Monoclonal antibody against prolactin and its preparation method and application

Abstract

The application discloses a kind of anti-prolactin protein monoclonal antibody and its preparation method and application, comprising: C58A / C174A double mutation and PEG epitope shielding are carried out to prolactin to obtain immunogen;Pre-injection chemical cross-linking macroprolactin induces mouse immunological tolerance;Using hierarchical immunization strategy, PEG modification and unmodified prolactin mutant are combined with different adjuvants to immunize mouse in turn;High specificity hybridoma is obtained by three-channel parallel negative screening;The variable region of screening antibody is transplanted to the human IgG3 skeleton of CH2 domain, and recombination antibody is obtained.The antibody prepared by the application does not recognize macroprolactin, and can effectively exclude the interference of rheumatoid factor and heterophilic antibody due to the deletion of Fc segment CH2 domain.The prolactin detection kit established based on the antibody has a sensitivity of 0.38 ng / mL, a cross-reaction rate with macroprolactin is less than 0.1%, a signal fluctuation under RF interference is less than 3%, and a batch variation coefficient is less than 6.8%, to provide a detection means with high accuracy and high reliability for hyperprolactinemia diagnosis.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and immunoassay, and in particular to a monoclonal antibody against prolactin, its preparation method, and its application. Background Technology

[0002] Prolactin (PRL) is a single-chain polypeptide hormone secreted by the lactotroph cells of the anterior pituitary gland, with a molecular weight of approximately 23 kDa. In the human body, it participates in various physiological processes, including mammary gland development, lactation initiation, immune regulation, stress response, and maintenance of reproductive function. Abnormally elevated serum PRL levels (hyperprolactinemia) are a common endocrine disorder in clinical practice. Its causes include pituitary prolactinoma, drug effects, and hypothalamic-pituitary axis dysfunction. Long-term hyperprolactinemia can lead to menstrual disorders, infertility, and galactorrhea in women, and decreased sexual function and infertility in men, severely reducing patients' quality of life. Therefore, accurate quantitative detection of serum PRL is of significant clinical value for differentiating the causes of hyperprolactinemia, assisting in the diagnosis of pituitary tumors, and monitoring treatment efficacy.

[0003] Currently, PRL detection mainly employs immunological methods, including chemiluminescent immunoassay, enzyme-linked immunosorbent assay (ELISA), and radioimmunoassay. The core raw material for these methods is anti-PRL monoclonal antibody. However, commercially available anti-PRL monoclonal antibodies generally suffer from the following technical defects: First, insufficient specificity, easily cross-reacting with macroprolactin (PRL dimer or multimer). Macroprolactin is a molecular weight variant of PRL (>100 kDa), with low immunogenicity but low biological activity. Traditional antibodies cannot distinguish between monomeric PRL and macroprolactin, leading to falsely elevated test results, causing misdiagnosis and unnecessary treatment. Second, susceptible to interference from heterophilic antibodies. Heterophilic antibodies are human anti-animal antibodies, widely present in the serum of some patients, and can bridge capture antibodies and detection antibodies, generating non-specific signals. Third, easily binding to rheumatoid factor (RF). RF recognizes the Fc fragment of IgG antibodies and is highly expressed in the serum of patients with autoimmune diseases such as rheumatoid arthritis. It can directly bind to detection antibodies or capture antibodies, interfering with test results. The aforementioned interfering factors lead to a high false positive rate and insufficient diagnostic specificity in existing PRL immunoassay reagents.

[0004] To address the aforementioned issues, there is an urgent need to develop a highly specific and interference-resistant anti-PRL monoclonal antibody and its detection system. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against prolactin, its preparation method, and its application, in order to solve at least some of the problems existing in the prior art.

[0006] Technical solution:

[0007] Firstly, this scheme provides a method for preparing a monoclonal antibody against prolactin, comprising the following steps:

[0008] Preparation of immunogen: Human prolactin protein was subjected to C58A / C174A double mutation, and polyethylene glycol was coupled to its surface lysine residues for epitope shielding modification to obtain PEG-modified prolactin mutants; the polyethylene glycol was maleimide-activated mPEG-MAL with a molecular weight of 5 kDa, the molar ratio of prolactin mutants to mPEG-MAL was 1:5, and each prolactin mutant molecule was coupled with an average of 3-4 polyethylene glycol chains;

[0009] Induction of immune tolerance: Mice were injected with chemically cross-linked macroprolactin to induce immune tolerance against the macroprolactin shared epitope; the chemically cross-linked macroprolactin was a mixture of human prolactin cross-linked into dimers and trimers using glutaraldehyde; the injection dose for inducing immune tolerance was 500 μg per mouse, administered via tail vein injection, 7 days before the first immunization;

[0010] Graded immunization: First, the prolactin mutant modified by PEG obtained in step (1) was used for primary and secondary immunization in combination with CpG ODN and QS-21 adjuvant. Then, the unmodified prolactin mutant was used for booster immunization in combination with aluminum adjuvant. Finally, the unmodified prolactin mutant was used for shock immunization.

[0011] Cell fusion and screening: Spleen cells from immunized mice were fused with myeloma cells. Hybridoma cells were screened using a three-channel parallel negative screening ELISA system. Channel A was coated with human prolactin monomer as a positive selector, channel B was coated with macroprolactin as a negative selector, and channel C was coated with human FcγRIIa protein and human IgG1 Fc fragment as a negative selector. Hybridoma cells that were positive in channel A and negative in both channels B and C were selected for cloning to obtain hybridoma cell lines that secrete anti-prolactin monoclonal antibodies.

[0012] (5) Antibody engineering: The antibody heavy chain variable region and light chain variable region genes were amplified from the hybridoma cell line obtained in step (4), and they were respectively linked to the human IgG3 hinge region-CH3 domain expression vector that lacks the CH2 domain. The vector was then transfected into host cells for expression to obtain a recombinant monoclonal antibody with the Fc segment lacking the CH2 domain.

[0013] Preferably, the specific procedure for graded immunization in step (3) is as follows: On day 1, PEG-modified prolactin mutant-CpG ODN-QS-21 complex is injected subcutaneously at multiple points, with a dose of 50 μg / animal based on prolactin; on day 14, the same complex is injected intraperitoneally at 50 μg / animal; on day 28, a mixture of unmodified prolactin mutant and aluminum adjuvant is injected intraperitoneally at 50 μg / animal; on day 35, unmodified prolactin mutant at 25 μg / animal is injected via tail vein without adjuvant.

[0014] Preferably, the conditions for the three-channel parallel negative screening ELISA system in step (4) are as follows: channel A is coated with human prolactin monomer at 2 μg / mL, channel B is coated with macroprolactin at 5 μg / mL, and channel C is coated with human FcγRIIa protein and human IgG1 Fc fragment at 2.5 μg / mL each; the hybridoma supernatant is added to the three channels simultaneously, channel A is detected by HRP-goat anti-mouse IgG, and a colorimetric A450 value greater than 1.0 is considered positive, while the A450 values ​​of channels B and C are both less than 0.2 and are considered negative.

[0015] Preferably, in the human IgG3 hinge region-CH3 domain expression vector with the CH2 domain missing described in step (5), the antibody heavy chain is composed of mouse VH and human hinge region-CH3 domain, and the CH2 exon is completely missing; the antibody light chain is composed of mouse VL and human κ chain constant region; the expression vector is transfected into CHO-S cells and purified using Protein L.

[0016] Secondly, this scheme also provides an anti-prolactin monoclonal antibody obtained according to the preparation method described in the first aspect, wherein the heavy chain constant region of the antibody lacks the CH2 domain, the molecular weight is 120±10 kDa, it does not bind to Fcγ receptor, C1q and rheumatoid factor, and is not recognized by heterophilic antibodies; the anti-prolactin monoclonal antibody can also be secreted by the hybridoma cell line described in the third aspect.

[0017] Thirdly, this scheme also provides a hybridoma cell line obtained by screening in step (4) of the first aspect, wherein the hybridoma cell line is selected from at least one of 1B7, 3E4 or 2H9.

[0018] Fourthly, this solution also provides a prolactin detection kit, comprising the monoclonal antibody described in the second aspect, wherein the monoclonal antibody comprises a pair of paired antibodies, one of which serves as a capture antibody and the other as a detection antibody; the paired antibody pair is: the capture antibody is antibody 1B7 or its recombinant form that recognizes the 10-25th amino acid epitopes of prolactin, and the detection antibody is antibody 2H9 or its recombinant form that recognizes the 175-190th amino acid epitopes of prolactin; or the two can be interchanged.

[0019] Preferably, the kit is a double-antibody sandwich ELISA kit, wherein the capture antibody is coated on a solid-phase carrier at 5 μg / mL, and the antibody-labeled horseradish peroxidase is detected. The kit also includes blocking buffer, standards, washing buffer and colorimetric solution, wherein the blocking buffer contains 5% BSA, 2% normal sheep serum and 1% mouse IgG.

[0020] Fifthly, the application of the preparation method according to the first aspect in the preparation of prolactin detection reagents is also provided.

[0021] Beneficial effects: This invention, through epitope shielding immunity, immune tolerance induction, and Fc domain deletion engineering, successfully prepared a highly specific anti-prolactin monoclonal antibody that can effectively eliminate interference from macroprolactin, heterophile antibodies, and rheumatoid factor, thereby improving the accuracy and reliability of prolactin immunoassay. Attached Figure Description

[0022] Figure 1 This is a flowchart of the overall preparation method of the present invention. Detailed Implementation

[0023] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0024] Example 1: Preparation and performance verification of anti-prolactin monoclonal antibody

[0025] 1. Materials and Reagents

[0026] Human prolactin (PRL) recombinant protein (purchased from R&D Systems, purity >95%), glutaraldehyde, mPEG-MAL (molecular weight 5 kDa), CpG ODN 1826 (synthetic), QS-21, aluminum adjuvant, BALB / c mice (6-8 weeks old, female), SP2 / 0 myeloma cells, HAT / HT selection medium, HRP-goat anti-mouse IgG, human FcγRIIa recombinant protein, human IgG1 Fc fragment (self-made), Protein L purified packing material, CHO-S cells, pFUSEd-ΔCH2 expression vector.

[0027] 2. Preparation of immunogens

[0028] 2.1 Expression and purification of PRL mutants

[0029] The cDNA encoding human PRL(1-199) was site-directed to introduce C58A and C174A mutations, and Strep-tag II was fused to the N-terminus. The resulting clone was then cloned into the pET-28a vector and transformed into E. coli BL21(DE3). After IPTG induction, Strep-Tactin was purified by affinity chromatography. SDS-PAGE showed a single band with a molecular weight of approximately 23 kDa and a purity >95%.

[0030] 2.2 PEG Epitope Shielding Modification

[0031] The purified PRL mutant (1 mg / mL) was dissolved in PBS (pH 7.2), and mPEG-MAL (5 mg / mL) was added to a final molar ratio of 1:5. The reaction was carried out at room temperature in the dark for 2 hours. The reaction solution was concentrated by ultrafiltration (10 kDa cutoff) to remove free PEG. MALDI-TOF mass spectrometry showed that each PRL molecule was coupled with 3.5 ± 0.5 PEG chains.

[0032] 2.3 Preparation of macroprolactin

[0033] Natural PRL (1 mg / mL) was incubated with 0.05% glutaraldehyde at room temperature for 2 hours. The reaction was terminated by adding glycine. The fractions with molecular weights of 45-70 kDa (a mixture of dimers and trimers) were collected by Superdex 200 gel filtration chromatography, and the protein concentration was determined by the BCA method.

[0034] 3. Induction of immunity and tolerance in mice

[0035] Ten BALB / c mice were selected and divided into an experimental group (n=8) and a control group (n=2). Seven days before immunization, the experimental group received a tail vein injection of 500 μg / mouse of macroprolactin (dissolved in 200 μL PBS); the control group received an equal volume of PBS. The immunization schedule is as follows:

[0036] Day 1: PEG-modified PRL mutant (50 μg) + CpG ODN (10 μg) + QS-21 (5 μg), mixed and then PBS was added to 100 μL, and the mixture was injected subcutaneously at multiple sites.

[0037] Day 14: Same as Day 1, but changed to intraperitoneal injection;

[0038] Day 28: Unmodified PRL mutant (50 μg) + aluminum adjuvant (50 μL), intraperitoneal injection;

[0039] Day 35: Unmodified PRL mutant (25 μg, dissolved in 100 μL PBS), administered via tail vein injection.

[0040] On day 38, blood was collected from the orbital rim to measure serum titers. ELISA (coated with natural PRL monomer) showed that the titers in the experimental group were all >1:100,000, while the titers in the control group were approximately 1:50,000.

[0041] 4. Cell fusion and three-channel negative screening

[0042] 4.1 Integration

[0043] Spleen cells (1×10⁻⁶) were taken from the experimental group. 8 (number) and SP2 / 0 cells (2×10) 7 (1) were fused with PEG 1500 and seeded on 10 96-well plates. After HAT screening for 10 days, the medium was replaced with HT medium.

[0044] 4.2 Establishment of a Three-Channel Screening System

[0045] Channel A (positive screening): Coated with 2 μg / mL human PRL monomer, incubated overnight at 4°C;

[0046] Channel B (megaprolactin crossover): Coated with 5 μg / mL megaprolactin, incubated overnight at 4°C;

[0047] Channel C (heterophile antibody / RF cross): a mixture coated with human FcγRIIa (2.5 μg / mL) and human IgG1 Fc fragment (2.5 μg / mL).

[0048] Each channel is sealed with 5% BSA and then kept on standby.

[0049] 4.3 Screening

[0050] 100 μL of hybridoma supernatant was added to channels A, B, and C, and incubated at 37°C for 30 minutes. HRP-goat anti-mouse IgG (1:5000) was added to channel A, and A450 was measured after color development. A total of 960 wells were screened, yielding 138 positive wells with A450 > 1.0. Among these, 9 wells had A450 < 0.2 in channel B and A450 < 0.15 in channel C. Cells from these 9 positive wells were amplified and subjected to limiting dilution cloning. After each cloning, the three-channel screening method was used again, ultimately obtaining three stable antibody-secreting hybridomas, named 1B7, 3E4, and 2H9. After three months of continuous in vitro passage and liquid nitrogen cryopreservation and thawing, the antibody secretion capacity remained stable.

[0051] 5. Antibody pairing and affinity assay

[0052] 5.1 Epitope Competition Experiment

[0053] Biomembrane interferometry (BLI, ForteBio Octet RED96) was used for assays. PRL monomers were biotinylated and immobilized on an SA sensor, which then bound to 1B7, 3E4, and 2H9, respectively. After saturation, another antibody was added. Results showed that 1B7 and 2H9 recognized non-overlapping epitopes, while 3E4 partially overlapped with both. 1B7 and 2H9 were selected for further studies.

[0054] 5.2 Affinity Measurement

[0055] EC50 was determined by ELISA: 1B7 was serially diluted (0.01-100 ng / mL) for coating, and a fixed concentration of PRL (10 ng / mL) was added. The binding signal was then detected. The EC50 of 1B7 was 8.2 ng / mL, and the EC50 of 2H9 was 12.5 ng / mL, both meeting the requirement of ≤10-20 ng / mL.

[0056] 6. Antibody engineering (CH2 deletion in the Fc segment)

[0057] 6.1 Cloning of variable region genes

[0058] Total RNA was extracted from 1B7 and 2H9 hybridoma cells (Trizol method) and reverse transcribed into cDNA. The VH and VL genes were amplified using universal primers, and the PCR products were ligated into a T-vector and sequenced. In 1B7, VH represents the mouse IgG2a subtype, and VL represents the κ subtype; in 2H9, VH represents IgG1, and VL represents the κ subtype.

[0059] 6.2 Construction of expression vector

[0060] A human IgG3 hinge region-CH3 domain gene (completely deleting the CH2 exon) was synthesized and cloned into the pFUSE vector. The 1B7VH gene was ligated upstream of the human IgG3 hinge region-CH3 domain by overlap PCR, and the 1B7VL gene was ligated upstream of the human κ chain constant region (CL). The pFUSE-1B7H-ΔCH2 and pFUSE-1B7L vectors were constructed, respectively. The 2H9 expression vector was also constructed.

[0061] 6.3 Transfection and Expression

[0062] CHO-S cells were co-transfected with the 1B7 heavy and light chain vectors using the PEI transfection method (suspension culture). 24 hours post-transfection, puromycin (10 μg / mL) was added for selection. After propagation in a stabilization tank, cells were cultured for 7 days in serum-free medium (CD CHO, Gibco). The supernatant was collected and purified by Protein L affinity chromatography. SDS-PAGE showed that under reducing conditions, the heavy chain was approximately 40 kDa (the intact IgG heavy chain is approximately 55 kDa, reduced due to CH2 deletion), and the light chain was approximately 25 kDa; under non-reducing conditions, it was approximately 120 kDa. The purified antibody was named 1B7-ΔCH2. 2H9-ΔCH2 was prepared similarly.

[0063] 6.4 Antibody Function Validation

[0064] Detection of FcγR binding: ELISA plates were coated with human FcγRIIa, and 1B7-ΔCH2 or wild-type mouse IgG1 was added for binding detection. The results showed that wild-type IgG1 bound significantly (A450>1.0), while 1B7-ΔCH2 did not bind (A450<0.05).

[0065] Binding with rheumatoid factor: Collect RF-positive serum (200 IU / mL), coat an ELISA plate with 1B7-ΔCH2 or human IgG1 Fc fragment, add RF serum, and detect binding. No binding was observed with 1B7-ΔCH2, while binding with human IgG1 Fc was significant (A450>1.5).

[0066] Binding with heterophilic antibodies: Detection was performed using commercially available interfering serum (Scantibodies, HBR-3) containing heterophilic antibodies. 1B7-ΔCH2 and 2H9-ΔCH2 were added to the sandwich ELISA, and no significant increase in signal was observed (relative deviation <5%).

[0067] 7. Reagent kit construction and performance validation

[0068] 7.1 Kit Components

[0069] Coated plates: 1B7-ΔCH2 was dissolved in carbonate buffer (pH 9.6) at 5 μg / mL, coated overnight at 4°C, and blocked with 5% BSA + 2% normal sheep serum + 1% mouse IgG.

[0070] Detection antibody: 2H9-ΔCH2-HRP (modified sodium periodate method labeling, labeling molar ratio 1:4);

[0071] Standards: Recombinant human PRL monomer (sec-HPLC purity >98%, amino acid analysis determined), prepared at concentrations of 0, 0.5, 1, 2.5, 5, 10, 25, 50, 100, and 150 ng / mL;

[0072] Washing solution: PBS-T (0.05% Tween-20);

[0073] Developing solution: TMB;

[0074] Termination solution: 2M H2SO4.

[0075] 7.2 Methodological Validation

[0076] (1) Sensitivity and linear range

[0077] The zero standard was measured 20 times repeatedly, and the limit of detection was calculated as mean + 3SD, yielding a limit of detection of 0.38 ng / mL. The standard curve (four-parameter fitting) showed good linearity in the range of 0.5–120 ng / mL (R² = 0.998). A hook effect appeared beyond the upper end of this range (150 ng / mL), and a linear range of 0.5–100 ng / mL is recommended.

[0078] (2) Precision

[0079] Intra-batch variation: Three concentration samples (low (2 ng / mL), medium (20 ng / mL), and high (80 ng / mL)) were taken, and 20 wells of each sample were measured in the same batch. The CVs were 6.2%, 4.8%, and 4.5%, respectively.

[0080] Inter-batch variation: For three different batches of the kit, each batch was used to measure the three concentrations mentioned above in 10 wells, and the CVs were 7.5%, 5.9%, and 5.3%, respectively.

[0081] (3) Recovery rate

[0082] When known amounts of PRL standards (final concentrations of 2, 20, and 80 ng / mL) were added to human serum matrix, the recoveries were 96%, 105%, and 92% respectively, with an average of 97.7%, when the measured values ​​were divided by the theoretical values.

[0083] (4) Dilution linearity

[0084] High-value serum samples (measured PRL 128 ng / mL) were serially diluted 1:2, 1:4, 1:8, 1:16, and 1:32 using sample dilution buffer. The measured values, after dilution factor correction, were 132, 126, 124, 119, and 115 ng / mL, respectively. The correlation coefficient R with the theoretical value was... 2 =0.996.

[0085] (5) Specificity

[0086] Cross-reactivity test: hGH, hPL, and hTSH were measured at 100 ng / mL, and the signals were all below zero standard + 2 SD, with a cross-reactivity rate of <0.01%. Macroprolactin (100 ng / mL PRL equivalent) was measured, and the signal was equivalent to monomeric PRL at 0.08 ng / mL, with a cross-reactivity rate of <0.1%.

[0087] (6) Anti-interference capability

[0088] Heterophilic antibody interference: When HBR blocking agent (1:10) was added to the sample, the signal change was <5% compared with that without the blocking agent; while a commercially available imported kit showed a signal increase of 30-50% without the blocking agent.

[0089] RF interference: With the addition of RF-positive serum (200 IU / mL), the signal change of this kit is <3%; the signal of the control conventional antibody preparation (unmodified Fc) increases by >60%.

[0090] This embodiment successfully prepared anti-prolactin monoclonal antibodies (1B7-ΔCH2 and 2H9-ΔCH2) based on epitope shielding immunity, immune tolerance induction, and Fc domain deletion engineering, and constructed a double-antibody sandwich ELISA kit. The kit has a sensitivity of 0.38 ng / mL, a linear range of 0.5-120 ng / mL, a recovery rate of 92%-105%, an intra-assay CV ≤ 6.2%, an inter-assay CV ≤ 7.5%, and a cross-reactivity with macroprolactin < 0.1%. It also exhibits extremely low response to RF and heterophilic antibodies. All performance characteristics meet or exceed the original design specifications, providing a highly accurate diagnostic tool for clinical hyperprolactinemia.

[0091] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various equivalent transformations can be made to the technical solutions of the present invention, and these equivalent transformations all fall within the protection scope of the present invention.

Claims

1. A method for preparing a monoclonal antibody against prolactin, characterized in that, Includes the following steps: (1) Preparation of immunogen: Human prolactin protein was subjected to C58A / C174A double mutation, and polyethylene glycol was coupled to its surface lysine residues for epitope shielding modification to obtain PEG-modified prolactin mutant; the polyethylene glycol was maleimide activated mPEG-MAL with a molecular weight of 5 kDa, the molar ratio of prolactin mutant to mPEG-MAL was 1:5, and each prolactin mutant molecule was coupled with an average of 3-4 polyethylene glycol chains; (2) Induction of immune tolerance: mice were injected with chemically cross-linked macroprolactin to induce immune tolerance against the shared epitope of macroprolactin; the chemically cross-linked macroprolactin was a mixture of human prolactin cross-linked into dimers and trimers using glutaraldehyde, the injection dose for inducing immune tolerance was 500 μg per mouse, the injection method was tail vein injection, and the injection time was 7 days before the first immunization; (3) Graded immunization: First, the prolactin mutant modified by PEG obtained in step (1) was used in combination with CpG ODN and QS-21 adjuvant for primary and secondary immunization, then the unmodified prolactin mutant was used in combination with aluminum adjuvant for booster immunization, and finally the unmodified prolactin mutant was used for shock immunization. (4) Cell fusion and screening: Spleen cells of immunized mice were fused with myeloma cells, and hybridoma cells were screened by a three-channel parallel negative screening ELISA system. Channel A was coated with human prolactin monomer as a positive screening, channel B was coated with macroprolactin as a negative screening, and channel C was coated with human FcγRIIa protein and human IgG1 Fc fragment as a negative screening. Hybridoma cells that were positive in channel A and negative in channels B and C were selected for cloning to obtain hybridoma cell lines that secrete anti-prolactin monoclonal antibodies. (5) Antibody engineering: The antibody heavy chain variable region and light chain variable region genes were amplified from the hybridoma cell line obtained in step (4), and they were respectively linked to the human IgG3 hinge region-CH3 domain expression vector that lacks the CH2 domain. The vector was then transfected into host cells for expression to obtain a recombinant monoclonal antibody with the Fc segment lacking the CH2 domain.

2. The preparation method according to claim 1, characterized in that, The specific procedure for graded immunization in step (3) is as follows: On day 1, PEG-modified prolactin mutant-CpG ODN-QS-21 complex is injected subcutaneously at multiple points, with a dose of 50 μg / animal based on prolactin; on day 14, the same complex is injected intraperitoneally, with a dose of 50 μg / animal; on day 28, a mixture of unmodified prolactin mutant and aluminum adjuvant is injected intraperitoneally, with a dose of 50 μg / animal; on day 35, unmodified prolactin mutant is injected intravenously via the tail vein, with a dose of 25 μg / animal without adjuvant.

3. The preparation method according to claim 1, characterized in that, The conditions for the three-channel parallel negative screening ELISA system described in step (4) are as follows: channel A is coated with human prolactin monomer at 2 μg / mL, channel B is coated with macroprolactin at 5 μg / mL, and channel C is coated with human FcγRIIa protein and human IgG1 Fc fragment at 2.5 μg / mL each; the hybridoma supernatant is added to the three channels at the same time, channel A is detected by HRP-goat anti-mouse IgG, and a colorimetric A450 value greater than 1.0 is considered positive, while the A450 values ​​of channels B and C are both less than 0.2 and are considered negative.

4. The preparation method according to claim 1, characterized in that, In the human IgG3 hinge region-CH3 domain expression vector with the CH2 domain missing described in step (5), the antibody heavy chain is composed of mouse VH and human hinge region-CH3 domain, with the CH2 exon completely missing; the antibody light chain is composed of mouse VL and human κ chain constant region; the expression vector is transfected into CHO-S cells and purified using Protein L.

5. The anti-prolactin monoclonal antibody obtained by the preparation method according to claim 1 or 4, characterized in that, The antibody lacks the CH2 domain in its heavy chain constant region, has a molecular weight of 120±10 kDa, does not bind to Fcγ receptors, C1q, and rheumatoid factor, and is not recognized by heterophilic antibodies.

6. A hybridoma cell line, characterized in that, The hybridoma cell line obtained by screening in step (4) of claim 1 is selected from at least one of 1B7, 3E4 or 2H9.

7. An anti-prolactin monoclonal antibody, characterized in that, It is secreted by the hybridoma cell line of claim 6, or prepared by the recombinant expression method of step (5) of claim 1.

8. A prolactin detection kit, characterized in that, The monoclonal antibody comprising the monoclonal antibody of claim 5 or 7, wherein the monoclonal antibody comprises a pair of paired antibodies, wherein one is a capture antibody and the other is a detection antibody; wherein the paired antibody pair is: the capture antibody is antibody 1B7 or a recombinant form thereof that recognizes the 10-25th amino acid epitopes of prolactin, and the detection antibody is antibody 2H9 or a recombinant form thereof that recognizes the 175-190th amino acid epitopes of prolactin; or the two are interchangeable.

9. The prolactin detection kit according to claim 8, characterized in that, The kit is a double-antibody sandwich ELISA kit, wherein the capture antibody is coated on a solid-phase carrier at 5 μg / mL, and the antibody-labeled horseradish peroxidase is detected. The kit also includes blocking buffer, standards, washing buffer and colorimetric solution. The blocking buffer contains 5% BSA, 2% normal sheep serum and 1% mouse IgG.

10. The application of the preparation method according to any one of claims 1-4 in the preparation of prolactin detection reagents.

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