CN115838692A - Anti-human mutant erythropoietin mouse monoclonal antibody, application and human recombinant erythropoietin stimulant detection method - Google Patents

Abstract

The invention relates to the technical field of antigen-antibody proteins, in particular to an anti-human mutant erythropoietin mouse monoclonal antibody, application and a human recombinant erythropoietin stimulant detection method. The preservation number of the hybridoma cell strain is CGMCC No.45302, and the mouse monoclonal antibody secreted and expressed by the hybridoma cell strain can specifically recognize the human mutant erythropoietin p.Arg193AspfsTer28. The bidirectional immune purification combined protein immunoblotting technology established by the mouse monoclonal antibody can be used as an excitant detection method of recombinant erythropoietin in human urine samples, effectively distinguishes wild erythropoietin, mutant erythropoietin and recombinant erythropoietin in human urine samples, directly and accurately detects the recombinant erythropoietin in urine samples from various groups of people, eliminates false positive risks, simultaneously optimizes an excitant detection process, and makes up the defects of the existing world anti-excitant field on recombinant erythropoietin detection.

Description

Anti-human mutant erythropoietin mouse monoclonal antibody, application and human recombinant erythropoietin stimulant detection method

Technical Field

The invention relates to the technical field of antigen-antibody proteins, in particular to an anti-human mutant erythropoietin mouse monoclonal antibody, application and a human recombinant erythropoietin stimulant detection method.

Background

Human recombinant erythropoietin (rEPO) is an excitant with a high abuse rate for athletes in endurance projects because the rEPO can stimulate the generation of red blood cells so as to improve the oxygen carrying capacity of human bodies.

Recent studies have found that the human EPO gene polymorphism c.577del occurs in east Asia population at 0.5-1%, and this polymorphism produces mutant erythropoietin p.Arg193AspfsTer28 (VAR-EPO). The VAR-EPO contains all amino acid structures of wild erythropoietin (WT-EPO), and at the same time, the C-terminal is extended by 26 amino acids, so that the EPO monoclonal antibody which is commercially produced at present can recognize both WT-EPO and rEPO, and VAR-EPO.

The molecular weight of the VAR-EPO is increased by 3.1kDa compared with that of the WT-EPO, the VAR-EPO is close to that of the rEPO, the rEPO and the VAR-EPO cannot be distinguished by adopting the existing protein immunoblotting stimulant detection method, and the risk of reporting the false positive of the rEPO exists in the stimulant detection. For these reasons, although the world anti-excitant agency has revised the procedures and criteria for detection of EPO, no effective method for detecting agonists has been established for low doses of EPO from the population carrying the EPO gene polymorphism c.577del, thereby affecting the fairness and justice of agonist detection.

Disclosure of Invention

The invention aims to solve the technical problem of providing an anti-human mutant erythropoietin mouse monoclonal antibody, application and a human recombinant erythropoietin stimulant detection method.

The technical scheme for solving the technical problems is as follows: the invention provides a hybridoma cell strain for secreting an anti-human mutant erythropoietin mouse monoclonal antibody, the Latin literature name of the hybridoma cell strain is Mus muscu l us, the hybridoma cell strain is named as mouse hybridoma cells in a classified manner, the preservation number is CGMCC No.45302, the preservation unit is the China general microbiological culture Collection center, the address is No. 3 of Xilu No. 1 of Xingyang district, beijing City, and the preservation date is 2022, 8 months and 26 days.

The invention also provides an anti-human mutant erythropoietin mouse monoclonal antibody, which is produced by the hybridoma cell strain.

Furthermore, the human mutant type erythropoietin is human mutant type erythropoietin p.Arg193AspfsTer28, and the amino acid sequence of the human mutant type erythropoietin is shown as SEQ ID NO. 1.

The invention also provides a polypeptide antigen for preparing the mouse monoclonal antibody, and the amino acid sequence of the polypeptide antigen is shown in SEQ ID NO. 3.

The invention also provides application of the mouse monoclonal antibody in detection of the human recombinant erythropoietin stimulant.

The invention also provides a detection method of the human recombinant erythropoietin stimulant, and the mouse monoclonal antibody is adopted to detect the human recombinant erythropoietin stimulant in a sample to be detected.

Further, when the sample to be detected only contains the human mutant type erythropoietin and/or the wild type erythropoietin, the detection result is negative; and when the sample to be detected contains the human recombinant erythropoietin stimulant, the detection result is positive.

Further, the detection method is a bidirectional immune purification combined protein immunoblotting method, and comprises at least one round of reverse immune purification, forward immune purification and protein immunoblotting steps.

Further, each round of the reverse immune purification comprises the following steps: mixing a sample to be detected or the supernatant solution obtained in the previous round with the mouse monoclonal antibody, and then sequentially carrying out the steps of incubation, adding immunomagnetic beads, incubation and magnetic bead enrichment to obtain the reverse immunopurification supernatant solution in the current round.

The invention also provides application of the mouse monoclonal antibody in preparing a reagent for detecting the human EPO gene polymorphism c.577del.

The invention has the beneficial effects that:

(1) The anti-VAR-EPO mouse monoclonal antibody can specifically identify VAR-EPO, can specifically and efficiently combine trace VAR-EPO in urine samples, and does not identify WT-EPO and rEPO at the same time;

(2) The stimulant detection method of rEPO can distinguish WT-EPO, VAR-EPO and rEPO in human urine samples by adopting the bidirectional immune purification combined protein immunoblotting technology established by the antibody, directly and accurately detect rEPO in urine samples from various crowds, and eliminate false positive risks;

(3) The rEPO detection method provided by the invention optimizes the excitant detection process and makes up for the defects of the existing world anti-excitant field on rEPO detection.

(4) The anti-VAR-EPO mouse monoclonal antibody of the present invention can be used for preparing a detection kit of human EPO gene polymorphism c.577del, thereby being used for screening the gene polymorphism.

Drawings

FIG. 1 is a non-denaturing SDS-PAGE electrophoresis of an antibody according to the invention;

FIG. 2 is a SDS-PAGE electrophoresis of the denaturation of the antibody according to the invention;

FIG. 3 is an immunoblot image obtained by detecting each sample by using a bidirectional immunopurification combined protein immunoblot and a forward immunopurification combined protein immunoblot, respectively, in the present invention.

Detailed Description

The principles and features of this invention are described below in conjunction with specific embodiments and figures, the examples given are intended to illustrate the invention and are not intended to limit the scope of the invention.

The invention designs and synthesizes a peptide segment as an antigen based on the C-terminal differential amino acid chain of the VAR-EPO and the WT-EPO, obtains a hybridoma cell strain by adopting a hybridoma cell technology, and obtains the anti-VAR-EPO mouse monoclonal antibody by expression and purification. The antibody is the first mouse monoclonal antibody which specifically recognizes the VAR-EPO in the world, can specifically and efficiently combine with trace VAR-EPO in urine samples, and does not recognize WT-EPO and rEPO at the same time. The bidirectional immune purification combined protein immunoblotting method established by the antibody can distinguish WT-EPO, VAR-EPO and rEPO in human urine samples, directly and accurately detect rEPO in urine samples from various populations, eliminate false positive risks, optimize an excitant detection process and make up the defects of the world anti-excitant field on rEPO detection.

Specifically, the rEPO stimulant detection method adopts a bidirectional immune purification combined protein immunoblotting method, and comprises at least one round of reverse immune purification, forward immune purification and protein immunoblotting steps. The steps of each round of reverse immune purification are as follows: mixing a sample to be detected or the supernatant solution obtained in the previous round with the mouse monoclonal antibody, and then sequentially carrying out the steps of incubation, adding immunomagnetic beads, incubation and magnetic bead enrichment to obtain the reverse immunopurification supernatant solution in the current round.

The determination mode of the detection result is that when the sample to be detected only contains VAR-EPO and/or WT-EPO, the detection result is negative; when the sample to be detected contains rEPO, the detection result is positive.

Wherein, the ELISA plate eluent of the sample to be detected is used for detecting rEPO. When a trail or a band appears at the rEPO position above the WT-EPO band, the result of the detection is positive if the sample contains rEPO, and when no trail or band is detected at the rEPO position above the WT-EPO band, the result of the detection is negative if the sample does not contain rEPO.

For negative detection results, the specific genotype carried by the magnetic bead eluent can be further judged by combining with an electrophoresis strip of the magnetic bead eluent. When the magnetic bead eluent detects a strip at the position of VAR-EPO, the sample contains VAR-EPO coded by EPO gene polymorphism c.577del; when no electrophoretic band appeared, it was shown that VAR-EPO was not contained. Specific detection steps and judgment of detection results can refer to specific embodiments.

The human recombinant erythropoietin stimulant detection method provided by the invention detects rEPO in urine samples by adopting a two-way immune purification combined protein immunoblotting method, can detect trace VAR-EPO in urine, and the minimum detection limit of the method can reach 0.1pg/mL through experimental verification.

The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 preparation of antigen

Since VAR-EPO has 26 amino acids added to the C-terminal of WT-EPO based on the inclusion of all the amino acids and glycosyl structures of WT-EPO, in order to prepare monoclonal antibodies which only recognize VAR-EPO but not WT-EPO and rEPO, the invention designs and synthesizes polypeptide antigen based on the C-terminal differential amino acid chains of VAR-EPO and WT-EPO on the basis of fully considering the immune specificity.

The amino acid sequence of the VAR-EPO amino acid chain is shown in SEQ ID NO. 1; wherein the amino acid sequence of the C-terminal differential amino acid chain of the VAR-EPO and the WT-EPO is shown in SEQ ID NO. 2, and the amino acid sequence of the C-terminal differential amino acid chain of the VAR-EPO and the WT-EPO is shown in SEQ ID NO. 2: GDDDQVCPPGHIHHLPHQHCLCHTLPRHS.

The antigen polypeptide is designed and synthesized by Beijing Yiqiao Shenzhou, and the design process fully considers the factors of immunogenicity, specificity, hydrophilicity and hydrophobicity, surface accessibility, space conformation and the like. The amino acid sequence of the antigen is shown in SEQ ID NO. 3, and the amino acid sequence of SEQ ID NO. 3: CHTLPRHSGGGSHTLPRHS.

The synthesized antigen polypeptide is coupled with virus-like particles (VLP) and is prepared into antigen which can be used for animal immunization.

Example 2 preparation of anti-VAR-EPO murine monoclonal antibody hybridoma cell line and murine monoclonal antibody

The antigen prepared in example 1 was used to prepare a murine monoclonal antibody, and the specific experimental procedures were as follows:

(1) Animal immunization

Balb/c mice were immunized by injection with antigen. The immunization method is that the immunogen is mixed with an equal amount of adjuvant and injected into the abdomen of the mouse in a plurality of subcutaneous injection points, wherein the injection dosage of the immunizing antigen of each mouse is 50ug. After the first immunization, the second immunization was performed at 2-week intervals, and the third immunization was performed at 3-week intervals.

(2) Blood sampling and potency detection

After the three immunizations, blood was taken to determine the serum titer. The serum titer is respectively determined by rEPO and recombinant VAR-EPO, and the specific steps are as follows:

one week after the third immunization, 50-60ul of blood is taken through orbital venous plexus of mice, and after standing overnight at 4 ℃, upper serum is centrifugally separated for titer detection; taking a proper amount of protein to be detected, diluting the protein to 5ug/mL by using a coating buffer solution, adding 100 mu L of the protein into each hole of a 96-hole plate by using a single-channel pipettor, slightly beating the plate to uniformly mix the samples, sealing the samples by using a preservative film, and coating the samples at 4 ℃ overnight; washing the plate 1 time by using a washing solution according to 200 mu L/hole, and buckling and drying the ELISA plate; then sealing the enzyme label plate by sealing liquid according to 300 mu L/hole, and sealing for 1 hour at room temperature; washing the plate with washing solution at 300 μ L/well for 2 times, adding sample (adding sample diluted by gradient and sample diluent at 100 μ L/well), adding detection antibody at the same time, adding into 96-well plate at 100 μ L/well, and acting at room temperature for 2 hr; washing the plate with washing solution at a rate of 200 μ L/hole for 3 times, adding color developing solution at a rate of 200 μ L/hole, and standing at room temperature for 12min; adding stop solution into 50 mu L/hole to stop reaction; and (3) detecting by using a microplate reader: the measurement wavelength was 450nm.

After the determination, mice with high titer in serum reacting with the recombinant VAR-EPO and no reaction with rEPO were selected, and the mice were boosted once with the immunogen, and 3 days later, spleens were taken for fusion.

(3) Fusion and screening

Taking the immunized mouse spleen cells, mixing the immunized mouse spleen cells with SP2/0 myeloma cells according to the proportion of 1, and fusing by an electrofusion method to obtain hybridoma cells. Respectively adopting rEPO (1 mu g/mL) and recombinant VAR-EPO (1 mu g/mL &0.1 mu g/mL) as coating antigens, measuring cell supernatant by an ELISA method, selecting VAR-EPO high-low concentration positive and rEPO negative holes, and continuously cloning for 2-3 times by limiting dilution to obtain a hybridoma cell strain which stably secretes the mouse monoclonal antibody, wherein the number of the hybridoma cell strain is 22TJY2-MM07.

(4) Hybridoma cell culture and antibody purification

Washing the Protein A affinity chromatographic column with ultrapure water, and then balancing with a balance buffer solution; and (3) loading the hybridoma cell supernatant obtained by culturing into an affinity chromatographic column, and leaching with an equilibrium buffer solution after loading. Eluting with an elution buffer, collecting an elution peak, neutralizing with a Tris buffer, and desalting to PBS7.4 to obtain the purified antibody.

After purification, the antibody was verified by native SDS-PAGE, which is shown in FIG. 1, and native SDS-PAGE, which is shown in FIG. 2.

Example 3 detection of rEPO in urine samples

In this embodiment, a method combining bidirectional immune purification and western blotting is used for detection, and the specific detection steps are as follows:

(1) Sample ultrafiltration concentration

Taking 15mL of urine sample to be detected, adding 1.5mL of Tris-HCl buffer solution (3.75M, pH7.4), fully mixing and adjusting the pH value of the sample, centrifuging for 20 minutes, and centrifuging for 4000g. After centrifugation, the supernatant was poured into a 15mL 10kD ultrafiltration centrifuge tube (Millipore), concentrated by ultrafiltration to 200. Mu.L-500. Mu.L, and replaced with a PBS buffer system, and the concentrated sample was transferred to a 1.5mL low-adsorption centrifuge tube.

(2) Reverse immunopurification

To the concentrated sample, 1. Mu.g of the murine monoclonal antibody prepared in example 2 was added, mixed well, and incubated at room temperature for 1 hour. After the incubation, 50. Mu.L of washed Anti-Mouse IgG magnetic beads (Dynabeads M-280 sheet Anti-Mouse IgG, invitrogen) were added to the sample, mixed well and incubated at 4 ℃ for 90 minutes. After incubation, the sample is placed on a magnetic frame to enrich the magnetic beads.

The supernatant solution was recovered, and 1. Mu.g of the murine monoclonal antibody prepared in example 2 was added to the supernatant solution again, mixed well, and incubated at room temperature for 1 hour. After the incubation, 50. Mu.L of washed Anti-mouse IgG magnetic beads were added to the sample, mixed well and incubated overnight at 4 ℃.

After incubation is finished, the sample is placed on a magnetic frame to enrich magnetic beads, and the magnetic beads and the supernatant are respectively collected.

After washing the enriched magnetic beads 3 times, 25. Mu.L of SDS loading buffer was added and incubated at 95 ℃ for 5 minutes to elute EPO protein. After incubation, the EP tube was cooled, centrifuged briefly, and placed in a magnetic rack for sample loading.

It should be noted that, in the actual detection, the reverse immune purification can be performed in multiple rounds, and each round of reverse immune purification is performed by repeating the above experimental steps on the supernatant obtained from the previous reverse immune purification.

(3) Forward immunopurification

Transferring the supernatant recovered in the step (2) to a 0.5mL 30kD ultrafiltration centrifugal tube (Millipore), performing ultrafiltration concentration to about 20 mu L, and transferring to an ELISA plate (Stemcell) coated with an EPO antibody for incubation for 2 hours at 37 ℃. After the incubation was completed, the ELISA plate was washed, and incubated for 5 minutes at 95 ℃ with 20. Mu.L of SDS loading buffer to elute EPO protein. After incubation, the ELISA plate was cooled and ready for loading.

(4) Western blotting method

And (3) respectively carrying out double protein immunoblotting on the magnetic bead eluent in the step (2) and the ELISA plate eluent in the step (3).

SDS-PAGE gel electrophoresis was performed using 10% Bis-Tris albumin gel (Invitrogen) in combination with SDS MOPS buffer system at a constant voltage of 200V for 75 minutes. Proteins were transferred to 0.45 μm PVDF membrane (Millipore) using Towbin transfer buffer in conjunction with semi-dry transfer for 60 min at 1.0mA/cm 2. After transfer, the PVDF membrane was placed in 5% LFM/PBS and sealed for 60 minutes. After blocking, the PVDF membrane was rinsed with PBS, and 20mL of primary antibody solution diluted with 1% LFM/PBS (1. Mu.g/mL, MAB2871, R & D) was added and incubated overnight at 4 ℃.

After the first anti-incubation, the PVDF membrane is washed by PBS and washed for 3 times, a second western blotting is carried out by adopting a semi-dry transfer method, the EPO antibody combined on the first PVDF membrane is transferred to the second PVDF membrane, the transfer buffer system is 0.7% acetic acid solution, the transfer condition is 0.72mA/cm < 2 >, and the transfer time is 10 minutes. After transfer, the PVDF membrane was placed in 5% LFM/PBS and sealed for 60 minutes. After blocking, the PVDF membrane was rinsed with PBS and then 40mL of a Biotin-labeled Secondary Antibody solution (0.65. Mu.g/mL, goat anti-Mouse IgG (H + L) Secondary Antibody Biotin, invitrogen) diluted with 1% LFM/PBS was added and incubated at room temperature for 60 minutes. After the incubation was completed, the PVDF membrane was rinsed and washed 3 times with PBS, and 40mL of Streptavidin-labeled HRP solution (0.25. Mu.g/mL, streptavidin Protein HRP, thermo) diluted with 1% LFM/PBS was added and incubated at room temperature for 60 minutes. After the incubation was completed, the PVDF membrane was washed with PBS and washed 5 times, and then developed with HRP chemiluminescent substrate developing reagent (SuperSignal West Femto).

(5) Analysis of detection results

Detecting whether the sample contains VAR-EPO encoded by EPO gene polymorphism c.577del or not by the band of magnetic bead eluent; the sample can be further tested for the presence of rEPO by combining the bands of the ELISA plate eluate. The results of the same sample ELISA plate eluate and magnetic bead eluate were analyzed in combination, and the detection results are shown in table 1:

TABLE 1

In Table 1, a to g respectively show the following results:

a: c.577del which does not carry EPO gene polymorphism and is rEPO negative;

b: EPO-free polymorphism c.577del, EPO-positive;

c: c.577del which does not carry EPO gene polymorphism, and is EPO positive;

d: EPO gene polymorphism c.577del heterozygotes, rhepo negative;

e, EPO gene polymorphism c.577del homozygote, rEPO negative;

EPO gene polymorphism c.577del heterozygote, rEPO positive;

g: EPO gene polymorphism c.577del homozygote, rEPO positive.

Comparative example 1

And detecting each sample by adopting forward immune purification combined with protein immunoblotting to obtain an immunoblotting result.

The test methods of example 3 and comparative example 1 were used to test each sample to verify the efficacy of the method of detecting an rEPO agonist of the present invention.

Wherein, the samples to be detected are known, the electrophoresis band of each sample is shown in fig. 3, wherein, the electrophoresis band number and the detection method are shown in table 2:

TABLE 2

As can be seen from FIG. 3, in the case of blank urine samples 1 to 4 to which rEPO was not added, only WT-EPO bands and not rEPO bands were detected in the detection results of the eluate from the ELISA plates by both the forward immuno-purification and the double immuno-purification of the present invention (1 and 2) in the detection of the population carrying the non-EPO gene polymorphism c.577del. When detecting EPO gene polymorphism c.577del heterozygous carrier population (3 and 4), only forward immune purification is adopted, and both a WT-EPO band is detected in an ELISA plate eluent and a band is displayed above the band; however, the results of the ELISA plate eluents detected by the inventive double-directional immunopurification showed only WT-EPO band, and no upper band. It can be seen that when only the forward immunopurification is adopted, even if the urine sample is not added with rEPO, the VAR-EPO contained in the urine sample can cause the detection to be positive by the EPO gene polymorphism c.577del heterozygous carrier population, so that the detection result is inaccurate; the result of the bidirectional immune purification detection shows that rEPO is negative, which shows that the method can effectively distinguish VAR-EPO and rEPO generated by carrying EPO gene polymorphism c.577del, so that the detection result is accurate.

In the urine samples 5 to 8 to which rEPO was added, whether only forward immune purification or the two-way immune purification method of the present invention was adopted, the bands were detected above the WT-EPO band in both the urine samples of the non-EPO gene polymorphism c.577del carrying population and the urine samples of the EPO gene polymorphism c.577del heterozygous carrying population, indicating that rEPO can be efficiently detected by the detection method of the present invention.

For samples 2 'and 4', the magnetic bead eluate was detected by the double immuno-purification method of the present invention, and it was found that, in the case of the population carrying the non-EPO gene polymorphism c.577del, the method of the present invention did not detect a band, and in the case of the population carrying the EPO gene polymorphism c.577del, the method of the present invention detected a band, indicating that the detection method of the present invention can effectively detect VAR-EPO.

The experimental results show that the anti-VAR-EPO mouse monoclonal antibody has high specificity and good affinity for VAR-EPO, and the bidirectional immune purification combined with the Western immunoblotting can effectively distinguish WT-EPO, VAR-EPO and rEPO, so that the samples from EPO gene polymorphism c.577del carrying population can be prevented from being misjudged as containing rEPO. Meanwhile, for EPO gene polymorphism c.577del carrying people, if rEPO is contained in urine sample, rEPO can also be detected, and omission can be effectively prevented.

Therefore, for the population with completely unknown genotype, no matter what genotype the population carries, the mouse monoclonal antibody and the bidirectional immune purification method can effectively judge whether the urine sample contains rEPO at one time, follow-up investigation is not needed, and the detection efficiency and accuracy are effectively improved.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (10)

1. A hybridoma cell strain for secreting an anti-human mutant erythropoietin mouse monoclonal antibody is characterized in that the Latin literature name of the hybridoma cell strain is Mus musculus, the preservation number is CGMCC No.45302, and the preservation unit is China general microbiological culture Collection center.

2. An anti-human mutant erythropoietin murine monoclonal antibody produced by the hybridoma cell line of claim 1.

3. The anti-human mutant erythropoietin murine monoclonal antibody according to claim 2, wherein the human mutant erythropoietin is human mutant erythropoietin p.arg193aspfster28, and the amino acid sequence thereof is shown as SEQ ID NO. 1.

4. A polypeptide antigen for use in the preparation of a murine monoclonal antibody according to claim 2 or 3, wherein the amino-selected sequence of said polypeptide antigen is as shown in SEQ ID No. 3.

5. Use of the murine monoclonal antibody of claim 2 or 3 in the detection of human recombinant erythropoietin agonist.

6. A method for detecting a human recombinant erythropoietin agonist, which comprises detecting the human recombinant erythropoietin agonist in a sample to be tested by using the murine monoclonal antibody according to claim 2 or 3.

7. The method of claim 6, wherein when the sample to be tested contains only human mutant erythropoietin and/or wild-type erythropoietin, the test result is negative; and when the sample to be detected contains the human recombinant erythropoietin stimulant, the detection result is positive.

8. The method of claim 6, wherein the method is a combination of two-way immunopurification and western blotting, and comprises at least one of reverse immunopurification, forward immunopurification, and western blotting.

9. The method of claim 8, wherein the steps of reverse immunopurification in each round are: mixing a sample to be detected or the supernatant solution obtained in the previous round with the mouse monoclonal antibody, and then sequentially carrying out the steps of incubation, adding immunomagnetic beads, incubation and magnetic bead enrichment to obtain the reverse immunopurification supernatant solution in the current round.

10. Use of a murine monoclonal antibody according to claim 2 or 3 for the preparation of a reagent for the detection of the human EPO gene polymorphism c.577del.

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