CN111499746B

CN111499746B - High-affinity rabbit monoclonal antibody for human interleukin-2 and application thereof

Info

Publication number: CN111499746B
Application number: CN202010349876.2A
Authority: CN
Inventors: 娄阳; 吴海; 陈嘉琛
Original assignee: Yourui Seth Wuhan Biotechnology Co ltd
Current assignee: Yourui Seth Wuhan Biotechnology Co ltd
Priority date: 2020-04-28
Filing date: 2020-04-28
Publication date: 2020-11-24
Anticipated expiration: 2040-04-28
Also published as: CN111499746A

Abstract

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. The invention successfully develops the anti-human interleukin-2 rabbit monoclonal antibodies A and B with high affinity by using a single B cell screening and culturing technology, proves 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 enzyme-linked immunoassay kit of the double-antibody sandwich method developed by using the antibody 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 human interleukin-2 protein in serum and cell culture medium samples, and has important significance in clinical diagnosis and scientific research application.

Description

High-affinity rabbit monoclonal antibody for human interleukin-2 and application thereof

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 (IL-2, hereinafter referred to as "IL-2"), also known as T cell growth factor (TCRF), consists of 133 amino acids and has a molecular weight of 15.4 KDa. IL-2 is synthesized from a precursor protein of 153 amino acids. The precursor protein is used at the N-terminus (i.e., amino-terminus) for a 20 amino acid hydrophobic secretory signal peptide sequence as compared to mature IL-2; after secretion to the extracellular space, the signal peptide is cleaved off, resulting in the mature IL-2 protein. IL-2 proteins possess a pair of disulfide bonds between cysteine residues at 58 and 105, which are critical for the biological activity of IL-2.

In the human body, IL-2 protein is synthesized and secreted mainly by T lymphocytes after being stimulated by antigenic substances or mitogens; in addition, B lymphocytes, natural killer cells (NK cells) and monocyte-macrophages are also capable of producing IL-2. Besides producing IL-2 protein, these immune cells can produce certain biological responses to IL-2 protein, such as the requirement of a certain amount of IL-2 protein for the growth of T lymphocytes in vitro to continue stimulation, the induction and enhancement of cytotoxic effects of T lymphocytes and macrophages under the stimulation of IL-2, and the like.

Since IL-2 induces and enhances cytotoxic activity, IL-2 has found widespread use in the study of certain diseases, particularly in the treatment of tumors. For example, IL-2 alone or in combination with LAK cells has been used for tumor therapy to achieve a certain therapeutic effect. IL-2 has also been used in the treatment of viral infections, immunodeficiency diseases and autoimmune diseases. However, IL-2 can cause adverse reactions in the treatment process, such as fever, vomiting, capillary leak syndrome, water-salt metabolism disorder, renal, hepatic, cardiac and pulmonary dysfunction, and the like, so that the detection of the change of the IL-2 level in the serum of a patient has very important clinical significance in the clinical treatment process of applying IL-2.

Furthermore, the abnormal production or expression of IL-2 has a close relationship with clinical diseases, and the measurement of the IL-2 level in human peripheral blood, urine or culture supernatant of human activated lymphocytes 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 IL-2 protein level is 3.5-6.5 ng/L (ELISA method), which belongs to a lower level, so that the development of a high-sensitivity IL-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 IL-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 human IL-2 protein which is expressed by recombination, or a human IL-2 protein secreted by cells, or an IL-2 protein in human serum.

In summary, the advantages and positive effects of the invention are:

the immunogen for preparing the human IL-2 rabbit monoclonal antibody is human IL-2 full-length protein which is expressed by in vitro recombination of escherichia coli and proved to have bioactivity; the preparation method is a monoclonal antibody development technology based on single B lymphocyte screening and culture.

The invention successfully develops high-affinity anti-human interleukin-2 rabbit monoclonal antibodies A and B by using a single B cell screening and culturing technology, and the affinity constant of the combination of the antibody A and the recombinant expression human IL-2 is 8.99X 10^-10M, antibody B and recombinant expression of human IL-2 binding affinity constant of 2.93X 10^-10And M. 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 enzyme-linked immunoassay kit of the double-antibody sandwich method developed by using the antibody 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 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 IL-2 rabbit monoclonal antibodies A and B;

FIG. 3 is a standard curve of an enzyme-linked immunoassay method based on human IL-2 rabbit monoclonal antibodies A and B;

FIG. 4 is a graph showing the detection of the level of human IL-2 secreted from cells by an enzyme-linked immunoassay based on human IL-2 rabbit monoclonal antibodies A and B;

FIG. 5 is a serum peak recovery experiment based on an enzyme-linked immunoassay established with human IL-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 IL-2 rabbit monoclonal antibodies A and B;

FIG. 7 is a thermostability assay for human IL-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 IL-2 Rabbit monoclonal antibodies

1. Animal immunization: in order to obtain the rabbit monoclonal antibody for identifying the human IL-2 protein, the invention takes the recombinant human IL-2 protein produced by offshore biotechnology limited as immunogen to immunize a New Zealand white rabbit. Immunizing each big white rabbit by 200ug, mixing immunogen with equal amount of complete Freund adjuvant to prepare emulsifier for the first immunization, performing subcutaneous multipoint injection on abdomen and back, mixing 100ug of immunogen with equal amount of incomplete Freund adjuvant at intervals of 3 weeks to prepare emulsifier, performing subcutaneous multipoint injection on abdomen and back, performing boosting immunization twice, measuring serum titer by ELISA (enzyme-linked immuno sorbent assay) after three times of immunization, taking rabbits with high serum titer, performing subcutaneous multipoint injection once by 200ug of immunogen, and taking spleen 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 supernatants were identified as positive clones by antigen-coated ELISA. After the positive clone cells are collected and lysed, RNA is extracted and reverse transcribed into cDNA by a conventional method. Naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (VH and VL) were amplified from the corresponding positively cloned cDNA by PCR 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 IL-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 vector used is 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 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 PCR primer with the homologous sequence consistent with the carrier at the tail part is utilized, and the heavy chain gene primer sequence is as follows: 5'-tgaattcgagctcggtacccATGGAGACTGGGCTGCGCTG-3' and 5'-gtagcctttgaccaggcagcCGCCTGAGTTCCACGACACC-3'; the light chain gene-based amplification primers are as follows: 5'-tgaattcgagctcggtaccc ATGGACACGAGGGCCCCCAC-3' and 5'-acacacacgatggtgactgGTTTCTCGTAGTCTGCTTTGC-3', the light and heavy chain variable region genes (VH and VL) of the obtained rabbit monoclonal antibody are amplified; the PCR reaction system is as follows: at 95 ℃ for 3min, then carrying out 25 cyclic reactions at 95 ℃ for 1min, 56 ℃ for 1min and 68 ℃ for 0.5 min; finally, 10min at 95 ℃.

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 IL-2 monoclonal antibody, identifying, packaging, and storing at-20 deg.C.

Example 2 antibody screening and identification

After obtaining a plurality of human IL-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: the affinity of the obtained human IL-2 rabbit monoclonal antibody was preliminarily determined by using a Gator biomolecule interaction analyzer from Probe Life. Wherein the material used was recombinant human IL-2 protein at a concentration of 527nM and the antibody obtained at a concentration of 333 nM; by comparing the affinities of the individual antibodies, the antibody with the better affinity is selected from the group.

2) Identification of antigen recognition epitopes: the human IL-2 rabbit monoclonal antibody obtained in the invention and the human IL-2 protein antigen recognition epitope are identified. Testing the epitope determinant recognized by the obtained antibody by using a pairing reaction of a Gator biomolecule interaction analyzer of Probe Life company; wherein the material used was recombinant human IL-2 protein at a concentration of 527nM and the antibody obtained at a concentration of 333 nM; two antibodies recognizing different epitope determinants are selected from the two antibodies by analyzing the data of the pairing between the two antibodies.

According to the affinity of a plurality of human IL-2 rabbit monoclonal antibodies and the primary identification and screening results of antigen recognition epitopes, two rabbit monoclonal antibodies A and B with high affinity are obtained; and the affinity of the finally selected antibody was precisely determined using Biacore 3000 biomolecule interaction analyzer from GE (see FIG. 2); wherein the selected antibody is used at a concentration of 333 nM; and after conditioning, the recombinant human IL-2 protein was diluted to 5 different concentrations, 64, 32, 16, 8, 4nM respectively; the affinity of the human IL-2 rabbit monoclonal antibody A finally obtained is 8.99X 10^-10M; human IL-2 Rabbit monoclonal antibody B with 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 regions 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 regions 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 IL-2 protein rabbit monoclonal antibodies A and B

The double antibody sandwich enzyme-linked immunoassay method comprises the following specific steps: (1) coating the capture antibody, anti-human IL-2 protein rabbit monoclonal antibody A with carbonate buffer (pH9.4,0.05M), and incubating overnight at 4 ℃; washing the plate with a washing solution; (2) blocking with phosphate buffer (pH7.2,0.05M) containing 5% bovine serum albumin, 0.05% Tween-20; (3) diluting a standard sample (recombinant human IL-2 protein) and 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 an enzyme label plate, incubating for 1 hour under a normal temperature condition, and washing the plate by using a washing solution; (3) adding an anti-human IL-2 protein rabbit monoclonal antibody B which is diluted by phosphate buffer containing 1% of bovine serum albumin and 0.05% of Tween-20 and is marked by long-chain biotin (NHS-LC-biotin) into a plate, incubating for 1 hour under normal temperature conditions, and then washing the plate by using a washing solution; (4) adding avidin-labeled Horse Radish Peroxidase (HRP) diluted by phosphate buffer containing 1% of bovine serum albumin and 0.05% of Tween-20, incubating for 1 hour under normal temperature, and washing the plate by using a washing solution; (5) adding TMB developing solution, developing at normal temperature for 10min, adding oxalic acid to stop developing, measuring absorbance at 450nm and 630nm, and subtracting OD630 from OD450 to obtain corrected absorbance.

The biotin labeling treatment method comprises the following steps: preparing a 1mg/ml solution of an anti-human IL-2 protein rabbit monoclonal antibody B; preparing NHS-LC-biotin into a solution with the concentration of 60mg/ml by DMSO; taking 200ul of 1mg/ml anti-human IL-2 protein rabbit monoclonal antibody B solution, and adding 10ul of 60mg/ml NHS-LC-biotin solution; after mixing, the mixture was left at room temperature for 30 minutes, and 50ug of 500mM pH9.0 Tris solution was added to terminate the reaction; finally, a large amount of 1 XPBS buffer, pH7.4, was added and centrifuged through a spin column with an exclusion limit of 30kD, to remove excess biotin molecules and to allow equilibration of the buffer system.

The conditions of the enzyme-linked immunoassay method of the double antibody sandwich method are groped: by an orthogonal test method, the background and the light absorption value of the recombinant protein are comprehensively considered, the coating concentration of the anti-human IL-2 protein rabbit monoclonal antibody A is selected to be 5ug/ml, and the detection concentration of the biomarker anti-human IL-2 protein rabbit monoclonal antibody B is selected to be 5 ug/ml.

The sensitivity of the enzyme-linked immunoassay method of the double antibody sandwich method is as follows: the added sample was recombinant human IL-2 protein, which was diluted with a phosphate buffer containing 1% bovine serum albumin, 0.05% Tween-20 at concentrations of 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 an addition amount of 25 ul/well, respectively. The Log of the concentration of human IL-2 protein (Log10) is plotted as the abscissa and the corrected absorbance value (OD450-OD630) is plotted as the ordinate (see FIG. 3). And the lowest human IL-2 protein concentration of which the light absorption value average value is more than three times that of the blank control is taken as the sensitivity of the enzyme-linked immunoassay method of the double-antibody sandwich method. The experimental result shows that the detection sensitivity of the established enzyme-linked immunoassay method based on the anti-human IL-2 protein rabbit monoclonal antibodies A and B and establishing a double-antibody sandwich method reaches 1 pg/ml.

Example 4 detection of the Performance of rabbit monoclonal antibodies A and B against human IL-2 protein

1. Human peripheral blood lymphocyte (PBMC) secretion human IL-2 protein detection assay

The literature indicates that human peripheral blood lymphocytes (PBMC) can secrete human IL-2 protein after being stimulated by relevant stimulating factors (PMA and PHA), so that the anti-human IL-2 protein rabbit monoclonal antibody in the patent can be proved to recognize the IL-2 protein in a natural state by measuring the secretion of the human IL-2 protein in PBMC culture supernatant.

According to the literature, in 6-well cell culture plates, 400X 10 seeds were seeded into each well⁶PBMC cultured for 24 hr, and then supernatant added with 25ng/ml PMA and 1ug/ml PHA; control wells were not stimulated with PMA and PHA. After further culturing for 24 hours, the supernatant was collected and the human IL-2 protein level in the cell culture supernatant was determined by the double antibody sandwich ELISA method established in example 3.

The results are shown in FIG. 4, and show that the endogenous human IL-2 protein secreted by the stimulated PBMCs can be identified by establishing a double-antibody sandwich enzyme-linked immunoassay method based on the anti-human IL-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 IL-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 IL-2 protein rabbit monoclonal antibodies A and B in example 3, and the serum recovery rate of human IL-2 protein was calculated from the difference between the actual detected concentration and the theoretically calculated concentration.

The results are shown in figure 5, and the recovery rate of the human IL-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 IL-2 protein rabbit monoclonal antibodies A and B, which is calculated according to the results in figure 5, so that the detection method is high in detection accuracy and strong in anti-interference capability.

3. Detection of specificity for anti-human IL-2 protein rabbit monoclonal antibodies A and B

In this example, five kinds of interleukin proteins (human IL-1beta, human IL-4, human IL-9, human IL15, and human IL-25 protein) similar to human IL-2 protein were used to detect the specificity of anti-human IL-2 protein rabbit monoclonal antibodies A and B, the method used was a double-antibody sandwich enzyme-linked immunoassay method, and the concentration of all standard proteins was 10 ng/ml.

The results are shown in fig. 6, and show that the double-antibody sandwich enzyme-linked immunoassay method based on the anti-human IL-2 protein rabbit monoclonal antibodies A and B has no cross reaction to other interleukin proteins, which proves that the anti-human IL-2 protein rabbit monoclonal antibodies A and B have high specificity to the human IL-2 protein.

4. Testing of thermal stability of anti-human IL-2 protein Rabbit monoclonal antibodies A and B

In the embodiment, anti-human IL-2 protein rabbit monoclonal antibodies A and B are placed in an incubator at 37 ℃, sampled on days 3, 5, 7 and 14 respectively, and then detected on human IL-2 standard protein by a double-antibody sandwich enzyme-linked immunoassay method; and (3) comparing the antibody samples treated at 37 ℃ for different time with the antibody samples not treated at 37 ℃, establishing a standard curve, and detecting the thermal stability of the anti-human IL-2 protein rabbit monoclonal antibodies A and B.

The results are shown in FIG. 7, and show that the two rabbit anti-IL-2 monoclonal antibodies of the invention have less than 1% effect on the detection sensitivity and linear range after being treated at 37 ℃ for 14 days, which proves that the rabbit monoclonal antibodies A and B for anti-human IL-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

<210> 1

<211> 217

<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

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 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

165 170 175

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 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 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 Val Thr Val Ser Ser

115

<210> 15

<211> 9

<212> PRT

<213> light chain complementarity determining region CDR1(Rabbit monoclonal antibody B) of 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 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 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 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 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 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

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 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 complementarity determining regions CDR1, CDR2 and CDR3 on the light chain of the 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; 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.

2. 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.

3. 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.

4. 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;

5. Use of the high affinity rabbit monoclonal antibody against human interleukin-2 according to any one of claims 1 to 4 for establishing a high sensitivity enzyme-linked immunoassay for human interleukin-2 for non-diagnostic purposes.

6. Use according to claim 5, characterized in that: the enzyme-linked immunoassay method is a double-antibody sandwich enzyme-linked immunoassay method.

7. Use according to claim 6, 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.

8. Use of a high affinity rabbit monoclonal antibody against human interleukin-2 according to any one of claims 1 to 4 in the preparation of a reagent or kit for detecting human interleukin-2, said human interleukin-2 comprising recombinantly expressed human IL-2 protein, or human IL-2 protein secreted from cells, or IL-2 protein in human serum.