CN111499746A - High-affinity rabbit monoclonal antibody for human interleukin-2 and application thereof - Google Patents
High-affinity rabbit monoclonal antibody for human interleukin-2 and application thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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- 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
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/52—Assays involving cytokines
- G01N2333/54—Interleukins [IL]
- G01N2333/55—IL-2
Abstract
The invention belongs to the field of biotechnology, and particularly relates to a high-affinity rabbit monoclonal antibody aiming at human interleukin-2 and application thereof, wherein a single B cell screening and culturing technology is used for successfully developing high-affinity anti-human interleukin-2 rabbit monoclonal antibodies A and B, and the high-affinity anti-human interleukin-2 rabbit monoclonal antibodies A and B prove that the two antibodies can identify different antigenic determinants on the surface of human interleukin-2 protein and can be used for developing a double-antibody sandwich method enzyme-linked immunoassay kit, and the double-antibody sandwich method enzyme-linked immunoassay kit developed by using the antibodies has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, and the detection sensitivity is 1pg/ml (1 ng/L).
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-affinity rabbit monoclonal antibody for human interleukin-2 and application thereof.
Background
Human interleukin-2 protein (I L-2, hereinafter referred to as "I L-2"), also known as T cell growth factor (TCRF), consists of 133 amino acids, with a molecular weight of 15.4 KDa. I L-2 is synthesized from a precursor protein of 153 amino acids compared to mature I L-2, the precursor protein is used at the N-terminus (i.e. the amino-terminus) for a hydrophobic secretory signal peptide sequence of 20 amino acids, which is cleaved after secretion extracellularly to yield the mature I L-2 protein.
In human body, I L-2 protein is synthesized and secreted mainly by T lymphocyte after stimulated by antigen substance or mitogen, and in addition, B lymphocyte, natural killer cell (NK cell) and monocyte-macrophage can also produce I L-2, these immunocyte can produce certain biological reaction to I L-2 protein besides I L-2 protein, for example, T lymphocyte can need a certain amount of I L-2 protein to stimulate continuously for growth in vitro, T lymphocyte and macrophage can induce and enhance its cytotoxic action under the stimulation of I L-2, etc.
I L-2 has been widely used in the research of some diseases, especially tumor treatment, because I L-2 can induce and enhance cytotoxic activity, for example, I L-2 alone or in combination with L AK cells and the like has achieved certain curative effect in tumor treatment, I L-2 has also been applied in the treatment of viral infection, immunodeficiency diseases and autoimmune diseases, but I L-2 also causes some adverse reactions in the treatment process, such as fever, vomiting, capillary leakage syndrome, water-salt metabolism disorder and abnormal functions of kidney, liver, heart and lung, and therefore, it has very important clinical significance in detecting the change of I L-2 level in the serum of patients in the clinical treatment process of I L-2.
In addition, I L-2 generation or expression abnormality has a close relation with clinical diseases, and the level of I L-2 in human peripheral blood, urine or human activated lymphocyte culture supernatant can be used as a method for diagnosing, prognosing and observing diseases such as tumor, cardiovascular diseases, liver diseases, lupus erythematosus, leprosy, AIDS and the like, and can also be used for early diagnosis of rejection after organ transplantation, in the serum of normal people, the reference range of the level of I L-2 protein is 3.5-6.5 ng/L (E L ISA method), which belongs to a lower level, so that the development of a high-sensitivity I L-2 protein detection methodology has very important significance.
Disclosure of Invention
The invention provides a high-affinity rabbit monoclonal antibody aiming at human interleukin-2 and application thereof, aiming at solving part of problems in the prior art or at least relieving part of problems in the prior art.
The invention is realized in such a way that a high affinity rabbit monoclonal antibody against human interleukin-2 is characterized in that: the rabbit monoclonal antibody is rabbit monoclonal antibody A or rabbit monoclonal antibody B;
the sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain of the rabbit monoclonal antibody A are respectively the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
the sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain of rabbit monoclonal antibody B are respectively the amino acid sequences shown in SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17.
Further, the sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain of the rabbit monoclonal antibody A are the amino acid sequences shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10, respectively;
the sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the rabbit monoclonal antibody B heavy chain are respectively the amino acid sequences shown in SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20.
Further, the light chain variable region sequence of the rabbit monoclonal antibody A is an amino acid sequence shown in SEQ ID NO. 3;
the sequence of the variable region of the light chain of the rabbit monoclonal antibody B is an amino acid sequence shown as SEQ ID NO. 13;
further, the sequence of the heavy chain variable region of the rabbit monoclonal antibody A is an amino acid sequence shown as SEQ ID NO. 4;
the sequence of the heavy chain variable region of the rabbit monoclonal antibody B is an amino acid sequence shown as SEQ ID NO. 14;
further, the light chain sequence of the rabbit monoclonal antibody A is an amino acid sequence shown as SEQ ID NO. 1; the amino acid sequence of the heavy chain of the rabbit monoclonal antibody A is SEQ ID NO. 2;
the sequence of a light chain of the rabbit monoclonal antibody B is an amino acid shown as SEQ ID NO. 11; the heavy chain sequence of the rabbit monoclonal antibody B is amino acid shown as SEQ ID NO. 12.
Further, in the rabbit monoclonal antibodies a and B, the light chain constant region is a kappa chain and the heavy chain constant region is IgG1 type.
Further, rabbit monoclonal antibody a and rabbit monoclonal antibody B bind to different epitopes on the surface of human I L-2.
The application of the high-affinity rabbit monoclonal antibody aiming at the human interleukin-2 in establishing an enzyme-linked immunoassay method of the human interleukin-2 with high sensitivity is disclosed.
Further, the enzyme-linked immunoassay method is a double-antibody sandwich enzyme-linked immunoassay method.
Further, in the double-antibody sandwich enzyme-linked immunoassay method, the capture antibody is a rabbit monoclonal antibody A, and the detection antibody is a rabbit monoclonal antibody B labeled by biotin.
The application of the high-affinity rabbit monoclonal antibody aiming at the human interleukin-2 in the preparation of a reagent or a kit for detecting the human interleukin-2, wherein the human interleukin-2 comprises a recombinant expressed human I L-2 protein, or a human I L-2 protein secreted by cells, or an I L-2 protein in human serum.
In summary, the advantages and positive effects of the invention are:
the immunogen for preparing the human I L-2 rabbit monoclonal antibody is human I L-2 full-length protein which is expressed in vitro by using escherichia coli in a recombinant mode and proved to have biological activity, and the preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture.
The invention successfully develops the anti-human interleukin-2 rabbit with high affinity by using a single B cell screening and culturing technologyThe affinity constant of the combination of the monoclonal antibodies A and B, the antibody A and the recombinant expression human I L-2 is 8.99X 10-10M, antibody B and recombinant expression human I L-2 combined affinity constant of 2.93X 10-10M. and prove that the two antibodies can identify different antigenic determinants on the surface of the human interleukin-2 protein, and can be used for developing a double-antibody sandwich enzyme-linked immunoassay kit, the double-antibody sandwich enzyme-linked immunoassay kit developed by utilizing the antibodies has the advantages of high specificity, strong anti-interference capability, high detection sensitivity, good stability and the like, and the detection sensitivity is 1pg/ml (1 ng/L). the establishment of the methodology provides a kit capable of stably detecting the trace level of the human interleukin-2 protein in serum and cell culture medium samples, and has important significance in clinical diagnosis and scientific research application.
Drawings
FIG. 1 is a schematic representation of the construction of a parent plasmid containing mammalian cells (e.g., CHO, HEK293, etc.) containing rabbit monoclonal antibody heavy chain constant region (FIG. 1a) and light chain constant region (FIG. 1 b);
FIG. 2 is an affinity assay for human I L-2 rabbit monoclonal antibodies A and B;
FIG. 3 is a standard curve of an enzyme-linked immunoassay method based on human I L-2 rabbit monoclonal antibodies A and B;
FIG. 4 is a graph showing the detection of the level of human I L-2 secreted from cells by an enzyme-linked immunoassay based on human I L-2 rabbit monoclonal antibodies A and B;
FIG. 5 is a serum peak recovery experiment based on the enzyme-linked immunoassay established with human I L-2 rabbit monoclonal antibodies A and B;
FIG. 6 shows specificity tests (anti-interference tests) of an enzyme-linked immunoassay method established based on human I L-2 rabbit monoclonal antibodies A and B;
FIG. 7 is a thermostability assay for human I L-2 rabbit monoclonal antibodies A and B.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the equipment and reagents used in the examples and test examples are commercially available without specific reference. The specific embodiments described herein are merely illustrative of the invention and are not intended to be limiting.
The proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, plants) using recombinant techniques.
The invention discloses a high-affinity rabbit monoclonal antibody aiming at human interleukin-2 and application thereof, which are shown in the following embodiments.
Example 1 preparation of human I L-2 Rabbit monoclonal antibody
1. Animal immunization, in order to obtain a rabbit monoclonal antibody for identifying human I L-2 protein, recombinant human I L-2 protein produced by Haihai offshore biotechnology limited is used as immunogen to immunize New Zealand white rabbits, each white rabbit is immunized by 200ug, the immunogen and an equal amount of complete Freund adjuvant are mixed to prepare an emulsifier for the first immunization, the emulsifier is prepared by subcutaneous multipoint injection on the abdomen and back, 100ug of the immunogen and an equal amount of incomplete Freund adjuvant are taken at intervals of 3 weeks, the emulsifier is prepared by subcutaneous multipoint injection on the abdomen and back, the boosting immunization is carried out twice, the serum titer is determined by an E L ISA method after three times of immunization, the rabbit with high serum titer is taken, the boosting immunization is carried out once by subcutaneous multipoint injection by 200ug of the immunogen, and the spleen is taken after three days.
2. Spleen cells were isolated using standard procedures common in the art.
3. B lymphocyte sorting: the method is described in the previous Chinese patent of the applicant, with the application number of CN201910125091.4, and is named as a method for efficiently separating single antigen-specific B lymphocytes from spleen cells.
4. Cloning of genes encoding rabbit monoclonal antibodies
The cultured B cell supernatant is identified with antigen-coated E L ISA, the positive cloned cell is collected and cracked, RNA is extracted and reverse transcribed into cDNA by a conventional method, and naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and V L) are amplified from the cDNA corresponding to the positive cloned cell by a PCR method and sequenced to determine the sequence.
5. Production and purification of monoclonal antibodies
In order to obtain a plurality of rabbit monoclonal antibodies for identifying human I L-2 protein, the invention clones and expresses heavy chain and light chain genes of the obtained rabbit monoclonal antibodies, and the method comprises the following specific steps:
1) the mammalian expression vectors used are shown in fig. 1, where pRB322Ori and f1Ori are replication promoters in e.coli, Ampcillin is a plasmid resistance gene, CMV promoter is a promoter in eukaryotes, SV40_ PA _ terminator is a tailing signal, and Heavy chain constant (fig. 1a) and L light chain constant (fig. 1b) are rabbit Heavy chain constant and light chain constant region sequences, respectively.
2) Carrying out conventional linearization treatment on mammalian cell expression parent plasmids containing a heavy chain constant region (figure 1a) and a light chain constant region (figure 1b) of the rabbit monoclonal antibody by using NheI and XbaI restriction enzymes respectively;
3) the light and heavy chain variable region genes (VH and V L) of the obtained rabbit monoclonal antibody are amplified by utilizing PCR primers with homologous sequences consistent with the vector at the tail parts, wherein the heavy chain gene primer sequences are 5'-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3' and 5'-gtagcctttgaccaggcagcCGCCTGAGTTCCACGACACC-3', the light chain gene amplification primers are 5'-tgaattcgagctcggtacccATGGACACGAGGGCCCCCAC-3' and 5'-acacacacgatggtgactgGTTTCTCGTAGTCTGCTTTGC-3', the PCR reaction system is that the temperature is 95 ℃ for 3min, then 25 circulation reactions are carried out according to the following conditions, the temperature is 95 ℃ for 1min, the temperature is 56 ℃ for 1min, the temperature is 68 ℃ for 0.5min, and finally the temperature is 95 ℃ for 10 min.
4) Purifying the amplified PCR product, and respectively constructing heavy chain and light chain variable region genes into corresponding mammalian expression vectors in a homologous recombination mode;
5) after sequencing verification, the expression plasmids containing the light and heavy chain genes of the corresponding rabbit monoclonal antibody are transfected into 293 cells together;
6) after 72-96 hours of transfection, cells were removed by centrifugation, and the obtained culture supernatant was purified with protein a affinity gel resin;
7) purifying to obtain recombinant rabbit anti-human I L-2 monoclonal antibody, identifying, packaging, and storing at-20 deg.C.
Example 2 antibody screening and identification
After obtaining a plurality of human I L-2 rabbit monoclonal antibodies, firstly, carrying out primary identification and screening on the antibodies, including identification of antibody affinity and identification of antigen recognition epitopes, and specifically comprising the following steps:
1) identification of antibody affinity, namely performing primary identification on the antibody affinity of the obtained human I L-2 rabbit monoclonal antibody, and performing primary determination on the affinity of the obtained antibody by using a Gator biomolecule interaction analyzer of Probe L ife company, wherein the used material is recombinant human I L-2 protein, the used concentration is 527nM, the obtained antibody concentration is 333nM, and the antibody with better affinity is selected from the antibodies by comparing the affinity of each antibody.
2) Identification of antigen recognition epitope human I L-2 rabbit monoclonal antibody obtained in the invention and human I L-2 protein antigen recognition epitope are identified, the obtained antibody is subjected to pairing reaction by using a Gator biomolecule interaction analyzer of Probe L ife company to test the recognized epitope determinant thereof, wherein the used material is recombinant human I L-2 protein, the used concentration is 527nM, the obtained antibody concentration is 333nM, and two antibodies recognizing different epitope determinants are selected by analyzing the pairing data between the two antibodies.
According to the primary identification and screening results of the affinity and antigen recognition epitope of a plurality of human I L-2 rabbit monoclonal antibodies, two rabbit monoclonal antibodies A and B with high affinity are obtained, the finally selected antibody affinity is accurately determined by using a Biacore3000 biomolecule interaction analyzer of GE company (see figure 2), wherein the selected antibody concentration is 333nM, the recombinant human I L-2 protein is diluted by 5 different concentrations which are 64, 32, 16, 8 and 4nM respectively after condition exploration, and the finally obtained human I L-2 rabbit monoclonal antibody A has the affinity of 8.99X 10-10M, human I L-2 Rabbit monoclonal antibody B has an affinity of 2.93X 10-10M。
The amino acid sequence of the light chain of the rabbit monoclonal antibody A obtained in the embodiment is SEQ ID NO. 1; the heavy chain amino acid sequence is SEQ ID NO 2; the amino acid sequence of the light chain variable region is SEQ ID NO. 3; the amino acid sequence of the heavy chain variable region is SEQ ID NO. 4; the amino acid sequences of the light chain complementarity determining region CDR1, CDR2 and CDR3 are SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7, respectively; the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO 8, SEQ ID NO 9 and SEQ ID NO 10, respectively.
The light chain amino acid sequence of the rabbit monoclonal antibody B obtained in this example is SEQ ID NO. 11; the heavy chain amino acid sequence is SEQ ID NO 12; the amino acid sequence of the light chain variable region is SEQ ID NO 13; the amino acid sequence of the heavy chain variable region is SEQ ID NO. 14; the amino acid sequences of the light chain complementarity determining region CDR1, CDR2 and CDR3 are SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17, respectively; the amino acid sequences of the heavy chain complementarity determining regions CDR1, CDR2 and CDR3 are SEQ ID NO 18, SEQ ID NO 19 and SEQ ID NO 20, respectively.
Example 3 establishment of double-antibody sandwich enzyme-linked immunoassay based on anti-human I L-2 protein rabbit monoclonal antibodies A and B
The enzyme-linked immunoassay method of the double-antibody sandwich method comprises the specific steps of (1) coating a capture antibody and an anti-human I L-2 protein rabbit monoclonal antibody A by using a carbonate buffer solution (pH9.4,0.05M), incubating overnight at 4 ℃, washing a plate by using a washing solution, (2) sealing by using a phosphate buffer solution (pH7.2,0.05M) containing 5% of bovine serum albumin and 0.05% of Tween-20, (3) diluting a standard sample (recombinant human I L-2 protein), a sample to be detected by using a phosphate buffer solution containing 1% of bovine serum albumin and 0.05% of Tween-20, adding the diluted sample into the plate, incubating for 1 hour at normal temperature, then washing the plate by using the washing solution, (3) adding an anti-human I L-2 protein rabbit monoclonal antibody B labeled by long-chain biotin (NHS-L C-biotin) diluted by using a phosphate buffer solution containing 1% of bovine serum albumin and 0.05% of Tween-20 into the plate by using a washing solution, adding a washing solution containing 4% of the washing solution, adding a washing solution, diluting by using a peroxidase, and then adding a light absorption enzyme (HRP) to the washing solution, and measuring the absorbance value after the absorbance of the diluted by using a normal-5 nm-5 and the absorbance of the HRP (HRP) diluted by using a chromogenic solution after the diluted by adding the diluted fluorescent dye solution (1-5 nm) for 1 nm) to obtain the absorbance of the absorbance.
The biotin labeling treatment method comprises the steps of preparing a 1mg/ml solution of an anti-human I L-2 protein rabbit monoclonal antibody B, preparing a 60mg/ml solution of NHS-L C-biotin by using DMSO, adding 10ul of a 60mg/ml solution of NHS-L C-biotin into 200ul of a 1mg/ml solution of the anti-human I L-2 protein rabbit monoclonal antibody B, uniformly mixing, placing at room temperature for 30 minutes, adding 50ug of a 500mM ph9.0 solution of Tris, stopping reaction, finally adding a large amount of 1X PBS buffer solution with pH7.4, centrifuging by using a centrifugal column with the exclusion limit of 30KD, and removing redundant biotin molecules and balancing a buffer solution system.
The conditions of the enzyme-linked immunoassay method of the double-antibody sandwich method are groped, namely the coating concentration of the anti-human I L-2 protein rabbit monoclonal antibody A is selected to be 5ug/ml and the detection concentration of the biomarker anti-human I L-2 protein rabbit monoclonal antibody B is selected to be 5ug/ml by an orthogonal test method and comprehensively considering the background and the light absorption value of recombinant protein.
The sensitivity of the enzyme-linked immunoassay method of the double-antibody sandwich method is that the added sample is recombinant human I L-2 protein, the sample is diluted by phosphate buffer containing 1% bovine serum albumin and 0.05% Tween-20, the concentrations are respectively 500pg/ml,250pg/ml,125pg/ml,62.5pg/ml,31.25pg/ml,15.625pg/ml,7.813pg/ml,3.906pg/ml,1.953pg/ml,0.977pg/ml,0.488pg/ml,0.244pg/ml,0.122pg/ml,0.061pg/ml,0pg/ml, and the sample addition amount is 25 ul/ml, the absorbance value (34 og 56) of the concentration of the human I L-2 protein is used as the horizontal coordinate, the corrected value (OD450-OD630) of the 3982-2 protein is used as the longitudinal coordinate, the average value of absorbance of the enzyme-linked immunosorbent assay method is plotted, and the absorbance value of the blank antibody of the enzyme-linked immunosorbent assay method is established based on the ELISA method, the result of the double-linked immunosorbent assay method, the absorbance detection result of the double-antibody sandwich protein is that the absorbance of the antibody is greater than the optimal absorbance of the optimal detection method of the double-antibody detection method, and the detection method of the double-enzyme-antibody detection method is based on the detection method.
Example 4 detection of the Performance of rabbit monoclonal antibodies A and B against human I L-2 protein
1. Human peripheral blood lymphocyte (PBMC) secretion human I L-2 protein detection assay
The literature indicates that human peripheral blood lymphocytes (PBMC) can secrete human I L-2 protein after being stimulated by relevant stimulating factors (PMA and PHA), so that the anti-human I L-2 protein rabbit monoclonal antibody in the patent can identify the natural I L-2 protein by measuring the secretion of human I L-2 protein in PBMC culture supernatant.
According to the literature, in 6-well cell culture plates, 400X 10 seeds were seeded into each well6After 24 hours of culture, the PBMC is cultured, 25ng/ml PMA and 1ug/ml PHA are added into the supernatant, the control wells are not stimulated by PMA and PHA, the supernatant is collected after 24 hours of culture, and the level of human I L-2 protein in the cell culture supernatant is detected by the double antibody sandwich enzyme-linked immunoassay method established in example 3.
The results are shown in FIG. 4, and show that the endogenous human I L-2 protein secreted by PBMC after stimulation can be identified by establishing a double-antibody sandwich enzyme-linked immunoassay method based on anti-human I L-2 protein rabbit monoclonal antibodies A and B.
2. Serum peak recovery experiment
The assay is used to detect analytical method or immunoassay accuracy, helps to account for the percent loss of analyte, and can detect the effect of interfering substances on the assay.
In this example, a known amount of standard human I L-2 protein was added to sera obtained from different persons, and the concentration thereof was determined by the double antibody sandwich enzyme-linked immunoassay method based on anti-human I L-2 protein rabbit monoclonal antibodies A and B in example 3, and the serum recovery rate of human I L-2 protein was calculated by the difference between the actual detected concentration and the theoretically calculated concentration.
The results are shown in figure 5, and the results are calculated according to the results in figure 5, and the recovery rate of human I L-2 protein in serum is 90-120% (the average recovery rate is 106%) by the double-antibody sandwich enzyme-linked immunoassay based on the anti-human I L-2 protein rabbit monoclonal antibodies A and B, which proves that the detection method has high detection accuracy and strong anti-interference capability.
3. Detection of specificity of rabbit monoclonal antibodies A and B against human I L-2 protein
In the embodiment, five kinds of interleukin proteins (human I L-1 beta, human I L-4, human I L-9, human I L15 and human I L-25 proteins) similar to human I L-2 protein are used for detecting the specificity of the rabbit monoclonal antibodies A and B of the anti-human I L-2 protein, the adopted method is a double-antibody sandwich method enzyme-linked immunoassay method, and the concentration of all standard proteins is 10 ng/ml.
The results are shown in FIG. 6, and the results show that the double-antibody sandwich enzyme-linked immunoassay method based on the anti-human I L-2 protein rabbit monoclonal antibodies A and B has no cross reaction with other interleukin proteins, and prove that the anti-human I L-2 protein rabbit monoclonal antibodies A and B have high specificity with respect to human I L-2 protein.
4. Testing of thermal stability of anti-human I L-2 protein Rabbit monoclonal antibodies A and B
In the embodiment, anti-human I L-2 protein rabbit monoclonal antibodies A and B are placed in a 37 ℃ incubator, sampled on days 3, 5, 7 and 14 respectively, and then detected for human I L-2 standard protein by a double-antibody sandwich enzyme-linked immunoassay method, antibody samples treated at 37 ℃ for different time are compared with antibody samples not treated at 37 ℃, a standard curve is established, and the thermal stability of the anti-human I L-2 protein rabbit monoclonal antibodies A and B is detected.
The results are shown in FIG. 7, and show that the two rabbit anti-I L-2 monoclonal antibodies of the present invention have less than 1% effect on detection sensitivity and linear range after being treated at 37 ℃ for 14 days, which proves that the rabbit monoclonal antibodies A and B against human I L-2 protein have strong thermal stability.
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 and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Yougui saisi (Wuhan) Biotechnology Ltd
<120> high affinity rabbit monoclonal antibody against human interleukin-2 and application thereof
<160>20
<170>SIPOSequenceListing 1.0
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<212>PRT
<213> light chain amino acid sequence of Rabbit monoclonal antibody A (Rabbit monoclonal antibody A)
<400>1
Ala Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val
1 5 10 15
Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Arg
20 25 30
Asp Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys
35 40 45
Leu Leu Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg
50 55 60
Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp
65 70 75 80
Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Val Tyr
85 90 95
Ser Ser Thr Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val
100 105 110
Lys Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala
115 120 125
Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys
130 135 140
Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln
145 150 155 160
Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys
165 170 175
Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn
180 185 190
Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val
195 200 205
Val Gln Ser Phe Asn Arg Gly Asp Cys
210 215
<210>2
<211>454
<212>PRT
<213> heavy chain amino acid sequence of Rabbit monoclonal antibody A (Rabbit monoclonal antibody A)
<400>2
Gln Ser Val Ala Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Thr Thr Tyr Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala
35 40 45
Ile Ile Tyr Ile Asp Gly Thr Thr Tyr Tyr Gly Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Arg Ile Ile
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Lys Trp Tyr
85 90 95
Val Gly Glu Met Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala Pro Cys Cys
115 120 125
Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu Val Lys Gly
130 135 140
Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Thr Leu Thr
145 150 155 160
Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser Ser Gln Pro Val Thr
180 185 190
Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys Val Asp Lys Thr Val
195 200 205
Ala Pro Ser Thr Cys Ser Lys Pro Met Cys Pro Pro Pro Glu Leu Pro
210 215 220
Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Thr Leu
225 230 235 240
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
245 250 255
Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr Ile Asn Asn Glu Gln
260 265 270
Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln Gln Phe Asn Ser Thr
275 280 285
Ile Arg Val Val Ser Thr Leu Pro Ile Ala His Gln Asp Trp Leu Arg
290 295 300
Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys Ala Leu Pro Ala Pro
305 310 315 320
Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln Pro Leu Glu Pro Lys
325 330 335
Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu Ser Ser Arg Ser Val
340 345 350
Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro Ser Asp Ile Ser Val
355 360 365
Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn Tyr Lys Thr Thr Pro
370 375 380
Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Ser
385 390 395 400
Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val Phe Thr Cys Ser Val
405 410 415
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Ile Ser Arg
420 425 430
Ser Pro Gly Lys Trp Ile Leu Ala Ile Pro Arg Arg Ile Arg Gln Gly
435 440 445
Leu Glu Leu Thr Leu Leu
450
<210>3
<211>113
<212>PRT
<213> amino acid sequence of light chain variable region of Rabbit monoclonal antibody A (Rabbit monoclonal antibody A)
<400>3
Ala Asp Ile Val Met Thr Gln Thr Pro Ala Ser Val Glu Ala Ala Val
1 5 10 15
Gly Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Gln Ser Val Tyr Arg
20 25 30
Asp Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Lys
35 40 45
Leu Leu Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg
50 55 60
Phe Lys Gly Ser Gly Ser Gly Thr Gln Phe Thr Leu Thr Ile Ser Asp
65 70 75 80
Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr Val Tyr
85 90 95
Ser Ser Thr Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu Val Val Val
100 105 110
Lys
<210>4
<211>113
<212>PRT
<213> amino acid sequence of heavy chain variable region of Rabbit monoclonal antibody A (Rabbit monoclonal antibody A)
<400>4
Gln Ser Val Ala Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro
1 5 10 15
Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Thr Thr Tyr Ala
20 25 30
Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Ala
35 40 45
Ile Ile Tyr Ile Asp Gly Thr Thr Tyr Tyr Gly Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu Arg Ile Ile
65 70 75 80
Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Lys Trp Tyr
85 90 95
Val Gly Glu Met Asp Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser
100 105 110
Ser
<210>5
<211>11
<212>PRT
<213> Rabbit monoclonal antibody A light chain complementarity determining region CDR1(Rabbit monoclonal antibody A)
<400>5
Gln Ser Val Tyr Arg Asp Asn Tyr Leu Ala Trp
1 5 10
<210>6
<211>11
<212>PRT
<213> Rabbit monoclonal antibody A light chain complementarity determining region CDR2(Rabbit monoclonal antibody A)
<400>6
Leu Ile Tyr Gly Thr Ser Thr Leu Ala Ser Gly
1 5 10
<210>7
<211>12
<212>PRT
<213> Rabbit monoclonal antibody A light chain complementarity determining region CDR3(Rabbit monoclonal antibody A)
<400>7
Ser Tyr Val Tyr Ser Ser Thr Ile Asp Asn Ala Phe
1 5 10
<210>8
<211>9
<212>PRT
<213> Rabbit monoclonal antibody A heavy chain complementarity determining region CDR1(Rabbit monoclonal antibody A)
<400>8
Phe Ser Leu Thr Thr Tyr Ala Met Ser
1 5
<210>9
<211>18
<212>PRT
<213> Rabbit monoclonal antibody A heavy chain complementarity determining region CDR2(Rabbit monoclonal antibody A)
<400>9
Trp Ile Ala Ile Ile Tyr Ile Asp Gly Thr Thr Tyr Tyr Gly Ser Trp
1 5 10 15
Ala Lys
<210>10
<211>13
<212>PRT
<213> Rabbit monoclonal antibody A heavy chain complementarity determining region CDR3(Rabbit monoclonal antibody A)
<400>10
Tyr Phe Cys Ala Lys Trp Tyr Val Gly Glu Met Asp Leu
1 5 10
<210>11
<211>215
<212>PRT
<213> light chain amino acid sequence of Rabbit monoclonal antibody B (Rabbit monoclonal antibody B)
<400>11
Ala Ile Glu Met Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly
15 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Asp Ile Glu Thr Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Ile Ala Ser Gly Val Ser Ser His Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Ser Tyr Ala Ser Gly
85 90 95
Tyr Trp Asn Gln Ala Phe Gly Gly Gly Thr Glu Val Val Val Arg Gly
100 105 110
Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln
115 120 125
Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe
130 135 140
Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr
145 150 155 160
Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr
165170 175
Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His
180 185 190
Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln
195 200 205
Ser Phe Asn Arg Gly Asp Cys
210 215
<210>12
<211>441
<212>PRT
<213> heavy chain amino acid sequence of Rabbit monoclonal antibody B (Rabbit monoclonal antibody B)
<400>12
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly Ser
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Gly Gly
20 25 30
Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Ser Arg His Thr Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Val Lys Ile
65 70 75 80
Thr Ser Pro Thr Ile Glu Asp Thr Ala Thr Tyr TyrCys Ala Arg Val
85 90 95
Ser Ser Gly Asp Gly Leu Tyr Ala Phe Asp Pro Trp Gly Pro Gly Thr
100 105 110
Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser Val Phe Pro
115 120 125
Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly
130 135 140
Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr Trp Asn
145 150 155 160
Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val Arg Gln
165 170 175
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr Ser Ser
180 185 190
Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn Thr Lys
195 200 205
Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Met Cys Pro
210 215 220
Pro Pro Glu Leu Pro Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
225 230 235 240
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
245 250 255
Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr Trp Tyr
260 265 270
Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg Glu Gln
275 280 285
Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile Ala His
290 295 300
Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His Asn Lys
305 310 315 320
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg Gly Gln
325 330 335
Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu Glu Leu
340 345 350
Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe Tyr Pro
355 360 365
Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu Asp Asn
370 375 380
Tyr Lys Thr Thr Pro Thr Val Leu Asp Ser Asp Gly Ser Tyr Phe Leu
385 390 395 400
Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly Asp Val
405 410 415
Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
420 425 430
Lys Ser Ile Ser Arg Ser Pro Gly Lys
435 440
<210>13
<211>111
<212>PRT
<213> amino acid sequence of light chain variable region of Rabbit monoclonal antibody B (Rabbit monoclonal antibody B)
<400>13
Ala Ile Glu Met Thr Gln Thr Pro Ser Pro Val Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ala Ser Glu Asp Ile Glu Thr Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Arg Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Ile Ala Ser Gly Val Ser Ser His Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Leu Glu Cys
65 70 75 80
Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Cys Thr Ser Tyr Ala Ser Gly
85 90 95
Tyr Trp Asn Gln Ala Phe Gly Gly Gly Thr Glu Val Val Val Arg
100 105 110
<210>14
<211>118
<212>PRT
<213> amino acid sequence of heavy chain variable region of Rabbit monoclonal antibody B (Rabbit monoclonal antibody B)
<400>14
Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly Ser
1 5 10 15
Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Ser Thr Gly Gly
20 25 30
Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly
35 40 45
Ile Ile Ser Arg His Thr Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60
Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Val Lys Ile
65 70 75 80
Thr Ser Pro Thr Ile Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Val
85 90 95
Ser Ser Gly Asp Gly Leu Tyr Ala Phe Asp Pro Trp Gly Pro Gly Thr
100 105 110
Leu ValThr Val Ser Ser
115
<210>15
<211>9
<212>PRT
<213> light chain complementarity determining region CDR1(Rabbit monoclonal antibody B) of the heavy chain variable region of Rabbit monoclonal antibody B
<400>15
Glu Asp Ile Glu Thr Tyr Leu Ala Trp
1 5
<210>16
<211>10
<212>PRT
<213> light chain complementarity determining region CDR2(Rabbit monoclonal antibody B) of the heavy chain variable region of Rabbit monoclonal antibody B
<400>16
Ile Tyr Ser Gly Ser Thr Ile Ala Ser Gly
1 5 10
<210>17
<211>14
<212>PRT
<213> light chain complementarity determining region CDR3(Rabbit monoclonal antibody B) of the heavy chain variable region of Rabbit monoclonal antibody B
<400>17
Cys Thr Ser Tyr Ala Ser Gly Tyr Trp Asn Gln Ala Phe Gly
1 5 10
<210>18
<211>9
<212>PRT
<213> heavy chain complementarity determining region CDR1(Rabbit monoclonal antibody B) of the heavy chain variable region of Rabbit monoclonal antibody B
<400>18
Ile Asp Leu Ser Thr Gly Gly Val Ser
1 5
<210>19
<211>18
<212>PRT
<213> heavy chain complementarity determining region CDR2(Rabbit monoclonal antibody B) of the heavy chain variable region of Rabbit monoclonal antibody B
<400>19
Trp Ile Gly Ile Ile Ser Arg His Thr Asn Thr Tyr Tyr Ala Ser Trp
1 5 10 15
Ala Lys
<210>20
<211>17
<212>PRT
<213> heavy chain complementarity determining region CDR3(Rabbit monoclonal antibody B) of the heavy chain variable region of Rabbit monoclonal antibody B
<400>20
Tyr Tyr Cys Ala Arg Val Ser Ser Gly Asp Gly Leu Tyr Ala Phe Asp
1 5 10 15
Pro
Claims (10)
1. A high affinity rabbit monoclonal antibody to human interleukin-2, characterized by: the rabbit monoclonal antibody is rabbit monoclonal antibody A or rabbit monoclonal antibody B;
the sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain of the rabbit monoclonal antibody A are respectively the amino acid sequences shown in SEQ ID NO. 5, SEQ ID NO. 6 and SEQ ID NO. 7;
the sequences of complementarity determining regions CDR1, CDR2 and CDR3 on the light chain of rabbit monoclonal antibody B are respectively the amino acid sequences shown in SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17.
2. The high affinity rabbit monoclonal antibody to human interleukin-2 of claim 1, wherein: the sequences of the complementarity determining regions CDR1, CDR2 and CDR3 on the heavy chain of the rabbit monoclonal antibody A are respectively the amino acid sequences shown in SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10;
the sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the rabbit monoclonal antibody B heavy chain are respectively the amino acid sequences shown in SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20.
3. The high affinity rabbit monoclonal antibody to human interleukin-2 of claim 1, wherein: the sequence of the light chain variable region of the rabbit monoclonal antibody A is an amino acid sequence shown in SEQ ID NO. 3; the sequence of the variable region of the light chain of the rabbit monoclonal antibody B is an amino acid sequence shown in SEQ ID NO. 13.
4. The high affinity rabbit monoclonal antibody to human interleukin-2 of claim 1, wherein: the sequence of the heavy chain variable region of the rabbit monoclonal antibody A is an amino acid sequence shown in SEQ ID NO. 4; the sequence of the heavy chain variable region of the rabbit monoclonal antibody B is an amino acid sequence shown in SEQ ID NO. 14.
5. The high affinity rabbit monoclonal antibody to human interleukin-2 of claim 1, wherein: the light chain sequence of the rabbit monoclonal antibody A is an amino acid sequence shown in SEQ ID NO. 1; the amino acid sequence of the heavy chain of the rabbit monoclonal antibody A is SEQ ID NO. 2;
the sequence of a light chain of the rabbit monoclonal antibody B is an amino acid shown as SEQ ID NO. 11; the heavy chain sequence of the rabbit monoclonal antibody B is amino acid shown as SEQ ID NO. 12.
6. The rabbit monoclonal antibody with high affinity for human interleukin-2 according to any one of claims 1-5, wherein said rabbit monoclonal antibody A and rabbit monoclonal antibody B bind to different epitopes on the surface of human I L-2.
7. The use of the high affinity rabbit monoclonal antibody against human interleukin-2 of claim 6 in the establishment of a highly sensitive enzyme-linked immunoassay for human interleukin-2.
8. Use according to claim 7, characterized in that: the enzyme-linked immunoassay method is a double-antibody sandwich enzyme-linked immunoassay method.
9. Use according to claim 8, characterized in that: in the double-antibody sandwich enzyme-linked immunoassay method, the capture antibody is a rabbit monoclonal antibody A, and the detection antibody is a rabbit monoclonal antibody B marked by biotin.
10. Use of the high affinity rabbit monoclonal antibody against human interleukin-2 according to any one of claims 1 to 5 in the preparation of a reagent or kit for detecting human interleukin-2, wherein said human interleukin-2 comprises a recombinantly expressed human I L-2 protein, or a cell secreted human I L-2 protein, or a human serum I L-2 protein.
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| CN113248610A (en) * | 2021-05-14 | 2021-08-13 | 厦门柏慈生物科技有限公司 | Interleukin 2 binding molecules, derivatives thereof, kits, methods of production and uses thereof |
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