CN111793135A

CN111793135A - Antibody pair for detecting RANKL content in serum and application thereof

Info

Publication number: CN111793135A
Application number: CN202010913877.5A
Authority: CN
Inventors: 刘静; 程勇; 刘继来; 王征
Original assignee: Langfang Tian Guang Biological Technology Co ltd
Current assignee: Langfang Tian Guang Biological Technology Co ltd
Priority date: 2020-05-11
Filing date: 2020-09-03
Publication date: 2020-10-20
Also published as: CN113150158A

Abstract

The application provides an antibody pair for detecting the content of RANKL in serum and application thereof, the RANKL antigen is divided into an N-terminal (SEQ ID NO:2) sequence and a C-terminal (SEQ ID NO:3) sequence, mice are immunized by the two sequences respectively, the antibody pair AntiRANKL _ N and AntiRANKL _ C with high specificity and high sensitivity are screened, the content of RANKL in serum can be effectively determined by the antibody pair, and further, the diagnosis and the treatment of osteoporosis are facilitated.

Description

Antibody pair for detecting RANKL content in serum and application thereof

Technical Field

The application relates to the field of medicines, in particular to an antibody pair for detecting the RANKL content in serum and application thereof.

Background

Osteoporosis is an age-related chronic systemic metabolic disease characterized primarily by decreased bone mass, destruction of bone microarchitecture, and increased bone fragility. The disease has become a global public health problem due to its increased morbidity and mortality. In view of the high incidence of osteoporosis and its serious consequences, the pathogenesis of osteoporosis has been studied intensively.

RANKL protein is considered to be an essential biomolecule in the activation and proliferation process of osteoclasts, and has three subtypes and all of them can promote osteoclast proliferation. RANKL can be expressed by various cells, and the RANKL expressed by cartilage tissues is regulated by 1, 25(OH)2D3 BMP2, a Wnt/b-catenin signal pathway, attracts osteoclast precursors to aggregate and absorb newly formed redundant bone, and prevents bone sclerosis; whereas RANKL expression in bone cells is associated with stress stimulation; RANKL expressed by B cells and T cells in inflammatory diseases is probably involved in immune response and B cell maturation process. Studies have found that serum RANKL levels are significantly elevated in women over the age of 57, which is consistent with the appearance of postmenopausal osteoporosis. Kim et al believe that RANKL induces persistent calcium concussion by mediating the reactive oxygen pathway, ultimately promoting monocyte differentiation into osteoclasts. Xu, et al, similarly suggest that osteoclast differentiation can be induced by modulating the RANKL/OPG ratio. Taken together, RANKL plays an important role in the process of osteoclast differentiation. Besides, RANKL can activate mature osteoclast, prolong the survival time of the osteoclast and enhance the bone absorption capacity. Since inhibiting RANKL by using related antibodies, peptides and natural compounds can prevent the formation and function of osteoclasts, RANKL is considered as a potential target for diagnosing and treating osteoporosis, and since RANKL is related to the occurrence and development of osteoporosis, the detection of RANKL content in serum is also the direction of research. However, no paired antibodies have been reported for clinical detection of RANKL by far.

Disclosure of Invention

In order to solve the technical problems, the application provides an antibody pair for detecting the RANKL content in serum and application thereof.

The technical scheme adopted by the application is as follows: the application provides an antibody pair for detecting the content of RANKL in serum, which comprises a monoclonal antibody AntiRANKL _ N combined with an N-terminal epitope of the RANKL, namely an amino acid sequence shown in SEQ ID NO. 2, and a monoclonal antibody AntiRANKL _ C combined with a C-terminal epitope of the RANKL, namely an amino acid sequence shown in SEQ ID NO. 3, wherein the monoclonal antibody AntiRANKL _ N comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 7 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 11, and the AntiRANKL _ C comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 17 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 21.

Further, the light chain amino acid sequence of the monoclonal antibody AntiRANKL _ N comprises an LCDR1 shown in SEQ ID NO. 4 or an amino acid sequence at least 90% homologous with the LCDR1, an LCDR2 shown in SEQ ID NO. 5 or an amino acid sequence at least 90% homologous with the LCDR2, and an LCDR3 shown in SEQ ID NO. 6 or an amino acid sequence at least 90% homologous with the LCDR 3; the amino acid sequence of the heavy chain of the monoclonal antibody AntiRANKL _ N comprises HCDR1 shown in SEQ ID NO. 8 or an amino acid sequence at least 90% homologous with the HCDR1, HCDR2 shown in SEQ ID NO. 9 or an amino acid sequence at least 90% homologous with the HCDR2, and HCDR3 shown in SEQ ID NO. 10 or an amino acid sequence at least 90% homologous with the HCDR 3.

Further, the light chain amino acid sequence of the monoclonal antibody AntiRANKL _ C comprises an LCDR1 shown in SEQ ID NO. 14 or an amino acid sequence at least 90% homologous with the LCDR1, an LCDR2 shown in SEQ ID NO. 15 or an amino acid sequence at least 90% homologous with the LCDR2, and an LCDR3 shown in SEQ ID NO. 16 or an amino acid sequence at least 90% homologous with the LCDR 3; the heavy chain amino acid sequence of the monoclonal antibody AntiRANKL _ C comprises HCDR1 shown in SEQ ID NO. 18 or an amino acid sequence at least 90% homologous with the HCDR1, HCDR2 shown in SEQ ID NO. 19 or an amino acid sequence at least 90% homologous with the HCDR2, and HCDR3 shown in SEQ ID NO. 20 or an amino acid sequence at least 90% homologous with the HCDR 3.

Furthermore, the monoclonal antibodies AntiRANKL _ N and AntiRANKL _ C are IgG type monoclonal antibodies, one is a capture antibody, and the other is a detection antibody.

Further, the AntiPF4_ N is a capture antibody, and the AntiPF4_ C is a detection antibody.

Further, the detection marker of the detection antibody is one of detection marker protein, a fluorescent group and a radioactive isotope.

Further, the detection marker protein of the detection antibody comprises horseradish peroxidase, alkaline phosphatase, luciferase and beta-galactosidase, and the detection antibody and the detection marker protein are expressed in a recombination mode.

Further, the detection method for detecting the antibody includes enzyme-linked immunosorbent assay (ELISA), chemiluminescence detection assay, western blot detection assay and Immunohistochemistry (IHC) assay.

Further, the antibody pair is an antibody fragment selected from the group consisting of Fab, F (ab ') 2, Fab', scFv and di-scFv that bind RANKL.

In a second aspect, the present application provides a DNA molecule encoding the antibody of

claim

1 or 2 or 3.

In a third aspect of the present application, there is provided an application of any one of the above antibody pairs in the preparation of a kit for detecting RANKL content in serum.

The application has the advantages and positive effects that: the RANKL antigen is divided into an N-end (SEQ ID NO:2) sequence and a C-end (SEQ ID NO:3) sequence, mice are immunized by the two sequences respectively, antibody pairs AntiRANKL _ N and AntiRANKL _ C with high specificity and high sensitivity are screened, and the content of RANKL in serum can be effectively determined by the antibody pairs, so that the diagnosis and treatment of osteoporosis are facilitated.

In addition to the technical problems addressed by the present application, the technical features constituting the technical solutions, and the advantages brought by the technical features of the technical solutions described above, other technical problems solved by the present application, other technical features included in the technical solutions, and advantages brought by the technical features will be described in further detail below.

Drawings

FIG. 1 is a schematic diagram of the detection of antibody pairs of AntiRANKL _ N and AntiRANKL _ C provided in the examples of the present application;

FIG. 2 is an electrophoretogram of pcDNA3.1-NL-LFc vector constructed as provided in the examples of the present application;

FIG. 3 is an electrophoretogram of pcDNA3.1-NH-HFc vector constructed as provided in the examples of the present application;

FIG. 4 is an electrophoretogram of the purified recombinant AntiRANKL _ N protein provided in the examples of the present application;

FIG. 5 is an electrophoretogram of pcDNA3.1-CL-LFc vector constructed as provided in the examples of the present application;

FIG. 6 is an electrophoretogram of the pcDNA3.1-CL-HFc vector constructed as provided in the examples of the present application;

FIG. 7 is an electrophoretogram of recombinant AntiRANKL _ C protein purification provided in the examples of the present application;

FIG. 8 is a schematic diagram of the verification process of the kit prepared by the antibody pair of AntiRANKL _ N and AntiRANKL _ C provided in the examples of the present application;

FIG. 9 is a first measurement curve of dose-response curve validation in performance index assessment provided in example four of the present application;

FIG. 10 is a second measurement curve of dose-response curve verification in performance index assessment provided in example four of the present application;

FIG. 11 is a third measurement curve of dose-response curve validation in performance index assessment provided in example four of the present application;

fig. 12 is a dose-response curve graph of RANKL detection by the kit prepared from the antibody of the present application and the control kit in clinical diagnosis provided by the embodiments of the present application.

Detailed Description

The present application will be described in further detail with reference to the following drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the relevant invention and not restrictive of the invention. It should be noted that, for convenience of description, only the portions related to the present invention are shown in the drawings.

It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict. The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.

The first embodiment is as follows: preparation of monoclonal antibodies AntiRANKL _ N and AntiRANKL _ C

1. The method comprises the following steps of (1) analyzing the antigenicity of the protein of RANKL (SEQ ID NO:1), dividing the RANKL antigen into two sequences of an N end (SEQ ID NO:2) and a C end (SEQ ID NO:3), respectively synthesizing an N end epitope (SEQ ID NO:2) of RANKL and a C end epitope (SEQ ID NO:3) of PF4, respectively coupling KLH, and obtaining an N end immunogen and a C end immunogen; mice were post-immunized, and were 8-week-old female Balb/c mice.

2. Animal immunization: an N-terminal immunogen and a C-terminal immunogen are adopted to immunize female Balb/C mice with age of 8 weeks respectively, and 100ug of antigen is used to immunize the mice 4 times with an interval of 4 weeks each time.

3. Cell fusion: killing the mouse by breaking the neck, placing the mouse in a 75% ethanol solution for 5-10min, taking out the spleen under aseptic conditions, extruding and slightly grinding spleen cells by using a sterilized ground glass slide, counting the spleen cells, mixing the immunized mouse spleen cells and mouse myeloma cells according to the ratio of 1:1, washing the mixture for 1 time by using a serum-free incomplete culture solution in a 50ml centrifuge tube, centrifuging the mixture for 10min at 1000rpm/min, discarding supernatant, sucking residual liquid by using a pipette (in order to avoid influencing the concentration of PEG), and slightly flicking the bottom of the centrifuge tube to ensure that cell precipitation is slightly loosened.

4. 1ml of 50% PEG (pH 8.0) preheated to 40 ℃ was added to the mixture in 60 seconds by means of a pipette while gently stirring.

5. Adding 20-30ml of preheated incomplete culture medium in 90s with a 10ml pipette (terminating PEG action); standing at 20-27 deg.C for 10 min.

6. Centrifuging at 1000r/min for 5 min, and removing supernatant;

7. adding 5ml HAT culture medium, gently sucking the precipitated cells, suspending and mixing, and supplementing HAT culture medium containing peritoneal macrophages to 80-100 ml.

8. Subpackaging 96-well cell culture plate (with feeder cell layer in the well plate) with 0.1-0.15ml per well (or subpackaging 24-well plate with 1.0-1.5ml per well), placing the culture plate at 37 deg.C and 6% CO₂In the incubatorAnd (5) nourishing.

9. After 5 days, 1/2 medium was replaced with HAT medium

10. Changing out HAT culture medium after 7-10 days;

11. and (3) frequently observing the growth condition of the hybridoma cells, sucking out supernatant when the hybridoma cells grow to a pore bottom area of 1/10 or more, detecting the activity (positive clone) of the hybridoma cells by ELISA, screening the hybridoma cells with high antibody titer, carrying out 3-4 times of subcloning to ensure that the hybridoma cells are subjected to amplification culture when the monoclonal antibody is formed, injecting ascites cells, and purifying the antibody.

Example two: the monoclonal antibody obtained in example 1 was verified

(1) Respectively coating the RANKL complete antigen (SEQ ID NO:1), the N-terminal antigen (SEQ ID NO:2) of RANKL and the C-terminal antigen (SEQ ID NO:3) of RANKL, wherein the coating concentration is 2ug/ml, the temperature is 4 ℃ for 2 hours, washing the plate for 2 times by using 0.9% NaCl washing liquid, patting to be dry, adding 200 mu l of sealing liquid (0.5mol PBS + 1% BSA + 2.5% sucrose), sealing for 2 hours at 37 ℃, pouring all liquid in the plate hole, and patting to be dry;

(2) different volumes of AntiRANKL _ N and AntiRANKL _ C obtained in example 1 (shown in Table 1) were added and reacted at 37 ℃ for 1 hour;

(3) washing the plate with 0.9% NaCl washing solution for 3 times, and drying;

(4) adding a secondary antibody of goat anti-mouse IgG marked by HRP, and reacting for 1 hour at 37 ℃;

(5) washing the plate with 0.9% NaCl washing solution for 5 times, and drying;

(6) finally, adding substrate solution luminol, measuring the luminous value of the luminol, and measuring the result as shown in table 1:

TABLE 1

As can be seen from Table 1, AntiRANKL _ N specifically binds to the N-terminal antigen of RANKL (SEQ ID NO:2) and not to the C-terminal antigen of RANKL (SEQ ID NO: 3). AntiRANKL _ C specifically binds to the C-terminal antigen of RANKL (SEQ ID NO:3) but not to the N-terminal antigen of RANKL (SEQ ID NO: 2).

Example three: sequencing of monoclonal antibodies AntiRANKL _ N and AntiRANKL _ C and preparation of corresponding antibodies by hybridoma cells

1. Total RNA of the AntiRANKL _ N hybridoma in example 1 is extracted, a cDNA synthesis reverse transcription kit is adopted, RNA is used as a template to synthesize first-strand cDNA, and then cDNA is used as a template to amplify the variable region gene of monoclonal antibody AntiRANKL _ N. And (3) carrying out T/A cloning on the variable region PCR product sequence, then selecting a positive bacterial colony for sequencing, and carrying out amino acid translation analysis on the sequencing result.

The results show that the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of AntiRANKL _ N monoclonal antibody are respectively listed in SEQ ID NO. 4, 5 and 6. In addition, the light chain variable region gene containing the variable region and the coded amino acid sequence are shown as SEQ ID NO: 12 and 7.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody kl _ N single antibody heavy chain variable region are set forth in SEQ id no:8, 9 and 10. In addition, the heavy chain variable region gene comprising the above variable region and the encoded amino acid sequence are shown in SEQ ID N0:13 and 11.

Optimizing the light chain variable region gene and the heavy chain variable region gene of the AntiRANKL _ N monoclonal antibody, cloning the light chain variable region gene into a pcDNA3.1-LFc vector, and constructing the pcDNA3.1-NL-LFc vector, as shown in figure 2; cloning the heavy chain variable region gene into pcDNA3.1-HFc vector, constructing pcDNA3.1-NH-HFc vector, as shown in FIG. 3, in this example, pcDNA3.1-LFc and pcDNA3.1-HFc already contain human light chain constant region and heavy chain constant region, co-transfecting pcDNA3.1-NL-LFc and pcDNA3.1-NH-HFc into CHO-S cell according to 1:1, recombinantly expressing anti-RANKL N-terminal antibody AntiRANKL _ N, as shown in FIG. 4, collecting antibody AntiRANKL _ N.

2. Total RNA of the AntiRANKL _ C hybridoma in example 1 is extracted, a cDNA synthesis reverse transcription kit is adopted, RNA is used as a template to synthesize first-strand cDNA, and then cDNA is used as a template to amplify the variable region gene of the monoclonal antibody AntiRANKL _ C. And (3) carrying out T/A cloning on the variable region PCR product sequence, then selecting a positive bacterial colony for sequencing, and carrying out amino acid translation analysis on the sequencing result.

The results showed that the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region of AntiRANKL _ C monoclonal antibody are shown in SEQ ID NO:14, 15 and 16, respectively. In addition, the light chain variable region gene containing the variable region and the coded amino acid sequence are shown as SEQ ID NO: 22 and 17.

The amino acid sequences of HCDR1, HCDR2 and HCDR3 of the antibody kl _ C single antibody heavy chain variable region are set forth in SEQ id no:18, 19 and 20. In addition, the heavy chain variable region gene comprising the above variable region and the encoded amino acid sequence are shown in SEQ ID Nos. 0:23 and 21.

Optimizing the light chain variable region gene and the heavy chain variable region gene of the AntiRANKL _ C monoclonal antibody, cloning the light chain variable region gene into a pcDNA3.1-LFc vector, and constructing the pcDNA3.1-CL-LFc vector, as shown in FIG. 5; cloning the heavy chain variable region gene into pcDNA3.1-HFc vector, constructing pcDNA3.1-CH-HFc vector, as shown in FIG. 6, in this example, pcDNA3.1-LFc and pcDNA3.1-HFc already contain human light chain constant region and heavy chain constant region, co-transfecting pcDNA3.1-CL-LFc and pcDNA3.1-NH-CFc into CHO-S cell according to 1:1, recombinantly expressing anti-RANKL C end antibody AntiRANKL _ C, as shown in FIG. 7, collecting antibody AntiRANKL _ C.

Example four: application of AntiRANKL _ N and AntiRANKL _ C antibody pair in chemiluminescence method diagnostic kit

According to the existing production process of a chemiluminescence method diagnostic reagent, the AntiRANKL _ C obtained in the embodiment 3 is used as a capture antibody to be coated to form an RANKL coated plate, the AntiRANKL _ N mark obtained in the embodiment 3 is used as a binding antibody to be coated to form an RANKL enzyme conjugate, the RANKL antigen is prepared into a series of calibrators (0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 400pg/ml and 800pg/ml) to form a complete kit, and a series of verifications are carried out on the kit.

1. Performance verification index

By referring to the technical requirements of the existing RANKL kit in the market, a performance verification index for the RANKL detection reagent is formulated.

(1) Minimum limit of detection

Should not be higher than 40 pg/ml.

(2) Linearity of dose-response curve

Within the linear range of 0 pg/ml-800 pg/ml, the linear correlation coefficient (r) of the dose-response curve should be not less than 0.9900.

(3) Accuracy of

The recovery rate detected by the kit should be in the range of 0.85-1.15.

(4) Precision degree

Precision (CV) should be no greater than 10%.

2. Experimental configuration

(1) RANKL coated plate configuration

AntiRANKL _ C was added to the coating buffer (0.05mol PBS) as capture antibody at a ratio of 2 ug/ml. Adding 100ul of the buffer solution into each blank luminescent plate hole, standing overnight at 4 ℃, taking out, washing for 2 times by using a plate washing solution (0.9% NaCl), adding 200ul of a confining solution (0.5mol PBS + 1% BSA + 2.5% sucrose) into each hole, standing overnight at 4 ℃, taking out, discarding the liquid in the plate, and drying to prepare the RANKL coated plate.

(2) RANKL enzyme conjugate configurations

A. The enzyme bound to the detection antibody is one of horseradish peroxidase, alkaline phosphatase, luciferase and beta-galactosidase.

B. In this example, the antibody rankl _ N was labeled with HRP, 1mg/ml antibody rankl _ N was taken and activated with acetic acid solution for 1mgHRP, and the antibody and HRP were mixed in a ratio of 1:1, reversing and mixing uniformly, and adding sodium borohydride to terminate the reaction after 2 hours to obtain the AntiRANKL _ N-HRP conjugated antibody.

C. AntiRANKL _ N-HRP was added as a conjugated antibody to an enzyme diluent (50Mmol Tris + 1% BSA) at 1/1000.

(3) Calibrator arrangement

The RANKL antigen was diluted in six gradients (0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 400pg/ml, 800pg/ml) with antigen diluent (0.5mol PBS + 1% BSA).

(4) Recovery rate standard solution preparation

The RANKL antigen is diluted to 600pg/L by using an antigen diluent (0.5mol PBS + 1% BSA) to serve as a standard solution, and 30-40pg/L of normal human serum is selected to serve as a low-value sample.

(5) Quality control product configuration

The RANKL antigen was diluted with antigen diluent (0.5mol PBS + 1% BSA) to QC1(150 pg/ml. + -. 10%), QC2(650 pg/ml. + -. 10%).

3. Sample application operation, as shown in FIG. 8

(1) Respectively taking 50 mu l of each of the configured RANKL calibrator, the recovery rate sample and the quality control product (3) to (5) and adding the RANKL calibrator, the recovery rate sample and the quality control product into the corresponding coated plate hole (1) of the configuration, then taking 50 mu l of the configured RANKL enzyme conjugate (2) and adding the RANKL enzyme conjugate into the corresponding coated plate hole (1) of the configuration, and oscillating the RANKL enzyme conjugate for 30 seconds by an oscillator to fully and uniformly mix the RANKL calibrator, the recovery rate sample and the quality control product;

(2) covering a sealing plate film, and incubating for 60 minutes at 37 ℃;

(3) taking out, washing the plate 5 times with an application washing solution (0.5mol PBS + 0.025% T-20);

(4) 100ul of substrate solution (purchased from ThermoFisher T2142) was added to each well;

(5) chemiluminescence intensity (RLU) was measured using a Zhongshengbuck BHP9504 chemiluminescence apparatus.

4. Performance index assessment

(1) Minimum limit of detection

The relative luminescence intensity of 20-well zero calibrators (0pg/ml) was measured in parallel, the mean value and standard deviation SD of the luminescence values were calculated, and the corresponding concentration value when the mean value +2 × SD of the luminescence values was calculated from the dose-response curve, which was the lowest detection limit, and the results are shown in table 2.

TABLE 2

As can be seen from Table 2, the minimum detection limit was 15.07pg/ml, which was not higher than 40pg/ml, and the detection was satisfactory.

(2) Linearity of dose-response curve

The reference samples (S0-S5, concentration of 0pg/ml, 50pg/ml, 100pg/ml, 200pg/ml, 400pg/ml and 800pg/ml) of the double-well assay kit are determined in parallel three times, the dose-response curve of the kit is fitted by a four-parameter method, and the dose-response curve of the three times of determination is shown in figure 9, figure 10 and figure 11. Measured dose-response curvesLinear correlation coefficient (R)²) As shown in table 3.

TABLE 3

	First measurement	Second measurement	The third measurement
				Linear correlation coefficient R²	0.9991	0.9996	0.9996

As can be seen from FIGS. 9, 10 and 11, and Table 3, the RANKL antigen curve of the concentration range of 0-800pg/ml was measured three times in succession, and the linear correlation coefficient R was determined²Are all larger than 0.9990, which meets the index.

(3) Accuracy of

Preparing a standard solution (600pgl/L) from the RANKL antigen according to a volume ratio of 1: 9 were added to low value samples of known concentration (30-40pg/L), tested on the kit and the recovery R was calculated according to the following formula, as shown in Table 4.

In the formula: r-recovery rate;

v-volume of standard solution added;

V₀-volume of low value samples;

c is the detection concentration of the low-value sample after being added into the standard solution;

C₀-the detected concentration of the low value sample;

cs-concentration of standard solution.

TABLE 4

CS(pmol/L)	C0(pmol/L)	Sample C luminescence value	C(pmol/L)	Recovery (%)
					600	32.9	136839	91.325	102.9

As can be seen from Table 4, the accuracy of the accuracy specimen prepared by the RANKL international standard substance is continuously detected for three times, and the result accuracy is within the range of 95-105%, and meets the index within the requirement of 90-110%.

(4) Precision degree

Each 10 wells of the quality control Q1 and the quality control Q2 were measured in parallel, and CV was calculated according to the following equation, and the results are shown in Table 5.

In the formula:

average value of concentration values determined by quality control

SD-Standard deviation of concentration value measured by quality control Material

TABLE 5

As can be seen from Table 5, the results of parallel measurements of QC1 and QC2 performed three times in succession, both CV (%) were not greater than 10% and met the criteria.

In the embodiment, the performance indexes of the RANKL detection reagent such as the minimum detection limit, the dose-response curve, the accuracy and the precision meet the requirements of the prior art and are superior to the existing methodology reagent in the market.

Example five: clinical testing

40 human serum samples were randomly selected, and the antibodies of the present application were used to prepare kits and control RANKL kits for detection, with the results shown in Table 6.

TABLE 6

And dose-response curves were plotted according to table 6, as shown in figure 12.

As shown in FIG. 12, the correlation R between the detection concentration results of the kit prepared based on the antibody of the present application and the control kit (purchased from Kyoto Kanganzi Biotech Co., Ltd.) is shown²The value is 0.9969, which shows that the detection result of the kit prepared by the antibody pair of the application is consistent with that of a control kit.

The embodiments of the present invention have been described in detail, but the description is only for the preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the application of the present invention should fall within the scope of the patent coverage of the present invention.

Sequence listing

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Ser

<210>11

<211>123

<212>PRT

<213> mouse (Mus musculus)

<400>11

Gln Val Gln Leu Gln Gln Pro Gly Ser Val Leu Val Arg Pro Gln Ser

1 5 10 15

Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser Trp

20 25 30

Met His Trp Ala Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

35 40 45

Glu Ile His Pro Asn Ser Gly Asn Thr Asn Tyr Ser Asp Lys Phe Lys

50 55 60

Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Val

65 70 75 80

Asp Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Lys

85 90 95

Ser Ser Glu Ser Ile Leu Tyr Ser Asn Lys Leu Thr Thr Val Val Ser

100 105 110

Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

115 120

<210>12

<211>10

<212>PRT

<213> mouse (Mus musculus)

<400>12

Lys Ser Val Ser Thr Ser Gly Tyr Ser Tyr

1 5 10

<210>13

<211>3

<212>PRT

<213> mouse (Mus musculus)

<400>13

Leu Val Ser

1

<210>14

<211>12

<212>PRT

<213> mouse (Mus musculus)

<400>14

Gln His Ile Arg Glu Leu Thr Arg Ser Glu Gly Gly

1 5 10

<210>15

<211>111

<212>PRT

<213> mouse (Mus musculus)

<400>15

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Tyr Arg Ala Ser Lys Ser Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Ile His Trp Asn Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Val Ser Asn Leu Glu Ser Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln His Ile Arg

85 90 95

Glu Leu Thr Arg Ser Glu Gly Gly Pro Ser Trp Lys Ser Asn Val

100 105 110

<210>16

<211>8

<212>PRT

<213> mouse (Mus musculus)

<400>16

Gly Phe Thr Phe Ser Ser Tyr Ala

1 5

<210>17

<211>7

<212>PRT

<213> mouse (Mus musculus)

<400>17

Ile Asn Ser Gly Gly Arg Thr

1 5

<210>18

<211>14

<212>PRT

<213> mouse (Mus musculus)

<400>18

Ala Arg Val Pro Thr Ala Thr Glu Gly Tyr Ser Met Asp Tyr

1 5 10

<210>19

<211>120

<212>PRT

<213> mouse (Mus musculus)

<400>19

Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met Thr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Ser Gly Gly Arg Thr Phe Tyr Pro Gly Ser Val Arg

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu

65 70 75 80

Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala

85 90 95

Arg Val Pro Thr Ala Thr Glu Gly Tyr Ser Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210>20

<211>336

<212>DNA

<213> mouse (Mus musculus)

<400>20

tccatcgtga tgactcagac accaaagttc ctcttggtgt ccgccggtga ccgcgtgaca 60

atcacatgca agcccagcca gagcctggtg agtagcaacg gcaacacata cctggcttgg 120

tatcagcaaa aacctggaca gtccccaaaa ctgctgatct actacgctag caaccgatac 180

actggagtgc cagaccggtt cactggtagt ggttatggca ccgacttcac ctttactatt 240

agcaccgtcc aggctgagga tctggctgta tatttctgtc agcaggacta ctcttcccca 300

cttacattcg gcgctgggac aaagctggaa ctcaag 336

<210>21

<211>369

<212>DNA

<213> mouse (Mus musculus)

<400>21

caagtccaac ttcaacagcc tggctctgta ctggttagac cacagtctgt gaagctttca 60

tgtaaggcct caggttatac attcacaagc tcttggatgc actgggcaaa acagagacca 120

ggtcagggtc tggagtggat cggagaaatt caccccaatt cagggaatac taattacagc 180

gacaagttca aaggtaaggc cactttgacc gtggacaaaa gtagcagcac tgcttacgtg 240

gatctcagct ccctgacctc tgaagactct gctgtctatt attgtaagtc atccgaatcc 300

atcctctata gtaacaagtt gactactgtc gttagctggg gtcagggtac cacactgact 360

gtgtctagc 369

<210>22

<211>333

<212>DNA

<213> mouse (Mus musculus)

<400>22

gatatcgtac tgactcagtc tccagcatct ttggccgtga gtctgggcca acgtgctaca 60

attagctacc gggcaagtaa gtctgtctct acctcagggt atagttatat tcattggaac 120

cagcaaaagc caggccagcc tccccgactg ctgatctacc tggtgagcaa cctcgaaagc 180

ggcgttcctg ctaggttttc cggctctggg tctggcaccg actttacatt gaatattcac 240

cccgtcgaag aggaggatgc tgctacctac tattgtcagc atattagaga gctgacaaga 300

tctgaaggtg gacctagttg gaagtctaac gtg 333

<210>23

<211>360

<212>DNA

<213> mouse (Mus musculus)

<400>23

gaggtcaaac ttgtagaaag cggaggggga ctggtgaaac ctggtggcag cctgaaactg 60

agctgcgcca cctcaggctt cactttttct agctatgcta tgacatgggt tcgccagaca 120

cccgagaaac gcctggagtg ggtggcatct atcaactccg gaggccgcac cttttaccca 180

gggagtgtcc gcgggcgttt cacaatatct agagataacg ctcgcaatat tctgtatctg 240

cagatgagct ctctgaggtc cgaggacact gccatgtatt attgtgccag agtgcctacc 300

gccactgaag gatactccat ggactattgg ggccagggga ccagcgtgac agtctctagc 360

Claims

1. An antibody pair for detecting the content of RANKL in serum is characterized by comprising a monoclonal antibody AntiRANKL _ N combined with an N-terminal epitope of RANKL, namely an amino acid sequence shown in SEQ ID NO. 2, and a monoclonal antibody AntiRANKL _ C combined with a C-terminal epitope of RANK, namely an amino acid sequence shown in SEQ ID NO. 3, wherein the monoclonal antibody AntiRANKL _ N comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 7 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 11, and the AntiRANKL _ C comprises a light chain variable region of the amino acid sequence shown in SEQ ID NO. 17 and a heavy chain variable region of the amino acid sequence shown in SEQ ID NO. 21.

2. The antibody pair for detecting the RANKL content in serum of claim 1, wherein the light chain amino acid sequence of the monoclonal antibody AntiRANKL _ N comprises LCDR1 shown in SEQ ID NO. 4, LCDR2 shown in SEQ ID NO. 5 and LCDR3 shown in SEQ ID NO. 6; the heavy chain amino acid sequence of the monoclonal antibody AntiRANKL _ N comprises HCDR1 shown in SEQ ID NO. 8, HCDR2 shown in SEQ ID NO. 9 and HCDR3 shown in SEQ ID NO. 10.

3. The antibody pair for detecting the RANKL content in serum of claim 2, wherein the light chain amino acid sequence of the monoclonal antibody AntiRANKL _ C comprises LCDR1 shown in SEQ ID NO. 14, LCDR2 shown in SEQ ID NO. 15 and LCDR3 shown in SEQ ID NO. 16; the heavy chain amino acid sequence of the monoclonal antibody AntiRANKL _ C comprises HCDR1 shown in SEQ ID NO. 18, HCDR2 shown in SEQ ID NO. 19 and HCDR3 shown in SEQ ID NO. 20.

4. The antibody pair for detecting the content of RANKL in serum according to claim 3, wherein the monoclonal antibodies AntiRANKL _ N and AntiRANKL _ C are IgG type monoclonal antibodies, one is a capture antibody and the other is a detection antibody.

5. The antibody pair for detecting the level of RANKL in serum according to claim 4, wherein the AntiRANKL _ N is a capture antibody and the AntiRANKL _ C is a detection antibody.

6. The antibody pair for detecting the RANKL content of serum of claim 4, wherein the detectable label of the detectable antibody is one of a detectable labeled protein, a fluorophore, and a radioisotope.

7. The antibody pair for detecting the RANKL content of serum of claim 6, wherein the detection marker proteins of the detection antibody comprise horseradish peroxidase, alkaline phosphatase, luciferase and β -galactosidase, and wherein the detection antibody is recombinantly expressed with the detection marker proteins.

8. The antibody pair for detecting the RANKL content of serum of claim 7, wherein said antibody is detected by a method comprising an enzyme linked immunosorbent assay, a chemiluminescent assay, a western blot assay, or an immunohistochemical assay.

9. An antibody pair for detecting RANKL content in serum as claimed in claim 1, wherein said antibody pair is selected from the group consisting of antibody fragments of Fab, F (ab ') 2, Fab', scFv and di-scFv that bind RANKL.

10. A DNA molecule encoding the antibody of claim 1 or 2 or 3.

11. Use of the antibody of claims 1-10 for the preparation of a kit for the detection of RANKL content in serum.