CN113621060B

CN113621060B - OPG antibody pair and application thereof

Info

Publication number: CN113621060B
Application number: CN202010376562.1A
Authority: CN
Inventors: 吴迪; 楼炜; 程勇
Original assignee: Zhejiang Ruishuo Biotechnology Co ltd
Current assignee: Zhejiang Ruishuo Biotechnology Co ltd
Priority date: 2020-05-07
Filing date: 2020-05-07
Publication date: 2023-07-04
Anticipated expiration: 2040-05-07
Also published as: CN113621060A

Abstract

The invention relates to the field of biological detection, and discloses an OPG antibody pair and application thereof. An OPG antibody pair comprising a capture antibody and a detection antibody, the light chain of the capture antibody comprising three nucleotide sequences of SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and the heavy chain of the capture antibody comprising three nucleotide sequences of SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10; the light chain of the detection antibody comprises three nucleotide sequences of SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, and the heavy chain of the capture antibody comprises three nucleotide sequences of SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20. The invention solves the problems of larger OPG antigen and larger antigen preparation difficulty in the prior art.

Description

OPG antibody pair and application thereof

Technical Field

The invention relates to the field of biological detection, in particular to an OPG antibody pair and application thereof.

Background

Osteoporosis is an age-related chronic systemic metabolic disease characterized primarily by reduced bone mass, damaged bone microstructure, and increased bone fragility. The disease has become a global public health problem due to its increased morbidity and mortality. Currently, the world population with osteoporosis has more than 10.2 billion, the number is expected to rise to 13.6 billion in 2030, fracture patients caused by osteoporosis will reach 289 000, and the economic burden of each year is as high as billions. Korean statistics in 2011 show: of 50% of people over 50 years old suffer from osteoporosis, 70% are postmenopausal women. The situation in China is also optimistic, and the research taking-2.0 SD as a diagnosis standard in 2016 shows that: the population of osteoporosis over 40 years of age nationwide is about 1.4 hundred million, accounting for 24.62% of the total population. In view of the high incidence of osteoporosis and its serious consequences, intensive research into its pathogenesis has been conducted.

OPG is the first protein found on the OPG/RANK/OPG signal pathway, and OPG was found in the random clone sequencing process of mice in 1997 by Simonet et al. OPG was then shown to be a tumor necrosis factor receptor consisting of 7 domains and 3 functional regions, the N-terminal D1-D4 region of which is directly involved in inhibiting osteoclast action, the D5 and D6 regions mediating cytotoxicity, and the D7 region being essential for maintaining its function. OPG can bind to two ligands, namely: the key factor OPG in the process of osteoclast differentiation and TNF related apoptosis inducing ligand TRAIL in immune monitoring system. Therefore, as pseudo-ligands for OPG and TRAIL, OPG can inhibit apoptosis induced by TRAIL in addition to inhibiting binding of OPG to RANK, blocking osteoblast-induced differentiation of osteoclast precursor cells, and regulating osteoclast function [6 ]. The research shows that OPG can induce the disassembly of the pseudo-feet of the osteoclast through Ca-p38-MAPK signal path and other ways, thereby protecting the cortical bone. Liu et al believe that OPG can induce apoptosis of osteoclasts and osteoclast precursor cells via the Fas/FasL pathway. In animal experiments, researchers found that highly expressed OPG stiffened bone and the bone marrow cavity disappeared, while distinct trabeculae were also visible in the cartilage tissue that was not fully resorbed. High concentrations of OPG have an important role in maintaining bone mass in human trials.

Disclosure of Invention

In order to solve the problems of large OPG antigen and large antigen preparation difficulty in the prior art, the invention provides an OPG antibody pair and application thereof.

The invention adopts the following technical scheme: an OPG antibody pair comprising a capture antibody and a detection antibody, the light chain of the capture antibody comprising three nucleotide sequences of SEQ ID No. 4, SEQ ID No. 5, SEQ ID No. 6, and the heavy chain of the capture antibody comprising three nucleotide sequences of SEQ ID No. 8, SEQ ID No. 9, SEQ ID No. 10; the light chain of the detection antibody comprises three nucleotide sequences of SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, and the heavy chain of the capture antibody comprises three nucleotide sequences of SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20.

The capture antibody comprises a light chain variable region with an amino acid sequence of SEQ ID NO. 7 and a heavy chain variable region with an amino acid sequence of SEQ ID NO. 11.

The detection antibody comprises a light chain variable region with an amino acid sequence of SEQ ID NO. 17 and a heavy chain variable region with an amino acid sequence of SEQ ID NO. 21.

The detection label of the detection antibody is an enzyme, a fluorescent group or a radioisotope.

The capture antibody and the detection antibody are IgG type monoclonal antibodies.

The capture antibody and the detection antibody are any one antibody of Fab, F (ab ') 2, fab', scFv or di-scFv.

The OPG antibody pair is expressed in a recombinant way with a detection marker protein.

The detection marker protein is selected from any one of root peroxidase, alkaline phosphatase, luciferase or beta-galactosidase.

The OPG antibody pair detects OPG content in a biological sample by an immunological assay.

In another aspect, the invention provides a kit comprising said OPG antibody pair.

In a further aspect, the invention provides the use of said antibody pair in the detection of OPG content.

The invention adopts a double antibody sandwich method, the N end and the C end of an antigen are respectively immunized with a mouse to obtain a paired antibody, the OPG antigen is divided into two sequences of the N end (SEQ ID NO: 2) and the C end (SEQ ID NO: 3), the two sequences are respectively immunized with the mouse, and the OPG antibody pair with high specificity and high sensitivity is screened. The capture antibody binds to the N-terminal epitope of OPG and the detection antibody binds to the C-terminal epitope of OPG. The invention overcomes the technical resistance of the prior art that the antigen preparation difficulty is large, and has the advantages of rapidness and accuracy.

Drawings

FIG. 1 is a diagram showing the construction result of pcDNA3.1-NH-HFc vector.

FIG. 2 is a diagram showing the construction result of pcDNA3.1-NL-LFC vector.

FIG. 3 is a recombinant capture antibody protein purification profile.

FIG. 4 is a diagram showing the construction result of pcDNA3.1-CH-HFc vector.

FIG. 5 is a diagram showing the construction result of pcDNA3.1-CL-LFC vector.

FIG. 6 is a purification map of recombinant detection antibody protein.

FIG. 7 is a linear graph of the concentration results of the assay of the present invention versus the concentration of the control kit.

Wherein: lane M: DNA Marker in FIG. 1; lane 1: pcDNA3.1- -HFc Plasmid; lane 2:NH; lane M: DNA Marker in FIG. 2; lane 1: pcDNA3.1-NL-LFC Plasmid; lane 2:NL; lane M in FIG. 3: a Marker; lane 1: capturing the antibody; lane M: DNA Marker in FIG. 4; lane 1: pcDNA3.1-CH-HFc Plasmid; lane 2:CH; lane M: DNA Marker in FIG. 5; lane 1: pcDNA3.1-CL-LFC Plasmid; lane 2, CL; lane M in FIG. 6: a Marker; lane 1: the antibodies were detected in pg/ml on the abscissa and RLU on the ordinate in FIG. 7

Detailed Description

Antibody preparation (hybridoma)

a) The N-terminal epitope of OPG (SEQ ID NO: 2) and the C-terminal epitope of OPG (SEQ ID NO: 3) were synthesized separately, and the mice were immunized after coupling KLH, respectively, and the mice were female Balb/C mice of 8 weeks of age.

b) The immunization method comprises the following steps: 100ug of antigen was immunized 4 times, 4 weeks apart.

c) Cell fusion: the immunized mouse spleen cells and the mouse myeloma cells are mixed together according to the proportion of 1:1, the mixture is washed 1 time by serum-free incomplete culture solution in a 50ml centrifuge tube, the mixture is centrifuged at 1000rpm/min for 10 minutes, the supernatant is discarded, residual liquid is sucked by a suction tube (in order to avoid influencing the concentration of PEG), and the bottom of the centrifuge tube is gently flicked to slightly loosen cell sediment.

d) 1ml of 50% PEG (pH 8.0) preheated to 40℃was added with gentle stirring over 60s using a pipette.

e) 20-30ml of pre-warmed incomplete medium (stopping PEG action) was added over 90s with a 10ml pipette; standing at 20-27deg.C for 10 min.

f) Centrifuging at 1000r/min for 5 min, and discarding supernatant

g) 5ml of HAT medium was added, the precipitated cells were gently aspirated, suspended and mixed well, and then the HAT medium containing macrophages in the abdominal cavity was added to 80-100ml.

h) Packing 96-well cell culture plate (feeder cell layer in the well plate) with 0.1-0.15ml per well (or packing 24-well plate with 1.0-1.5ml per well), and placing the culture plate at 37deg.C and 6% CO ₂ Culturing in an incubator.

i) After 5 days 1/2 medium was exchanged with HAT medium

j) Exchanging the HT medium for 7-10 days;

k) The growth of hybridoma cells is frequently observed, and when the hybridoma cells grow to more than 1/10 of the area of the bottom of the well, the supernatant is aspirated and the activity is detected by ELISA. Clones of pure effective titers were finally obtained by multiple rounds of screening and limiting dilution.

The obtained cloned antibodies were screened for N-terminal antigen (SEQ ID NO: 2) and C-terminal antigen (SEQ ID NO: 3), respectively. Finally, the best-pair antibody pair of the capture antibody and the detection antibody is obtained, wherein the capture antibody can specifically bind to the N-terminal antigen (SEQ ID NO: 2) of OPG, but not to the C-terminal antigen (SEQ ID NO: 3) of OPG. The detection antibody specifically binds to the C-terminal antigen of OPG (SEQ ID NO: 3) but does not bind to the N-terminal antigen of OPG (SEQ ID NO: 2).

OPG holoantigen (SEQ ID NO: 1) was coated and verified by chemiluminescence.

The N-terminal antigen of OPG (SEQ ID NO: 2) was coated and detected by chemiluminescence.

The C-terminal antigen of OPG (SEQ ID NO: 3) was coated and detected by chemiluminescence.

Coating OPG holoantigen (SEQ ID NO: 1), OPG N-terminal antigen (SEQ ID NO: 2) and OPG C-terminal antigen (SEQ ID NO: 3) respectively, adding capture antibodies and detection antibodies with different volumes, adding secondary antibodies for reaction, and finally adding substrate solution, and detecting the luminescence values, wherein the results are shown in the following table:

the above table confirms that the capture antibodies in the antibody pairs of the invention are able to recognize the N-terminal antigen and not the C-terminal antigen. The detection antibodies in the antibody pair of the invention can recognize the C-terminal antigen and can not recognize the N-terminal antigen.

After sequencing hybridomas of the capture antibodies in the obtained antibody pairs, the light chain variable region sequence (SEQ ID NO: 7) and the heavy chain variable region (SEQ ID NO: 11) of the antibody capture antibodies are obtained. After optimizing the sequence, the light chain is cloned into pcDNA3.1-LFC vector to obtain pcDNA3.1-NL-LFC. The heavy chain was cloned into pcDNA3.1-HFc vector to obtain pcDNA3.1-NH-HFc. The pcDNA3.1-LFC and pcDNA3.1-HFc already contain human light chain constant regions and heavy chain constant regions. Wherein the nucleic acid sequence of NL (light chain) is SEQ ID NO. 12 and the nucleic acid sequence of NH (heavy chain) is SEQ ID NO. 13. pcDNA3.1-NL-LFC and pcDNA3.1-NH-HFc were co-transfected into CHO-S cells at 1:1, and the N-terminal antibody capture antibody against OPG was recombinantly expressed.

After sequencing hybridomas of detection antibodies in the obtained antibody pairs, the light chain variable region sequence (SEQ ID NO: 17) and the heavy chain variable region sequence (SEQ ID NO: 21) of the antibody detection antibody are obtained. After optimizing the sequence, the light chain is cloned into pcDNA3.1-LFC vector to obtain pcDNA3.1-CL-LFC. The heavy chain was cloned into pcDNA3.1-HFc vector to obtain pcDNA3.1-CH-HFc. The pcDNA3.1-LFC and pcDNA3.1-HFc already contain human light chain constant regions and heavy chain constant regions. Wherein the nucleic acid sequence of CL (light chain) is SEQ ID NO. 22, and the nucleic acid sequence of NH (heavy chain) is SEQ ID NO. 23. pcDNA3.1-CL-LFC and pcDNA3.1-CH-HFc were co-transfected into CHO-S cells at 1:1, and the C-terminal antibody detection antibody against OPG was expressed recombinantly.

The preparation and verification of the antibody pair participation sandwich method chemiluminescence kit are as follows:

according to the existing chemiluminescent diagnostic reagent production process, the detection antibody of the invention is used as a capture antibody to be coated to be prepared into an OPG coated plate, the capture antibody label of the invention is used as a binding antibody to be prepared into an OPG enzyme conjugate, and the OPG antigen is prepared into a series of calibration products (0 pg/ml, 12.5pg/ml, 25pg/ml, 50pg/ml, 100pg/ml and 200 pg/ml) to form a complete kit and carry out a series of verification.

Referring to the technical requirements of the existing OPG kit in the market, the performance verification index of the OPG detection reagent is formulated

1. Performance index

1.1 minimum detection limit

Should not be higher than 10pg/ml.

1.2 linearity of dose-response curve

The linear correlation coefficient (r) of the dose-response curve should be not less than 0.9900 in the linear range of 0pg/ml to 200 pg/ml.

1.3 accuracy

The recovery rate is detected by the kit and is in the range of 0.85-1.15.

1.4 precision

Precision (CV) should be not more than 10%.

2. Inspection method

2.1 minimum detection limit

Relative luminescence intensity of 20-well zero calibrator (0 pg/ml) was measured in parallel, and the luminescence value average was calculated

And standard deviation SD, the luminescence value was calculated as +.>

The corresponding concentration value is the lowest detection limit, and the result meets the requirement of 1.1.

2.2 linearity of dose-response curve

The reference substances (S0-S5, the concentrations are 0pg/ml, 12.5pg/ml, 25pg/ml, 50pg/ml, 100pg/ml, 200 pg/ml) of the compound well measuring kit are sequentially subjected to fitting by four parameters, and the correlation coefficient (r) of the dose-response curve meets the requirement of 1.2.

2.3 accuracy

OPG antigen was formulated as standard solution (150 pgl/L) at a volume ratio of 1:9 is added to a low value sample (5-10 pg/L) of known concentration, the detection is performed on the kit, and the recovery rate R is calculated according to the formula (1), and the result is in accordance with the regulation of 1.3.

Wherein: r is the recovery rate;

v-the volume of added standard solution;

V ₀ -a volume of low value sample;

c, detecting concentration of the low-value sample after the low-value sample is added into the standard solution;

C ₀ -detection concentration of low value samples;

cs—concentration of standard solution.

2.4 precision

And measuring 10 holes of each of the quality control product Q1 and the quality control product Q2 in parallel, and calculating CV according to a formula respectively, wherein the result meets the specification of 1.4.

Wherein:

average value of measured concentration value of quality control material

Standard deviation of SD-quality control material measured concentration value

3. Experimental configuration

3.1OPG coated plate configuration

The detection antibody was used as capture antibody and added to the coating buffer (0.05 mol PBS) at a rate of 1 ug/ml. 100ul of each blank light-emitting plate hole is added overnight at 4 ℃, taken out, washed 2 times with plate washing liquid (0.9% NaCl), 200ul of blocking liquid (0.5mol PBS+1%BSA+2.5% sucrose) is added to each hole, taken out overnight at 4 ℃, the liquid in the plate is discarded, and the OPG coated plate is prepared after drying.

3.2OPG enzyme conjugate configuration

The capture antibody was added as a binding antibody to the enzyme dilution (50Mmol Tris+1%BSA) at a ratio of 1/1500.

3.3 calibration Material configuration

OPG antigen was diluted with antigen diluent (0.5 mol PBS+1% BSA) for 6 gradients (0 pg/ml, 12.5pg/ml, 25pg/ml, 50pg/ml, 100pg/ml, 200 pg/ml).

3.4 recovery Standard solution configuration

OPG antigen was diluted to 150pg/L with antigen diluent (0.5 mol PBS+1% BSA) and 5-10pg/L human serum was used as a standard solution and as a low-value sample.

3.5 quality control article configuration

OPG antigen was diluted with antigen diluent (0.5 mol PBS+1% BSA) to QC1 (20 pg/ml.+ -. 10%), QC2 (170 pg/ml.+ -. 10%).

4. Sample application operation

1. Respectively taking the 3.3-3.5OPG calibrator, the accuracy specimen, the quality control product and 50 mu L of each of the quality control products, adding the 3.3-3.5OPG calibrator and the quality control product into the holes of the corresponding coating plate of the 3.1OPG coating plate, and then taking 50 mu L of the 3.2OPG enzyme conjugate, adding the 3.2OPG enzyme conjugate into each hole oscillator, oscillating for 30 seconds, so that the mixture is fully and uniformly mixed.

2. The membrane was covered and incubated at 37℃for 60 minutes.

3. The plate was removed and washed 5 times with application of wash solution (0.5mol PBS+0.025%T-20).

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

5. Chemiluminescent intensity (RLU) was measured using a well-known hundred gram BHP9504 chemiluminescent instrument.

5. Performance index validation results

5.1 minimum detection limit

The lowest detection limit is 3.706pg/ml, which is not higher than 10pg/ml and meets the index.

5.2 linearity of dose-response curve

	First time measurement	Second measurement	Third measurement
				Linear correlation coefficient R	0.9997	0.9996	0.9997

And (3) continuously measuring the OPG antigen curve with the concentration range of 0-200pg/ml for three times, wherein the linear correlation coefficient r is larger than 0.9990, and the OPG antigen curve meets the index.

5.3 accuracy

And detecting the OPG accuracy specimen, wherein the accuracy of the result is within the range of 95-105% and within the requirement of 90-110%, and the OPG accuracy specimen meets the index.

5.4 precision

And the result of continuous three-time parallel measurement of QC1 QC2 is not more than 10%, and meets the index.

The performance indexes such as the accuracy, the uniformity and the like of the dose-response curve of the OPG kit meet the requirements of the prior art.

10 serum was selected and tested using the kit and control OPG kit, and the correlation was compared as follows.

As can be seen from FIG. 7, the correlation R between the concentration detection result of the present invention and the concentration detection result of the control kit ² 0.9980.

Amino acid sequence listing and CDR regions

OPG sequence (SEQ ID NO: 1)

MNNLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQLLCDKCPPGTYLKQHCTAKWKT VCAPCPDHYYTDSWHTSDECLYCSPVCKELQYVKQECNRTHNRVCECKEGRYLEIEFCL KHRSCPPGFGVVQAGTPERNTVCKRCPDGFFSNETSSKAPCRKHTNCSVFGLLLTQKG NATHDNICSGNSESTQKCGIDVTLCEEAFFRFAVPTKFTPNWLSVLVDNLPGTKVNAESV ERIKRQHSSQEQTFQLLKLWKHQNKDQDIVKKIIQDIDLCENSVQRHIGHANLTFEQLRSL MESLPGKKVGAEDIEKTIKACKPSDQILKLLSLWRIKNGDQDTLKGLMHALKHSKTYHFPK TVTQSLKKTIRFLHSFTMYKLYQKLFLEMIGNQVQSVKISCL

N-terminal epitope of OPG (SEQ ID NO: 2)

MNNLLCCALVFLDISIKWTTQETFPPKYLHYDEETSHQ

C-terminal epitope of OPG (SEQ ID NO: 3)

HSKTYHFPKTVTQSLKKTIRFLHSFTMYKLYQKLFLEMIGNQVQSVKISCL

Capture antibodies

Light chain CDR regions

LCDR1(SEQ ID NO:4)QGIYNY

LCDR2(SEQ ID NO:5)YTS

LCDR3(SEQ ID NO:6)QQYSKFPWT

Light chain variable region sequence (SEQ ID NO: 7):

DIQMTQTTSSLSASLGDRVTISCSASQGIYNYLNWFQQKPDGTVKLLIYYTSSLHSGVPSR FSGSGSGTDFSLTISNLEPEDIATYYCQQYSKFPWTFGGGTNVEIK

heavy chain CDR regions

HCDR1(SEQ ID NO:8)GYTFTDYS

HCDR2(SEQ ID NO:9)IHTETGEP

HCDR3(SEQ ID NO:10)GRSFSYGNLRAMDY

Heavy chain variable region sequence (SEQ ID NO: 11):

QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWIHTETGEPT YADDFKGRFAFSLETSANTAYLQINNLKNEDTATYFCGRSFSYGNLRAMDYWGQGTSVT VSS

antibody light chain nucleic acid sequence SEQ ID NO. 12

GATATCCAGATGACACAAACTACATCATCCTTGAGCGCCTCTTTGGGGGATCGTGTTA CAATATCCTGTTCAGCTTCCCAAGGGATCTATAATTACCTGAATTGGTTTCAGCAGAA GCCTGACGGCACCGTGAAGTTGCTGATCTACTATACCAGCAGCTTGCACTCCGGTGT GCCATCACGTTTCTCAGGCTCTGGGAGTGGTACTGATTTTAGCTTGACTATTTCTAAT TTGGAGCCCGAAGATATCGCAACATATTACTGTCAGCAGTACTCTAAATTCCCTTGGA CATTCGGAGGCGGCACCAATGTGGAGATTAAG

Antibody heavy chain nucleic acid sequence SEQ ID NO. 13

CAGATTCAACTGGTGCAGAGTGGGCCTGAGTTGAAGAAACCAGGGGAGACAGTCAA AATCAGTTGCAAGGCCAGTGGATACACATTTACTGATTATAGTATGCATTGGGTCAAA CAGGCTCCTGGCAAGGGCCTGAAATGGATGGGATGGATCCATACTGAAACCGGCGA ACCAACTTATGCCGATGACTTTAAGGGACGGTTCGCATTTTCACTGGAAACCTCAGCC AATACCGCTTATCTGCAGATAAATAATCTCAAAAATGAAGATACCGCTACCTACTTCTG TGGCAGAAGTTTCTCATATGGGAATCTTAGAGCCATGGATTATTGGGGCCAGGGAAC CTCCGTCACAGTCTCAAGT

Detection antibodies

LCDR1(SEQ ID NO:14)QSVSDN

LCDR2(SEQ ID NO:15)YAS

LCDR3(SEQ ID NO:16)QQSNSWPYT

Light chain variable region sequence (SEQ ID NO: 17):

DIVLTQSPATLSVTPGDSVSLSCRQSQSVSDNLHWYHQKSHESPRLLIKYASQSISGIPSR FSGSGSGTDFTLSINNVETEDFGMYFCQQSNSWPYTFGGGTKLEIK HCDR1(SEQ ID NO:18)GFNIKDTY

HCDR2(SEQ ID NO:19)IYPALGNT

HCDR3(SEQ ID NO:20)AREEDYSWYFDV

heavy chain variable region sequence (SEQ ID NO: 21):

QVQLEGSGGGLVQPGGSMLSCTASGFNIKDTYIHWVKQRPEQGLEWIGRIYPALGNTKY DPKFQDKATITADTSSNSAYLHLNSLTSEDTAVYYCAREEDYSWYFDVWGAGTTVTVSS antibody light chain nucleic acid sequence SEQ ID NO. 22

GACATAGTGCTCACTCAATCTCCTGCAACACTTTCTGTCACCCCCGGCGATAGCGTCT CTCTGAGCTGTCGTCAGTCACAGTCTGTTAGTGACAACCTTCATTGGTACCACCAGAA GTCTCACGAAAGCCCACGGCTGTTGATCAAGTACGCATCTCAGTCTATTTCCGGTATA CCATCTAGGTTCAGCGGATCTGGGAGTGGCACCGATTTTACTCTGAGCATCAACAAT GTAGAGACTGAAGATTTCGGTATGTACTTTTGCCAACAGAGTAACTCATGGCCTTATA CATTTGGCGGTGGAACAAAACTGGAGATCAAG

Antibody heavy chain nucleic acid sequence SEQ ID NO. 23

CAAGTTCAATTGGAGGGCTCAGGTGGAGGACTTGTACAACCTGGTGGTTCCATGCTC TCATGTACCGCCTCCGGCTTTAATATCAAAGATACATATATCCACTGGGTAAAACAGC GTCCTGAGCAAGGCCTGGAATGGATCGGACGCATTTATCCAGCCCTTGGCAATACAA AGTATGACCCTAAGTTTCAGGACAAGGCCACAATCACCGCAGACACTAGTTCTAACTC AGCCTACCTTCACCTCAACTCATTGACCTCTGAAGATACAGCAGTGTACTATTGTGCC AGGGAGGAGGACTACTCCTGGTACTTTGACGTCTGGGGTGCTGGCACTACCGTAAC CGTTAGTTCC。

Sequence listing

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Ser Gly Ser Gly Thr Asp Phe Ser Leu Thr Ile Ser Asn Leu Glu Pro

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Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile His Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Asn Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Gly Arg Ser Phe Ser Tyr Gly Asn Leu Arg Ala Met Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 12

<211> 321

<212> DNA/RNA

<213> Artificial sequence (Artificial sequence)

<400> 12

gatatccaga tgacacaaac tacatcatcc ttgagcgcct ctttggggga tcgtgttaca 60

atatcctgtt cagcttccca agggatctat aattacctga attggtttca gcagaagcct 120

gacggcaccg tgaagttgct gatctactat accagcagct tgcactccgg tgtgccatca 180

cgtttctcag gctctgggag tggtactgat tttagcttga ctatttctaa tttggagccc 240

gaagatatcg caacatatta ctgtcagcag tactctaaat tcccttggac attcggaggc 300

ggcaccaatg tggagattaa g 321

<210> 13

<211> 363

<212> DNA/RNA

<213> Artificial sequence (Artificial sequence)

<400> 13

cagattcaac tggtgcagag tgggcctgag ttgaagaaac caggggagac agtcaaaatc 60

agttgcaagg ccagtggata cacatttact gattatagta tgcattgggt caaacaggct 120

cctggcaagg gcctgaaatg gatgggatgg atccatactg aaaccggcga accaacttat 180

gccgatgact ttaagggacg gttcgcattt tcactggaaa cctcagccaa taccgcttat 240

ctgcagataa ataatctcaa aaatgaagat accgctacct acttctgtgg cagaagtttc 300

tcatatggga atcttagagc catggattat tggggccagg gaacctccgt cacagtctca 360

agt 363

<210> 14

<211> 6

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Gln Ser Val Ser Asp Asn

1 5

<210> 15

<211> 3

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

Tyr Ala Ser

1

<210> 16

<211> 9

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 16

Gln Gln Ser Asn Ser Trp Pro Tyr Thr

1 5

<210> 17

<211> 107

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 17

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

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Gln Ser Gln Ser Val Ser Asp Asn

20 25 30

Leu His Trp Tyr His Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Gln Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Asn Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Met Tyr Phe Cys Gln Gln Ser Asn Ser Trp Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 18

<211> 8

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

Gly Phe Asn Ile Lys Asp Thr Tyr

1 5

<210> 19

<211> 8

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 19

Ile Tyr Pro Ala Leu Gly Asn Thr

1 5

<210> 20

<211> 12

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 20

Ala Arg Glu Glu Asp Tyr Ser Trp Tyr Phe Asp Val

1 5 10

<210> 21

<211> 118

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 21

Gln Val Gln Leu Glu Gly Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Met Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr

20 25 30

Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly

35 40 45

Arg Ile Tyr Pro Ala Leu Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln

50 55 60

Asp Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Ser Ala Tyr Leu

65 70 75 80

His Leu Asn Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Glu Asp Tyr Ser Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 22

<211> 321

<212> DNA/RNA

<213> Artificial sequence (Artificial sequence)

<400> 22

gacatagtgc tcactcaatc tcctgcaaca ctttctgtca cccccggcga tagcgtctct 60

ctgagctgtc gtcagtcaca gtctgttagt gacaaccttc attggtacca ccagaagtct 120

cacgaaagcc cacggctgtt gatcaagtac gcatctcagt ctatttccgg tataccatct 180

aggttcagcg gatctgggag tggcaccgat tttactctga gcatcaacaa tgtagagact 240

gaagatttcg gtatgtactt ttgccaacag agtaactcat ggccttatac atttggcggt 300

ggaacaaaac tggagatcaa g 321

<210> 23

<211> 354

<212> DNA/RNA

<213> Artificial sequence (Artificial sequence)

<400> 23

caagttcaat tggagggctc aggtggagga cttgtacaac ctggtggttc catgctctca 60

tgtaccgcct ccggctttaa tatcaaagat acatatatcc actgggtaaa acagcgtcct 120

gagcaaggcc tggaatggat cggacgcatt tatccagccc ttggcaatac aaagtatgac 180

cctaagtttc aggacaaggc cacaatcacc gcagacacta gttctaactc agcctacctt 240

cacctcaact cattgacctc tgaagataca gcagtgtact attgtgccag ggaggaggac 300

tactcctggt actttgacgt ctggggtgct ggcactaccg taaccgttag ttcc 354

Claims

1. The OPG antibody pair is characterized by comprising a capture antibody and a detection antibody, wherein three CDR sequences of a light chain variable region of the capture antibody are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and three CDR sequences of a heavy chain variable region of the capture antibody are respectively shown as SEQ ID NO. 8, SEQ ID NO. 9 and SEQ ID NO. 10; the three CDR sequences of the light chain variable region of the detection antibody are respectively shown as SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, and the three CDR sequences of the heavy chain variable region of the detection antibody are respectively shown as SEQ ID NO. 18, SEQ ID NO. 19 and SEQ ID NO. 20; the light chain variable region of the capture antibody is shown as SEQ ID NO. 7, and the heavy chain variable region is shown as SEQ ID NO. 11; the light chain variable region of the detection antibody is shown as SEQ ID NO. 17, and the heavy chain variable region is shown as SEQ ID NO. 21.

2. The OPG antibody pair of claim 1, wherein the capture antibody and the detection antibody are any one of IgG type monoclonal, fab, F (ab ') 2, fab', scFv, or di-scFv.

3. The OPG antibody pair according to claim 1 or 2, wherein the OPG antibody pair detects OPG content in a biological sample by immunological detection.

4. A kit comprising the OPG antibody pair of claim 1 or 2.

5. Use of the antibody pair of claim 1 or 2 in the detection of OPG content, said use not involving diagnosis and treatment of disease.