WO2020253187A1 - 瘦素免疫原、杂交瘤细胞、单克隆抗体、多克隆抗体及应用 - Google Patents

瘦素免疫原、杂交瘤细胞、单克隆抗体、多克隆抗体及应用 Download PDF

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WO2020253187A1
WO2020253187A1 PCT/CN2019/127876 CN2019127876W WO2020253187A1 WO 2020253187 A1 WO2020253187 A1 WO 2020253187A1 CN 2019127876 W CN2019127876 W CN 2019127876W WO 2020253187 A1 WO2020253187 A1 WO 2020253187A1
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leptin
immunogen
monoclonal antibody
antibody
detection
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PCT/CN2019/127876
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English (en)
French (fr)
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周密
林宇剑
钱纯亘
胡鹍辉
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深圳市亚辉龙生物科技股份有限公司
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Priority to EP19933626.4A priority Critical patent/EP3988564A4/en
Priority to US17/424,484 priority patent/US20220144912A1/en
Publication of WO2020253187A1 publication Critical patent/WO2020253187A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/26—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against hormones ; against hormone releasing or inhibiting factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the invention relates to a leptin immunogen, hybridoma cells, monoclonal antibodies, polyclonal antibodies and applications.
  • Leptin is a single-chain protein with 167 amino acid residues encoded by the human ob gene. After it is secreted from adipocytes into plasma, the 21 amino acid residues at the amino terminus are removed under the action of signal peptidase. Therefore, the total length of leptin present in plasma is 146 amino acid residues and the molecular weight is about 16kDa. Leptin has good hydrophilicity. About 80% of leptin in plasma is bound to plasma proteins, and only a small part exists in free form.
  • Leptin exerts biological effects through specific receptors and corresponding signal transduction systems that are widely present in different tissues and organs such as the central nervous system, fat, heart, liver, lung, kidney, and pancreas, and participates in the metabolism of sugar, fat and energy. adjust.
  • leptin The level of leptin will be reduced accordingly, indicating that leptin may be used as an indicator for evaluating the condition of patients with malignant tumors and monitoring the efficacy before and after medication; in normal pregnancy, the level of serum leptin will gradually increase with the progress of pregnancy, and the level of leptin will also increase when hypertension occurs Therefore, leptin has certain clinical value in judging the condition of pregnancy-induced hypertension.
  • leptin is related to a variety of acute and chronic diseases. As people deepen their understanding of it, it can be better applied in clinical practice.
  • the detection method of leptin mainly depends on the specific binding ability of leptin antibody and leptin in the test sample. Therefore, the specificity and sensitivity of anti-leptin antibodies are very important for the detection of leptin content.
  • the anti-leptin monoclonal antibody prepared from the leptin immunogen has better specificity and higher sensitivity.
  • a hybridoma cell that secretes a monoclonal antibody against leptin with better specificity and higher sensitivity, a monoclonal antibody, and a preparation method and application thereof are also provided.
  • a leptin immunogen including a polypeptide whose amino acid sequence is shown in SEQ ID NO.1.
  • the above-mentioned leptin immunogen includes a polypeptide whose amino acid sequence is shown in SEQ ID NO. 1.
  • the anti-leptin monoclonal antibody prepared from the leptin immunogen can specifically recognize the amino acid at positions 41 to 60 of the amino terminal of leptin The residues have high affinity and sensitivity with leptin.
  • the leptin immunogen further includes a carrier protein, and the carrier protein is coupled to the polypeptide.
  • a method for preparing hybridoma cells that secrete anti-leptin monoclonal antibodies includes the following steps:
  • a hybridoma cell secreting an anti-leptin monoclonal antibody is prepared by the method for preparing a hybridoma cell secreting an anti-leptin monoclonal antibody.
  • An anti-leptin monoclonal antibody is secreted by the aforementioned hybridoma cells that secrete anti-leptin monoclonal antibodies.
  • a leptin immunogen including a polypeptide whose amino acid sequence is shown in SEQ ID NO.2.
  • the leptin immunogen further includes a carrier protein, and the carrier protein is coupled to the polypeptide.
  • a preparation method of anti-leptin polyclonal antibody characterized in that it comprises the following steps:
  • the animal is immunized with the above-mentioned leptin immunogen to obtain the polyclonal antibody against leptin.
  • An anti-leptin polyclonal antibody prepared by the above-mentioned preparation method of anti-leptin polyclonal antibody.
  • a leptin detection reagent includes at least one of the above-mentioned anti-leptin monoclonal antibody and the above-mentioned anti-leptin polyclonal antibody.
  • a leptin detection kit includes the above leptin detection reagent.
  • One embodiment of the present invention provides a leptin immunogen.
  • the anti-leptin monoclonal antibody secreted by hybridoma cells prepared using the leptin immunogen can specifically recognize the epitope at the amino terminus of leptin. With strong affinity and high sensitivity, it can be used to prepare leptin detection reagents, prepare leptin detection test papers, and prepare leptin detection kits.
  • the leptin immunogen includes a polypeptide whose amino acid sequence is as shown in SEQ ID NO.1.
  • the amino acid sequence shown in SEQ ID NO. 1 is: RINDISHTQSVSSKQKVTGL.
  • the above-mentioned leptin immunogen is designed for the amino terminus of leptin.
  • the amino acid sequence shown in SEQ ID NO. 1 is identical to the amino acid at positions 41 to 60 in the full-length amino acid sequence of human leptin, and is identical to the carboxyl group of leptin. The end distance is larger.
  • the antibody obtained after immunizing animals with the above-mentioned leptin immunogen including the polypeptide shown in SEQ ID NO.1 can specifically recognize the amino terminus of leptin with strong affinity, avoiding interference with other antibodies' recognition and binding of leptin The carboxyl end.
  • the leptin immunogen further includes a carrier protein, and the carrier protein is coupled to a polypeptide with an amino acid sequence as shown in SEQ ID NO.1.
  • the coupling of carrier protein and polypeptide as immunogen is beneficial to stimulate helper T cells and further induce B cell immune response.
  • the carrier protein is selected from one of hemocyanin (KLH), bovine serum albumin, chicken ovalbumin (OVA), rabbit serum albumin and fibrinogen.
  • the carrier protein is selected from one of hemocyanin and bovine serum albumin.
  • An embodiment of the present invention also provides a preparation method of hybridoma cells secreting anti-leptin monoclonal antibody, the preparation method is step S110 to step S150.
  • Step S110 Immunize the animal with an immunogen to obtain spleen cells of the immunized animal.
  • the immunogen is the leptin immunogen in any of the above embodiments. Further, the immunogen includes a polypeptide whose amino acid sequence is shown in SEQ ID NO. 1.
  • the immunogen was mixed with Freund's complete adjuvant and fully emulsified, then injected into the mouse body and continuously strengthened the immunization, and the blood was taken to test the serum titer, and the titer was selected to reach 10 5 to 10 6 From the spleen of the mouse, the spleen cells of the immunized animal are obtained.
  • the dose of the immunogen for the first immunization is 90 ⁇ g to 120 ⁇ g
  • the dose of the immunogen for the last immunization is 30 ⁇ g to 60 ⁇ g
  • the interval of immunization is 10 days to 21 days.
  • the dose of the immunogen for the first immunization is 100 ⁇ g to 110 ⁇ g
  • the dose of the immunogen for the last immunization is 40 ⁇ g to 50 ⁇ g
  • the interval of immunization is 14 to 21 days.
  • step S130 spleen cells are fused with myeloma cells, and then positive fusion cells are obtained by screening.
  • spleen cells and mouse myeloma cells are subjected to cell fusion, and then selectively cultured and screened to obtain positive fusion cells.
  • amino acid sequence shown in SEQ ID NO. 1 is used to screen positive fusion cells that secrete anti-leptin antibodies.
  • Step 150 The positive fusion cells are subcloned to obtain the hybridoma cells secreting the anti-leptin monoclonal antibody.
  • the limiting dilution method is used for subcloning to obtain hybridoma cells capable of stably secreting anti-leptin monoclonal antibodies.
  • the leptin immunogen of any of the above embodiments is used as the immunogen to immunize animals, and the obtained hybridoma cells can stably secrete anti-leptin monoclonal antibodies, and The monoclonal antibody can specifically recognize the epitope at the amino terminal of the leptin, and has high affinity and specificity for the leptin.
  • An embodiment of the present invention also provides a hybridoma cell secreting anti-leptin monoclonal antibody.
  • the hybridoma cell is prepared by the method for preparing the hybridoma cell secreting anti-leptin monoclonal antibody.
  • the anti-leptin monoclonal antibody secreted by the hybridoma cells was designated LEP-Ab-01.
  • LEP-Ab-01 can specifically recognize the surface antigen of the amino terminal of leptin, with high affinity and high specificity.
  • the present invention also provides an anti-leptin monoclonal antibody, which is secreted by hybridoma cells prepared by the above-mentioned method for preparing anti-leptin monoclonal antibody-secreting hybridoma cells.
  • the monoclonal antibody is LEP-Ab-01.
  • LEP-Ab-01 is the monoclonal antibody with the highest titer secreted by hybridoma cells prepared according to the above-mentioned method for preparing anti-leptin monoclonal antibody-secreting hybridoma cells.
  • the above-mentioned anti-leptin monoclonal antibody can specifically recognize the amino terminal of human leptin, has high affinity and specificity for human leptin, and the recognition site is far away from the carboxyl terminal of leptin to avoid interference
  • Other antibodies recognize and bind to the carboxy terminus of leptin. It can be widely used in the field of leptin detection, such as the preparation of detection reagents, detection test papers, detection kits, etc. for detecting leptin. Compared with traditional monoclonal antibodies, it has significant advantages in terms of specificity, sensitivity and detection rate.
  • the aforementioned anti-leptin monoclonal antibody can be used as a capture antibody.
  • the capture antibody is used to capture the antigen in the sample to be tested.
  • the aforementioned anti-leptin monoclonal antibody can also be used as a detection antibody.
  • the detection antibody is used to bind to the captured antigen for detection.
  • An embodiment of the present invention also provides another leptin immunogen.
  • the polyclonal antibody prepared by using the leptin immunogen can specifically recognize the epitope at the carboxyl end of leptin, with better specificity, higher sensitivity, and It is used to prepare leptin detection reagents, prepare leptin detection test papers, and prepare leptin detection kits.
  • the leptin immunogen includes a polypeptide with an amino acid sequence as shown in SEQ ID NO.2.
  • the amino acid sequence shown in SEQ ID NO. 2 is: SCHLPWASGLETLDSLGGVLEASGY.
  • the above-mentioned leptin immunogen including the polypeptide shown in SEQ ID NO. 2 is designed for the carboxyl end of leptin, and the amino acid sequence shown in SEQ ID NO. 2 is consistent with the 116 position of the human leptin full-length amino acid sequence.
  • the amino acid at position ⁇ 140 is the same, and the distance from the amino terminal of leptin is larger.
  • the antibody obtained by immunizing animals with the above-mentioned leptin immunogen including the polypeptide shown in SEQ ID NO. 2 can specifically recognize the carboxyl end of leptin with strong affinity, and avoid interference with other antibodies' recognition and binding of leptin Amino terminal.
  • the leptin immunogen also includes a carrier protein, and the carrier protein is coupled to a polypeptide whose amino acid sequence is as shown in SEQ ID NO.2.
  • the coupling of carrier protein and polypeptide as immunogen is beneficial to stimulate helper T cells and further induce B cell immune response.
  • the carrier protein is selected from one of hemocyanin (KLH), bovine serum albumin, chicken ovalbumin (OVA), rabbit serum albumin and fibrinogen.
  • the carrier protein is selected from one of hemocyanin and bovine serum albumin.
  • One embodiment of the present invention also provides a method for preparing an anti-leptin polyclonal antibody.
  • the preparation method includes step S210 to step 230.
  • Step S210 Immunize the animal with the immunogen to obtain the immunized animal.
  • the immunogen includes a polypeptide whose amino acid sequence is shown in SEQ ID NO.2. Further, the immunogen also includes a carrier protein coupled to a polypeptide whose amino acid sequence is as shown in SEQ ID NO.2.
  • rabbits are immunized with immunogen for multiple times to obtain immunized rabbits.
  • the dose of immunogen for each immunization is 1 mg to 2 mg, and the interval between immunizations is 10 to 14 days.
  • the dose of the immunogen for each immunization is 1 mg to 1.5 mg.
  • immune sites include, but are not limited to, under the skin of the back, under the skin of the abdomen, under the skin of the armpits, and under the skin of the limbs.
  • Step S230 Extract and purify the polyclonal antibody against leptin from the immunized animal.
  • polyclonal antibodies against leptin are separated and purified from the blood of immunized animals.
  • the method for preparing the above-mentioned anti-leptin polyclonal antibody uses a polypeptide including an amino acid sequence as shown in SEQ ID NO. 2 as an immunogen to obtain an anti-leptin polyclonal antibody.
  • One embodiment of the present invention also provides an anti-leptin polyclonal antibody, which is prepared by the above-mentioned anti-leptin polyclonal antibody preparation method. Further, the polyclonal antibody is LEP-Ab-02.
  • the above-mentioned polyclonal antibody can specifically recognize multiple epitopes of leptin, especially the epitope located at the carboxyl terminal of leptin, and has high specificity and affinity for natural leptin.
  • the polyclonal antibody can be applied in the field of leptin detection. For example, it can be used to prepare leptin detection reagents, prepare leptin detection test papers, and prepare leptin detection kits.
  • an embodiment of the present invention provides a leptin detection reagent.
  • the leptin detection reagent includes at least one of the aforementioned anti-leptin monoclonal antibody and the aforementioned anti-leptin polyclonal antibody. Further, the leptin detection reagent includes the aforementioned anti-leptin monoclonal antibody and the aforementioned anti-leptin polyclonal antibody. Furthermore, leptin detection reagents include LEP-Ab-01 and LEP-Ab-02.
  • one of the aforementioned anti-leptin monoclonal antibody and the aforementioned anti-leptin polyclonal antibody is coated with magnetic particles to form a capture antibody, then the anti-leptin monoclonal antibody and the aforementioned
  • the other of the polyclonal antibodies against leptin is labeled to form a detection antibody.
  • the methods commonly used in the art can be used for labeling, such as fluorescein labeling method, enzyme labeling method, biotinylation labeling method, and colloidal gold labeling method.
  • acridine ester is used as the label.
  • the chemiluminescence immunoassay system using acridinium ester has a wide linear range of 0.5ng/mL-100ng/mL, and compared with enzyme-labeled materials, acridinium ester is more cost-effective.
  • the leptin detection reagent includes at least one of the anti-leptin monoclonal antibody and the anti-leptin polyclonal antibody. It also has good specificity and sensitivity when detecting leptin.
  • An embodiment of the present invention also provides a leptin test paper.
  • the leptin detection test paper includes a solid phase carrier and a capture antibody coated on the solid phase carrier.
  • the capture antibody is selected from one of the aforementioned anti-leptin monoclonal antibodies and the aforementioned anti-leptin polyclonal antibodies.
  • the solid phase carrier is a cellulose membrane.
  • the leptin detection test paper further includes a detection antibody selected from one of the labeled anti-leptin monoclonal antibody and the labeled anti-leptin polyclonal antibody.
  • the capture antibody is the above-mentioned anti-leptin polyclonal antibody
  • the detection antibody is the above-mentioned labeled anti-leptin monoclonal antibody.
  • the leptin detection test paper includes at least one of the anti-leptin monoclonal antibody and the anti-leptin polyclonal antibody, and also has good specificity and sensitivity when detecting leptin.
  • An embodiment of the present invention also provides a leptin detection kit, which includes the above-mentioned leptin detection test paper or the above-mentioned leptin detection reagent.
  • the leptin detection kit further includes other detection reagents.
  • Other test reagents can be set according to test needs. For example, leptin standards, buffers.
  • the leptin detection kit includes a detection reagent or test paper containing at least one of the anti-leptin monoclonal antibody and the anti-leptin polyclonal antibody. Therefore, it has good specificity and sensitivity when detecting leptin.
  • the leptin detection kit compared with the plate-type magnetic microparticle chemiluminescence analysis kit that selects FITC as the labeling material, the leptin detection kit has good repeatability and stable results. Compared with the latex enhanced immune turbidity kit using surface functional groups, the leptin detection kit has high sensitivity, high specificity, and is not easily affected by blood lipid concentration.
  • the immunogen 1 prepared in Example 1 and Freund's complete adjuvant were mixed in equal amounts and fully emulsified to immunize 3 BALB/c mice. Each BALB/c mouse was immunized three times. The injection volume of the first two immunogen 1 and Freund's complete adjuvant for each mouse was 100 ⁇ g, and the interval between the first two immunizations was 14 days. The third immunization was carried out three days before the cell fusion: 50 ⁇ g of immunogen 1 was mixed with an equal amount of Freund's incomplete adjuvant and fully emulsified to immunize mice. Then the immunized BALB/c mice were kept for three days to obtain immunized mice.
  • the coating antigen of the ELISA plate is a polypeptide whose amino acid sequence is shown in SEQ ID NO.1. Pick OD450>1.5 well cells as positive fusion cells.
  • step (3) The positive cell fusion cells obtained in step (3) were subcloned by the limiting dilution method and cloned three times to obtain 7 hybridoma cells capable of stably secreting anti-leptin monoclonal antibodies, numbered 1 respectively ⁇ 7. Among them, the monoclonal antibody secreted by the hybridoma cell number 1 is referred to as LEP-Ab-01.
  • the immunogen was diluted 10-fold, respectively, 102 times, 103 times, 104 times, 105 times, 106 times, using indirect ELISA titers, part of the detection results shown in Table 1 below.
  • Table 1 "1#” represents the group corresponding to the hybridoma cell number 1
  • “2#” represents the group corresponding to the hybridoma cell number 2, and so on.
  • ammonium sulfate precipitation and Protein A affinity chromatography purification step (5) were used to obtain ascites in each group. Specifically: the ascites obtained in step (5) were respectively subjected to ammonium sulfate precipitation: ammonium sulfate powder was added to the ascites of each group under stirring conditions, and a saturated solution of ammonium sulfate of each group was formed within 5-10 minutes.
  • the primary purified product was passed through the Protein A affinity chromatography column equilibrated with 1 ⁇ PBS, and then washed with 1 ⁇ PBS to remove the contaminant proteins not bound to the medium, and finally eluted with 0.1M glycine, and the eluate was collected and purified
  • the monoclonal antibodies of the following groups Identification by SDS-PAGE showed that the purity of the purified monoclonal antibodies in each group was above 98%.
  • Example 1 Animal immunization: The immunogen 2 prepared in Example 1 and Freund's complete adjuvant were mixed in equal amounts and fully emulsified, and then injected into 2 New Zealand white rabbits. The injection amount of immunogen 2 for each rabbit was 1 mg. After an interval of 14 days, the booster immunization was carried out: the immunogen 2 and the Freund's incomplete adjuvant were mixed in equal amounts and fully emulsified, and then the immunization injection was carried out. The injection amount of the immunogen 2 for each rabbit was 1 mg. Three booster immunizations were performed, and the immunogen 2 and Freund's incomplete adjuvant for each booster were 1 mg.
  • step (1) Use ammonium sulfate precipitation and Protein A affinity chromatography purification step (1) to obtain purified polyclonal antibodies. Specifically: Centrifuge the venous blood obtained in step (1) to obtain serum, and then subject the serum to ammonium sulfate precipitation: Add ammonium sulfate powder to the ascites of each group under stirring conditions, and form ammonium sulfate saturation within 5-10 minutes Solution. Continue stirring for 20 minutes, then centrifuge at 10000 ⁇ g for 10 minutes at 4°C, discard the supernatant, suspend the precipitate in 2 times the volume of the precipitate in 1 ⁇ PBS, and dialyze in 1 ⁇ PBS overnight to remove the ammonium sulfate to obtain the primary Purification.
  • the leptin standard (purchased from Abcam) was used as the antigen to be detected, the double antibody sandwich ELISA method was used, and the labeled monoclonal antibody secreted by the tumor cells prepared in Example 2 was used as the detection antibody, and the test antibody was prepared in Example 3.
  • the polyclonal antibody is a capture antibody to perform a pairing test. The pair of antibodies with the highest detection signal is used as the most suitable antibody pair, and the human serum with known leptin content is used for retesting and confirming.
  • LEP-Ab-01 recognizes the amino terminus of leptin
  • LEP-Ab-02 recognizes the carboxy terminus of leptin.
  • Nanomagnetic bead coating Take 50mg of carboxylated magnetic particles (1 ⁇ m in size) suspension, magnetically separate, leave the precipitate, then resuspend in 20mM, pH5.5 MES buffer, add 1mL of freshly prepared 10mg /mL EDC aqueous solution, activate the carboxyl groups on the surface of the magnetic beads, add 4 mg of LEP-Ab-02 prepared in Example 2, suspend at room temperature for 6 hours, magnetically separate, remove the supernatant, and use 100 mM pH 8.0 Tris containing 2% BSA Resuspend the buffer to 1 mg/mL to obtain coated magnetic particles, which are divided into 5 mL/bottle and stored at 4°C for later use.
  • leptin calibrator use buffer (40mM Tris-Cl, 0.5% BSA, 1% NaCl, pH 8.0) to prepare leptin standard at concentrations of 0ng/mL, 20ng/mL, 60ng /mL, each bottle of 0.5mL aliquoted and freeze-dried, stored at 4°C for later use.
  • the leptin determination kit prepared in Example 5 uses a chemiluminescence analyzer as a detection tool, and the double antibody sandwich method is used for performance evaluation. That is to say, the sample to be tested or the leptin standard and the magnetic particles coated with the anti-leptin polyclonal antibody (ie LEP-Ab-02) are reacted for 10 minutes, and then magnetic separation is performed. Then, an acridinium-labeled anti-leptin monoclonal antibody (ie, acridinium-labeled LEP-Ab-01) was added, and after reacting for 10 minutes, magnetic separation was performed.
  • acridinium-labeled anti-leptin monoclonal antibody ie, acridinium-labeled LEP-Ab-01
  • the sensitivity of the leptin detection kit of Example 5 is calculated to be 0.5 ng/mL.
  • the linear correlation coefficient r 0.9995. According to the experimental method recommended by CLSI EP17-A, it is calculated that the linear range of the leptin detection kit of Example 5 for the detection of leptin samples is: 0.5ng/ml-100ng/mL.
  • leptin detection kit is used for detection, and the intra-assay and inter-assay differences of the leptin detection kit are calculated.
  • the calculation results show that the intra-assay and inter-assay differences of the leptin detection kit of Example 5 are both less than 5%.
  • the interfering substances are bound bilirubin, free bilirubin, hemoglobin, ascorbic acid, cholesterol and glycerides, and the ratio of the mass of the interfering substances to the mass of the mixed serum is 1:20.
  • the leptin content of the mixed serum and the mixed serum after adding different interfering substances are respectively determined, and the deviation of the leptin content of the mixed serum and the mixed serum added with the interfering substance is calculated, and the acceptable range is ⁇ 10%.
  • the calculation results show that the interference of the leptin detection kit of Example 5 has reached the document standard of NCCLS, and can be used for accurate assessment of the leptin status in clinical laboratories.
  • Determination method Take the leptin calibrator, quality control product and the interfering experimental samples in Example 6.
  • the performance evaluation method is the same as that in Example 6.
  • the experimental operation was performed and the results were calculated according to the instructions of the kit, and the evaluation results of the leptin determination kit in Example 5 and Example 6 were compared.
  • a commercially available human leptin peptide fragment (amino acid residues 22 to 56, the specific amino acid sequence is shown in SEQ ID NO. 3, namely VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQK) was used as an immunogen to prepare a monoclonal antibody, denoted as C-Ab-10.
  • C-Ab-10 a monoclonal antibody
  • the method for preparing C-Ab-10 is roughly the same as the method for preparing LEP-Ab-01 in Example 2.
  • the immunogen of Comparative Example 2 uses commercially available human leptin peptide fragments (position 22 ⁇ Amino acid residue at position 56).
  • the leptin standard (purchased from Abcam) was used as the detected antigen, and the labeled C-Ab-10 and labeled LEP-Ab-01 were used as the detection antibodies.
  • the indirect ELISA method was used to compare the titers. The test results are as follows:
  • a commercially available murine leptin peptide fragment (116-130 amino acid residues, the specific sequence is shown in SEQ ID NO. 4, namely SCSLPQTSGLQKPES) was used as an immunogen to prepare a polyclonal antibody, denoted as LEPC-Ab-01.
  • LEPC-Ab-01 and LEP-Ab-02 as capture antibodies, and paired experiments with detection antibody LEP-Ab-01, using leptin standard and human serum containing high concentration of leptin as the detected antigen, respectively Double antibody sandwich ELISA method for testing, the testing results are as follows:

Abstract

提供一种瘦素免疫原、杂交瘤细胞、单克隆抗体、多克隆抗体及应用。该瘦素免疫原包括氨基酸序列如SEQ ID NO.1所示的多肽。由该瘦素免疫原制备得到的抗瘦素的单克隆抗体的特异性较好、灵敏度较高。

Description

瘦素免疫原、杂交瘤细胞、单克隆抗体、多克隆抗体及应用 技术领域
本发明涉及一种瘦素免疫原、杂交瘤细胞、单克隆抗体、多克隆抗体及应用。
背景技术
瘦素(Leptin)是人的ob基因编码的含有167个氨基酸残基的单链蛋白质，其由脂肪细胞分泌到血浆后，在信号肽酶的作用下除去氨基端的21个氨基酸残基。因此，血浆中存在的瘦素全长为146个氨基酸残基，分子量约为16kDa。瘦素具有较好的亲水性，在血浆中大约80％的瘦素与血浆蛋白质结合，只有少部分以游离形式存在。瘦素通过与广泛存在于中枢神经***、脂肪、心、肝、肺、肾、胰腺等不同组织和器官的特异性受体和相应的信号传导***发挥生物效应，参与糖、脂肪和能量代谢的调节。
研究发现，在很多生理和病理情况下，人的瘦素水平会发生变化，对许多能量代谢有关疾病的发生、发展、预后和用药疗效检测都具有非常重要的临床指导价值。例如，肥胖者的血清瘦素浓度均高于非肥胖者，因此体内瘦素水平对肥胖的预防和治疗具有一定积极意义；恶性肿瘤患者血清中瘦素水平显著高于正常对照，经治疗后瘦素水平会相应降低，表明瘦素可能作为评价恶性肿瘤患者病情和用药前后疗效监控的指标；在正常妊娠中血清瘦素水平会随妊娠进展逐渐上升，妊娠高血压发生时瘦素水平也呈上升趋势，因此，瘦素对判断妊娠高血压病情具有一定临床价值。总之，瘦素与多种急慢性疾病相关，随着人 们对其认识的加深，可以将其更好地应用于临床。
目前，瘦素的检测方法主要依赖于瘦素抗体与检测样本中的瘦素的特异性结合能力。因此，抗瘦素抗体的特异性和灵敏度对于瘦素含量的检测十分重要。
但是，目前市场上特异性较好、灵敏度较高的抗瘦素抗体还较少，不能满足市场的需求。
发明内容
基于此，有必要提供一种瘦素免疫原，由该瘦素免疫原制备得到的抗瘦素的单克隆抗体的特异性较好、灵敏度较高。
此外，还提供一种分泌特异性较好、灵敏度较高的抗瘦素的单克隆抗体的杂交瘤细胞、单克隆抗体及其制备方法和应用。
一种瘦素免疫原，包括氨基酸序列如SEQ ID NO.1所示的多肽。
上述瘦素免疫原包括氨基酸序列如SEQ ID NO.1所示的多肽，由该瘦素免疫原制备得到的抗瘦素的单克隆抗体，能够特异性识别瘦素氨基端的41位～60位氨基酸残基，与瘦素的亲和力和灵敏度较高。
在其中一个实施例中，所述瘦素免疫原还包括载体蛋白，所述载体蛋白与所述多肽偶联。
一种分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法，包括以下步骤：
用免疫原免疫动物，得到免疫后的动物的脾细胞，其中所述免疫原为上述瘦素免疫原；
将所述脾细胞与骨髓瘤细胞融合，然后筛选得到阳性融合细胞；及
对所述阳性融合细胞进行亚克隆，得到所述分泌瘦素单克隆抗体的杂交瘤细胞。
一种分泌抗瘦素单克隆抗体的杂交瘤细胞，由上述分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法制得。
一种抗瘦素的单克隆抗体，由上述分泌抗瘦素单克隆抗体的杂交瘤细胞分泌。
一种瘦素免疫原，包括氨基酸序列如SEQ ID NO.2所示的多肽。
在其中一个实施例中，所述瘦素免疫原还包括载体蛋白，所述载体蛋白与所述多肽偶联。
一种抗瘦素的多克隆抗体的制备方法，其特征在于，包括以下步骤：
用上述的瘦素免疫原免疫动物，得到所述抗瘦素的多克隆抗体。
一种抗瘦素的多克隆抗体，由上述抗瘦素的多克隆抗体的制备方法制得。
上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的至少一种在制备瘦素检测试剂、制备瘦素检测试纸或制备瘦素检测试剂盒中的应用。
一种瘦素检测试剂，包括上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的至少一种。
一种瘦素检测试剂盒，包括上述瘦素检测试剂。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使本发明公开内容更加透彻全面。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本发明一实施方式提供了一种瘦素免疫原，使用该瘦素免疫原制备的杂交瘤细胞分泌的抗瘦素的单克隆抗体能够特异性识别瘦素氨基端的抗原表位，并且与瘦素的亲和力强、灵敏度较高，能够应用于制备瘦素检测试剂、制备瘦素检测试纸、制备瘦素检测试剂盒。
具体地，该瘦素免疫原包括氨基酸序列如SEQ ID NO.1所示的多肽。如SEQ ID NO.1所示的氨基酸序列为：RINDISHTQSVSSKQKVTGL。上述瘦素免疫原针对瘦素的氨基端进行设计，如SEQ ID NO.1所示的氨基酸序列与人的瘦素全长氨基酸序列的41位～60位的氨基酸相同，且与瘦素的羧基端距离较大。以包括氨基酸序列如SEQ ID NO.1所示的多肽的上述瘦素免疫原免疫动物后得到的抗体，能够特异性地识别瘦素的氨基端且亲和力强，避免干扰其他抗体识别和结合瘦素的羧基端。
在其中一个实施例中，瘦素免疫原还包括载体蛋白，载体蛋白与氨基酸序列如SEQ ID NO.1所示的多肽偶联。载体蛋白与多肽偶联作为免疫原有利于刺激辅助性T细胞，进一步诱导B细胞免疫反应。具体地，载体蛋白选自血蓝蛋白(KLH)、牛血清白蛋白、鸡卵白蛋白(OVA)、兔血清白蛋白及纤维蛋白原中的一种。优选地，载体蛋白选自血蓝蛋白及牛血清白蛋白中的一种。
本发明一实施方式还提供了一种分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法，该制备方法步骤S110～步骤S150。
步骤S110、用免疫原免疫动物，得到免疫后的动物的脾细胞。
具体地，免疫原为上述任一实施例中的瘦素免疫原。进一步地，免疫原包括氨基酸序列如SEQ ID NO.1所示的多肽。
在其中一个实施例中，将免疫原与弗式完全佐剂混合并充分乳化后，注射入小鼠体内并不断加强免疫，取血检测血清效价，取效价达到10 5～10 6的小鼠的 脾脏，得到免疫后的动物的脾细胞。
具体地，首次免疫的免疫原的剂量为90μg～120μg，末次免疫的免疫原的剂量为30μg～60μg，免疫的间隔时间为10天～21天。进一步地，首次免疫的免疫原的剂量为100μg～110μg，末次免疫的免疫原的剂量为40μg～50μg，免疫的间隔时间为14天～21天。
步骤S130、将脾细胞与骨髓瘤细胞融合，然后筛选得到阳性融合细胞。
具体地，将脾细胞与小鼠骨髓瘤细胞进行细胞融合，然后经过选择性培养和筛选，得到阳性融合细胞。
具体地，利用SEQ ID NO.1所示的氨基酸序列筛选分泌抗瘦素抗体的阳性融合细胞。
步骤150、阳性融合细胞进行亚克隆，得到所述分泌抗瘦素单克隆抗体的杂交瘤细胞。
具体地，采用有限稀释法进行亚克隆，得到能够稳定分泌抗瘦素单克隆抗体的杂交瘤细胞。
上述分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法，采用上述任一实施例的瘦素免疫原作为免疫原免疫动物，得到的杂交瘤细胞能够稳定分泌抗瘦素的单克隆抗体，且该单克隆抗体能够特异性识别瘦素氨基端的表位，对瘦素的亲和力高、特异性高。
本发明一实施方式还提供了一种分泌抗瘦素单克隆抗体的杂交瘤细胞。该杂交瘤细胞由上述分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法制得。该杂交瘤细胞分泌出抗瘦素的单克隆抗体记为LEP-Ab-01。其中，LEP-Ab-01能够针对性地识别瘦素的氨基端的表面抗原，具有高亲和力、高特异性。
本发明还提供一种抗瘦素的单克隆抗体，该单克隆抗体由上述分泌抗瘦素 单克隆抗体的杂交瘤细胞的制备方法制备得到的杂交瘤细胞分泌。进一步地，该单克隆抗体为LEP-Ab-01。LEP-Ab-01是按照上述分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法制备得到的杂交瘤细胞分泌的效价最高的单克隆抗体。
上述抗瘦素的单克隆抗体能够特异性识别人的瘦素的氨基端，对人的瘦素的亲和力高、特异性高，且识别的位点与瘦素的羧基端距离较大，避免干扰其他抗体识别和结合瘦素的羧基端。可广泛应用于瘦素检测领域，如用于制备检测瘦素的检测试剂、检测试纸、检测试剂盒等。在特异性、灵敏度和检测率等方面较之传统的单克隆抗体具有显著的优势。
具体地，上述抗瘦素的单克隆抗体可以作为捕获抗体。捕获抗体用于捕获待测样品中的抗原。上述抗瘦素的单克隆抗体也可以作为检测抗体。检测抗体用于与被捕获的抗原结合，以便检测。
本发明一实施方式还提供了另一种瘦素免疫原，使用该瘦素免疫原制备得到的多克隆抗体能够特异性识别瘦素羧基端的抗原表位，特异性较好、灵敏度较高，能够应用于制备瘦素检测试剂、制备瘦素检测试纸、制备瘦素检测试剂盒。
具体地，该瘦素免疫原包括氨基酸序列如SEQ ID NO.2所示的多肽。如SEQ ID NO.2所示的氨基酸序列为：SCHLPWASGLETLDSLGGVLEASGY。
经研究发现，瘦素羧基端106位～120位、116位～130位、126位～140位等氨基酸残基具有明显的生理功能，具有明显活性。包括氨基酸序列如SEQ ID NO.2所示的多肽的上述瘦素免疫原针对瘦素的羧基端设计，如SEQ ID NO.2所示的氨基酸序列与人的瘦素全长氨基酸序列的116位～140位的氨基酸相同，并且与瘦素氨基端距离较大。以包括氨基酸序列如SEQ ID NO.2所示的多肽的上述瘦素免疫原免疫动物得到的抗体，能够特异性地识别瘦素的羧基端且亲和力 强，避免干扰其他抗体识别和结合瘦素的氨基端。
在其中一个实施例中，瘦素免疫原还包括载体蛋白，载体蛋白与氨基酸序列如SEQ ID NO.2所示的多肽偶联。载体蛋白与多肽偶联作为免疫原有利于刺激辅助性T细胞，进一步诱导B细胞免疫反应。具体地，载体蛋白选自血蓝蛋白(KLH)、牛血清白蛋白、鸡卵白蛋白(OVA)、兔血清白蛋白及纤维蛋白原中的一种。优选地，载体蛋白选自血蓝蛋白及牛血清白蛋白中的一种。
本发明一实施方式还提供了一种抗瘦素的多克隆抗体的制备方法，该制备方法包括步骤S210～步骤230。
步骤S210、用免疫原免疫动物，得到免疫后的动物。
具体地，该免疫原包括氨基酸序列如SEQ ID NO.2所示的多肽。进一步地，该免疫原还包括与氨基酸序列如SEQ ID NO.2所示的多肽偶联的载体蛋白。
在其中一个实施例中，用免疫原多次免疫兔，得到免疫后的兔。每次免疫时免疫原的剂量为为1mg～2mg，免疫的间隔时间为10天～14天。优选地，每次免疫时免疫原的剂量为为1mg～1.5mg。当然，免疫的部位包括但不限于背部皮下、腹部皮下、腋窝皮下、四肢皮下。
步骤S230、从免疫后的动物中提取并纯化抗瘦素的多克隆抗体。
具体地，从免疫后的动物的血液中分离和纯化抗瘦素的多克隆抗体。
上述抗瘦素的多克隆抗体的制备方法采用包括氨基酸序列如SEQ ID NO.2所示的多肽作为免疫原，获得抗瘦素的多克隆抗体。
本发明一实施方式还提供一种抗瘦素的多克隆抗体，该多克隆抗体由上述抗瘦素的多克隆抗体的制备方法制备得到。进一步地，该多克隆抗体为LEP-Ab-02。
上述多克隆抗体能够特异性识别瘦素的多个抗原表位，特别是能识别位于 瘦素的羧基端的抗原表位，对天然瘦素的特异性高、亲和力强。该多克隆抗体能够应用于瘦素检测领域。例如，能够用于制备瘦素检测试剂、制备瘦素检测试纸、制备瘦素检测试剂盒等。
上述抗瘦素的单克隆抗体及上述抗瘦素的多克隆抗体中的至少一种在制备瘦素检测试剂、制备瘦素检测试纸或制备瘦素检测试剂盒中的应用。
具体地，本发明一实施方式提供了一种瘦素检测试剂。该瘦素检测试剂包括上述抗瘦素的单克隆抗体及上述抗瘦素的多克隆抗体中的至少一种。进一步地，瘦素检测试剂包括上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体。更进一步地，瘦素检测试剂包括LEP-Ab-01和LEP-Ab-02。
在其中一个实施例中，上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的其中一种与磁微粒一起包被，形成捕获抗体，则抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的另一种被标记，形成检测抗体。当然，可以采用本领域常用的手段进行标记，例如荧光素标记法、酶标记法、生物素化标记法、胶体金标记法。在其中一个实施例中，选用吖啶酯作为标记。用吖啶酯的化学发光免疫分析***线性范围宽为0.5ng/mL-100ng/mL，并且与酶标材料相比，吖啶酯更节约成本。
上述瘦素检测试剂包括上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的至少一种。在检测瘦素时，也具有良好的特异性及灵敏度。
本发明一实施方式还提供了一种瘦素检测试纸。该瘦素检测试纸包括固相载体及包被在固相载体上的捕获抗体。该捕获抗体选自上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的一种。具体地，固相载体为纤维素膜。
进一步地，该瘦素检测试纸还包括检测抗体，该检测抗体选自被标记的上述抗瘦素的单克隆抗体和被标记的上述抗瘦素的多克隆抗体中的一种。更进一 步地，捕获抗体为上述抗瘦素的多克隆抗体，检测抗体为被标记的上述抗瘦素的单克隆抗体。将捕获抗体设为上述抗瘦素的多克隆抗体，能够提高上述该瘦素检测试纸的检测灵敏度和特异性。
上述瘦素检测试纸包括上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的至少一种，在检测瘦素时，也具有良好的特异性及灵敏度。
本发明一实施方式还提供了一种瘦素检测试剂盒，该包括上述瘦素检测试纸或上述瘦素检测试剂。
在其中一个实施例中，瘦素检测试剂盒还包括其他检测试剂。其他检测试剂可以根据检测需要设置。例如瘦素标准品、缓冲液。
上述瘦素检测试剂盒包括含有上述抗瘦素的单克隆抗体和上述抗瘦素的多克隆抗体中的至少一种的检测试剂或检测试纸。因此，在检测瘦素时，具有良好的特异性及灵敏度。另外，与选择FITC作为标记材料的板式磁性微粒子化学发光分析试剂盒相比，上述瘦素检测试剂盒的重复性好、结果稳定。与利用表面官能团的胶乳增强免疫比浊试剂盒相比，上述瘦素检测试剂盒的灵敏度高、特异性高、不易受血脂浓度影响。
具体实施例
以下结合具体实施例进行详细说明。实施例中采用药物和仪器如非特别说明，均为本领域常规选择。实施例中未注明具体条件的实验方法，按照常规条件，例如文献、书本中所述的条件或者生产厂家推荐的方法实现。
实施例1
免疫原的制备
(1)委托生工生物工程(上海)股份有限公司合成氨基酸序列如SEQ ID  NO.1所示的多肽和氨基酸序列如SEQ ID NO.2所示的多肽。如SEQ ID NO.1所示的氨基酸序列为：RINDISHTQSVSSKQKVTGL；如SEQ ID NO.2所示的氨基酸序列为：SCHLPWASGLETLDSLGGVLEASGY。
(2)通过蛋白偶联技术，将氨基酸序列如SEQ ID NO.1所示的多肽与牛血清白蛋白偶联，得到用于制备抗瘦素的单克隆抗体的免疫原，记作免疫原1。
(3)通过蛋白偶联技术，将氨基酸序列如SEQ ID NO.2所示的多肽与牛血清白蛋白偶联，得到用于制备抗瘦素的多克隆抗体的免疫原，记作免疫原2。
实施例2
抗瘦素的单克隆抗体的制备
(1)将实施例1制备的免疫原1与弗式完全佐剂等量混合充分乳化后免疫3只BALB/c小鼠，每只BALB/c小鼠共免疫三次。每只小鼠前两次的免疫原1和弗式完全佐剂的注射量均为100μg，前两次的免疫间隔为14天。在细胞融合之前三天进行第三次免疫：将50μg免疫原1与等量弗式不完全佐剂混合充分乳化后免疫小鼠。然后继续将免疫后的BALB/c小鼠饲养三天，得到免疫后的小鼠。
(2)细胞融合：取免疫后的小鼠的脾细胞，并与预先培养好的Sp2/0骨髓瘤细胞(购自美国典型培养物保藏中心，简称ATCC)在PEG的作用下进行融合，得到融合细胞。
(3)选择性培养和筛选：使用HAT培养基在96孔细胞培养板上培养融合细胞，7天后取融合细胞的上清进行ELISA检测。其中，酶标板的包被抗原为氨基酸序列如SEQ ID NO.1所示的多肽。挑取OD450﹥1.5孔内细胞作为阳性融合细胞。
(4)细胞克隆：对步骤(3)得到的阳性细胞融合细胞采用有限稀释法进 行亚克隆，克隆3次，得到7株能够稳定分泌抗瘦素的单克隆抗体的杂交瘤细胞，分别编号1～7。其中，编号1的杂交瘤细胞分泌的单克隆抗体记作LEP-Ab-01。
(5)腹水制备：8周龄的BALB/c小鼠注射石蜡10天后随机分为5组，按照一株杂交瘤细胞对应一组BALB/c小鼠分配，将步骤(4)得到的各株杂交瘤细胞扩大培养后注入对应组的BALB/c小鼠的腹部，7天后从各组BALB/c小鼠的腹部分别采集富含抗体的腹水。
(6)腹水效价检测：
将免疫原1分别稀释10倍、10 2倍、10 3倍、10 4倍、10 5倍、10 6倍，使用间接ELISA检测腹水效价，部分检测结果如下表1所示。表1中，“1#”表示编号1的杂交瘤细胞所对应的组别，“2#”表示编号2的杂交瘤细胞所对应的组别，其他依次类推。
表1
Figure PCTCN2019127876-appb-000001
(7)纯化鉴定：采用硫酸铵沉淀和Protein A亲和层析纯化步骤(5)得到各组腹水。具体地：将步骤(5)得到的腹水分别进行硫酸铵沉淀：在搅拌条件 下，将硫酸铵粉末分别加入各组的腹水中，5min-10min内形成各组的硫酸铵饱和溶液。；继续搅拌20min，然后4℃，10000×g离心10min，弃去上清液，将沉淀悬浮于2倍沉淀物体积的1×PBS中，并在1×PBS中过夜透析，除去硫酸铵，从而得到初级纯化物。
然后将初级纯化物过1×PBS平衡后的Protein A亲和层析柱，再用1×PBS洗去未与介质结合的杂蛋白，最后用0.1M甘氨酸洗脱，收集洗脱液，得到纯化后的各组的单克隆抗体。经SDS-PAGE鉴定，纯化的各组的单克隆抗体的纯度在98％以上。
实施例3
抗瘦素的多克隆抗体的制备
(1)动物免疫：将实施例1制备的免疫原2与弗式完全佐剂等量混合充分乳化后注射2只新西兰大白兔，每只兔的免疫原2的注射量均为1mg。间隔14天后，进行加强免疫：将免疫原2与弗式不完全佐剂等量混合充分乳化后进行免疫注射，每只兔的免疫原2的注射量均1mg。进行三次加强免疫，每次加强免疫的免疫原2与弗式不完全佐剂均为1mg。每次加强免疫之前，采耳动脉血1ml检测抗体效价，至效价不再升高时取静脉血，以备分离纯化抗瘦素的多克隆抗体。
(2)纯化：采用硫酸铵沉淀和Protein A亲和层析纯化步骤(1)得到纯化后的多克隆抗体。具体地：将步骤(1)得到的静脉血离心，得到血清，然后将血清进行硫酸铵沉淀：在搅拌条件下，将硫酸铵粉末分别加入各组的腹水中，5min-10min内形成硫酸铵饱和溶液。继续搅拌20min，然后4℃，10000×g离心10min，弃去上清液，将沉淀悬浮于2倍沉淀物体积的1×PBS中，并在1× PBS中过夜透析，除去硫酸铵，得到初级纯化物。
然后将初级纯化物过1×PBS平衡后的Protein A亲和层析柱，再用1×PBS洗去未与介质结合的杂蛋白，最后用0.1M甘氨酸洗脱，收集洗脱液，得到纯化后的多克隆抗体，记为LEP-Ab-02。
实施例4
抗瘦素的单克隆抗体和多克隆抗体的配对
以瘦素标准品(购自Abcam)为被检测的抗原，采用双抗体夹心ELISA法，以标记的实施例2制备的交瘤细胞分泌的单克隆抗体为检测抗体，以实施例3制备得到的多克隆抗体为捕获抗体进行配对试验，以检测信号最高的一对抗体作为最适抗体对，并用已知瘦素含量的人血清进行复测确认。
结果表明，与LEP-Ab-02最适配对的为LEP-Ab-01。LEP-Ab-01识别瘦素的氨基端，LEP-Ab-02识别瘦素的羧基端。
实施例5
制备瘦素检测试剂盒
(1)纳米磁珠包被：取50mg羧基化的磁微粒(粒径为1μm)悬浮液，磁分离，留沉淀，然后使用20mM，pH5.5 MES缓冲液重悬，加入1mL新鲜制备的10mg/mL的EDC水溶液，活化磁珠表面羧基，加入4mg实施例2制备的LEP-Ab-02，室温下混悬6h，磁分离，去上清，用含2％BSA的100mM pH8.0的Tris缓冲液重悬到1mg/mL，得到包被好的磁颗粒，按5mL/瓶分装，4℃保存备用。
(2)吖啶酯标记的抗瘦素的单克隆抗体的制备：取50ul 25mg/mL的实施 例3制备的兔源抗瘦素的单克隆抗体(LEP-Ab-01)，加入150ul 0.1M pH9.0的碳酸盐缓冲液，混匀，然后加入1.5μL 5mg/mL的吖啶酯混匀，室温下避光反应，1.5h后取出，用5mL GE Desalting预装柱脱盐处理，先使用TBS平衡层析柱，然后加入反应后的吖啶酯溶液，收集蛋白峰样品保存，按5mL/瓶分装，4℃保存备用。
(3)瘦素定标品的制备：用缓冲液(40mM Tris-Cl，0.5％BSA，1％NaCl，pH8.0)将瘦素标准品配制成浓度为0ng/mL、20ng/mL、60ng/mL，每瓶0.5mL分装冻干，4℃保存备用。
实施例6
瘦素检测试剂盒的性能评价：
将实施例5制备的瘦素测定试剂盒以化学发光测定仪为检测工具，以双抗夹心法进行性能评价。即将待测样品或瘦素标准品和抗瘦素的多克隆抗体(即LEP-Ab-02)包被的磁颗粒反应10min后，进行磁分离。然后加入吖啶酯标记的抗瘦素的单克隆抗体(即吖啶酯标记的LEP-Ab-01)，反应10min后，进行磁分离。然后依次加入化学发光预激发液、化学发光激发液进行发光反应，最后记录发光强度，从而绘制标准曲线，R 2大于0.99，并根据标准曲线计算出待测样品的瘦素含量。
灵敏度的检测：
参照CLSI EP17-A文件推荐实验方法，计算得到实施例5的瘦素检测试剂盒的灵敏度为0.5ng/mL。
线性的检测：
用实施例5的瘦素检测试剂盒和浓度为0.5ng/mL、20ng/mL、40ng/mL、 60ng/mL、80ng/mL和100ng/mL标准品做线性分析，计算得到线性相关系数r＝0.9995。根据CLSI EP17-A文件推荐的实验方法计算得出实施例5的瘦素检测试剂盒对瘦素样品检测的线性范围为：0.5ng/ml～100ng/mL。
精密度测定：
取浓度为20ng/mL及60ng/mL两个瘦素样本(由瘦素标准品配制，瘦素标准品购自Abcam)，每个样本每个浓度各做3个平行，用三批实施例5的瘦素检测试剂盒进行检测，计算瘦素检测试剂盒批内及批间差。
计算结果表明实施例5的瘦素检测试剂盒批内及批间差均小于5％。
干扰性实验：
取浓度为20ng/mL及60ng/mL两个瘦素样本(由瘦素标准品配制，瘦素标准品购自Abcam)然后分别将两个瘦素分为六组，其中的五组分别添加不同的干扰物，剩余的一组作为对照。添加的干扰物为结合胆红素、游离胆红素、血红蛋白、抗坏血酸、胆固醇和甘油酯，干扰物的质量与混合血清的质量之比为1：20。然后分别测定混合血清及添加了不同干扰物后的混合血清的瘦素含量，计算混合血清与添加干扰物的混合血清的瘦素含量的偏差，以±10％为可接受范围。
计算结果表明，实施例5的瘦素检测试剂盒的干扰性均达到NCCLS的文件标准，可用于临床实验室瘦素状况的准确评估。
对比例1
在目前市场上瘦素含量的测定大多是酶联免疫吸附法，本实验比对血清瘦素测定采用美国DSL 10-23100 Human leptin ELISA试剂盒。
测定方法：取瘦素校准品、质控品及实施例6中的干扰性实验样本，性能 评估方法同实施例6。根据该试剂盒说明书进行实验操作并对结果进行计算，对比实施例5和实施例6中瘦素测定试剂盒评估结果。
计算结果表明，对比例1中美国DSL 10-23100 Human leptin ELISA试剂盒与瘦素测定试剂盒结果符合率达到90％，一致性好。经过性能评估，瘦素测定试剂盒在灵敏度及抗干扰性均优于比对试剂。
对比例2
采用市售的人瘦素肽段(22位～56位氨基酸残基，具体氨基酸序列如SEQ ID NO.3所示，即VPIQKVQDDTKTLIKTIVTRINDISHTQSVSSKQK)作为免疫原制备单克隆抗体，记为C-Ab-10。具体地，制备C-Ab-10的方法与实施例2制备LEP-Ab-01的方法大致相同，其不同在于，对比例2的免疫原为采用市售的人瘦素肽段(22位～56位氨基酸残基)。
以瘦素标准品(购自Abcam)为被检测抗原，分别以标记的C-Ab-10和标记的LEP-Ab-01作为检测抗体，采用间接ELISA法进行效价对比，检测结果如下：
Figure PCTCN2019127876-appb-000002
Figure PCTCN2019127876-appb-000003
结果显示，使用市售免疫原制得的单抗效价低于LEP-Ab-01，本案的免疫原制得的单抗性能更好。
对比例3
采用市售的鼠源瘦素肽段(116位～130位氨基酸残基，具体序列如SEQ ID NO.4所示，即SCSLPQTSGLQKPES)作为免疫原制备多克隆抗体，记为LEPC-Ab-01。将LEPC-Ab-01与LEP-Ab-02分别作为捕获抗体，与检测抗体LEP-Ab-01进行配对实验，分别以瘦素标准品和含有高浓度瘦素的人血清作为被检测抗原，采用双抗体夹心ELISA法进行检测，检测结果如下：
Figure PCTCN2019127876-appb-000004
结果显示，使用市售免疫原制得的多抗LEPC-Ab-01与LEP-Ab-01的组合测得的效价低于本案LEP-Ab-02与LEP-Ab-01的组合，本案的免疫原制得的多抗与LEP-Ab-01的组合性能更好。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对 上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种瘦素免疫原，其特征在于，包括氨基酸序列如SEQ ID NO.1所示的多肽。
  2. 根据权利要求1所述的瘦素免疫原，其特征在于，所述瘦素免疫原还包括载体蛋白，所述载体蛋白与所述多肽偶联。
  3. 一种分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法，其特征在于，包括以下步骤：
    用免疫原免疫动物，得到免疫后的动物的脾细胞，其中所述免疫原为权利要求1或2所述的瘦素免疫原；
    将所述脾细胞与骨髓瘤细胞融合，然后筛选得到阳性融合细胞；及
    对所述阳性融合细胞进行亚克隆，得到所述分泌瘦素单克隆抗体的杂交瘤细胞。
  4. 一种分泌抗瘦素单克隆抗体的杂交瘤细胞，其特征在于，由权利要求3所述的分泌抗瘦素单克隆抗体的杂交瘤细胞的制备方法制得。
  5. 一种抗瘦素的单克隆抗体，其特征在于，由权利要求4所述的分泌抗瘦素单克隆抗体的杂交瘤细胞分泌。
  6. 一种瘦素免疫原，其特征在于，包括氨基酸序列如SEQ ID NO.2所示的多肽。
  7. 根据权利要求1所述的瘦素免疫原，其特征在于，所述瘦素免疫原还包括载体蛋白，所述载体蛋白与所述多肽偶联。
  8. 一种抗瘦素的多克隆抗体，其特征在于，制备所述抗瘦素的多克隆抗体的步骤包括：
    用权利要求6～7任一项所述的瘦素免疫原免疫动物，得到所述抗瘦素的多 克隆抗体。
  9. 权利要求4所述的抗瘦素的单克隆抗体和权利要求8所述的抗瘦素的多克隆抗体中的至少一种在制备瘦素检测试剂、制备瘦素检测试纸或制备瘦素检测试剂盒中的应用。
  10. 一种瘦素检测试剂盒，其特征在于，包括权利要求5所述的抗瘦素的单克隆抗体和权利要求8所述的抗瘦素的多克隆抗体中的至少一种。
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