CN112111463A

CN112111463A - Hybridoma cell strain DOX-6A10, monoclonal antibody, preparation and application thereof

Info

Publication number: CN112111463A
Application number: CN202011168153.9A
Authority: CN
Inventors: 林冠峰; 吴英松; 卞论
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2020-10-27
Filing date: 2020-10-27
Publication date: 2020-12-22

Abstract

The invention provides a hybridoma cell strain DOX-6A10, a monoclonal antibody, a preparation process and application thereof. The hybridoma cell strain DOX-6A10 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020147. The DOX monoclonal antibody uses the traditional fusion technology of an immune mouse and hybridoma cells, then positive cell strain clones are screened by ELISA, monoclonal cell culture is carried out, and finally the DOX monoclonal antibody is obtained. The DOX monoclonal antibody is different from a commonly used polyclonal antibody directly extracted from immune mouse serum, can avoid the influence of irrelevant antibodies, can be directly coated and used, and fills the blank of the preparation process of the DOX monoclonal antibody. The kit for detecting the blood concentration of DOX by applying the competitive method prepared from the DOX monoclonal antibody can immediately detect the blood concentration level of DOX of a target object by using the DOX standard substance with known concentration and the blood sample of a patient with unknown DOX concentration to competitively bind with the monoclonal antibody.

Description

Hybridoma cell strain DOX-6A10, monoclonal antibody, preparation and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a hybridoma cell strain DOX-6A10, a DOX monoclonal antibody generated by the hybridoma cell strain DOX-6A10, a preparation process of the DOX monoclonal antibody, and a kit for detecting the blood concentration of DOX by a DOX monoclonal antibody competition method.

Background

Doxorubicin (DOX), also known as Doxorubicin, is an anthracycline antibiotic extracted from the fermentation broth of a streptomyces baumannii variant. Doxorubicin can inhibit the transcription synthesis of DNA and RNA, has a broad spectrum of anti-tumor effects, but is highly toxic and can cause severe cardiotoxicity and liver damage in long-term use. Therefore, in the treatment of tumors with DOX, it is necessary to constantly monitor the blood level of DOX in a patient to ensure that the patient receives as little toxic side effects of DOX as possible while being effectively treated for DOX. The individual metabolic rates of tumor patients at different times are different, and even if the same dosage is used in each chemotherapy, the blood concentration level of DOX in the patients is different after each chemotherapy.

The commonly used detection techniques for blood concentration include spectrophotometry, gas chromatography, high performance liquid chromatography and immunological methods. The immunological method includes radioimmunoassay, enzyme immunoassay, fluoroimmunoassay, and immunochemiluminescence. The enzyme-linked immunosorbent assay has the characteristics of high sensitivity, strong specificity, and qualitative and quantitative properties.

There is no report in the prior art relating to the development of DOX monoclonal antibodies. The ELISA method is difficult to detect small molecule drugs such as DOX. Because the ELISA measurement range is small, a trace amount of small molecule drugs contain a large amount of antigen epitopes, and the detection of the ELISA method is easy to exceed the peak value. In addition, the small molecule drug has few types of antigen epitopes, so that the sandwich ELISA method is difficult to be used for immunoassay, and finally the detection concentration of the existing DOX detection kit is inaccurate.

If the DOX monoclonal antibody can be prepared, the DOX monoclonal antibody can be combined with an enzyme-linked immunosorbent assay to quickly and accurately detect the blood concentration level of DOX in a human body, so that the problems in the prior art are solved. Therefore, aiming at the defects of the prior art, the invention provides a hybridoma cell strain DOX-6A10, a DOX monoclonal antibody generated by the hybridoma cell strain DOX-6A10 and a kit for detecting the blood concentration of DOX by a DOX monoclonal antibody competition method, which are necessary to overcome the defects of the prior art.

Disclosure of Invention

The invention aims to avoid the defects of the prior art and provides a hybridoma cell strain DOX-6A10, a DOX monoclonal antibody generated by the hybridoma cell strain DOX-6A10, a preparation process and a kit for detecting the blood concentration of DOX by a DOX monoclonal antibody competition method. The DOX monoclonal antibody combined enzyme immunity protein method can quickly and accurately detect the blood drug concentration level of DOX in a human body, and has the characteristics of high sensitivity, strong specificity, and qualitative and quantitative properties. The DOX concentration detection kit developed on the basis can quickly and effectively monitor the blood concentration of the target object.

In order to achieve the purpose, the invention adopts the following technical scheme:

the hybridoma cell strain DOX-6A10 provided by the invention is preserved in China center for type culture Collection (CCTCC for short); the preservation unit address is Wuhan university collection center, Wuhan university school, eight paths 299 # Wuhan university in Wuchang district, Wuhan City, Hubei China; the preservation number is CCTCC NO: C2020147, and the preservation time is 2020, 8 and 31 days.

The invention provides a DOX monoclonal antibody secreted and produced by the hybridoma cell strain DOX-6A 10. The DOX monoclonal antibody is of an IgM type, and light chains are kappa chains.

The invention also provides the application of the DOX monoclonal antibody in the preparation of a kit for detecting the blood concentration of DOX by a competition method.

The invention also provides a kit for detecting the blood concentration of DOX, which adopts the monoclonal antibody to detect by a competition method and contains the DOX monoclonal antibody. In particular to a kit for detecting the blood concentration of DOX by a competitive method with a DOX monoclonal antibody.

The invention also provides a method for preparing the DOX monoclonal antibody, which comprises the following steps:

s1, preparation of immunogen: respectively dissolving carrier protein KLH and medicament DOX for standby, performing buffer solution replacement on a KLH solution by using an interception column in an EP (ethylene propylene) tube, adding EDC solution and NHS solution into the EP tube to activate KLH, performing buffer solution replacement on the KLH solution by using the interception column in the EP tube, and then adding DOX solution for coupling to prepare DOX-KLH immunogen.

S2, preparing hybridoma cells: immunizing in a BALB/c mouse by using a DOX-KLH immunogen, strengthening immune splenocytes, performing fusion culture on the immune splenocytes and myeloma cells, screening out positive hybridoma cell strains by an ELISA method, performing subcloning, and finally obtaining a hybridoma cell strain DOX-6A10DOX monoclonal antibody of a hybridoma cell strain DOX-6A10 which stably secretes an anti-DOX monoclonal antibody.

The anti-adriamycin monoclonal antibody secreted by the hybridoma cell strain DOX-6A10 is identified as an IgM type, and light chains are kappa chains.

S3, preparation of monoclonal antibody: the hybridoma cell strain DOX-6A10 is used for inducing proliferation in a BALB/c mouse to prepare the DOX monoclonal antibody.

Specifically, the specific operation steps of step S1 are:

s11, dissolving the KLH freeze-dried powder by using pure water to prepare a KLH solution with the concentration of 10mg/mL for later use; 2mg of DOX powder was weighed, and dissolved in 50mM MES buffer solution having a pH of 5.0 to prepare a DOX solution having a concentration of 5mg/mL for use.

S12, the KLH solution was buffer-exchanged with a 50mM MES buffer solution having a pH of 5.0 to a 50mM MES buffer solution having a pH of 6.5 using a trap column.

S13, restoring EDC and NHS to room temperature, adding MES buffer solution to dissolve, preparing 10mg/mL solution, adding 50. mu.L EDC solution and 112.8. mu.L NHS solution into the EP tube collected with KLH solution in step S12, and slightly shaking to activate for 30 min.

S14, the activated KLH solution was subjected to buffer exchange using a trap column, and 50mM MES buffer solution having a pH of 6.5 was exchanged with 10mmol/L PBS buffer solution having a pH of 7.4.

S15, adding 200 mu L of DOX solution supernatant into the EP tube collected with the KLH solution in the step S14, and shaking the reaction for 120min to complete the coupling.

And S16, dialyzing the coupled immunogen with 10mmol/L PBS buffer solution with the pH value of 7.4, replacing the dialyzate every 8h for 3 times, and finally obtaining the purified DOX-KLH immunogen.

Specifically, the specific operation steps of step S2 are:

s21, immunizing BALB/c mice by using DOX-KLH immunogen prepared in the step S1, and specifically: and in the primary immunization, fully emulsifying the DOX-KLH immunogen with an equal volume of Freund complete adjuvant, and injecting the DOX-KLH immunogen and an equal volume of Freund complete adjuvant into a female BALB/c mouse body of 6-8 weeks old at multiple points in a subcutaneous manner, wherein the dosage of the DOX-KLH immunogen is 200 mu g/mouse, and the DOX-KLH immunogen and the equal volume of Freund incomplete adjuvant are fully emulsified and injected into the abdominal cavity of the BALB/c mouse every 2 weeks, and the dosage of the immunogen is 200 mu g/mouse.

On day 7 after the fourth immunization, BALB/c mice were subjected to venous blood collection, and serum titer was measured, and 3 days before cell fusion, serum titer of 1.0X 10 was selected⁵The BALB/c mice were splenic boosted with DOX-KLH immunogen at 200. mu.g/mouse.

S22, preparing DOX-KLH immune spleen cells, which specifically comprises the following steps: killing the BALB/c mouse which is boosted and immunized in the step S21, infiltrating the mouse with 75% alcohol, taking out spleen with disinfected scissors and tweezers, removing fat and connective tissues, washing with 75% alcohol, leaching with serum-free DMDM culture medium, placing on a screen, fully grinding with a rubber head of a sterile injector, flushing spleen cells into a sterile centrifuge tube through the serum-free IMDM culture medium, centrifuging at 1500rpm for 3min, finally, re-suspending with the serum-free IMDM culture medium, counting, and adjusting the cell concentration to 2 x 10⁸And (4) per mL, as DOX-KLH immune spleen cells.

S23, myeloma cell SP2/0 and DOX-KLH immune spleen cell prepared in the step S22 are fully and uniformly mixed according to the volume ratio of 1:7, the mixture is centrifuged at 1500rpm for 3min, supernatant is discarded, the mixture is placed on a water bath at 37 ℃, preheated 1mL of PEG-1500 is slowly added within 1min, the mixture is shaken while adding, and after the addition is finished, the fusion effect is stopped by preheated 20mL of serum-free IMDM culture medium.

Centrifuging at 1500rpm for 3min, discarding supernatant, suspending the precipitate with HAT complete medium, adding into 96-well plate at 300 μ L/well, standing at 37 deg.C and 5% CO₂The cell culture chamber of (2) for cell culture.

S24, screening positive hybridoma cells and clones, specifically: DOX is used as a coating antigen, 0.05mol/L CB buffer solution with the pH value of 9.6 is used for diluting to 1 mu g/ml, 100 mu L/hole is added into an enzyme label plate, the enzyme label plate is coated at 4 ℃ overnight and then dried, gelatin-PBS buffer solution with the mass volume ratio of 1% is used for sealing, 300 mu L/hole is used for sealing at 4 ℃ overnight and then dried for standby.

And (3) taking the supernatant of the cell culture solution subjected to fusion culture in the step S23, adding an enzyme label plate in a 100 mu L/hole, placing the mixture at 37 ℃ for incubation for 60min, washing the mixture with 0.01mol/L PBST buffer solution containing Tween-20, patting the mixture dry, adding 100 mu L/hole HRP-labeled goat-anti-mouse secondary antibody, placing the mixture at 37 ℃ for incubation for 60min, washing the mixture dry, adding 100 mu L/hole TMB color development solution, placing the mixture at 37 ℃ for dark color development for 10min, and adding 50 mu L/hole 1mol/L HCl to terminate the reaction.

And (3) inoculating the positive hybridoma cell strain detected by the ELISA method to an HT incomplete culture medium, and performing subcloning for at least 3 times by using a limiting dilution method to obtain the positive hybridoma with the monoclonal cell strain positive rate of 100%.

Specifically, the specific operation steps of step S3 are: selecting BALB/c mice 8-10 weeks old, injecting 0.5ml liquid paraffin into the abdominal cavity, injecting 3 x 10 liquid paraffin into the abdominal cavity 7-14 days later⁵～4×10⁵Respectively extracting ascites by using an injector and marking after 7-10 days for the positive hybridoma cells prepared in the step S24; purifying by affinity chromatography Protein A Sepharose Fast Flow to obtain DOX monoclonal antibody, desalting by Sepharose S-200 molecular sieve, and determining the purity of the monoclonal antibody by SDS-PAGE, wherein the purity is over 90%.

Specifically, the specific operation steps of step S12 are: and (3) placing the 100kD cut-off column into an EP tube, adding 500 mu L of the KLH solution obtained in the step S11 into the cut-off column, centrifuging for 10min at 9500rpm in a centrifuge precooled at 4 ℃, discarding waste liquid in the EP tube, adding 300 mu L of 50mM MES buffer solution with the pH value of 6.5 into the cut-off column, centrifuging again, and repeating for 5-6 times.

The trap column was placed in a new EP tube, centrifuged at 3000rpm for 3min, 200. mu.L of 50mM MES buffer pH 6.5 was washed and allowed to stand for 5min, then the trap column was again placed upside down in an EP tube in which KLH was collected and centrifuged at 3000rpm for 3 min.

The specific operation steps of step S14 are as follows: and (3) placing the 100kD trapping column into an EP tube, adding the activated KLH solution into the trapping column, centrifuging for 10min at 9500rpm in a 4-DEG C precooled centrifuge, discarding the waste liquid in the EP tube, adding 300 mu L of 10mmol/L PBS buffer solution with the pH value of 7.4 into the trapping column, centrifuging again, and repeating for 5-6 times.

The trap column was placed in a new EP tube, centrifuged at 3000rpm for 3min, 200. mu.L of 10mmol/L PBS buffer pH 7.4 was washed and allowed to stand for 5min, then the trap column was again placed upside down in an EP tube containing KLH and centrifuged at 3000rpm for 3 min.

At present, no DOX monoclonal antibody exists. The DOX monoclonal antibody uses the traditional immune mouse and hybridoma cell fusion technology, then positive cell strain clones are screened by ELISA, monoclonal cell culture is carried out, and finally the DOX monoclonal antibody is obtained. The DOX monoclonal antibody is different from the commonly used polyclonal antibody which is directly extracted from the serum of an immune mouse, can avoid the influence of irrelevant antibodies, and can be directly coated and used. Because the cells for producing the monoclonal antibody are all from the replication and propagation of the initial positive hybridoma cell, the same genes are expressed in the same cells, the various characteristics of the antibody are consistent, and the characteristic that the hybridoma cells can be propagated infinitely is utilized, the purpose of producing the DOX monoclonal antibody in large quantities is achieved, and the blank of the preparation process of the DOX monoclonal antibody is filled.

The invention provides a kit for detecting the blood concentration of DOX by using a DOX monoclonal antibody to perform a competition method. The kit can immediately detect the blood concentration level of DOX in a target object by using the DOX standard substance with known concentration and the target object blood sample with unknown DOX concentration to compete and combine with the monoclonal antibody. The kit can achieve the purpose of rapidly and effectively monitoring the blood concentration of DOX in the target object.

Drawings

The invention is further illustrated by means of the attached drawings, the content of which is not in any way limiting.

FIG. 1 is a graph showing the standard curve of the competitive assay for measuring the blood concentration of DOX in example 2 of the present invention.

Fig. 2 is a graph of the measured concentration of a serum sample of known concentration fitted with a DOX plasma concentration kit according to example 2 of the present invention.

In FIG. 1, the X-axis represents the concentration of the standard substance, and the Y-axis represents the measurement signal value.

In fig. 2, the X-axis is the nominal sample concentration and the Y-axis is the measured concentration of the DOX plasma concentration kit on the sample.

Biological preservation description: the hybridoma cell strain DOX-6A10 is preserved in China center for type culture Collection (CCTCC for short); the preservation unit address is Wuhan university collection center, Wuhan university school, eight paths 299 # Wuhan university in Wuchang district, Wuhan City, Hubei China; the preservation number is CCTCC NO of C2020147; the preservation time is 2020, 8, 31 days.

Detailed Description

The invention is further illustrated by the following examples.

Example 1.

The hybridoma cell strain DOX-6A10 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: C2020147. The DOX monoclonal antibody secreted by the hybridoma cell strain DOX-6A10 is identified to be an IgM type, and light chains are kappa chains.

The hybridoma cell strain DOX-6A10 and the DOX monoclonal antibody secreted and generated by the hybridoma cell strain DOX-6A10 are prepared by the following steps:

DOX (Doxorubicin), also known as Doxorubicin, is an anthracycline antibiotic extracted from the fermentation broth of a streptomyces baumannii variant. DOX can inhibit the transcription and synthesis of DNA and RNA, has a broad-spectrum antitumor effect, but has high toxicity, and can cause serious cardiotoxicity and liver damage after long-term use. Therefore, during the treatment of tumors with DOX, the level of DOX plasma levels in a patient must be strictly monitored at all times.

KLH (Keyhole limpet hemocyanin) is free blue copper-containing respiratory protein with high molecular weight existing in hemolymph of certain mollusks and arthropods, has a certain configuration, has the molecular weight of 450000-1300000 generally, can be connected with other molecules, and is often used as an immunogen carrier in the field of immunization.

EDC (1-ethyl- (3-dimethyl amino propyl) carbodiimide hydrochloride) belongs to a carbodiimide series condensing agent. The reaction mechanism of the compound as a dehydrating agent is as follows: firstly, carboxyl reacts with carbodiimide to generate intermediate O-acyl isothiourea, and ester group is introduced to activate carboxylic acid. Then reacting with amine to generate the target product amide and urea. The activated carboxylic acid can react with another molecule of carboxylic acid to generate anhydride, and the anhydride and the amine reaction liquid obtain amide. At the same time, the activated carboxylic acid rearranges to form the N-acylurea as a by-product. EDC can greatly improve condensation efficiency under the action of NHS (N-hydroxysuccinimide), and is beneficial to the proceeding of coupling reaction.

Myeloma cells have an unlimited dividing ability, and since the cells are not highly differentiated cells, they have a strong dividing ability, while other tumor cells have a weak dividing ability, and therefore, they are often fused with B lymphocytes during the preparation of monoclonal antibodies, and the monoclonal antibodies are rapidly proliferated in large quantities. The hybridoma capable of stably secreting the DOX monoclonal antibody prepared by fusing the immune splenocytes and the myeloma cells has the characteristic of unlimited proliferation, is favorable for the massive proliferation of the DOX monoclonal antibody, and can realize the large-scale production of the DOX monoclonal antibody.

The DOX monoclonal antibody of the embodiment uses the traditional fusion technology of an immune mouse and a hybridoma cell, then positive cell strain clones are screened by ELISA, monoclonal cell culture is carried out, and finally the DOX monoclonal antibody is obtained. The DOX monoclonal antibody is different from the commonly used polyclonal antibody which is directly extracted from the serum of an immune mouse, can avoid the influence of irrelevant antibodies, and can be directly coated and used. Because the cells for producing the monoclonal antibody are all from the replication and propagation of the initial positive hybridoma cell, the same genes are expressed in the same cells, the various characteristics of the antibody are consistent, and the characteristic that the hybridoma cells can be propagated infinitely is utilized, the purpose of producing the DOX monoclonal antibody in large quantities is achieved, and the blank of the preparation process of the DOX monoclonal antibody is filled.

Wherein, the specific preparation process of the immunogen in the step S1 is as follows:

and S11, dissolving the KLH freeze-dried powder by using pure water to prepare a KLH solution with the concentration of 10mg/mL for later use. 2mg of DOX powder was weighed, and dissolved in 50mM MES buffer solution having a pH of 5.0 to prepare a DOX solution having a concentration of 5mg/mL for use.

S12, placing a 100kD cut-off column into an EP tube, adding 500 mu L of the KLH solution obtained in the step S11 into the cut-off column, centrifuging the solution at 9500rpm for 10min in a 4 ℃ precooled centrifuge, discarding the waste liquid in the EP tube, adding 300 mu L of 50mM MES buffer solution with the pH value of 6.5 into the cut-off column, centrifuging again, and repeating the steps for 5-6 times.

S14, placing a 100kD trapping column into an EP tube, adding the activated KLH solution into the trapping column, centrifuging the solution in a 4 ℃ precooled centrifuge at 9500rpm for 10min, discarding the waste liquid in the EP tube, adding 300 mu L of 10mmol/L PBS buffer solution with the pH value of 7.4 into the trapping column, centrifuging again, and repeating for 5-6 times.

S15, adding 200. mu.L of DOX solution supernatant to the EP tube collected with KLH solution in the step S14, and slightly shaking the reaction for 120min to complete the coupling.

Wherein, the specific preparation process of the hybridoma in the step S2 is as follows:

s21, BALB/c mice were immunized with DOX-KLH immunogen prepared in step S1 above. And in the primary immunization, fully emulsifying the DOX-KLH immunogen with an equal volume of Freund complete adjuvant, and injecting the DOX-KLH immunogen and an equal volume of Freund complete adjuvant into a female BALB/c mouse body of 6-8 weeks old at multiple points in a subcutaneous manner, wherein the dosage of the DOX-KLH immunogen is 200 mu g/mouse, and the DOX-KLH immunogen and the equal volume of Freund incomplete adjuvant are fully emulsified and injected into the abdominal cavity of the BALB/c mouse every 2 weeks, and the dosage of the immunogen is 200 mu g/mouse.

S22, preparing DOX-KLH immune spleen cells. Killing BALB/c mice boosted in the step S21, soaking the mice with 75% alcohol, taking out spleens with disinfected scissors and tweezers, removing fat and connective tissues, washing with 75% alcohol, rinsing with serum-free IMDM medium, placing the mice on a screen, and using sterile IMDM medium to perform sterile filtrationFully grinding rubber head of the injector, washing spleen cells into a sterile centrifuge tube through a serum-free IMDM culture medium, centrifuging at 1500rpm for 3min, finally resuspending with the serum-free IMDM culture medium, counting, and adjusting cell concentration to 2 × 10⁸And (4) per mL, as DOX-KLH immune spleen cells.

The HAT complete medium used in this example consisted of IMDM medium containing 100. mu. mol/L hypoxanthine, 0.4. mu. mol/L aminopterin, 16. mu. mol/L thymidine, 15% (v/v) fetal bovine serum.

S24, screening positive hybridoma cells and cloning. DOX is used as a coating antigen, 0.05mol/L CB buffer solution with the pH value of 9.6 is used for diluting to 1 mu g/ml, 100 mu L/hole is added into an enzyme label plate, the enzyme label plate is coated at 4 ℃ overnight and then dried, gelatin-PBS buffer solution with the mass volume ratio of 1% is used for sealing, 300 mu L/hole is used for sealing at 4 ℃ overnight and then dried for standby.

The positive hybridoma cell strain detected by an ELISA method is inoculated to an HT incomplete culture medium, multiple subcloning is carried out by using a limiting dilution method, the hybridoma cell strain with the positive rate of 100 percent and stably secreting a DOX monoclonal antibody is obtained, liquid nitrogen is frozen and stored after the expansion culture, the hybridoma cell strain is named DOX-6A10, the cell strain is preserved in China center for type culture collection No. 9/4 in 2020, and the preservation number is CCTCC NO: C2020147.

The DOX monoclonal antibody secreted by the hybridoma cell strain is identified as an IgM type, and light chains are kappa chains.

Wherein, the specific preparation process of the DOX monoclonal antibody in the step S3 is as follows:

selecting BALB/c mice 8-10 weeks old, injecting 0.5ml liquid paraffin into the abdominal cavity, injecting 3 x 10 liquid paraffin into the abdominal cavity 7-14 days later⁵～4×10⁵Respectively extracting ascites by using an injector and marking after 7-10 days for the positive hybridoma cells prepared in the step S24; the ascites fluid was centrifuged at 12000rpm for 10min and the supernatant collected and purified using Protein A (available from GE Co.): filling the filler into a chromatographic purification glass column, connecting the purification column with a Bio-rad normal pressure purifier, flushing 5 column volumes by using an equilibrium buffer solution (configured according to a scheme in the specification), diluting the ascites to be purified by 10 times by using an equilibrium solution, then loading at the flow rate of 0.5ml/min, continuing flushing with the equilibrium solution to a base line after loading, eluting an antibody by using an eluent, collecting a peak tip signal sample, neutralizing the eluted antibody by using a neutralization solution, and measuring the concentration of the antibody by using a Smart Spec plus nucleic acid protein determinator of Bio-rad, wherein the concentration of the antibody is 2.52 mg/ml. Purified antibodies were stored at-20 degrees.

The DOX monoclonal antibody uses the traditional immune mouse and hybridoma cell fusion technology, then positive cell strain clones are screened by ELISA, monoclonal cell culture is carried out, and finally the DOX monoclonal antibody is obtained. The DOX monoclonal antibody is different from the commonly used polyclonal antibody which is directly extracted from the serum of an immune mouse, can avoid the influence of irrelevant antibodies, and can be directly coated and used. Because the cells for producing the monoclonal antibody are all from the replication and propagation of the initial positive hybridoma cell, the same genes are expressed in the same cells, the various characteristics of the antibody are consistent, and the characteristic that the hybridoma cells can be propagated infinitely is utilized, the purpose of producing the DOX monoclonal antibody in large quantities is achieved, and the blank of the preparation process of the DOX monoclonal antibody is filled.

Example 2.

The DOX monoclonal antibody can play a role in preparing a kit for detecting the blood concentration of DOX by a competition method.

The kit for detecting the blood concentration of DOX adopts a monoclonal antibody to carry out detection by a competition method, contains the DOX monoclonal antibody of the invention, and particularly relates to the kit for detecting the blood concentration of DOX by the competition method of the DOX monoclonal antibody. Because DOX is reduced as a surface antigen of a small molecule drug, the DOX concentration in a blood sample of a target object is detected by using a DOX standard with a known concentration to compete with the blood sample of the target object with an unknown DOX concentration to bind a monoclonal antibody.

The following is a description of specific embodiments: the application of the DOX monoclonal antibody in the prepared DOX time-resolved fluoroimmunoassay quantitative analysis kit.

DOX-KLH labeled antigen

1mg of DOX-KLH conjugated antigen in a mass ratio of 5: 1 and 0.2mg of europium labeling reagent in labeling buffer (50mmol/L Na)₂CO₃pH 9.0), shaking overnight at room temperature, purifying with Sephadex G-50 gel chromatography column the next day, collecting eluate (1 ml/tube), measuring fluorescence signal value tube by tube, combining peak tubes, adding 10% BSA protectant, and storing at-20 deg.C.

2. Preparation of calibration article

1.5% BSA standard diluent (50mmol/L Tris-HCl, 0.9% NaCl, 0.1% NaN)₃0.01% Tween-20, 1.5% BSA, pH 7.8) to make a series of DOX calibrator solutions: 0ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000 ng/mL.

3. Microporous plate coated antibody

The DOX monoclonal antibody was dissolved in the coating solution (50mmol/L Na) at a concentration of 3.5. mu.g/ml₂CO₃pH 9.0), adding 100. mu.L of buffer solution to the reaction well, coating the reaction well with shaking at 37 ℃ for 2 hours, discarding the coating solution in the well, and adding blocking solution (50mmol/L Tris-HCl, 0.1% BSA, 4% trehalose, 0.05% NaN)₃pH 7.2), 150 μ L/well, blocked overnight at 4 ℃. The next day, the wells were discarded and sealed, patted dry, evacuated, and stored at 4 ℃ for further use.

4. Procedure for the preparation of the

Firstly, taking out the kit from a refrigerator, after balancing for 15min at room temperature, adding 25 muL of a standard substance or a sample to be detected, 25 muL of DOX-KLH labeled antigen and 50 muL of an analysis buffer solution into a microporous plate, oscillating and incubating for 30min at 37 ℃, then washing the plate for 6 times by a plate washing machine, adding 100ul of an enhancement solution, oscillating and incubating for 5min at 37 ℃, distinguishing a fluorescence signal value of fluorescence immunoassay detection in Victor 31420 time, and calculating the DOX concentration of the sample to be detected from a standard curve.

The method for detecting DOX by the kit is characterized in that the detection basis is to label immunoreaction by a competition method, a calibration substance or a sample to be detected and a marked DOX-KLH are added into a microporous plate to competitively bind a DOX monoclonal antibody coated in the microporous plate, a standard curve is drawn by the detection signal value of the added calibration substance, and the DOX concentration of the sample to be detected is calculated by substituting the detection value of the sample to be detected into the curve. Wherein the sample to be tested comprises serum.

The europium-labeled reagent, the enhancement solution and the signal detection instrument used in the invention are purchased from Perkinelmer.

In the practical application of the kit for detecting the DOX blood concentration, the dilution concentration of the marker is 1:1000, and a series of DOX calibration sample solutions are prepared: 0ng/mL, 10ng/mL, 20ng/mL, 100ng/mL, 500ng/mL, 1000ng/mL, 2000 ng/mL. Working solutions of each concentration were tested in parallel 2 times and the results are shown in table 1.

TABLE 1

The principle of antigen detection by the competition method is that the antigen in a sample to be detected and a certain amount of enzyme-labeled antigen compete to be combined with a solid-phase antibody. FIG. 1 is a graph showing the standard curve of the competitive method for detecting DOX concentration, and as shown in FIG. 1, the more DOX content in the sample to be detected is, the less enzyme-labeled antigen is bound to the solid phase, the lighter the final color development is, and the weaker the measurement signal value is. The relationship between the measured signal values and the concentrations of the standards was fitted to the data in table 1 as a linear relationship of y + a + b x, with a-5.09023 and b-2.00956 for the curves.

Correlation coefficient r of the above-mentioned standard curve²0.99508, the detection method has the characteristics of high accuracy and high precision, and can meet the detection requirement of the DOX blood concentration.

10 clinical serum samples with known sample concentrations were tested using a DOX blood concentration kit, the marker dilution concentration was 1:1000, the measured signal values obtained by the test were calculated using the standard curve shown in FIG. 1, and the results are shown in Table 2.

TABLE 2

The result of comparing the actual concentration of the sample obtained by using the DOX blood concentration kit with the nominal sample concentration is shown in figure 2, and the correlation coefficient r²0.99916, the DOX blood concentration kit has better performance in detecting the DOX concentration in a serum sample and reliable detection result.

In the embodiment, the kit for detecting the blood concentration of DOX by using the competitive method of the DOX monoclonal antibody can immediately detect the blood concentration level of DOX in a target object by using the DOX standard with a known concentration and the target object blood sample with an unknown DOX concentration to competitively bind the monoclonal antibody in clinical application. The kit can quickly and effectively monitor the blood concentration of DOX in a target object, controls the use amount of the medicament on the basis of the blood concentration, customizes individual medicament administration schemes for different patients, reduces the toxic and side effects of the medicament to the lowest possible, and balances the effective blood concentration and the low toxic and side effects of the medicament.

The kit is used for detecting the blood concentration of DOX by using the DOX monoclonal antibody as a competition method, and because DOX is reduced as a surface antigen of a small molecule drug, the DOX standard substance with known concentration and the target blood sample with unknown DOX concentration are combined in a competition manner to achieve the purpose of detecting the DOX concentration in the target blood sample. The technical problem that the concentration of DOX in a blood sample of a target object cannot be accurately detected due to the fact that the DOX is used as a small molecule drug surface antigen in the prior art is solved.

The DOX monoclonal antibody is combined with an enzyme-immune protein method to serve as a kit for detecting the blood concentration of DOX by a competition method, can quickly and accurately detect the blood concentration level of DOX in a human body, and has the characteristics of high sensitivity, strong specificity, and qualitative and quantitative effects. The DOX concentration detection kit developed on the basis can quickly and effectively monitor the blood concentration of the target object.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The hybridoma cell strain DOX-6A10 is characterized in that: is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2020147.

2. The DOX monoclonal antibody secreted by the hybridoma cell line DOX-6A10 of claim 1.

3. The DOX monoclonal antibody secreted by the hybridoma cell strain DOX-6A10 according to claim 2, wherein the DOX monoclonal antibody is characterized in that: the DOX monoclonal antibody is of an IgM type, and light chains are kappa chains.

4. Use of a DOX monoclonal antibody according to claim 2 for the preparation of a kit for the competitive detection of blood levels of DOX.

5. A kit for detecting the blood concentration of DOX adopts monoclonal antibodies to detect by a competition method, and is characterized in that: comprising a DOX monoclonal antibody according to claim 2.

6. A method of making a DOX monoclonal antibody of claim 2 characterized by: is prepared by the following steps:

s1, preparation of immunogen: respectively dissolving carrier protein KLH and medicament DOX for later use, performing buffer solution replacement on a KLH solution by using an interception column in an EP (ethylene propylene glycol) tube, adding an EDC solution and an NHS solution into the EP tube to activate KLH, performing buffer solution replacement on the KLH solution by using the interception column in the EP tube again, and then adding a DOX solution for coupling to prepare a DOX-KLH immunogen;

s2, preparing hybridoma cells: immunizing in a BALB/c mouse by using a DOX-KLH immunogen, strengthening immune splenocytes, performing fusion culture on the immune splenocytes and myeloma cells, screening out positive hybridoma cell strains by an ELISA method for subcloning, and finally obtaining a hybridoma cell strain DOX-6A10 stably secreting an anti-DOX monoclonal antibody;

the anti-DOX monoclonal antibody secreted by the hybridoma cell strain DOX-6A10 is identified as an IgM type, and light chains are kappa chains;

7. The method for producing a DOX monoclonal antibody according to claim 6, wherein: the specific operation steps of step S1 are as follows:

s11, dissolving the KLH freeze-dried powder by using pure water to prepare a KLH solution with the concentration of 10mg/mL for later use; weighing 2mg of DOX medicinal powder, adding 50mM MES buffer solution with the pH value of 5.0 for dissolving, and preparing DOX solution with the concentration of 5mg/mL for later use;

s12, buffer exchange is carried out on the KLH solution by using a trapping column, and 50mM MES buffer solution with pH value of 5.0 is exchanged with 50mM MES buffer solution with pH value of 6.5;

s13, restoring EDC and NHS to room temperature, respectively adding MES buffer solution for dissolving to prepare solutions with the concentration of 10mg/mL, adding 50 μ L EDC solution and 112.8 μ L NHS solution into the EP tube collected with KLH solution in the step S12, and slightly shaking for activation for 30 min;

s14, buffer replacement is carried out on the activated KLH solution by using a trapping column, and 50mM MES buffer solution with the pH value of 6.5 is replaced by 10mmol/L PBS buffer solution with the pH value of 7.4;

s15, adding 200 mu L of DOX solution supernatant into the EP tube with the KLH solution collected in the step S14, and carrying out shake reaction for 120min to complete coupling;

8. The method for producing a DOX monoclonal antibody according to claim 6, wherein: the specific operation steps of step S2 are as follows:

s21, immunizing BALB/c mice by using DOX-KLH immunogen prepared in the step S1, and specifically: fully emulsifying the DOX-KLH immunogen and an isovolumetric Freund's complete adjuvant during primary immunization, and injecting the DOX-KLH immunogen and the isovolumetric Freund's incomplete adjuvant into a female BALB/c mouse body of 6-8 weeks old at multiple points in a subcutaneous manner, wherein the dosage of the DOX-KLH immunogen is 200 mu g/mouse, and the dosage of the DOX-KLH immunogen and the isovolumetric Freund's complete adjuvant are fully emulsified and injected into the abdominal cavity of the BALB/c mouse every 2 weeks, and the dosage of the immunogen is 200 mu g/mouse;

on day 7 after the fourth immunization, BALB/c mice were subjected to venous blood collection, and serum titer was measured, and 3 days before cell fusion, serum titer of 1.0X 10 was selected⁵The BALB/c mice are subjected to spleen boosting immunization by DOX-KLH immunogen, and the dosage of the DOX-KLH immunogen is 200 mu g/mouse;

s22, preparing DOX-KLH immune spleen cells, which specifically comprises the following steps: killing the BALB/c mouse which is boosted and immunized in the step S21, infiltrating the mouse with 75% alcohol, taking out spleen with disinfected scissors and tweezers, removing fat and connective tissues, washing with 75% alcohol, leaching with serum-free DMDM culture medium, placing on a screen, fully grinding with a rubber head of a sterile injector, flushing spleen cells into a sterile centrifuge tube through the serum-free IMDM culture medium, centrifuging at 1500rpm for 3min, finally, re-suspending with the serum-free IMDM culture medium, counting, and adjusting the cell concentration to 2 x 10⁸Per mL as DOX-KLH immune spleen cells;

s23, fully and uniformly mixing myeloma cells SP2/0 and DOX-KLH immune spleen cells prepared in the step S22 according to the volume ratio of 1:7, centrifuging at 1500rpm for 3min, removing supernatant, placing on a water bath at 37 ℃, slowly adding preheated 1mL of PEG-1500 within 1min, shaking while adding, and terminating the fusion action by preheated 20mL of serum-free IMDM culture medium;

centrifuging at 1500rpm for 3min, discarding supernatant, suspending the precipitate with HAT complete medium, adding into 96-well plate at 300 μ L/well, standing at 37 deg.C and 5% CO₂The cell culture box is used for carrying out cell culture;

s24, screening positive hybridoma cells and clones, specifically: using DOX as a coating antigen, diluting the DOX to 1 mu g/ml by using 0.05mol/L CB buffer solution with the pH value of 9.6, adding 100 mu L/hole of the diluted DOX into an enzyme label plate, coating the DOX at 4 ℃ overnight, then patting the DOX dry, sealing the DOX dry by using 1% gelatin-PBS buffer solution in mass volume ratio, sealing the DOX dry at 300 mu L/hole at 4 ℃ overnight, and then patting the DOX dry for later use;

taking the supernatant of the cell culture solution subjected to fusion culture in the step S23, adding an enzyme label plate in 100 mu L/hole, placing the cell culture solution at 37 ℃ for incubation for 60min, washing and patting the cell culture solution dry with 0.01mol/L PBST buffer solution containing Tween-20, adding 100 mu L/hole HRP-labeled goat-anti-mouse secondary antibody, placing the cell culture solution at 37 ℃ for incubation for 60min, washing and patting the cell culture solution dry, adding 100 mu L/hole TMB color development solution, placing the cell culture solution at 37 ℃ for dark color development for 10min, and adding 50 mu L/hole 1mol/L HCl to terminate the reaction;

9. The method for producing a DOX monoclonal antibody according to claim 6, wherein: the specific operation steps of step S3 are as follows: selecting BALB/c mice 8-10 weeks old, injecting 0.5ml liquid paraffin into the abdominal cavity, injecting 3 x 10 liquid paraffin into the abdominal cavity 7-14 days later⁵～4×10⁵Respectively extracting ascites by using an injector and marking after 7-10 days for the positive hybridoma cells prepared in the step S24; purifying with affinity chromatography Protein A Sepharose Fast Flow to obtain DOX monoclonal antibody, desalting with Sepharose S-200 molecular sieve, and determining the purity of the monoclonal antibody with SDS-PAGE (purity of 90%The above.

10. The method for preparing DOX monoclonal antibodies according to claim 7, wherein the specific operation steps of step S12 are as follows: putting a 100kD trapping column into an EP tube, adding 500 mu L of the KLH solution obtained in the step S11 into the trapping column, centrifuging for 10min at 9500rpm in a centrifuge precooled at 4 ℃, discarding waste liquid in the EP tube, adding 300 mu L of 50mM MES buffer solution with the pH value of 6.5 into the trapping column, centrifuging again, and repeating for 5-6 times;

placing the interception column into a new EP tube, centrifuging at 3000rpm for 3min, washing the interception column with 200 μ L50 mM MES buffer solution with pH of 6.5, standing for 5min, placing the interception column into the EP tube with KLH, and centrifuging at 3000rpm for 3 min;

the specific operation steps of step S14 are as follows: placing a 100kD trapping column into an EP tube, adding an activated KLH solution into the trapping column, centrifuging for 10min at 9500rpm in a 4-DEG C precooled centrifuge, discarding the waste liquid in the EP tube, adding 300 mu L of 10mmol/L PBS buffer solution with the pH value of 7.4 into the trapping column, centrifuging again, and repeating for 5-6 times;