB-2-F is preferably CGMCC No.45341.
The invention provides a polymyxin B monoclonal antibody, which is secreted and generated by a polymyxin B monoclonal antibody hybridoma cell strain C-PB-2-F according to the technical scheme. The polymyxin B monoclonal antibody provided by the invention has high sensitivity equivalent recognition capability to polymyxin B1 and B2, and has higher specificity.
Based on the above functions of the polymyxin B monoclonal antibody, the present invention further provides one or more of the following uses of the polymyxin B monoclonal antibody according to the above technical scheme: detecting polymyxin B at a non-diagnostic destination; preparing an immunochromatography test strip for detecting polymyxin B; preparing a magnetic particle immunodetection reagent of polymyxin B; preparing a homogeneous enzyme immunoassay kit for detecting polymyxin B. The present invention preferably detects the concentration of polymyxin B in blood or urine.
The invention also provides a magnetic particle immune detection reagent of polymyxin B, which comprises a magnetic particle working solution, an enzyme-labeled working solution and an indicator solution which are independently packaged;
the magnetic particle working solution comprises a combination body obtained by combining magnetic beads with the polymyxin B monoclonal antibody in the technical scheme;
the enzyme-labeled working solution comprises a conjugate obtained by coupling a polymyxin B hapten and a marker;
the indicator solution includes an indicator that includes a concentration of a marker for quantitative detection.
The magnetic beads of the invention are preferably paramagnetic magnetic beads, more preferably Dynal beads M280 Tosyl magnetic beads.
The present invention preferably employs 1-ethyl- (3-dimethylaminopropyl) carbodiimide as a coupling agent to couple polymyxin B hapten to a label, preferably alkaline phosphatase (ALP).
The indicator solution of the present invention is preferably AMPPD luminophore.
The invention also provides a preparation method of the magnetic particle immune detection reagent of polymyxin B, which comprises the following steps:
1: obtaining a magnetic particle working solution:
1-1: diluting the magnetic particles in borate buffer solution to obtain magnetic particle solution, adding polymyxin B specific antibody into the magnetic particle solution, and performing concussion reaction to obtain magnetic particle-antibody solution;
1-2: magnetically absorbing the magnetic particle antibody solution to remove supernatant, adding TBST buffer solution, reacting for 10-40 h, magnetically absorbing the obtained solution to remove supernatant, and adding TBST buffer solution to obtain magnetic particle working solution; the mass concentration of the magnetic particles in the magnetic particle working solution is 0.2-0.6 mg/mL;
2: and (3) obtaining an enzyme-labeled working solution:
2-1: dissolving the marker in PBS buffer solution to obtain a marker solution;
2-2: adding the polymyxin B hapten in the technical scheme into a marker solution, uniformly mixing, and then adding a coupling agent for coupling reaction to obtain a solution containing a conjugate; the mass ratio of the marker to the polymyxin B hapten to the coupling agent is 1: 0.2-1: 0.2-1;
2-3: dialyzing the solution containing the conjugate by using PBS buffer solution, and diluting the obtained dialyzate by using mixed buffer solution to obtain an enzyme-labeled working solution, wherein the concentration of the marker in the enzyme-labeled working solution is 0.1-2 mug/mL;
3: and (3) obtaining an indicator solution.
When the magnetic particle working solution is obtained, the magnetic particles are preferably diluted in borate buffer solution to obtain magnetic particle solution, polymyxin B specific antibody is added into the magnetic particle solution, and the magnetic particle-antibody solution is obtained through oscillation reaction. The mass ratio of the magnetic particles to the polymyxin B specific antibody is preferably 10-70: 1, more preferably 50:1. the temperature of the oscillation reaction is preferably 20-40 ℃, more preferably 37 ℃. The concentration of the borate buffer solution is preferably 20-100 mM, more preferably 50mM, and the pH value of the borate buffer solution is preferably 7-9, more preferably 8; the mass concentration of the magnetic particles in the magnetic particle solution is preferably 10-40 mg/mL, more preferably 25mg/mL.
After the magnetic particle-antibody solution is obtained, the invention preferably carries out magnetic attraction on the magnetic particle-antibody solution to remove supernatant, adds TBST buffer solution for reaction for 10-40 h, carries out magnetic attraction on the obtained solution to remove supernatant, and adds TBST buffer solution to obtain the magnetic particle working solution, wherein the mass concentration of the magnetic particles in the magnetic particle working solution is 0.2-0.6 mg/mL. The reaction time according to the invention is preferably 12 hours; the reaction temperature is preferably 20 to 40 ℃, more preferably 37 ℃. The mass concentration of the magnetic particles in the magnetic particle working solution is 0.4mg/mL. The operation of removing the supernatant by magnetic attraction is not particularly limited, and conventional steps in the art can be adopted.
In the case of obtaining the enzyme-labeled working solution, the present invention preferably dissolves the label in PBS buffer to obtain a label solution. The label of the invention is preferably alkaline phosphatase, and the mass concentration of the label in the label solution of the invention is preferably 0.5-2 mg/mL, more preferably 1mg/mL.
After the marker solution is obtained, the polymyxin B hapten according to the technical scheme is preferably added into the marker solution and uniformly mixed, and then a coupling agent is added for coupling reaction to obtain a conjugate; the mass ratio of the marker to the polymyxin B hapten to the coupling agent is 1: 0.2-1: 0.2 to 1. The coupling agent is preferably 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), and the coupling reaction time is preferably 0.5-4 h, more preferably 2h; the temperature of the coupling reaction is preferably 20 to 40 ℃, more preferably 37 ℃. The mass ratio of the marker to the polymyxin B hapten to the coupling agent is 1:1:1.
and (3) dialyzing the solution containing the conjugate by adopting a PBS buffer solution, and diluting the obtained dialyzate by using a mixed buffer solution to obtain an enzyme-labeled working solution, wherein the concentration of the marker in the enzyme-labeled working solution is 0.1-2 mug/mL. The molecular weight cut-off of the dialysis is preferably 7000-33000 Da, more preferably 15000Da. The mixed buffer solution of the present invention preferably comprises 50mMMES,0.9% NaCl 5mg/mL BSA and 1mM MgCl 2 . The pH value of the mixed buffer solution is preferably 6.0-7.0, more preferably 6.7.
The indicator solution of the present invention is preferably AMPPD luminophore. The source of the AMPPD luminescent liquid is not particularly limited, and the AMPPD luminescent liquid can be obtained by adopting products sold in the field.
The magnetic particle immunodetection reagent of polymyxin B is preferably prepared in an on-site manner or prepared finished products are stored at 2-8 ℃.
The invention also provides a use method of the magnetic particle immunity detection reagent of polymyxin B, which comprises the following steps: mixing a sample, a magnetic particle working solution and an enzyme-labeled working solution, incubating at 37 ℃, washing, adding an indicator solution for color development, and collecting 400-550 nm optical signals by adopting a photomultiplier.
The volume ratio of the sample, the magnetic particle working solution and the enzyme-labeled working solution is preferably 1:4:6, and particularly 10 mu L of the sample, 40 mu L of the magnetic particle working solution and 60 mu L of the enzyme-labeled working solution can be preferably selected. The specific step of the washing is not particularly limited, and the washing step when the magnetic particle immunodetection reagent is used is adopted in the field. The sample according to the present invention is preferably blood or urine, more preferably blood.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
Polymyxin B hapten (polymyxin B carboxyl derivative) was prepared as follows: the preparation route is shown in fig. 1-2:
60mg of polymyxin B and 20mL of toluene are stirred and dissolved, a solution prepared from 7.5mg of monomethyl succinate acyl chloride and 5mL of toluene is slowly added under the condition of heat preservation at 12 ℃, and the reaction is continued for 6 hours under the condition of 12 ℃. And adding 20mL of water after the reaction is finished, stirring for 20min, standing for layering, wherein the water layer is an aqueous solution of the intermediate 1. Cooling the intermediate 1 aqueous solution to 7 ℃, adjusting the pH value to 12 by liquid alkali, and carrying out hydrolysis reaction for 30min; adding toluene 20mL, standing for layering, adding dilute sulfuric acid into a water layer until the pH value reaches 4.0, mixing with ethanol, and distilling under reduced pressure for 2 times to obtain polymyxin B hapten.
The identification result of the nuclear magnetic hydrogen spectrum of the polymyxin B hapten is as follows:
HNMR(400MHZ, DMSO-d6):δ8.49-7.34 (m, 10H),7.19-7.14 (m, 5H), 5.80 (m, 1H), 4.94-4.63 (m, 8H), 4.42-4.15 (m, 12H), 4.11-3.81 (m, 18H), 3.80-3.55(m, 7H),3.39-3.27(m,7H), 3.10-2.56 (m, 9H), 2.50-2.05 (m, 9H), 1.18 (s, 3H), 0.96-0.91 (d, 12H)。
example 2
The preparation of the polymyxin B artificial antigen comprises the following specific steps:
1. taking 5mg of polymyxin B hapten prepared in example 1, and adding 1mL of DMSO to dissolve completely;
2. 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and dissolved in 100 mu L of water, and then added into the solution obtained in the step 1, and reacted for 1h at room temperature to obtain solution A;
3. weighing 20mg of bovine thyroglobulin and dissolving the bovine thyroglobulin in 5mL of PBS buffer solution to obtain solution B;
4. mixing the solution A and the solution B, and stirring for 2 hours at room temperature;
5. the solution obtained by mixing solution A and solution B was dialyzed 4 times against PBS buffer using a dialysis bag having a molecular weight cut-off of 15000Da, and the retentate was stored frozen at-20 ℃.
Example 3
The preparation of the polymyxin B monoclonal antibody adopts a hybridoma cell fusion technology, and comprises the following steps:
diluting the polymyxin B artificial antigen prepared in the example 2 to 1mg/mL by using PBS buffer solution, adding an equal volume of Freund's complete adjuvant, emulsifying completely, and performing primary immunization on the mice according to a dosage of 0.1 mg/mouse; after 4 weeks of interval, taking 1mg of the polymyxin B artificial antigen prepared in the example 2, mixing and emulsifying the polymyxin B artificial antigen with Freund's incomplete adjuvant in an equal volume, and performing secondary immunization on the mice according to a dosage of 0.1 mg/mouse;
after 3 days, the second immunized splenocytes were fused with Sp2/0 cells, and the supernatant of the fused cells was titered and competition determined using sheep anti-mouse IgG coated magnetic microsphere at 20 μg/mg (20 μg sheep anti-mouse IgG was added per 1mg magnetic microsphere), polymyxin B derivative-ALP.
Wherein: the preparation method of the polymyxin B derivative-ALP comprises the following steps:
1. taking 1mg of polymyxin B hapten prepared in example 1, adding 0.2mL of DMSO, and dissolving completely;
2. 2mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride is weighed and dissolved in 20 mu L of water, and added into the solution obtained in the step 1, and the solution is reacted for 1h at room temperature to obtain solution A;
3. weighing 5mg of alkaline phosphatase and dissolving in 5mL of PBS buffer to obtain solution B;
4. mixing the solution A and the solution B, and stirring for 2 hours at room temperature;
5. the solution obtained by mixing solution A and solution B was dialyzed 4 times against PBS buffer using a dialysis bag having a molecular weight cut-off of 15000Da, and the retentate was stored frozen at-20 ℃.
The method for determining the titer and the competition of the fused cell supernatant comprises the following steps: 50 mu L of fusion cell supernatant, 50 mu L of sample (sample containing different polymyxins) and 50 mu L of polymyxin B derivative-ALP are added into 50 mu L of goat anti-mouse IgG coated magnetic particle ball to react for 5min, cleaning is carried out, AMPPD substrate solution is added to react for 5min, and signal value is read, wherein the process is automatically completed by a full-automatic chemiluminescence instrument.
The screening results of the supernatant of the fused cells are shown in Table 1:
TABLE 1 screening results of supernatant of fused cells
Equivalence = S B1 /S B2 *100% refers to the extent of recognition of polymyxins B1 and B2 by antibodies under the same reaction conditions, with closer to 100% indicating better equivalence.
Specificity (crossover rate) = (1-S BE /S B1 ) 100% refers to the cross-over rate of antibody to polymyxin E, lower values indicating lower cross-over rates.
Sensitivity=s 0 /S B1 The reaction is antibody sensitivity, and a larger S/N indicates a higher sensitivity.
From table 1, it can be derived that: the recognition efficiency of cell supernatant C-PB-2-F on polymyxins B1 and B2 is close to 100%, the sensitivity is very high, and the crossing rate of polymyxin E is very low, so that a cell hole containing cell supernatant C-PB-2-F is selected for preparing mouse ascites.
Example 4
And cloning the cell hole containing the cell supernatant C-PB-2-F by using a limiting dilution method to obtain a monoclonal hybridoma cell strain C-PB-2-F capable of secreting the polymyxin B monoclonal antibody.
The preparation and purification method of the polymyxin B monoclonal antibody comprises the following steps:
reference is made to "Sun Anrui, li Xiaobing, wang Zhe. Method for preparing ascites from mouse hybridoma cells secreting monoclonal antibodies [ J ]. Chinese modern doctor, 2007, 45 (06Z): 2" for ascites preparation.
Purifying the collected ascites by using a Protein A/G purification method to obtain Protein A/G purification, and obtaining a polymyxin B monoclonal antibody which is marked as C-PB-2-F.
Example 5
The establishment of the method for detecting the blood concentration of polymyxin B by using a magnetic particle chemiluminescence method comprises the following steps:
1) 50mg Dynal beads M280 Tosyl magnetic beads are diluted in 2mL 50mM BB pH 8.0 buffer solution, 1mg of polymyxin B monoclonal antibody C-PB-2-F prepared in example 4 is added, and after uniform mixing, the mixture is subjected to shaking reaction at 37 ℃ for 8 hours.
2) The supernatant was removed by magnetic attraction, and TBST (50 mM Tris,0.9% NaCl,0.1% TW20, pH 7.4) was added and reacted at 37℃for 12 hours.
3) Removing supernatant by magnetic attraction, adding TBST, diluting to 0.4mg/mL, naming as magnetic particle working solution, and preserving at 2-8deg.C for use.
4) 1mg of ALP was dissolved in 1mL of PBS, 0.5mg of polymyxin B hapten (dissolved in 100. Mu.L of DMSO) was added, mixed well, 1mg of EDC solid was added, and mixed well at room temperature for 2h. Dialysis into PBS, 50mM MES 0.9% NaCl 5mg/mL BSA 1mM MgCl 2 pH6.7Diluted to 1 mug/mL and named as enzyme-labeled working solution.
5) The reaction procedure: 10 mu L of sample and 40 mu L of magnetic particle liquid working solution prepared in the step 3) and 60 mu L of enzyme-labeled working solution prepared in the step 4), incubating at 37 ℃ for 5min, cleaning, and adding AMPPD luminescent liquid for color development.
Calibration materials of polymyxin B at different concentrations of 0, 40, 200, 1000 and 5000ng/mL were prepared using a calf serum matrix, and the standard curves prepared are shown in table 2 and fig. 3, wherein X represents concentration and Y represents signal value in table 2 and fig. 3, and the right end point value of the abscissa of fig. 3 is 5000 instead of 500, and the pictures are not shown completely, and are described herein; and determining the precision and the minimum detection limit of the detection method and the cross reaction of polymyxin E, vancomycin, teicoplanin and linezolid, and the results are shown in tables 3 and 4, wherein the data in table 3 are calculated CV for 10 times of measurement of quality control products QC1 and QC2 respectively; loB is a negative sample measured 20 times and the average +2SD is calculated as the detection limit.
TABLE 2 calibration Curve data for polymyxin B blood concentration detection method by magnetic particle chemiluminescence method
TABLE 3 precision and sensitivity of method for detecting blood concentration of polymyxin B by magnetic particle chemiluminescence method
Cross-reactivity was calculated by adding different concentrations of cross-test substances to a polymyxin B sample at a concentration of 100ng/mL, and the results are shown in Table 4:
TABLE 4 magnetic particle chemiluminescence method polymyxin B plasma concentration detection method Cross-reacting polymyxin E, vancomycin, teicoplanin, and linezolid
Cross-reaction rate = (measured concentration-theoretical concentration)/concentration of added cross-species x 100%;
from the results of tables 3 to 4, it can be found that: the magnetic particle luminescence method polymyxin B determination reagent prepared by adopting the polymyxin B monoclonal antibody C-PB-2-F has high sensitivity, good repeatability and good specificity.
Example 6
Preparation of polymyxin B homogeneous enzyme immunoassay reagent
The polymyxin B homogeneous enzyme immunoassay reagent comprises two separately arranged reagents, and specifically comprises the following steps:
preparation of reagent R1: 20mM NAD,30mM glucose-6-phosphate (G6P), 55 mM Tris buffer at pH=8.0 was dissolved to prepare a homogeneous enzyme substrate; 0.05% of the anti-polymyxin B monoclonal antibody C-PB-2-F was added to the above homogeneous enzyme substrate.
Preparation of reagent R2: 0.05% of the prepared polymyxin B derivative-G6 PDH enzyme-labeled conjugate was added to Tris buffer at 0.1M ph=8.5, with a specific ratio of 0.05% in this example.
Wherein 600U G6PDH is adopted to dissolve the polymyxin B derivative-G6 PDH enzyme-labeled conjugate in 1mL PBS, 0.5mg of polymyxin B derivative-ALP (dissolved in 100 mu L DMSO) is added, and the preparation method of the polymyxin B derivative-ALP is the same as that of the embodiment 3, and is not repeated. Uniformly mixing, adding 1mg of EDC solid, and uniformly mixing for 2 hours at room temperature. Dialyzed into PBS, diluted to 5U/mL with 100mM Tris 0.9%NaCl 5mg/mL BSA pH8.5, and designated as R2 working solution.
Test procedure for 20. Mu.L sample +50. Mu. L R1 for 5min followed by addition of 50. Mu. L R2 for 5min and determination of OD 340 The rate of rise of the wavelength was set to Hitachi 7170.
Clinical 40 samples of polymyxin B urine concentration were selected and compared with the results of the measurement of total polymyxin B by the homogeneous enzyme method constructed in the present invention and the LC-MS method (see Liu X, yu Z, wang Y, et al Therapeutic drug monitoring of polymyxin B by LC-MS/MS in plasma and urine [ J ]. Bioanalysis, 2020, 12 (12)), and the results are shown in FIG. 4:
as can be seen from FIG. 4, the antibodies produced based on the present invention are establishedThe content of total polymyxin B measured by a homogeneous enzyme method has good correlation with LC-MS method, R 2 Is 0.9961, and meets clinical requirements.
From the above embodiments it can be derived that: the polymyxin B hapten provided by the invention can be used for preparing the specific antibody which has high sensitivity and good specificity and has equivalent recognition capability on polymyxin B1 and B2.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A polymyxin B hapten has a structure shown in a formula I:
formula I.
2. A polymyxin B artificial antigen, characterized in that it is obtained by coupling a carrier protein with the polymyxin B hapten of claim 1 using a coupling agent.
3. The polymyxin B artificial antigen of claim 2, wherein the carrier protein comprises bovine serum albumin, chicken ovalbumin, bovine thyroglobulin, human serum albumin, or rabbit serum albumin;
the coupling agent comprises 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide or carbonylimidazole.
4. The artificial antigen of polymyxin B of claim 2 or 3, wherein the molar ratio of polymyxin B hapten to carrier protein is (2-80): 1.
5. use of a polymyxin B hapten as defined in claim 1 or a polymyxin B artificial antigen as defined in any one of claims 2 to 4 for the preparation and/or detection of a polymyxin B specific antibody.
6. A hybridoma cell strain of a polymyxin B-specific antibody, characterized in that the polymyxin B artificial antigen of any one of claims 2-4 is used as an immunogen to immunize an animal; the hybridoma cell strain comprises a monoclonal hybridoma cell strain C-PB-2-F.
7. The hybridoma cell strain according to claim 6, wherein the preservation number of the monoclonal hybridoma cell strain C-PB-2-F is CGMCC No.45341.
8. A polymyxin B monoclonal antibody, wherein the polymyxin B monoclonal antibody is secreted by the polymyxin B monoclonal antibody hybridoma cell line C-PB-2-F of claim 6 or 7.
9. Use of a polymyxin B monoclonal antibody according to claim 8 in one or more of the following i-iv:
i: detecting polymyxin B at a non-diagnostic destination;
II: preparing an immunochromatography test strip for detecting polymyxin B;
III: preparing a magnetic particle immunodetection reagent of polymyxin B;
IV: preparing the polymyxin B homogeneous enzyme immunoassay kit.
10. The magnetic particle immune detection reagent of polymyxin B is characterized by comprising a magnetic particle working solution, an enzyme-labeled working solution and an indicator solution which are independently packaged;
the magnetic particle working solution comprises a combination body obtained by specifically combining magnetic beads with the polymyxin B monoclonal antibody of claim 8;
the enzyme-labeled working solution comprises a conjugate obtained by coupling a polymyxin B hapten and a marker;
the indicator solution includes an indicator for quantitatively detecting the concentration of the marker.
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